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The characterization of Rab11a and trafficking mechanisms of polymeric immunoglobulin receptor (pIgR) in lacrimal gland acinar cells (LGACs)

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The characterization of Rab11a and trafficking mechanisms of polymeric immunoglobulin receptor (pIgR) in lacrimal gland acinar cells (LGACs)

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Content USCSCHOOLOFPHARMACY THE CHARACTERIZATION OF RABllA AND TRAFFICKING MECHANISMS OF POLYMERIC IMMUNOGLOBULIN RECEPTOR (pigR) IN LACRIMAL GLAND ACINAR CELLS (LGACS) Copyright 2013 By Shi (Ben) Xu A Diss ertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA. In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PHARMACEUTICAL SCIENCES) May 2013 Shi (Ben) Xu Dedication This diss ertation is dedicated to my beloved parents, grandparents, and fiancee Hu Chengye cm;l!&Sil'). Your constant support behind me is the power helping me finish PhD studies. A happy fam ily is the most valuable treasure in my life. This disse rta tion is also a gift for my dear mentor Dr. Sarah Hamm-Alvarez. It is my honor to work with her for 6 whole years . Acknowledgments It is my fo rtune to work with my mentor Dr. Sarah Harn m-Alvarez for 6 years . She is a great mentor with the charm of personality: the markers of outsta nding scientist, excelle nt admini strator, fr iendly teacher and good mother all co-localize on her and have deeply impr essed me. Beyond sci entific knowledge, I have learned a lot fr om her in many aspects of life. It is my great experience to work under her guida nce: our efforts in these years finally produced two good research papers and one review article on peer-reviewed sci entific journals. Without her generous support, I would not be able to obtain the PhD degree in Phar maceutical Sciences, the M.S. degree in Regulatory Science, as well the Regulatory Aff airs Certification (RAC US, RAC EU). I must express my sincere thanks to my lovely mentor for the achievements I have made in these years. I would also like to thank Dr. Curtis Okamoto for all his guidance in the studies of Rablla and pigR. He kindly gave me valua ble edifications in science, and also helped me improve my writing skills. I would like to thank my other committee members, Dr. Judy Garner, Dr. Austin Mirchef f, Dr. Roger Duncan and Dr. Bangyan Stiles, for all their kind help and adv ice through the years . I am very thankful to all my lab-mates: we have worked together several years and been good fr iends. I would particularly thank Fra ncie Yarber for all her technical expertise on the lacrimal gland acinar cell culturing and for accommoda ting all the preps 11 to our schedules. She has been the lovely mom for our whole laboratory. I would like to thank Dr. Eunbyul Evans, Dr. Maria Edman, Dr. Guoyong Sun, Dr. Jiansong Xie, Dr. Lilian Chiang, Dr. Janette Contreras for their adv ice on many experiments. I would also thank my dear collea gues Zhen Meng, Pang-yu Hsueh, Srikanth Reddy Janga, Mihir Shah and Hua Pei. Our laboratory is like a big fam ily, and the days working with you is a beautiful piece of memory. I want to express my special thanks to Linlin Ma. We have worked together for 2 years on two papers on scien tific journa ls. As a smart student, she made solid efforts to accel erate our resea rch, and it is my great pleasure to instruct her during her Master 's studies. Last, but not least, I cannot thank my family enough for all their love and support over these years . My parents, Huang Yan CJ!iEf) and Xu Bingj un C%i.f;Ei), have been my strongest spiritual support and helped me overcome many difficulties. My grandparents, Huang Ben CJ!zls:) and Zhuang Youfang CJ±:f;!J35'), are my dearest fam ily members who influ enced me most deeply. My fiance e Hu Chen gye cm;l!&Bi!') is the most bea utiful color in my PhD studies. Her love and care accompa nied me fr om my academic years to career life. I owe all of them for every piece of achievement I made on the land of the United States. 111 Table of Contents List of Figures Abbreviations Abstract Chapter I. Introduction The morph ology and physiology of the lacrimal gland acinar cells Polymeric immunoglobulin receptor Associa tion of Rab11 a with the transcytotic pathway The regulated secr etory pathway in LGACs PKC-s involved in exocytosis in LGACs The pur poses of this study Chapter II. Materials and Methods Materials Prepara tion of primary LGAC and treatment conditions Production and purif ica tion of Adenovirus constructs Adenovirus transduction Baculovirus transduction Cell fr actionation Generation of rabbit B-e ell hybridomas for dlgA production and purification of dlgA Uptake and accumulation of dlgA Confocal microscope and image processing Live cell confocal fluorescence imaging Indirect immunof luorescence Uptake of antibody speci fically bound to plgR Quantification of �-hexosaminidase and hSC released by LGACs Co-Immu noprecipita tion Statistics Vll X XI 1 1 4 7 10 11 12 14 14 16 16 20 21 22 22 25 26 26 27 27 28 29 30 IV Chapter III. The characterization of Rablla in rabbit lacrimal gland acinar cells (LGACs) Endogenous Rablla in primary LGACs Over-expre ssion of EGFP-Rablla in primary LGACs EGFP-Rablla labels a membrane compa rtment that is distinct fr om early endosomes and lysosomes Rablla-enriched vesicles are distinct fr om the regulated secretory pathway plgR and dlgA are sorted into the Rablla-regulated transcytotic pathway Traffi cking ofRablla-enriched vesicles involves the MT network The traffi cking ofRablla-enriched vesicles associa ted with the F -actin network Chapter IV. The characterization of pigR traff icking in LGACs Expression of constructs of fluorescent protein fused to the plgR cytoplasmic domain in primary LGACs Traffi cking of plgR in the transcytotic pathway Basal-to-apical transport ofbasolaterally endocytosed plgR is dependent on the MT network Traffi cking of plgR in the regulated secretory pathway Myosin V c is associa ted with Rab3 D-enriched mature secretory vesicles plgR that enters the transcytotic pathway remains distinct fr om the regulated secr etory pathway The transcytotic pathway and the regulated secretory pathway distinctively media te apically-targeted traffi cking of plgR PKC-s is the common regulator for apical release of plgR through both the Rablla-regulated tra nscytotic pathway and the Rab3D-regulated secretory pathway Chapter V. Discussion: The mechanisms of pigR trafficking in LGACs 31 31 34 35 39 43 46 48 56 56 59 71 72 80 82 89 97 103 v Referen ces Appendices Protein sequences of EGFP-Rablla constructs Protein sequence ofhplgR-EGFP Protein sequence ofmCherry- Rab3D 118 126 126 127 127 VI Figure 1.1 Figure 1.2 Figure 2.1 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 3.8 Figure 3.9 Figure 3.10 Figure 4. 1 List of Figures Schematic diagram of the morph ology of lacrimal gland and primary LGACs Schematic structure of the human plgR Identification of rabbit dlgA produc ed by hybridoma Endogenous and over-expressed Rablla in LGACs The Ra bllb-enriched membrane compartment 1s not co localized with plgR The Ra blla-enriched membrane compartment 1s not co localized with early endocytotic or lysosomal markers The Ra blla-enriched membrane compa rtment is distinct fr om the Rab27b-enriched secr etory vesicles The expressiOn mCherr y-myosin Vb localization of Rab3 D of EGFP-Rablla constructs and tail did not significa ntly alter the The expression of EGFP-Rablla constructs did not signi ficantly alter the secr etory function of LGACs Tra nscytosis ofpl gR and dlgA is regulated by Rablla Rablla-enriched vesicles are associa ted with MTs and pl50 of the dynactin complex Rablla vesicle movements utilize the myosin Vb motor Myosin Vb plays a crucial terminal regulatory role m the Rablla-regulated tra nscytotic pathway Characterization of the hplgR-EGFP construct 3 6 24 32 34 36 38 40 42 44 47 50 54 58 Vll Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10 Figure 4.11 Figure 4.12 Figure 4.13 Figure 4.14 Goat and mouse anti-hSC antibodies distinguish hSC fr om endogenous rabbit SC Traffi cking of basola terally endocytosed plgR in LGACs Sheep anti-SC serum specifically recognizes plgR on the basola teral membrane of LGACs Rablla regulates the basal-to-apical transcytosis of plgR Nocodazole inhibited the basal-to-apical traffi cking of basola terally endocytosed plgR Characterization of the regulated secretory pathway in LGACs Characterization of mCherry- Rab3D expressed in LGACs The distribution of hplgR-EGFP in the regulated secretory pathway and the transcytotic pathway Syncollin-GFP-enriched vesicles mCherry-Rab 3D-enriched vesicles in resting LGACs and Syncollin-GFP-enriched vesicles and mCherry-Rab 3D-enriched vesicles in LGACs stimulated with CCh Characterization of over-expressed mCherry-myo sin Vb tail and EGFP-myosin V c tail in LGACs plgR endocytosed fr om the basolateral membrane enters the transcytotic pathway but does not intersect with the regulated secretory pathway CCh accelerates the SC release on the apical membrane of LGAC acini 60 63 65 68 70 73 74 77 79 80 84 87 91 V111 Figure 4.15 Figure 4.16 Figure 4.17 Figure 4.18 Figure 5.1 Apically-targe ted traffi cking of plgR is regulated by the transcytotic pathway and the regulated secr etory pathway distinctively Inhibition of basal-to-apical transcytosis of endogenous plgR by over-expression of PKC-s DN Inhibition of apical release of endogenous plgR by PKC-s DN Expression of PKC-s DN does not alter the localization of Rab3D or Rabll Working model of the traffi cking ofplgR and dlgA in LGACs 95 98 100 101 112 IX Ad APM BLM CA CCh dlgA DN DIC EGFP GDI hSC hplgR LG LGAC MDCK cell MT MTOC PCM PKC-s plgR RFP sc SDS-P AGE slgA YFP WT Abbreviations Adenovirus apical membrane basolateral membrane constitutiv ely active carbachol dimeric immunoglobulin A dominant nega tive diff erential interference microscopy enhanced green fluorescent protein GDP-dissoc iation inhibitor human secr etory component contrast human polymeric immu noglobulin receptor lacrimal gland lacrimal gland acinar cell Martin-Darby canine kidney cell microtubule microtubule orga nizing center Peter's Complete Medium protein kinase C-s polymeric immunoglobulin receptor red fluorescence protein secr etory component sodium dodecyl sulf ate polyacrylamide gel electrophor esis secr etory IgA yellow fluorescent protein wild type X Abstract This study chara cterizes the intracellular traffi cking pathway of polymeric immunoglobulin receptor (plgR) that mediates the basal-to-apical transcytosis of dimeric immunoglobulin A ( dlgA) in lacrimal gland acinar cells (LGACs ). This study demonstrates that the traffi cking of plgR to the apical plasma membrane in LGACs involves two distinct pathwa ys: the transcytotic pathway and the regulated secretory pathway. Rab3D and myosin V c fa cili tate the release of a distinct pool of plgR fr om the regulated secr etory pathway to the apical plasma membrane in a process stimulated by the muscarinic cholinergic agonist carbachol (CCh); whereas Rab27b, another regulator of the regulated secr etory pathway, is not assoc iated with the traffi cking of plgR. The transcytotic pathway, regulated by Rab lla and myosin Vb, is distinct fr om the regulated secr etory pathways, and serves as the main source of plgR transported constitutiv ely to the apical plasma membrane in unstimulated cells. Comparison of dlgA uptake in LGACs expressing wild type and domina nt negative (DN) EGFP-Rablla showed that the rapid exocytosis of dlgA was inhibited in acini expressing the DN mutant protein, which additi onally , redistributed subapical plgR. EGFP-Rablla DN also decreased the accumulation of basola terally endocytosed plgR in the subapical domain. The traffi cking of EGFP-Rablla-enriched vesicles was regulated by microtu bule-based and myosin Vb motors at distinct steps. My data shows that Rablla is a crucial regulator XI of dlgA traffi cking in primary acinar secretory epithelial cells and further support a role for microtu bules, cytoplasmic dynein, actin filaments and myosin Vb in the maintenance of the Rab 11 a compartment in specialized epithelial cells with exocrine secretory functions. plgR is known to media te epithelial cell transcytosis of dlgA and its release into mucosal secretions as part of the mucosal defense system of ocular surf ace. This study reveals the complexity of the traffi cking of plgR to the apical plasma membrane in specialized epithelial cells with exocrine secretory functions; in LGACs, traff icking of plgR involves both the transcytotic pathway and at least one arm of the regulated secretory pathway. By speci fically tracking basola terally endocytosed plgR, this study demonstrates that the plgR sorted into the Rab lla-regulated transcytotic pathway does not access the regulated secretory pathways assoc iated with myosin V c. However, previous work in LGACs that is expanded here has shown that a subpopulation of plgR resides in the Ra b3D-enriched secretory membrane compartment. Myosin Vb and myosin V c motors modulate release of proteins fr om the Rablla-regulated transcytotic pathway and the Ra b3D-regulated secr etory pathway in LGAC, respectively. Confocal fluorescence microscopy and biochemical assays showed that inhibition of myosin Vb and myosin V c fun ctions by over-expression of their dominant negative mutants each significa ntly but differen tially impaired aspects of xu apically-targeted plgR traffi cking and SC release, suggesting that these motors fun ction to regulate plgR traff icking in both the tra nscytotic and exocytotic pathwa ys. The mature secretory vesicle population labeled by Rab27b in LGACs was lar gely devoid of plgR cargo, suggesting that Rab3D-enriched mature secretory vesicles specifically carry a particular subset of cargo proteins from the trans-Golgi network to the apical plasma membrane. PKC-s DN inhibited the apical release of plgR via the Rablla-regulated transcytotic pathway and the Ra b3D-reg ulated secretory pathway. Our previous research also showed that PKC-s DN inhibited the apical release of syncollin-GFP via the Rab27b-regulated secretory pathway. These obser vations indicate that PKC-s is a common regulator for cargo release at the apical plasma membrane in LGACs of these 3 pathways, likely by regulating the reorganization of F-actin in the subapical domain. X111 Chapter 1: Introduction The morphology and physiology of the lacrimal gland acinar cells (LGACs) The lacrimal glands responsible for tear protein and fluid secretion are comprised of tubular secretory epithelium orga nized into lobes and ductal drain system. The ducts that originate fr om different lobes converge to form larger ducts that finally drain onto the ocular surf ace (Millar et a!., 1996; Walcott, 1998), see Figure 1.1. Lacrimal gland acinar cells (LGACs) are epithelial cells specialized for production, packaging and regulated secretion of diverse tear proteins. The plasma cells present in the interstitial spaces of the lacrimal gland secrete immunoglobulin A (IgA), which contributes to mucosal immunity and protects the ocular surf ace fr om infection (Franklin, 1989; Cameron et a!., 1995). The LGACs that account for 80% of the cells present in the lacrimal gland synt hesize and secrete tear-specific proteins and water (Walcott, 1998; Zoukhri, 2006). The LGACs are also responsible for transcytose the IgA secreted by the plasma cells fr om the interstitial space into the lumen of the gland (Walcott, 1998). The LGACs form tight junctions loca ted on the lateral membranes near the cell apices, thus they form a lumen surrounded by the apical plasma membrane (APM). Large secretory vesicles are loca ted in the subapical region of the LGACs (Rismondo et a!., 1994; Wang et a!., 2003; Jerdeva, G. et a!., 2005). F-actin networks are located beneath the plasma membranes, and particularly enriched benea th the APM (Da Costa et a!., 2003; I Jerdeva, G. et a!., 2005). In this study, primary LGACs isola ted fr om rabbit lacrimal glands are used as the model to study the phys iological functions of LGACs. After 2�3 days ' culture in vitro, primary LGACs aggregate and reconstitute acinar-like structure s: the APM gather to form lumena inside the acinar-like cell cluster, and most lumena have openings accessible to the culture media (Jerdeva, G. et a!., 2005). The basola teral plasma membrane (BLM) surrounds the acinar-like cell cluster (Jerdeva, G. et a!., 2005). 2 Figure 1.1 A B 30 BLM - -- _ . . , . -- - - - --·-- -- - -- � • Nucleus Ductal cells 20 Confocal plane Cell Lumena 3 Figure 1.1 Schematic diagram of the morphology of lacrimal gland and primary LGACs A) The diagram represents a lobe of the tubular secretory epithelium of the lacrimal gland. Secretory proteins and fluid released into the lumens of multip le acini drain into ducts . Ducts fr om different lobes converge to form larger ducts that finally drain onto the ocular surf ace. B) Schematic of acinar-like structures fo rmed by primary LGACs. Left: 3D structure, Right: 2D cross-sec tion of the 3D structure, represen ting the confocal plane fr om which the microscopic images were taken. APM: apical membrane, BLM: basola teral membrane, CJ: cell junction, L: lumen. Polymeric immunog lobulin rece ptor LGACs uti lize transcytotic mechanisms to transfer key proteins such as dimeric IgA ( dlgA) into the tear fluid. dlgA comprises two IgA monomers coupled by J chain, and synt hesized by plasma cells in the lacrimal gland interstitial space (Walcott, 1998). dlgA is highly abundant in tear fluid in the form of secretory IgA (slgA), i.e. dlgA associated with the secretory component (SC) (Wu et a!., 2006). slgA in tears produced by the LG is the predominant antibody protecting the ocular surf ace against antigenic challenge (Gudmun dsson et a!., 1985; Allansmith et a!., 1987) and is a critical element of mucosal immunity (Allansmith et a!., 1987; Childers et a!., 1989; Corthesy and Kraehenbuhl, 1999). Despite its central role in lacrimal gland and ocular surf ace physiolog y, the transcytotic mechanism that transfers slgA into the tears remains poorly unders tood. Basal-to-apical transcytosis of dlgA into mucosal fluids depends on the polymeric 4 immunoglobulin receptor (plgR) (Childers et a!., 1989; Mostov et a!., 1995; Corthesy and Kraehenbuhl, 1999; Rojas and Apodaca, 2002b ). plgR is expressed in various types of epithelial cells, e.g. hepatocytes (Perez et a!., 1989), intestinal epithelial cells (Bruno et a!., 201 0), parotid gland acinar cells (Carpenter et a!., 2004), and LGACs (Evans et a!., 2008). plgR is an Fe receptor which fa cilitates the secretion of IgA and IgM in epithelial cells. It is unique among Fe receptors for making only a single-trip across epithelial cells before being cleaved at the apical surface (Kaet zel, 2005). The N' -terminal extracellular domain of plgR, which is known as SC after proteolytic cleavage and release, is the ligand-binding portion of the recep tor. Among the 5 immunoglobulin-like domains in the extracellular domain of plgR, domain I of is both necessa ry and sufficient for binding polymeric IgA and IgM (Kaetz el, 2005). Seven sites for N-glycosylation have been confirmed in the immu noglobulin-like domains of human plgR (Kaet zel, 2005), but N-glycosy lation is not necessa ry for binding immunoglobulin ligands (Hamburger et a!., 2004). The C' -terminal cytoplasmic domain of plgR is responsible for downstream signaling: serine 664 and 726 on the cytoplasmic domain have been identified as crucial signals for the internalization and transcytosis of plgR (Hirt et a!., 1993; Okamoto et a!., 1994). 5 Figure 1.2 � N-glycosylation COOH Charlotte S. Kaetzel Immunological Reviews 2005 Vol. 206: 83-99 Figure 1.2 Schematic structure of the human plgR. (Kaetzel, 2005) The plgR is a single-transmembrane protein, with an extracellular ligand binding domain comprising five domains with homology to immunoglobulin variable regions. The three complementarity-dete1mining regions (CDRs) in domain 1 fmm a non-covalent binding surface for dlgA. During transcytosis, a disulfide bridge is formed between domain 5 of plgR and the Fca. region of dlgA. Peptide motifs in domains 3 and 4 cooperate to form a binding surface for the SpsA protein of Streptococcus pneumoniae. A peptide of unknown structure links domain 5 to the membrane-spanning region and contains site(s) for proteolytic cleavage of plgR to generate secretory component (SC). Seven N-glycosylation sites on domains 1, 2, 4, and 5 contribute to innate immune functions of SC. The cytoplasmic domain of plgR contains serine sites available for phosphorylation, and these sites provide highly conserved signals for intracellular sorting, endocytosis, and transcytosis In transfected Madin-Darby canine kidney (MDCK) cells, plgR appears to traffic from the trans-Golgi network to the BLM. With or without its bound ligand, plgR is basolaterally endocytosed and transported through a series of endosomal compartments to the APM (Apodaca et al., 1994; Barroso and Sztul, 1994; Mostov et al., 1995). At the APM, the plgR extracellular domain is proteolytically cleaved and released as either SC bound to dig A or fi·ee SC. However, plgR is not endogenously expressed in MDCK cells 6 without transf ection, and MDCK cells do not secrete other proteins through either transcytotic pathways or regulated secr etory pathways equivalent to those expressed in exocrine acinar cells. Therefore, primary LGAC represents a valuable model for studying the function of Rablla in regulating transcytotic mechanisms in specialized secr etory cells that are also robustly engaged in transcytosis of key mucosal defense proteins. The LG serves as the major source of tear slgA and SC (Gudmun dsson et a!., 1985). It has been shown that, unusually, LGACs divert some fr action of their plgR intracellularly to generate fr ee SC, which is stored in and released fr om Rab3D-enriched secretory vesicles in the subapical domain (Evans et a!., 2008). LGACs also respond to cholinergic secretagogue stimulation by increasing SC secretion through the secr etory and tra nscytotic mechanisms (Evans et a!., 2008). The relationship between these unique exocytotic pathways for SC and the constitutive transcytotic pathway that is responsible for binding interstitial dlgA and excreting slgA at the lumen is, however, not well-cha racterized in these cells previously. Associa tion of Rablla with the transcvt otic pathway Rab proteins constitute the largest fam ily of small monomeric GTPases, and they regulate intracellular traffi cking by recruiting membrane-tethering and docking fa ctors (Pfeffer, 2001). Rab GTPases cycle between active GTP-bound and inactive GDP-bound 7 fo rms to carry out their functions (S chwartz et a!., 2008). Such cycling is regulated by Rab GDP-dissoc iation inhibitors (GDis) (Shisheva et a!., 1999), Rab GTPase- activating proteins (GAPs), and Rab-GTP exchange fa ctors (GEFs) (van de Graaf et a!., 2006). Moreover, single amino acid substitutions is capable of generating mutant Rab proteins that are locked either in the GTP-bound or GDP-bound sta te. The Ra bll GTPases have 2 isof orms: Rablla and Rabllb, both of which are ubiquitou sly expressed in mammalian cells (Lapierr e et a!., 2003). Although Rablla and Rab 11 b share 90% identity in their amino acid sequences, they show crucial diff erences in their crystal structur es (Scapin et a!., 2006), and localize to different membrane compartments in MDCK and gastric parietal cells (Lapierre et a!., 2003). Rablla is thought to participate in the basola teral-to-apical transcytosis of plgR and dlgA in transfected MDCK cells expressing plgR, because S25N GDP-locked DN mutant Rablla strongly inhibits this process (Wang et a!., 2000). However, Rablla also appears to be involved in regulated secr etory processes in specialized cells: in bladder um brella cells, Rablla is associa ted with discoid al/fus iform exocytotic vesicles, and expression of Rablla DN inhibited regulated secretion from these vesicles (Khandelwal et a!., 2008). This study chara cterized the Rablla compartment in LGACs as well as its function in transcytosis and/or exocytosis, and also determined the roles of cytoskeletal filaments and their associated motors involved in Rablla-enriched vesicle traffi cking. Yeast 8 two-hybrid analysis has demonstra ted that the tail region of the myosin Vb motor directly interacts with Rabll-FIP2, a downstream eff ector of Rablla (Lapierre et a!., 2001; Hales et a!., 2002). Yeast two-hybrid analysis also showed that Rablla wildtype (WT) was capable of interacting with the myosin Vb tail, whereas Rablla DN was not (Lapierre et a!., 2001). Moreover, expression of the myosin Vb tail inhibited basola teral-to-apical transcytosis of dlgA in MDCK cells, trapping dlgA intracellularly (Lapierre et a!., 2001). Rablla and myosin Vb proved to be ess ential for the fo rmation of bile canaliculus in WIF-B9 cells (Wakab ayashi et a!., 2005) and for the recycling of the M4 muscarinic acetyl choline receptor in PC12 cells (Volpicelli et a!., 2002). Previous study also showed that Rablla-enriched compartments interact with microtubu les (MTs). In WIF-B9 cells, Rablla was assoc iated with an endosomal complex that traff icked on MTs but could not traverse the pericanalicular actin and fuse with the canalicular membrane (Wakab ayashi et a!., 2005) Based on these knowledge, we hypothe sized that both myosin Vb and the MT network might participate in the traffi cking ofRablla-enriched vesicles in LGACs. My study demonstrates that in LGACs, Rablla participates m the constitu tive transcytotic pathway, which is distinct fr om lysosomal pathway, Rab3D-regulated secretory pathway and Rab27b-regulated secr etory pathway. There is no evidence for Rablla's involvement in regulated secretion of LGACs, in contrast to research carried 9 out in bladder epithelial cells (Khandelwal et a!., 2008) and HCl-se creting gastric parietal cells (Duman et a!., 1999). This study also confirms, in acinar cells engaged functionally in transcytosis of dlgA to a mucosal surf ace, that Rablla plays an impor tant role in the mucosal immune system. The regulated secr etory pathway in LGACs Previous studies have shown that, the Rab GTPase Rab3D participate m the regulated secretions of various exocrine cells: Rab3D is a speci fic marker for zymogen granu les in gastric chief cells of rats and rabbits (Tang et a!., 1996). Overexpre ssion of Rab3D elevates regulated amylase secretion fr om mouse pancreatic acini (Ohnishi et a!., 1997), and DN Rab3D inhibits amylase release fr om mouse pancreatic acini (Chen et a!., 2002). In rat parotid glands, Rab3D localizes to zymogen granule membranes and is involved in regulated secretion of amylase (Raffa niello et a!., 1999). Previous study in our lab confirmed the direct interaction between Rab3D and plgR, and also showed that a pool of plgR and fr ee SC derived fr om plgR reside in Rab3D-enriched secr etory vesicles in the subapical domain, and the kinetics of SC release by LGACs fo llowing exposure to cholinergic agonist carbachol (CCh) exhibit a "burst" effect consi stent with rapid mobilization of a secr etory vesicles (Evans et a!., 2008). Thus in LGACs, plgR is present in the regulated secretory pathway, and can be cleaved intracellularly to create a pool of 10 SC available for rap id release by the disch arge of secretory vesicles. Therefore, it is reasonable to hypothe size that the traffi cking of plgR is differen tially regulated by the regulated secr etory and the transcytotic pathways in LGAC. Rab27b is another identif ied marker associated with regulated secretions in exocrine cells. Rab27b localizes to zymogen granu les and regulates exocytosis of amylase in pancreatic acinar cells, (Chen et a!., 2004). Rab27b also regulates amylase release fr om rat parotid acinar cells (Imai et a!., 2004). Previous study in our lab showed that, in LGACs, Rab27b speci fically labels a pool of large secr etory vesicles, which merge with the APM under the treatment of CCh (Chiang et a!., 2011) , and Rab27b-enriched secretory vesicles may originate fr om a sub-c ompartment of the Golgi or a sepa rate compartment working sequentially with the Golgi (Chiang et a!., 2012). Syncollin-GFP, an exogenous protein expressed in LG AC using an adenoviral vector, was fo und to be the cargo of Rab27b-enriched vesicles in LGACs (Chiang et a!., 201 1). However, the relation between plgR and the Rab27b-regulated secr etory pathway has not been characteriz ed in any previous study. PKC-s involved in exocvtosis in LGACs Our previous study has shown that protein kinase C-s (PKC-s) is involved in the exocytosis in LGACs. Over- expression of PKC-s signific antly decreased the 11 syncollin-GFP release into the culture superna tant, and this was the direct evidence showing the regulation of PKC-s on the regulated secretory pathway (Jerdeva et a!., 2005). Over-expre ssion of PKC-s also significantly decreased the SC released into the culture superna tant (Jerdeva et a!., 2005), indicating that PKC-s also regulates the intracellular traffi cking of plgR. However, the previous study did not cla rify whether PKC-s DN inhibited plgR traffi cking and SC release through the transcytotic pathway or the regulated secretory pathway. Therefore, I further investigated this previously unresolved question. The purp oses of this study The overall purp ose of this study is to charact erize the transcytotic and secretory pathways as well as their regulators involved in the traff icking of plgR in LGACs. This study identif ies the path taken by basola terally endocytosed plgR to reach the apical plasma membrane. This study identif ies different arms of the regulated secretory pathway, and the associa tion of plgR with various bio markers of the secretory pathway. This study also extends our knowledge of the cytoskeleton and motors that participate in the intracellular traff icking of plgR. This study specifically interrogates whether plgR sorted into the transcytotic pathway can access apically-targeted secr etory vesicles, thus providing the source of the 12 plg R/SC recovered in Rab3D-enriched mature secr etory vesicles. This analys is is ess ential to provide insig hts into the possible fun ction of the novel secreted pool of plgR and SC, specifically whether it is expected to be loaded with antibody as secretory IgA or present as fr ee SC. By selectively impairing transcytotic versus exocytotic traffi cking in LGAC, I show that the transcytotic pathway mediates basal-to-apical transport of plgR. However, the Rab3D-regulated secretory pathway appears to acquire plgR directly from the biosy nthetic pathway, and this pool of plgR remains inaccessible to basola terally endocytosed plgR or dlgA. Moreover, this study also shows that PKC-s regulates the traffi cking of plgR in both the transcytotic pathway and the regulated secr etory pathway. 13 Chapter II: Materials and Methods Materials Carbachol (CCh) was purchased fr om Sigma-Aldrich (St. Louis, MO, USA). Pepstatin A, tosyl-phenylalanyl -chloromethyl-keto ne, leupeptin, tosyl-lysyl-ch loromethyl ketone, soybean tryp sin inhibitor, and phenylmethane sulphanyl fluoride were also purchas ed fr om Sigma-Aldrich and were used in the protease inhibitor cocktail for preparation of cellular homogenates as previously described (Vilalta et a!., 1998). Alexa Fluor® 488-c onjugated goat anti-mouse, Alexa Fluor® 488-c onjugated donkey anti-rabbit, Alexa Fluor® 568-co njuga ted donkey anti-sheep, and Alexa Fluor® 568-co njugated goat anti-mouse seco ndary antibodies, Alexa Fluor® 647-c onjugated phalloidin, Tetramethyl Rhoda mine-Dextran 70000, Lysotracker® Red DND-99, and the Prolong® anti-fade Kit were purchas ed fr om Mole cular Probes/ Invitrogen (Carlsbad, CA, USA). Rabbit anti-Rablla serum was a kind gift fr om Dr. James Goldenring, Vanderbilt University. Goat polyclonal antibody to the extracellular domain (SC) of human plgR was purchased from R&D systems (Minnea polis, MN, USA). Mouse anti-green fluorescent protein (GFP) antibody, goat anti-myosin Vb antibody, pre-immune sheep serum and mouse IgG were purchased fr om Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-myosin V c antibody was raised against the tail region of myosin V c as previously described (Marchelletta et a!., 2008). Goat polyclonal antibody to the 14 extracellular domain of human plgR (anti-hSC) was purchas ed fr om R&D systems (Minnea polis, MN, USA). Mouse monoclonal anti-hSC antibody was purch ased fr om NeoMa rkers (Fremont, CA, USA). Mouse anti-green fluorescent protein (GFP) antibody, pre-immune sheep serum and mouse IgG were purchased fr om Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-red fluorescent protein (RFP) mouse monoclonal antibody that also recognizes mCherry was purcha sed fr om MBL (Woburn, MA, USA). Rabbit anti-Rab3D serum was raised against recombinant Rab3D expressed in bacteria as previously described (Evans et a!., 2008). Sheep anti-serum that recognizes the extracellular domain of plgR was prepared by Caprilogics (Hardwick, MA, USA) using SC fr om rabbit gall bladder bile as antigen. IRDy e®800-con jugated and IRDye®700-conjugated donkey anti-goat and goa t anti-mouse seco ndary antibodies, as well as IRDy e®800-conjugated Streptavidin, were purchas ed fr om Rockland (Gilbert sville, PA, USA). Blocking buffer was purchas ed fr om Li-COR Biosciences (Lincoln, NB, USA). Doxycycline was obtained fr om Clontech (Mo untain View, CA, USA). CellL ight 1M RFP-Ra b5a Bacmarn2.0 reagent was purchas ed fr om Molecular Probes/I nvitrogen (Carlsbad, CA, USA). Protein A/G -co njugated aga rose resm was purchas ed from Santa Cruz (S anta Cruz, CA, USA). EZ-Link® Sulfo-NHS-Biotin was purchas ed fr om Pierce/Thermo Scien tific (Rockford, FL, USA). Doxycycline was obtained fr om Clontech (Mou ntain View, CA, USA). Peter's Complete Media (PCM) 15 was prepared by mixing Ham's F-12 (Thermo Scie ntific, Rockford, FL, USA) and DMEM with 1.5 giL glucose (Mediatech, Manas sas, VA, USA) at a 1:1 ra tio and adding other reagents to the fo llowing final concentrations: penicillin 100 U/mL, streptomycin 0.1 mg/mL, linoleic acid 0.3 11M, n-butyric acid 2 mM, transferrin 5 11g/mL, insulin 5 11g/mL, sodium selenite 30 nM, hyd rocortisone 5 nM, laminin 4 fl g/mL, CCh 0.1 11M/mL and L-thy roxine 1 nM. All other reagents were the highest quality availa ble. Non-den atu ring lysis buffer was prepared as: 20 mM Tr is.HCl, 137 mM NaCl, 10% glycerol, 1% Trit on-X 100, 2 mM EDTA, pH�8. Prepara tion of primary LGAC and treatment conditions Female New Zealand White rabbits (1.8 -2.2 kg) were obtained fr om Irish Farms (Norco, CA). Isola tion of LGACs was in accordance with previously established protocol (Rismondo et al., 1994). LGACs cultured for 2-3 days in PCM aggregate into acinu s-li ke structures (Figure 3.1B) while individual cells within these structu res display distinct apical and basola teral domains and maintain secretory responses to CCh (Evans et al., 2008). Production and purification of Adenovirus constructs Human Rablla WT and S25N (DN) in the pEGFP-C2 plasmid, as well as the 16 plasmid mCherry- C1 -MyoVb-tail encoding rabbit myosin Vb tail fused with mCherry were kind gifts fr om Dr. James Goldenring, Vanderbilt University (Fan et a!., 2004). In these plasmids, enhanced Green Fluorescence Protein (EGFP) was fused to the N' terminus of human Rab 11 a. Human Rab 11 a has an identical protein sequence to rabbit Rab11a . The DN variant maintains a GDP-locked conformation, while the CA variant maintains a GTP-locked conforma tion. The pEGFP-C2 Rab11 a WT was digested with Nhe1 and Sail restriction enzymes at the 592 bp (5' end) and the 2046 bp (3 ' end) site of the pEGFP-C2 Rab11a WT vector, respec tively. The pEGFP-C2 Rab11 a WT was digested with Nhe1 and Eag1 restriction enzymes at the 592 bp (5' end) and the 1334 bp (3 ' end) site of the pEGFP-C2 Rab11a WT vector, respec tively. The pEGFP-C2 Rab11 a S20V was digested with Nhe1 and Sail restriction enzymes at the 592 bp (5' end) and the 2024 bp (3' end) site of the pEGFP-C2 Rab11a S20V vector, respec tively. The pEGFP-C2 Rab11a S25N was digested with Nhe1 and Sail restriction enzymes at the 592 bp (5' end) and the 2046 bp (3' end) site of the pEGFP-C2 Rab11 a S25N vector, respectively. The 1454 bp fr agment encoding EGFP-Rab11 a WT, the 742 bp fr agment encoding EGFP, the 1432 bp fr agment encoding EGFP-Rab11a S20V and the 1454 bp fr agment encoding EGFP-Rab11a S25N were subcloned into the pTRE- Shut tle2 vector fr om the AdenoX Te t-on® expression system kit (Clontech, Mountain View, CA) and further subcloned into the AdenoX System 1 viral DNA vector, respectively. The recombinant viral DNA 17 vectors lineariz ed with Pacl restriction enzyme were subsequently used to transfect the HEK-293- derived helper cell line, QBI in accordance to the manufacturer's protocol (Clontech, 2003). Ad EGFP-Rablla WT, Ad EGFP and Ad EGFP-Rablla DN (S25N) all require co-transduction with the Adeno-X Tet -On® regulatory virus and doxycycline induction for protein expression. For constructing Ad hplgR-EGFP, the open reading frame for human plgR precursor eDNA sequence was PCR amplified fr om cloning vector (Genbank Entry: BC1 10494.2) with the fo llowing primer pairs : Sense primer, 5 '- TA CTGCTAGCTCAACGGGAGAGAAGGAAGT GG-3 ', Anti-sense primer, 5 '-TA GACTCG AGATAG GCTTCCTGGGGGCCGTC -3'. The PCR product was subcloned into pCR® II-TOPO vector (Invitrogen, Carlsbad, CA, USA). The pCR® II-TOPO plgR was digested with Nhel and Xhol restriction enzymes, and The 2381bp fr agment encoding human plgR was inserted into pEGFP-N1 vector using these 2 enzyme clea vage sites. The pEGFP-N1 plgR was digested with Nhel and Notl restriction enzy mes. The 31 72bp fr agment encoding hplgR-EGFP was subcloned into the pTRE-Sh uttle2 vector fr om the AdenoX Tet -on® expression system kit (Clontech, Mountain View, CA), respectively; and further subcloned into the AdenoX System 1 viral DNA vector, respectively. The recombinant viral DNA vectors were lineariz ed with Pacl restriction enzyme and used to transfect the HEK-293-derived helper 18 cell line, QBI in accordance to the manufacturer's protocol. For constructing Ad mCherry-Ra b3D, mCherry with a 12-a a linker of 3 G-G-S-G sequence repeats was fused to the N' -terminal of rabbit Rab3D. The cloning vectors containing mCherry-Ra b3D sequence was sent to Vector Biolabs (Philadelphia, PA, USA) for customized construction of Adenovir us. Ad hplgR-EGFP and Ad mCherr y-Rab3D both req mre co-transduction with the Adeno-X Tet -On® regulatory virus and doxycycline induction for protein expression. To generate the adenoviral construct for myosin Vb tail, the entire open reading fr ame for mCherry-myo sin Vb tail was PCR amplified with the fo llowing primer pairs: Sense primer, 5 'taaaggccttac cgcca tgcattag3 '; Anti-sense primer, 5 'tttaggcctaccac aactagaa tgc3 '. The PCR product was digested with Stul and then inserted into adenoviral shuttle plasmid pDC311 (Microbix Biosystems Inc., Canada), resulting Ill pDC311 /mCherr y-MyoVb-tail. Doxycycline-inducible Ad EGFP-full-length-myosin Vc based on AdenoX Tet -On® system is described in our previous study (Marc helle tta et a!., 2008). Ad EGFP-myosin V c tail was constructed based on EGFP-myosin V c tail plasmid as a kind gift of Richard Cheney (University of North Carolina at Chapel Hill), as described in previous study in our lab (Marchelletta et a!., 2008). Ad PKC-s DN/GFP was a kind 19 gift fr om George King (Harvard University) and Darlene Dartt (Harvard University), and has been used in a previous study in our lab (Jerdeva et a!., 2005). Ad YFP-Rab27b was a kind gift fr om Dr. Serhan Karvar (Division of Gastrointestinal & Liver, University of Southern Calif orn ia). rAV cMv LifeAct-TagRFP was purch ased fr om Ibidi GmbH (Martinsried, Germany). All Ad vectors were amplified in QBI HEK293 cells according to established protocols (Wang et a!., 2003). Adenovirus transduction LGACs were transduced on the 2 n d day of culture. LGACs seeded in culture dishes were co-i ncubated for 2 hours at 37°C with each of the inducible Ad constructs and the Adeno-X Te t-On® regulatory virus, at a multip licity of infection (MOl) of 5 for each virus. The Adeno-X Tet -On® regulatory virus encodes a regulatory protein that recognizes the "reverse" Tet repressor (rTetR) upstream of the DNA sequences encoding fluorescent protein-f us ed plgR in our Ad constructs, and therefore ind uces the expression of fluorescent protein-fused plgR in LGACs. Doxycycline was added at a concentration of 0. 1 11g/mL (for Ad hplgR-EGFP) or I 11g/mL (for other inducible Ad constructs) after the removal of virus and replacement of culture medium. Ad mCherr y-myosin Vb tail, Ad EGFP-myosin V c tail, Ad PKC-s DN/GFP, Ad YFP-Rab27b and rAV cMv Lif eAct-TagRFP were constructs that lead to consti tutive 20 expression without induction of doxycycline. They were used alone at an MOl of 5 for 2 hours of incubation at 37°C for transduction. However, when these Ad constructs were used for co-transduction with the doxycyc line-inducible construc ts, the regulatory virus and doxycycline were also applied. After virus transduction, LGACs were cultured another 16-18 hours before analys is. For membrane isola tion or biotin-dlgA uptake assays, LGACs on the 2n d day of culture were collec ted by centrifuga tion at 50 x g for 5 minutes and re-suspended in culture medium at a density of 1.7 x 10 7 cells/mL. LGACs were co-i ncubated for 2 hours at 37°C with the Ad EGFP-Rablla WT or DN constructs and with the Adeno-X Te t-On® regulatory virus, at a multip licity of infection (MOl) of 5 for each virus, respectively. Then the cells were washed with fr esh medium, and reseeded in culture dishes at 5.4 x 10 6 cells/mL in fr esh medium. The cells were cultured with doxycycline as described above for another 16-18 hours before use. Baculovirus transduction Culture medium fr om LGACs on the 2n d day of culture was aspirated, and fr esh PCM containing CellL ight 1M RFP-Rab5a Bacmam 2.0 reagent was added to reach a final concentration of 30 parti cles per cell, as suggested by the manufacturer's protocol. The cells were gently shaken at 3TC for I hour to maximize transduction efficiency, and 21 then cultured for another 16-18 hour before analysis. Cell fr actionation �1 .60 x 10 8 LGACs were homogenized with a Dounce tissue homogenizer for 20 cycles in 2 mL homogenization buffer (250 mM sucrose, 1 mM EDT A, 3 mM imida zole, pH 7.4) with prote ase inhibitor cocktail (1 mM PMSF, 1. 75 11g/ml aprotinin, 2.5 11g/ml soybean tryp sin inhibitor, 1 11g/ml chymostatin, 1 11g/ml pepstatin A, 1 11g/ml leupeptin). The homogenate was centrifuged at 800 x g for 10 minutes. The post-nuclear superna tant (PNS) was centrifuged in a Sorvall® RC Ml2 0EX ultracentrif uge (Thermo Scie ntific, Rockford, FL, USA) at 15,000 rpm for 1 hour. The superna tant (Si) and pellets (Pi) re-suspended in homogenization buffer were further analyzed by Western blotting. Generation of rabbit B-cell hy bridomas for dlgA production and purifi cation of dlgA LG fr om 3 fe male �4 kg New Zealand White rabbits fr om Irish Farms (Norco, CA, USA) were collec ted, minced and digested to isola te the interstitial cells as in previously established methods (Guo et al., 2000). Interstitial cells were fr ozen and sent to Epitomics Inc. (Burlingame, CA, USA) for fusion with a proprietary rabbit myeloma cell line. The 3 hybridoma multi -clones with the highest dlgA yields were selected by ELISA. The coating and detection antibody used for analysis were goa t Anti -rabbit IgA and 22 HRP-c onjugated goat anti-rabbit IgG, respe ctively, both fr om Abeam (Cambridge, MA, USA). Each multi -clone was further expanded to 12 subclones, from which 9 subclones with the highest dlgA yields were selected. The rabbit dlgA produced by clone 13-9 was used in this study. Clone 13-9 hybridoma cells were cultured in RPMI 1640 medium, with cell density maintained between 1 x 10 5 and 1 x 10 6 cells/mL. The culture superna tant was concentrated by centrifuga tion at 3,000 x g with 100 kDa centrifugal filter (Millipore, Billerica, MA, USA), filtered through a 0.2 11m filter, and then loaded into a PBS-eq uilibrated HiPrep 1M 16/60 Sephacryl S-300 HR column (GE Healthcare, Pisca taway, NJ, USA) insta lled on a DuoFlow chromatography system (Bio- Rad, Hercules, CA, USA) via a 5 mL in jection loop. dlgA was eluted with PBS at a flow rate of 1 mL/min and monitored by UV absorbance at 280 nm. Fractions containing dlgA were pooled and concentrated by centrifugation at 3,000 x g with 100 kD centrifugal filter. The purity of dlgA was determined by SDS-PAGE, and the concentration of dlgA was measured using a NanoDrop Spec trophotometer (NanoDrop products, Wilmington, DE, USA) (Figure 2.1). 23 Figure 2.1 kDa M 1 2 460 268 238 171 117 71 55 41 - Figure 2.1 Identification of rabbit dlgA produced by hybridoma. Purified dlgA produced by hybridoma clone 13-9.1 mg purified dlgA was resolved on 6% acrylamide gels under non-reducing conditions, and analyzed by Coomassie Blue staining, as well as western blotting using primary goat anti-mouse IgA antibody, and secondary IRDye800-conj ugated donkey anti -goat anti body. M: HiMark pre-stained HMW protein standard (Invitrogen, Carlsbad, CA). Lane 1, western blotting; lane 2, Coomassie Blue staining 24 Uptake and accumulation of dlgA Purif ied dlgA was incubated with a 10 -fold excess of EZ-Link® Sulf o-NHS-Biotin reagent at room temperature for 30 minutes according to the manufacturer's protocol. Excessive reagent was removed by dialysis in PBS at 4 'C overnight. For each treatment condition, 2.88 x 10 8 LGACs on the 3 ' d day of culture were re-suspended in 1 mL binding medium (PCM containing 20 mM HE PES, 2 mM CaCh, 2 mM MgCh and 3% (m/v) BSA), and incubated with 300 11g/mL biotin-dlgA at 37'C with shaking for 15 minutes. The cells were washed with ice-cold PCM, and then incubated with/without an additional 30-mi nute chase in binding medium at 37'C . Cells were then washed with ice-cold PCM, re-suspended with 1 mL Ham's buffer containing 0.04 mg tryp sin, and incubated on ice with shaking for 1 hour to remove biotin-dlgA from the cell surf ace. Cells were again washed with ice-cold Ham's buffer, and then lysed with 0. 1% Triton X-100 dissolved in PBS containing the protease inhibitor cocktail. The lysates were centrifuged at 10,000 x g for 10 minutes to remove cell debris, and the protein concentrations of the supe rnatant were determined with the Bio-Rad Protein Assay Kit according to the manufacturer's pro tocol. Aliquots of each cell lysa te containing 100 11g of protein were mixed with non-reducing SDS-P AGE loading buffer and electrophore sed on 6% acrylamide gels under non-reducing conditi ons. Western blot membranes were probed with IRDy e®800-conjugated Streptavidin, scanned using an Odyssey® Imaging 25 System fr om LI-COR, and biotin-dlgA was quantifi ed with the Odyssey® 1.1 software . The relative recovery of biotin-dlgA in each treatment group was calculated as the biotin-dlgA signal strength divided by the biotin-dlgA signal of the 15-minute pulse group. Confocal microsco pe and image process ing Images were taken with a Zeiss LSM 510 Meta inverted confocal microscope equipped with Argon, red He/N e, and green He/N e lasers, using 63 x oil immersion objective lens NA 1.4 . For construction of 3D models of LGACs, images taken from consec utive confocal planes at 0.4 11m intervals were pro jected at the Y-axis using Zeiss LSM image examiner. Live cell confocal fluorescence imaging LGACs were cultured on 35 mm glass coverslip-bottomed dishes (MatTek, Ashland, MA, USA) coated with Matri gel for 2 days, and transduced with Ad constructs as detai led above. On the 3' d day of culture, the cells were mounted and imaged in a 37 ° C incubation chamber. For analysis of intracellular orga nelle movements in live cells (Movies), images fr om a single confocal plane were taken sequentially at a fixed time interval of 10 seconds. 26 Treatment conditions for LGACs transduced with EGFP-Rablla Adenoviral constructs were as fo llows: CCh, 100 11M for 15 minutes; nocodazole, 33 11M on ice for 5 minu tes, and then at 3TC for 2 hours ; latrunculin B, 10 11M for 30 minu tes; nocodazole plus latrunculin B, similar to nocodazole treatment, except for that latrunculin B was added into the media to reach a working concentration of 10 11M at 1. 5-hour of 3TC incubati on. Indirect immunof luorescence After treatments, LGACs cultured on Mat rigel 1M -coa ted glass coverslips were fixed with 4% paraformaldehyde in PBS for 15 minu tes, incubated with 50 mM NH4Cl in PBS for 5 minu tes, and then permea bilized with 0.1% Triton X-100 in PBS for 10 minutes. After extensive washing with PBS, cells were blocked with 1% BSA in PBS overnight at 4 ° C. LGACs were then incubated with the appropriate primary and seco ndary antibodies and Alexa Fluor®-647-conjugated phalloidin. Uptake of antibody specifically bound to plgR LGACs cultured in 12 -well plate were incubated on 1ce for 1 hour with sheep anti-SC serum 1: 20 diluted in binding medium (PCM containing 20 mM HE PES, 2 mM CaCh, 2 mM MgCh and 3% (m/v) BSA), or goat anti-human SC (hSC) antibody diluted 27 m binding medium (2 11g/ well). After extensive washing with PCM, LGACs were incubated at 3TC for the uptake and subsequent traffi cking of antibody specifically bound to plgR. At 0-minute, 15 -minute, 30-minute or 60-mi nute time point, LGACs were fixed and labeled for the obser va tion of indirect fluorescence. Quantif ica tion of 13- hexosa minidase and hSC released by LGACs LGACs grown on Matri gel 1M -coa ted 12 -well plates were transduced on the 2 n d day of cultu re. On 3 ' d day of culture, the old culture medium was aspirated and 500 11L fr esh PCM was added into each well. The LGACs were then incubated for 30 minutes in order to ad just cell conditions before further treatments. The secretion of P-hexosa minidase was quantifi ed using previously established methods (Andersson et al., 2006). The hSC released into culture supe rnatant during this period is quan tified as "background hSC release". The cells were then trea ted with 100 11M CCh or not (resting stage) for additional 30 minu tes, and the culture superna tant was extracted for quan tification of secretion. Cell pellets in the plates were dissolved with 0.5 M NaOH and quan tified by the BCA protein assay for normalizing the secretion of P-hexosa minidase and hSC to protein in the cell pellets. Harvested culture superna tant was concentrated� 10 times with Vivaspin- 500 concentrator (Littleton, MA, USA). Western blot membranes were probed with goat anti-hSC primary antibody, IRDye®800-conjugated donkey anti-goat 28 seco ndary antibody, scanned using an Odyssey® Imaging System fr om LI-COR, and hSC was quantifi ed with the Odyssey® 3 softwa re. The amount of SC in each treatment minus the background hSC release in the 30-mi nute pre-trea tment incubation indicated the net release of hSC during the 30-mi nute treatment. This is an impo rtant improvement for the method of quantif ying hSC described in a previous study (Jerdeva, G. et a!., 2005); by subtracting the background hSC release, the hSC release ind uced by mechanical stress and other confounding fa ctors during operation can be eliminated, providing a clean result of net release of SC during 30-mi nute treatment. The amount of hSC released was presented in relative recovery, which was normalized to the hSC release by LGACs transduced with rAV cMv LifeAct- TagRFP under CCh stimula tion. Co-immunoprecipita tion LGACs co-expressing hplgR-EGFP and mCherry-Ra b3D were collected at the 3' d day of culture by centrifuga tion. LGACs were lysed in non-denaturing lysis buffer with protease inhibitors by 30-mi nute agitation at 4°C. 1 mL lysis buffer was used for 2 x 10 7 cells/sample. Ly sate were centrifuged at 10,000 x g for 10 minutes to remove insoluble debris, and the post-nucleus supe rnatant was further pre-cleared with 40 11L (50% v/v) CL2B Sepharose beads twice. For capturing mCherry-Rab 3D, 20 11L (50% v/v) protein AIG agarose resin beads, 10 11L rabbit anti-Rab3D serum or pre-immune rabbit serum was added into the superna tant. For capturing plgR-EGFP, 20 11L (50% v/v) protein A/G 29 aga rose resin beads, 10 11L goa t anti-hSC antibody or pre-immune goat serum was added into the supe rnatant. The mixture was applied with end-to-end rotation at 4°C overnight. The beads were washed with lysis buffer for 3 times and mixed with 4x SDS-PAGE loading buffer containing reducing reagent dithiothreitol (DTT). The sample was analyzed by Western blotting: membranes were probed with mouse anti-mCherry and goat anti-hSC primary antibodies, as well as appropriate IRDye®-conjugated secondary antibodies. Quantification of mCherr y-Rab3D and plgR-EGFP was performed with the Odyssey® 3 software . Statistics: For quan tification of hSC released by LGACs, 5 repetitions were per form ed with each repetition fr om a sepa rate prepara tion. A one-sample Wilcoxon signed rank test was applied to compare the hSC released by different treatment groups with the control. Student's t-test based on unequal varia nces was applied to compare the hSC released by different groups at the resting stage. For immunofluore scence and western blots, a minimum of 3 repetitions were performed with each repetition fr om a sepa rate prepara tion. 30 Chapter III: The characterization of Rabll a in rabbit lac rimal gland acinar cells (LGACs) Endogenous Rablla in primary LGACs Expression of endogenous Rablla in LGACs was confirmed by Western blotting and immunof luorescence. Membrane compartments and cytosol fr om primary LGAC homogenate were separated by high-speed centrifuga tion. Endogenous Rab 11 isof orms reside primarily in membrane compartments (Pi) rather than in cytosol (Si) (Figure 3.1A). As an integral membrane protein with a single transmembrane domain, plgR was also enriched in the Pi fr action. Isolated primary LGACs form spherical or ovoid clusters by the 2n d day of culture, mimicking the acinar- like structu res present in situ in the LG. The lumenal cavity or cavi ties located in the center of these acinar-like structures ty pically have accessible opening(s) to the culture medium. Individual acinar cells establish histiotyp ic pola rity during the process of reform ing the acinar-like structu res: APM surround the lumena, whereas BLM fa ces the culture medium and adj acent cells (Figure 3.1B). Fluorophore -conjugated phalloidin, which labels F -actin, delineates the dense cortical actin network underlying the APM, outlining the posi tion of the lumen, and the less dense cortical actin network underlying the BLM (Evans et a!., 2008). Images taken from a single confocal plane within the reconstituted acinus reveal lumena, APM and BLM as 31 .bownin the schematic (Figure 3.1B). Figure 3.1 A E (a) (b) Lys.ate Pi Si kCla 1 2 3 """ 1 2 3 R•b11 - ·- ... . - �et� - � sow .. �EGFP. . Rl:tl>11� plgR � I Actin - -- 30 ··· � i£; . : , ..... ,. B c ··- q;? ) ·- -- .. -=-.__ --- c D 37 ,. ., ,. -RaD11 20 &Lffi .-7 ----.. , r"V !�l·-.. e-� 1 I $ '\ - l - - < · - r y -1- Confocal pl�ne Cell Lu1nena 32 Figure 3.1 Endogenous and over -expressed Rablla in LGACs. A) Cell lysate was fr actionated as described in Metho ds. The lysate, Pi and Si fr actions were analyzed by Western blotting using primary mouse anti-Rab11, mouse anti-actin antibodies and sheep anti-rabbit SC serum, as well as secondary IRDye® 700 goat anti-mouse antibody and Alexa Fluor® 680-conjugated donkey anti-sheep antibody. B) Schematic of acinar-like structures form ed by primary LGAC s. Left: 3D structure, Right: 2D cross-s ection of the 3D structure, representing the confocal plane fr om which the microscopic images were taken. APM: apical membrane, BLM: basola teral membrane, CJ: cell junction, L: lumen. C) LGACs were fixed, permea bilized, and labeled with primary rabbit anti-Rab11 a and sheep anti-rabbit SC serum, as well as secondary FITC-c onjugated donkey anti-rabbit, Alexa Fluor® 568-conjugated donkey anti-sheep antibodies and Alexa Fluor® 647-c onjugated phalloidin. Immunofluorescence was observed by confocal microscopy to localize Rab11a and pigR in LGACs. D) LGACs were co-transduced with the AdenoX Te t-On® regulatory virus and EGFP-Rab11a WT, exposed to doxycycline, and fixed and labeled with Alexa Fluor® 647-con jugated phalloidin prior to imaging. (E) LGACs were co-transduced with EGFP-Rab11 a WT (1) , EGFP-Rab11 a DN (2) and AdenoX Te t-On. Non-transduced LGACs were used as a control (3). After induction with 1 ll g/mL doxycycline, the cells were lysed with SDS-PAGE sample buffer, resolved by 10% SDS-P AGE and analyzed by Western blotting using primary mouse anti-Rab11 (a) and anti-GFP (b) antibodies, as well as secondary IRDye®700-conjugated goa t anti-mouse antibody. The signal intensity was quan tified with the Odyssey® 1.1 software . The schematic images in C) and D) show the location of APM, BLM and the lumena (L). In all panels: star, lumena; scale bar, 5 11m Confocal fluorescence microscopy of LGACs labeled with antibody to Rab11 a and pigR showed that endogenous Rab11a was enriched in the subapical domain beneath the APM, and that Ra b11 a was also colocalized with pigR (Figure 3.1C), suggesting Rab11a 's assoc iation with the intracellular traffi cking of pigR. In contrast, confocal microscopy did not show extensive co localization of Rab 11 b with pigR (Figure 3.2). This suggests that Rab 11 b labels a different membrane compartment than Rab 11 a, and that Rab 11 b-enriched compartment is not likely to be involved in the intracellular 33 traff icking of pigR. Figure 3.2 Figure 3.2 The Rabllb-enriched membrane compartment is not colocalized with plgR. LGACs were fixed, permeabiliz ed, and labeled with primary rabbit anti-Rabll b and sheep anti-rabbit SC senun, secondary FITC-con jugated donkey anti-rabbit, Alexa-Fluor-568®-con jugated donkey anti-sheep antibodies, and Alexa-Fluor-647®-con jugated phalloidin. *, lum ena; scale bar: 5!J m Over-exp ression of EGFP-Rablla in primary LGACs Ad EGFP-Rablla WT, Ad EGFP-Rablla DN, and Ad EGFP constructs are all capable of eff iciently transducing rabbit primary LGACs. Af ter induction with 1 !Jg/mL doxycycline overnight, the expre ssion of these recombi nant proteins was verified by western blotting using both anti-Rabll and anti-GFP antibodies (Figure 3.1E). The expre ssion levels of the fusion proteins were 13-fold of the endogenous Rabl l on average (shown in the same lane). The direct flu orescence of exogenously expre ssed EGFP-Rablla WT in LGACs was also analyz ed by conf ocal microscopy. EGFP-Rabll a-emiched vesicles were mostly localized to the subapical domain, beneath 34 the APM. The localization pattern of EGFP-Rablla was the same as that of endogenous Rablla (Figure 3.1C, D), suggesting that the over-expression of EGFP-Rablla does not affe ct its localization relative to that of endogenous Rabl la. Live cell imaging revealed rapid, directed movement of EGFP-Rablla-enriched small vesicles in the subapical domain, as well as tubules extending between the apical and basola teral domains of the cell. Theref ore, we hypot hesized that the subapical pool of EGFP-Rablla-enriched vesicles might participate in the basal-to-apical transcytosis of plgR. EGFP-Rablla labels a membrane compartment that is distinct fr om early endosomes and ly sosomes Direct and indirect fluorescence confocal microscopy was applied to charact erize the compartmental identity of the EGFP-Rablla-enriched vesicles in LGACs. Tetram ethylRhodamine (TMR)-Dextran 70000 was used as a marker for fluid-phase endocytosis in live LGACs. EGFP-Rablla-enriched vesicles did not appear to be accessible to TMR-Dextran (Figure 3.3A). This result suggests that Rablla-enriched vesicles have sele ctivity on cargoes, and are at least not accessible to the bulk endocytosed flui d-phase cargo. EGFP-Rablla-enriched vesicles were clea rly distinct fr om EEAl-e nriched early endosomes, which are large orga nelles (0.2�1 11m) existing in both the subapical and basolateral domains of LGACs (Figure 3.3B). Ly sotracker® 35 Red DND-99 .,.,.. used as a marker for the acidic ly.;osomes in live LGACs. EGFP-Rab lla.enriched vesic les clearly did not colocalize with ly.;osome s (Figure 3.3A), indicating their distinction from the ly.;osomal pathway. Figure 3.3 A B EG F P -Ra b1 1 a EEA 1 Ac t in Overlay • > • • • • � -- � � �.Jm !;. j.J r1 � � s ,m 5" -n 36 Figure 3.3 The Rabll a-enriched membrane compar tment is not colocalized with early endocytotic or lysosomal markers. (A) Live LGACs expressing EGFP-Rablla were incuba ted with 2 mg/ml TMR-dextran or with 50 nM Lysotracker® Red DND-99 for 1 hour. Live cells were observed by confocal fluorescence microscopy to localize EGFP-Rablla as well as the other fluorescent markers. Cellular ou tlines were obtained by comparing the respective image with the differential interference contrast (DIC) image. (B) LGACs expressing EGFP-Rablla were fixed, permea bilized and labeled with primary goat anti-EEAl antibodies, seco ndary Alexa-Fluor-568®-conjugated donkey anti-sheep antibody and Alexa-Fluor -647®-c onjugated phalloidin. *, lumena; scale bar: 5 fl m. 37 Figure 3.4 A 38 Figure 3.4 The Rablla-enriched membrane compartment is distinct from the Rab27b-enriched secretory vesicles. The fo llowing cells were subjected to confocal fluorescence microscopy. (A) Live LGACs expressing YFP-R ab27b alone. (B) Live LGACs co-expressing EGFP and YFP-R ab27b. (C) Live LGACs co-expressing EGFP-Rablla WT and YFP-R ab27b. (D) Live LGACs co-expressing EGFP-Rablla DN and YFP-Rab27b. Cellular outlines were obtained by comparing the respective image with the DIC image. *, lumena; scale bars: 5 11m (A) and 2 11m (B-D). Rablla-enriched vesicles are distinct fr om the regulated secretory pathway Rab27b has been identif ied as a marker of subapical secretory vesicles in LGACs (Chiang et a!., 201 1). In live LGACs, YFP-Rab27b labeled large secretory vesicles (�0.5 11m) loca ted in the suba pical domain (Figure 3. 4A), whereas EGFP-Rablla labeled a population of much smaller subapical vesicles in the same domain (Figure 3. 4C). Higher resolution images showed that YFP-R ab27b and EGFP-Rablla labeled two vesicle pools that exist in close spatial proximity but not to signi ficantly overlap (Figure 3.4C). Moreover, the co-expression of EGFP-Rablla WT, DN or EGFP did not alter the localization ofYFP-Rab27b-enriched secretory vesicles (Figure 3.4A, B, C, D). Rab3D has been identif ied as another marker for mature secretory vesicles in LGACs (Evans et a!., 2008) While some Rab3D-enriched secretory vesicles resided in the subapical domain, they were also detected extending into the central domain of the cell (Figure 3.5 A). EGFP-Rablla and endogenous Rab3D showed partial coloca lization in fixed cells but quite different distribution patterns (Figure 3.5 A). Similar to the case of YFP-Rab27b-enriched vesicles, the co-expression of EGFP-Rablla WT or DN, (Figure 39 3.5A,B,C) or EGFP (data not shown) did not seem to alter the localization of Rab3D-enriched secretory vesicles. Figure 3.5 A 8 c MyoVb tail Actin Overlay .. � D . * ' • * * * * ' ........... ........... ........... 51J m 51J m '51J m 40 Figure 3.5 The expression of EGFP-Rablla constructs and mCherry-myosin Vb tail did not significantly alter the localization of Rab3D. Non-transduced LGACs (A), LGACs expressing EGFP-Rab11a WT (B), EGFP-Rab11a DN (C) and mCherry-myosin Vb tail (D) were fixed, permea bilized, and labeled with primary rabbit anti-Rab3D serum, seco ndary Alexa-Fluor-568®-conjugated goat anti-rabbit antibody or Alexa-Fluor -488®- conjugated donkey anti-rabbit antibody, and Alexa-Fluor -647®-con jugated phalloidin. *, lumena; scale bar: 5f !m Carbachol (CCh) is a cholinergic agonist that accelerates the fusion of Rab27b and Rab3D-enriched secretory vesicles with the APM in acinar cells (Chen et a!., 2004; hnai et a!., 2004; Evans et a!., 2008; Chiang et a!., 201 1). The regulated secr etory pathway in primary LGACs can be stimulated by CCh (Jerdeva, G. et a!., 2005; Evans et a!., 2008; Chiang et a!., 2011) , and P-hexosa minidase has been identifi ed as a marker of the secretory fun ction in primary LGACs (Andersson et a!., 2006). Theref ore, the release of P-hexosa minidase and total protein by LGACs into the culture superna tant were both measured as an additional approach to investigate the potential impact of the expression of Ra b11a constructs on regulated secretions. The LGACs expressing EGFP-Rab11 a WT, DN or EGFP showed no signific ant diff erences in the secretion of P-hexosa minidase or total protein, either at the resting stage or under the treatment of CCh (Figure 3.6A, B). This result indicated that the expression of EGFP-Rab11a WT or DN did not signi ficantly alter the regulated secretions in LGACs. This result is also consi stent with the conclusions fr om confocal immunof luorescence microscopy, which indicated that Rab11a labels a unique membrane compartment distinct 41 fr om the regulated secr etory pathways in LGACs. Figure 3.6 250% 200% c A 0 -� 150% u "' 411 "' ·� 100% "' Gi a:: 50% 0% 350% 300% c 250% 0 "i B t; 200% "' VI "' 150% ·� "' Gi a:: 100% 50% 0% [3-hexosa minidase Secretion Non-transduced EGFP EGFP-Rab11a EGFP-Rab118 WT ON Total Protein Secretion Non-transduced EGFP EGFP-Rab11a EGFP-Rab11 a WT ON •CCh · • CCh + 42 Figure 3.6 The expression of EGFP-Rablla constructs did not significantly alter the secretory function of LGACs. LGACs seeded on Matr igel-coa ted 12 -well plates were co-transduced on the 2n d day of culture with Ad Tet -On®, as well as Ad EGFP-Rablla WT, DN or Ad EGFP construct. On the 3' d day of culture, non-transduced or transduced LGACs were incubated with fr esh medium ( 500 11l/ well), which was collected at the time point of 30 minutes, with or without 100 mM CCh. Cell pellets were dissolved in 0.5 M NaOH. The secretion of �-hexosaminidase (A) was quan tified and normalized to pellet protein for each sample. The total protein secretion (B) was quan tified with the Bio-Rad protein assay and normalized to pellet protein for each sample. The secretions of �-hexosaminidase and total protein under various conditions were divided by the basal secretion of non-trans duced cells, and are shown as relative secretion. plgR and dlgAare sorted into the Rablla-regulated transcvtotic pathway In fixed and non-permeabilized LGACs, plgR at the BLM is clea rly labeled by sheep anti-rabbit SC serum, whereas plgR is not detectab le intracellularly or at the APM (Figure 3.7 A). Analysis of plgR in fixed and permeabilized acini fo rmed by primary LGACs reveals BLM and also the intracellular membrane compartments containing plgR. Some plgR was detected in basola teral early endosomes labeled by RFP-Ra b5a (Figure 3.7B ). In LGACs, over-expressed EGFP-Rablla WT was colocalized with plgR in the subapical domain (Figure 3. 7C), similar to endogenous Rablla (Figure 3.1C), indicating that a subset of plgR enters the Rablla-enriched membrane compartment. Consistent with the concept that Rablla mediates the basal-to-apical transcytosis of basola terally-endocytosed plgR, over-expressed EGFP-Rablla DN signi ficantly reduced accumulation of plgR in the subapical domain (Figure 3. 7D). 43 Figure 3.7 B c I EGFP-Rab1 1 a ON 1----i S>t m E 2 1.6 � ·;;; 1: II> .. £ 1.2 II> ·� .. 0.8 1i ct: 0.4 0 lSmin pulse plgR 1----i * S>t m 15m in pulse+ 30min chase Actin '• 1----i '* S1-1 m Overlay '\! " ...,.._. * 5 >tm ' ._ _ 44 Figure 3.7 Transcytosis of plgR and dlgA is regulated by Rablla. (A) LGACs were fixed without permea biliza tion, and labeled with primary sheep anti-rabbit SC serum, seco ndary Alexa-Fluor -568®-conjugated donkey anti-sheep antibody, and Alexa-Fluor -647®-con jugated phalloidin. (B) LGACs transduced with CellLight® RFP-Ra b5a Bacmam 2.0 reagent were fixed, permea bilized and labeled with primary sheep anti-rabbit SC serum and secondary Alexa-Fluor- 488®- conjugated donkey anti-goat antibody, as well as Alexa-Fluor- 647®-c onjugated phalloidin. (C,D) LGACs expressing WT and DN EGFP-Rablla were fixed, permea bilized, and labeled with primary sheep anti-rabbit SC serum and secondary Alexa-Fluor -568®-co nj uga ted donkey anti-sheep antibody, as well as Alexa-Fluor -647-con jugated phalloidin. *, lumen; scale bar: 5 11m. (E) LGACs expressing EGFP-Rablla WT or DN were pulsed with 300 11g/ml biotin-dlgA for 15 minu tes, with or without a 30-minute chase period, as described in Materials and Methods. The recovery of biotin-dlgA is shown as relative intensity. * P<0.05 (two-sample t-test). Based on this obser vation, we hypot hesized that dysf unction of Rab lla might disrupt the Rablla-regulated transcytotic pathway, and result in intracellular accumulation of dlgA, the ligand of plgR, prior to its release at the APM. We used biotinylated rabbit dlgA as a tool to test this hypot hesis. Many lumena in reconstitu ted acini are open to the culture medium (Jerdeva, G. et a!., 2005). Thus, biotinylated dlgA bound to plgR on BLM (Figure 3. 7A) would be endocytosed and subseq uently transcytosed to the APM and released into the culture superna tant ; whereas if transcytotic pathway were blocked, the biotinylated dlgA would be trapped inside the cells. After a 15 -minute pulse with 300 11g/mL biotin-dlgA, LGACs expressing EGFP-Rablla DN showed about 60% more accumulation of biotin-dlgA in cell lysates relative to those expressing EGFP-Rablla WT; after an additional chase for 30 minu tes, the LGACs expressing EGFP-Rablla DN 45 showed roughly equivalent biotin-dlgA inside the cell relative to the cells expressing EGFP-Rablla WT (Figure 3.7E ). These data suggest that dysf unction of Rablla blocks biotin-dlgA in the transcytotic pathway, increasing the steady- state content as assessed at the end of the pulse labeling step. During the subseq uent chase period, it is possible that this accumulated biotin dlgA is degraded and/ or recycled. Traffi cking ofRablla-enriched vesicles involves the MT network LGACs have a MT network that is orga nized in a polarized mann er: Microtubule-organizing centers (MTOCs) containing y-tubu lin are located in the subapical domain beneath the lumena. MTs radiating fr om the MTOCs are abundant; some are intert wined in the subapical domain, others extend their plus-ends to the basal cytoplasm (Figure 3.8A,B). Since MTOCs and subapical MTs are located in regions rich in EGFP-Rablla-enriched vesicles, we hypot hesized that EGFP-Rablla-enriched vesicles might be transported on MTs or that their localization is dependent upon MTs. To test this hypot hesis, we disrupted the MT network in LGACs using nocodazole. Nocodazole signific antly reduced the number and length of MTs, and caused EGFP-Rablla-enriched vesicles to redistribute fr om the subapical to the basola teral domain (Figure 3.8B,C). The localization of EGFP-Rablla-enriched vesicles in the subapical domain therefore appeared to be dependent on the integrity of the MT network. 46 Figure 3.8 • EGFP-Rab1 1 a y-tub Actin Overlay _ -.r * " \·., * *·: _ ... � - � � � � 51J m 51J m 51J m 51J m - • EGFP-Rab11 a. a-t ub Actin Overlay . .... tr • .. ·:er . • �-]> r':{ ;_ . � · 1---1 1---1 1---1 1---1 51-J m 51-J m 51-J m 51-J m EGFP-Rab11 a a-tub Actin Overlay * * * * · ..- · - Noc ,fAr: . � � : . . �-.q - : . 1----i 1----i t--::1 1----i 5j.J m S j.Jm S!J m S j.Jm ·'I.. EGFP-Rab11 a p150 Actin o��rl ay ..... * .. * .. r> * .J .. •' * II - t - * "' • \"' . * -� . * .. * .. ' * · ... .. � � � � 51J m Sj.J m Sj.J m 51J m EGFP-Rab11 a p150 Actin Overlay .. * .. * * []- * � . * ...... * .. - ;.. -·4 .. .. ........ * * * * � � � � 51J m Sj.J m 51J m 51J m - * * * * 47 Figure 3.8 Rablla-enriched vesicles are associated with MTs and p150 of the dynactin complex. All images are 3D projections of images acquired fr om consecutive confocal planes. (A) LGACs expressing EGFP-Rablla were fixed, permea bilized and labeled with pnmary mouse anti-y-tubulin antibody and secondary Alexa-Fluor -568®-conjugated goat anti-mouse antibody, as well as Alexa-Fluor -647®-con jugated phalloidin. (B,C) LGACs expressing EGFP-Rablla were incubated on ice for 5 minu tes, and then incubated at 37°C without or with 33 11M nocodazole for 2 hours. The cells were fixed, permea bilized and labeled with primary mouse anti-u-tubulin (B), anti-pl50 antibodies (C) and secondary Alexa-Fluor -568®-conjugated goat anti-mouse antibody, as well as Alexa-Fluor -647®-con jugated phalloidin. *, lumen; scale bar: 5 11m; Con, Contro l, Noc: Nocodazole. Arrows show regions of coloca lization of pl5 0 and EGFP-Rablla before and after nocodazole-induced dispersal of the Rablla-enriched compa rtment. In fixed LGACs, EGFP-Rablla showed extensive coloca lization with pl5 0 (Figure 3. 8B,C), which is a subunit of the dynactin complex and a cytoplasmic dynein motor cofactor (Vaughan and Vallee, 1995; Wang et a!., 2003; Kardon and Vale, 2009). Although dispersed to the basolateral domain after nocodazole treatment, EGFP-Rablla retained its colocalization with pl50. This result indicates that the associa tion of the pl5 0 with Rablla 1s not dependent upon intact MTs, although the Rablla -pl5 0-enriched-vesicles likely require MTs for their movements or the maintenance of their subapical pool. The traffi cking ofRablla-enriched vesicles associa ted with the F-actin network Although nocodazole altered the distribution of EGFP-Rablla-enriched vesicles, live cell imaging showed that nocodazole did not inhibit all movements of these vesicles. This 48 result indicates that these vesicles may use machinery for their traffi cking other than MTs and their associa ted motors. Treatment with a combination of nocodazole and latrunculin B (a chemical which disrupts F-actin) inhibited essentially all movements of EGFP-Rablla-enriched vesicles, suggesting that the motility of Ra blla-enriched vesicles may be associa ted with both the MT and the F -actin network. The head domains of myosin motor engage actin and are responsible for mechanochemical coupling of ATP hydr olysis to movement, while the tail domains specifically interact with diverse cargoes. The over-expressed tail domain truncated construct satura tes the tail domain binding sites on interacting effector proteins, preventing cargo fr om interacting with endogenous intact myosin motors, and therefore acts as a dominant-negative construct. 49 Figure 3.9 A B Con Noc c EGFP Rab1 1a only w/ MyoVb tail y-tub >------< 5 �m MyoVb tail * .. * * .. * .. >------< 5 �m Actin Overlay ..,·.,) 4#1 .. � *' , * * .. ' , .. >------< >------< 5 �m .. , 5�m ... 50 Figure 3.9 Rablla vesicle movements utilize the myosin Vb motor. (A) LGACs expressing mCherr y-myosin Vb tail were fixed, permea bilized and labeled with primary mouse anti-y-tubulin antibody and seco ndary FITC-con jugated goa t anti-mouse antibody, as well as Alexa-Fluor- 647-c onjugated phalloidin. Arrows indicate MTOCs labeled by y-tu bulin. (B) LGACs co-expressing EGFP-Rablla and mCherr y-myosin Vb tail were treated or not with 33 11M nocodazole for 2 hours . Live cells were observed by confocal fluorescence microscopy to detect EGFP-Rablla and mCherr y-myosin Vb tail membrane compartments. (C) Live LGACs expressing EGFP-Rablla alone or co-expressing EGFP-Rablla and mCherr y-myosin Vb tail were observed by confocal fluorescence microscopy. The fluorescence signal fr om EGFP-Rablla and mCherr y-myosin Vb tail was overlaid for cells co-expressing both proteins. The white arrow indicates the current position of the EGFP-Rablla-enriched vesicle at each time point, green arrows indicate the positions at previous time points. *, lumena; scale bar: 5 11m. Based on the response ofRablla-enriched vesicles to actin depoly merization and the relationship between Rablla and myosin Vb demonstrated in other cells (Lapierre et a!., 2001; Volpicelli et a!., 2002; Fan et a!., 2004; Wakabayashi eta!., 2005; Prova nce et a!., 2008; Gardner et a!., 2011 ), we hypot hesized that over-expression of a myosin Vb tail construct might alter the distribution or motility of Rablla-enriched membranes in LGACs. When expr essed alone, the mCherr y-myosin Vb tail-enriched membrane compartment was highly concentrated around the MTOCs in the subapical domain (Figure 3.9A). When mCherr y-myosin Vb tail was co-expressed with EGFP-Rablla , it was completely colocalized with EGFP-Rablla vesicles (Figure 3.9 B), and also completely inhibited their dynamic movements in live cells (Figure 3. 9C). This result indicates that, apparently by competing with the endogenous myosin Vb motor, the mCherr y-myosin Vb tail completely inhibits the dynamic movements of these vesicles. 51 However, nocodazole partially dispersed the subapical pool of these immobile vesicles and induced their redistribution to the basolateral domain (Figure 3.9B). This confirms that Rablla-enriched vesicles are associated with both the MT and the F-actin networks. The expression of mCherr y-myosin Vb tail also significantly altered the localization of pl50, the subunit of dynein motor complex. Confocal fluorescence microscopy revealed that pl5 0 was highly concentrated in the subapical compartment labeled by mCherr y-myosin Vb tail, whereas pl5 0 was more evenly distributed in the subapical region of non-trans duced cells (Figs 3.10A, 3.8C). Moreover, the compartment enriched in mCherry-myo sin Vb tail was located in the periphery of the MTOCs labeled by y-tubu lin in the subapical region. As mentioned above, EGFP-Rablla was completely colocalized with mCherr y-myosin Vb tail. These findings suggest that myosin Vb and a MT-dependent motor uti lizing the dynactin complex, likely cytoplasmic dynein, are both associated with Rablla-enriched vesicles, which will accumulate around the MTOCs under the driving fo rce of the MT -dependent motor when their assoc iation with the F-actin network is disrupted by the mCherr y-myosin Vb tail. Remarkably, over-expression of mCherr y-myosin Vb caused plgR and endocytosed dlgA to accumulate in the subapical compa rtment surrounding the MTOCs labeled by mCherr y-myosin Vb tail (Figs 3.1C, 3.1 0B,C,D). This suggests that Rablla, myosin Vb, the dynactin complex and plgR are recruited into the same membrane compartment that 52 regulates the transcytosis of dlgA, and the traffi cking of this compa rtment involves both myosin Vb and the dynactin complex. 53 Figure 3.10 A p150 MyoVb tail Actin Overlay * * * - · · - . * - - * * * • * • , * , * * ,. ... �* t---i t---i t---i t---i s�.� m s�.� m s�.� m s�.� m 8 plgR MyoVb tail � ._ ._ �� ._ ._ * ._ >-----! ,____. s�.� m s�.� m c D E 54 Figure 3.10 Myosin Vb plays a crucial tenninal regulatory role in the Rabll a-regulated transcytotic pathway. (A,B) LGACs expressing mCherry-myosin Vb tail were fixed, permea bilized and labeled with (A) primary mouse anti-pl50 antibody or (B) primary sheep anti-rabbit SC serum. Samples were then incubated with seco ndary Alexa-Fluor -568®-conjugated goat anti-mouse or donkey anti-sheep antibodies and Alexa-Fluor -647®-con jugated phalloidin. (C,D) Non-transduced LGACs (C) or LGACs expressing mCherry- myosin Vb tail (D) were incubated with 200 11g/ml dlgA at 37°C for 1 hour. After washing with PCM, cells were fixed, permea bilized and labeled with primary goat anti-IgA antibody and secondary Alexa-Fluor -488®-c onjugated donkey anti-goat antibody, as well as Alexa-Fluor-647®-conjugated phalloidin. (E) Live LGACs co-expressing YFP-Rab27b and mCherry- myosin Vb tail were imaged by confocal fluorescence microscopy. Cellular outlines were obtained by comparing the respec tive image with the DIC image. *, lumena; scale bar: 5 11m. In contrast, over-expression of mCherry-myo sin Vb did not cause re-dis tribu tion of YFP-Rab27b-enriched or Rab3D-enriched secretory vesicles (Figs 3.10E, 3. 5D), nor did it alter the dyna mics of YFP-Rab27b-enriched secretory vesicles either at the resting stage or under CCh stimula tion. These results indicated that, the myosin Vb motor that regulates the traff icking of Rablla-enriched vesicles is clea rly not involved in the regulated secr etory pathway in the LGACs, thus further supporting our conclusion that the Rablla-regulated transcytotic pathway 1s distinct fr om the Ra b3D or Rab27b-regulated secretory pathwa ys. 55 Chapter IV: The characterization of plgR trafficking in LGACs Expre ssion of constructs of fluorescent protein fused to the plgR cyto plasmic domain in primary LGACs: Adenovirus (Ad) constructs are capable of transducing rabbit pnmary LGACs at efficiencies over 80% (Jerdeva et a!., 2005; Evans et a!., 2008; Chiang et a!., 2011; Xu et a!., 201 1). We constructed Ad encoding human plgR (hplgR) fused with EGFP at the C' terminus of the cytoplasmic domain of the hplgR (hplgR-EGFP) (Figure 4.1A). Ad hplgR-EGFP incorporates the Te t-On® promoter that enables regulation of the expressiOn level of hplgR-EGFP by doxycycline in the culture medium. Co-transduction of Ad hplgR-EGFP with Adeno-X Tet -On® fo llowed by doxycycline treatment induced the expression of hplgR-EGFP as verified by western blotting. As shown in Figure 4.1B, hplgR-EGFP was recognized by both anti-human SC (hSC) and anti-GFP antibodies (left and middle panels, white arrows). The hplgR-EGFP migrates as a doublet, a common fe ature of plgR expressed in a number of different cells due to different levels of glycosylation (Kuhn et a!., 1983; Deitcher et a!., 1986; Asano et a!., 1998; Ogura, 2005). In addition, as expected, cleaved hSC was recognized only by the anti-hSC antibody. The spec ificity of anti-hSC antibody for hplgR-EGFP and not the endogenous rabbit plgR is further shown in Figure 4.2A, B. hplgR-EGFP were mostly 56 localized to the subapical domain beneath the APM, as well as to the BLM, compara ble to endogenous pigR in non-trans duced cells (Figure 4.1C, D), suggesting that the fusion of hpigR with EGFP did not interfere with the appropriate localization of hpigR-EGFP. In addition, I have previously shown that endogenous pigR is extensively colocalized with exogenous EGFP-Rablla (Figure 3.1D); conversely, hpigR-EGFP also showed substantial coloca lization with endogenous Rab 11 (Figure 4.1E). 57 Figure 4.1 A N'-te rminal Dox 8 k D + 250 15 0 100 75 Extracellular domain lg-li k e domains Dox Dox Dox + Anti-GFP Anti-SC c D hplgR-EGFP Anti-hSC Actin * * * * * * * ..... ..... ,___. I ,___. I ,___. 5�m 5�m 5�m E hplgR-EGFP Rab11 Actin � � * * � � * � * - * � * - * ,____. ,____. ,___. 5 ° JJ m 5JJ m 5JJ m * * Cytoplasmic domain C'-terminal Dox Dox + Overlay * * Overlay * ... _... . *' * / \i ..... ,___. I 5�m Overlay ; ·* , .,__. � . m €) . � ' . �� · � .., * - 58 Figure 4.1 Characterization of the hplgR-EGFP construct. A) Schematic of the hplgR-EGFP construct, showing EGFP fused to the C' -terminal cytoplasmic domain of hplgR. B) LGACs were co-transduced with Ad hplgR-EGFP, and Adeno-X Te t-On®. After induction with 0.1 11g/mL doxycycline (+) or not (-) overnight, the cells were lysed with non-de naturing lysis buffer as described in Methods. Post-nuclear superna tant pre-cleared with CL2B beads was resolved by SDS-P AGE and analyzed by western blotting using primary goa t anti-SC or mouse anti-GFP antibodies, as well as secondary IRDye®800-co njuga ted donkey anti-goa t or IRDye®700-conjugated goat anti-mouse antibodies, respectively. White arrow, hplgR-EGFP; blue arrow, SC. C) and D) Non-transduced LGACs (C) or LGACs expressing hplgR-EGFP (D) were fixed, permea bilized, and labeled with primary goat anti-hSC antibody, as well as seco ndary Alexa Fluor ®-568-conjugated donkey anti-goat antibody and Alexa Fluor®-647- conjugated phalloidin. E) LGACs expressing hplgR-EGFP were fixed, permea bilized, and labeled with primary mouse anti-Rabll antibody, as well as seco ndary Alexa Fluor ®-568-conjugated goat anti-mouse antibody and Alexa Fluor®-647- conjugated phalloidin. White arrows, co localization between Red and Green; *, lumena; scale bar: 5 11m. Traffi cking of plgR in the transcvtotic pathway: Chapter III shows that Rablla regulates the transcytosis of plgR and dlgA in LGACs. To further investigate the transcytosis of plgR, we spec ifically labeled the pool of hplgR-EGFP at the BLM with its natural ligand, dlgA, or with an anti-hSC antibody to track the hplgR-EGFP as it was endocytosed fr om the BLM. Since virtually all the SC of apical plgR is enzy matically cleaved, the anti-SC antibody does not bind apical plgR; also since the antibody is added to live, unfixed acini cooled to 4 'C to impair endocytosis, it should be accessible only to plasma membrane, and not to intracellular pools of the hplgR-EGFP. 59 Figure4.2 A kD (a) Dox + 250 150 100 75 B c Non transduced LGACs Non transduced LGACs Dox kD (b) Dox Dox + 250 150 100 75 60 Figure 4.2 Goat and mouse anti-hSC antibodies distinguish hSC from endogenous rabbit SC. A) LGACs were co-transduced with Ad hplgR-EGFP and Adeno-X Tet -On®, with(+) or without(-) induction with 0.1 11g/mL doxycyc line. Superna tant fr om LGACs cultured overnight after transduction was concentrated using a Vivaspin-500 concentrator, and analyzed by western blotting, using primary goat anti -hSC antibody (a) or sheep anti-rabbit SC serum (b), and secondary IRDye®800-co njugated donkey anti-goat or Alexa Fluor ®-680-conjugated donkey anti-sheep antibodies, respec tively.. B) Live LGACs expressing hplgR-EGFP or non-trans duced LGACs were fixed, permea bilized and labeled with primary goat anti-hSC antibody, secondary Alexa Fluor ®-568-conjugated donkey anti-goat antibody and Alexa Fluor®-647-conjugated Phalloidin. C) Live LGACs expressing hplgR-EGFP or non-transduced LGACs were incubated with mouse anti-hSC antibody at 4 'C for 1 hour. After rinsing, cells were fixed, permea bilized and labeled with secondary Alexa-Fluor -568-conjugated goat anti-mouse antibody and Alexa Fluor®-647-con jugated phalloidin. White arrows, colocalization; *, lumena; scale bar: 5 11m. Rabbit dlgA can be theoretically used to label its recep tor, endogenous rabbit plgR. However, the complex form ed by fluorophore -con jugated rabbit dlgA and endogenous rabbit plgR does not generate sufficient fluorescent in tensity to yield a detectab le signal in direct fluorescence confocal microscopy (data not shown). However, direct fluorescence confocal microscopy showed that fluorophore -con jugated rabbit dlgA can be used to label hplgR-EGFP over-expressed by LGACs: LGACs over-expressing hplgR-EGFP were co-in cubated with Rhodamine -con jugated dlgA and an anti-hSC antibody that does not react with endogenous rabbit SC (Figure 4.2C) at 4 ° C. hplgR-EGFP on the BLM was clea rly labeled by anti-hSC antibody and Rhodami ne-conjugated dlgA, whereas hplgR-EGFP inside LGACs was not labeled because the antibodies could only access the cell surface (Figure 4.3A). The label was 61 exclusively basolateral at 0 min (prior to cell warming and internalization of receptor), and the immunof luorescence signal on the APM was negligible. After warming-up to 3TC to allow endocytosis and subseq uent traffi cking of the single cohort of dlgA-hp lgR-EGFP complexes and anti-hSC-hplgR-EGFP complexes, time-course experiments clearly showed that the anti-hSC-hplgR-EGFP complex colocalized with dlgA-hp lgR-EGFP complex at all time points (Figure 4.3A). This result suggests that the hplgR-EGFP labeled by anti-hSC antibody is endocytosed and traffi cked together with the dlgA-hp lgR-EGFP complex. This novel Ad plgR-EGFP construct and approach were thus able to resolve the post-endocytotic traffi cking of basola terally endocytosed plgR fr om the total plgR pool, allowing us to investigate the interrelati onship between plgR present in the tra nscytotic pathway and the regulated secretory pathway. However, because the LGACs need to be fixed to enable labeling anti-hSC antibody with fluorophore -con jugated seco ndary antibody, we could not 1mage a single acmus continuously for the entire post-endocytotic period, and therefore sepa rate LGAC samples were used for different time points. 62 Figure 4.3 A Surf ace label l'!ll in -.. 1!1!!!!1111 Omin 15 min Omin 63 Figure 4.3 Trafficking of basolaterally endocytosed plgR in LGACs. A) LGACs expressing hplgR-EGFP were co-i ncubated with 100 11g/mL Rhodami ne-conjugated rabbit dlgA and 4 11g/mL primary mouse anti-hSC antibody for 1 hour at 4 ,C . Subseq uently, LGACs were rinsed and incubated at 3T C, fixed at the time points indicated, and labeled with secondary Alexa Fluor ®-633-co njugated goa t anti-mouse antibody. Cellular outlines and the positions of lumena were obtained by comparing the respective fluorescent image with the differential interference contrast (DIC) image. White arrows, coloca lization between red, green and purp le B) Non-transduced LGACs were incubated with sheep anti-rabbit SC serum for 1 hour at 4 ,C , rinsed and incubated at 3T C. Cells were fixed at the time points indicated, permea bilized, and labeled with primary mouse anti-EEAl antibody, as well as secondary Alexa Fluor ®-488-c onjugated donkey anti-sheep and Alexa Fluor ®-568-conjugated goat anti-mouse antibodies, and Alexa-Fluor®-647-conjugated phalloidin. Actin labeled with Alexa Fluor®-647- conjugated phalloidin is displayed in purp le in the overlay image. The high magnification image displays a region of 10 x 10 11m fr om the yellow boxed image to the left. White arrows, co localization between red and green; *, lumena; scale bar: 5 11m. 64 Figure 4.4 A 0 !Jg/ml Anti-SC * * * n Actin * * * n overlay -. * * * n B Om in 15 min 30 min 60 min SC conce ntra tion 20 IJQ/ml 50 IJQ/ml Anti-SC * Anti-SC * * * n * n Actin * Actin * * * n * n Overlay * Overlay * * * n * n 100 IJQ/ml Anti-SC * n Actin * n Overlay * n 65 Figure 4.4 Sheep anti-SC serum specifically recognizes plgR on the basolateral membrane of LGACs. A) non-transduced LGACs were incubated with sheep anti-SC serum and purif ied rabbit SC at scaling up concentrations for 1 hour at 4 ,C . After extensive rinses, LGACs were fixed, permea bilized, and labeled with secondary Alexa-Fluor -488®- conjugated donkey anti-sheep antibody and Alexa-Fluor -647®-con jugated phalloidin. B) non-transduced LGACs were incubated with normal sheep serum for 1 hour at 4,C, rinsed and incubated at 3T C. Cells were fixed at time points indicated, permea bilized, and labeled with seco ndary Alexa-Fluor -488®- conjugated donkey anti-sheep and Alexa Fluor®-647-conjugated phalloidin. *, lumena; scale bar: 5 11m. Because hpigR-EGFP occupies one fluorescence filter channel for confocal microscopy, labeling endogenous rabbit pigR would enable the use of additional labels to track the post-endocytotic traff icking of pigR. I labeled endogenous rabbit pigR on the BLM with sheep anti-rabbit SC serum. In order to verify the spec ificity of anti-rabbit SC antibody in sheep serum for binding pigR on the BLM, SC purifi ed fr om rabbit bile was used as the competitor for antibody binding. Escalating concentrations of purifi ed rabbit SC significantly reduced the indirect fluorescent signal fr om sheep anti-rabbit SC on the BLM (Figure 4.4A), indicating that the sheep anti-rabbit SC antibodies bind to pigR with high speci ficity. When pre-immune sheep serum was used as a control, no endocytosed cargo with immunofluore scence was observed (Figure 4.4B); together, this obser vation plus the competition experiment suggest that Fe receptor does not non-specifically bind or endocytose the anti-SC antibodies. Theref ore, labeling of basolateral pigR with anti-SC antibodies provides a reliable method for tracking endocytosed endogenous pigR 66 and its post-endocy totic traffi cking. After warming to 37 ° C, LGACs exposed to anti-SC antibodies showed accumulation of endocytosed basola teral plgR in early endosomes labeled by EEAl by 15 minutes; at 30 minutes (not shown) and 60 minu tes, a subset of plgR still remained in the early endosomes (Figure 4.3B). However, at 30 minu tes, endocytosed plgR was also present in the subapical compartment labeled by EGFP-Rablla (Figure 4.5A), and endocytosed plgR continued to accumulate in this compartment by 60 minutes (Figure 4.5A). These obser vations indicate that basolaterally endocytosed plgR is eventually sorted fr om early endosomes into Rablla-enriched subapical vesicles, which are involved in the transcytotic pathway. 67 Figure 4.5 Surf ace labeling of endogenous plgR A 0 min 30 min 60 mi 8 Omin 30 mi • I EGk:l 1a WT * - - - * ,__. 5�m EGFP-Rab1 1a DN * * ,__. 5�m EGFP-Rab1 1a DN * D * ,__. 5�m EGFP-Rab1 1a DN D * ,__. 5�m Ant - - * - * - * ,__. 5�m Anti-SC * * ,__. 5 �m Anti-SC * D * ,__. 5�m Anti-SC D * ,__. 5�m Ov� � � � High Mag _(ij _ - � ' . .. � , � \ . :;; * . ' ,__. ' 5�m Overlay - ' - - ____ / � · * / * \ __ .. I I ·-- S 'lnn..... --- - - � Ov / , l!J ' , . . ' A. * ' .......... � 5�m Overlay L:J \ \ .._ .. ' �* I ' .......... 5�m �� . -. - - 1'-'t HighMr . - �hMag . ' \* • - 68 Figure 4.5 Rablla regulates the basal-to-apical transcytosis of plgR. LGACs expressing EGFP-Rablla WT (A) or EGFP-Rablla DN (B) were incubated with sheep anti-rabbit SC antiserum for 1 hour at 4 ,C, rinsed and incubated at 3T C. Cells were fixed at the time points indicated, permea bilized, and labeled with seco ndary Alexa Fluor ®-568-conjugated donkey anti-sheep antibody and Alexa Fluor®-647-conjugated phalloidin. Actin labeled with Alexa Fluor®-647- conjugated phalloidin is displayed in purp le only in the overlay image . The high magnification image displays a region of 12.5 x 12.5 11m fr om the yellow boxed image to the left. White arrows, coloca lization between red and green; *, lumena; scale bar: 5 11m. As shown in Figure 3.7, the domina nt negative (DN) S25N GDP-locked mutant form of Rab lla altered plgR distribution and inhibited the exocytosis of dlgA in LGACs, but it was unclear which plgR traffi cking pathway Rablla DN actu ally aff ected. In order to further investigate the role of Rablla in the transcytosis of plgR, I observed the traffi cking of basola terally endocytosed plgR in LGACs over-expressing EGFP-Rablla DN. EGFP-Rablla DN significa ntly reduced the amount of endocytosed basolateral plgR accumulating in the subapical region (Figure 4.5B), indicating the inhibition of basal-to-apical transport of endocytosed plgR. These observations further indicate that the basal-to-apical transport ofpl gR requires Rablla functionality. 69 Figure 4.6 A Surf ace labeling of endogenous plgR, Nocodazole 1"\IILI-�V L-L-1"\ I 1"\ W... llll 0 min .......... .......... .......... 5�m 5�m 5�m Anti-SC EEA1 Actin •· . .( ' .( 30 min -� .. .......... �-� ,___. ,___. 5�m . 5�m 5�m Anti-SC EEA1 Actin 60 min . � � · · . � � ,___. .......... ,___. 5�m 5 �m 5� m • . . 8 An t1 -SC Ra b11 Actin 0 min ·- ,___. ,___. ,___. 5�m 5�m 5�m 30 min Anti�sc • Ra b11 • Actin • • • 60 min � ,___. .......... ,___. 5�m 5�m 5�m •: � - . . �V'IIi;;i iiUJ j _J \ .......... .. 5�m Overlay \- <il!l . .'': "i. _ •. .. -co- .� .( " ... r � _·} · � \,.. -· -1" J ,___. :..1- ' 5 �m · -': 1.. . :' overlay . � - . ' � ·- . � \ ' .. � . . ' • . .. O V"er la.y .......- , . ) I. � � : -, ' ''- .£ . '. . · ,::........, -� 5 �m .:; oyerlay • • • .(• � - ' • • • . . I· •. . � � J �\. . . -� J? � 7m . "" 70 Figure 4.6 Nocodazole inhibited the basal-to-apical trafficking of basolaterally endocytosed plgR. A) Non-transduced LGACs were incubated with 33 11M Nocodazole and sheep anti-rabbit SC antiserum for 1 hour at 4 ,C , rinsed and incubated at 3T C. Cells were fixed at the time points indicated, permea bilized, and labeled with primary mouse anti-EEA1 antibody, seco ndary Alexa Fluor ®-488-co njugated donkey anti-sheep and Alexa Fluor ®-568-conjugated goat anti-mouse antibodies, as well as Alexa Fluor®-647- conjugated phalloidin. B) Non-transduced LGACs were incubated with 33 11M Nocodazole and sheep anti-rabbit SC antiserum for 1 hour at 4 ,C , rinsed and incubated at 3TC. Cells were fixed at the time points indicated, permea bilized, and labeled with primary mouse anti-Rabll antibody, seco ndary Alexa Fluor ®-488- conjugated donkey anti-sheep and Alexa Fluor ®-568-conjugated goat anti-mouse antibodies, as well as Alexa Fluor®-647-conjugated phalloidin. White arrows, coloca lization between red and green; *, lumena; scale bar: 5 11m. Basal-to-a pical transport of basolaterally endocvtosed plgR 1s dependent on the MT network As mentioned in Chapter III, disruption of the MT network by nocodazole caused the dispersion and redistribution of the subapical EGFP-Rablla-enriched vesicles (Figure 3.8). Theref ore, it is reasonable to hypothe size that the Rablla-regulated transcytotic pathway is dependent on the MT network. Confocal microscopy showed that, disruption of the MT by nocodazole inhibited the basal-to-apical traff icking of basola terally endocytosed plgR (Figure 4.6 A, B). As shown in Figure 4.6A, at the 30-minute and 60-minute time points, basola terally endocytosed plgR was still present in early endosomes in the basola teral domain. Disruption of the MT network did not noticeably reduce the coloca lization of basola terally endocytosed plgR with Rabll, but it caused Rab 11 -enriched vesicles containing basolaterally endocytosed plgR to stay in the 71 basolateral regwn at the 30-mi nute and 60-minute time points (Figure 4.6B), in comparison to the apically-targeted movements of Rab 11-e nriched vesicles containing basola terally endocytosed plgR in non-treated cells (Figure 4.5A, Figure 4.13 A). These obser vations indicate that, the sorting of plgR into Rablla-enriched vesicles is not dependent on the MT network; however, the Rablla-enriched vesicles containing basola terally endocytosed plgR are transported on the MT network for their basal-to-apical traffi cking, which is likely to be regulated by the dynein motor complex. Traffi cking of plgR in the regulated secretory pathway: Rab3D is a well-known marker of secretory vesicles in LGACs (Evans et a!., 2008) and other exocrine secr etory cells (Ohnishi et a!., 1996; Tian et a!., 2010). Immunofluorescence and detection by confocal fluorescence microscopy showed that endogenous Rab3D was also partially colocalized with y-adaptin (Figure 4.7), a subunit of the adaptor protein complex and a marker of late-Golgi or TGN traffi cking (Wong and Brodsk y, 1992; Chiang eta!., 2012). This suggests that the Rab3D-enriched membrane compartment originates fr om the biosy nthetic compartment in LGACs. 72 Figure 4.7 Rab3D y-adaptin Overlay High Mag • • D D ·u .. [� f . * . . · . ': .. .. .. ( ; � ! • * * • . . .. 1-i 1-i 1-i 5 �m 5 �m 5�m Figure 4.7 Characterization of the regulated secretory pathway in LGACs. A) LGACs were fixed, permea bilized and labeled with primary rabbit anti-Rab3D serum, primary mouse anti-y-adaptin antibody, secondary Alexa Flum®-488-con jugated donkey anti-rabbit and Alexa Fluot®-568-con jugated goat anti-mouse antibodies, as well as Alexa Fluor®-647-conjugated phalloidin. Actin stained with Alexa Fluor®-647-con jugated phalloidin is displayed in purple in the overlay image. High magnification image displays a region of 10 x 10 J..Lm 73 Figure 4.8 A Dox Dox Tet-On® Dox Dox Tet-On® Dox Dox Tet-On® kD + only + only + only 50 37 Anti-mCherry Anti-Rab3D Overlay 8 Non- Anti-Rab3D Actin Overlay * * * * * · * · transduced * * * * *· '* · LGACs * * * -,. * / * * ......... ......... ......... 5�m 5 �m 5 �m mCherry-Rab3D Anti-Rab3D Actin Overlay ·� ;..,., , * �, * .\� r, * - * - '-- .......... .......... .......... .......... «' 5�m 5 �m _. 5 �m _. 5 �m c IP: Anti- Control Anti- Control Anti- Control kD Rab3D serum Rab3D serum Rab3D serum ( a ) 250 IP with 150 anti-Rab3D � serum 100 Blot: Anti-hSC Anti-GFP Overlay 50 ....c: 37 Blot:Anti-mCherry Goat anti-rabbit IP: Anti- Control ( b ) kD hSC serum 250 IP with anti-hSC 150 � antibody 100 Blot: Anti-GFP 50 37 Blot: Anti-mCherry Anti-Rab3D Overlay 74 Figure 4.8 Characterization of mCherr y-Rab3D expressed in LGACs. A) LGACs were co-transduced with Ad mCherr y-Rab3D, and Adeno-X Tet- On®. After overnight induction with 0.1 11g/mL doxycycline (+) or not (-), the cells were lysed with the non-denaturing lysis buffer. As described in Methods, the post-nuclear superna tant pre-cleared with CL2B beads was resolved by 7.5% SDS-P AGE and analyzed by western blotting using primary anti-Rab3D rabbit antiserum and mouse anti-mCherry monoclonal antibody, as well as secondary IRDye®800- conjugated goat anti-rabbit (Green in the overlay image) and IRDye®700-con jugated goat anti-mouse (Red in the overlay image) antibodies. Blue arrow, mCherry-Ra b3D; green arrow, endogenous Rab3D. The expression level of mCherr y-Rab3D was 5-fold higher than endogenous Rab3D. B) LGACs expressing mCherry- Rab3D and non-transduced LGACs were fixed, permea bilized and labeled with primary rabbit anti-Rab3D serum, secondary Alexa Fluor ®-488- conjugated donkey anti-rabbit antibody and Alexa Fluor®-647-conjugated phalloidin. White arrow, coloca lization between red and green; *, lumena; scale bar: 5 11m. C) LGACs co-expressing mCherry- Rab3D and hpigR-EGFP were lysed with the non-denaturing lysis buffer, and immunopre cipi tation was performed as described in Metho ds. (a) Rabbit anti-Rab3D serum was used to immu noprecipi tate mCherry- Rab3D, and pre-immune rabbit serum was used as control. The immunoprecipitated mCherry- Rab3D and hpigR-EGFP were detected by Western blotting, using mouse anti-mCher ry, mouse anti-GFP and goat anti-hSC primary antibodies, as well as IRDye®700-conjugated goat anti-mouse (Red in overlay) and IRDye®800-co njugated donkey anti-goa t (Green in overlay) seco ndary antibodies. This image is repres entative for western blotting results from 3 prepa rations. Green arrow, hpigR-EGFP; blue arrow, mCherry- Rab3D; red arrow, rabbit Ig heavy chain . (b) goat anti-hSC antibody was used to immu noprecipi tate hpigR-EGFP, and pre-immune goat serum was used as control. The immunoprecipitated mCherry- Rab3D and hpigR-EGFP were detected by Western blotting, using mouse anti-mCh erry, mouse anti-GFP and goat anti-hSC primary antibodies, as well as IRDye®700-con jugated goat anti-mouse (Red in overlay) and IRDye®800-co njuga ted donkey anti-goat (Green in overlay) secondary antibodies. This image is repres entative for western blotting results fr om 3 prepara tions. Green arrow, hpigR-EGFP; blue arrow, mCherry- Rab3D In this study, I constructed an Ad vector that encodes rabbit Ra b3D with an mCherry fluorescent protein tag on the N' -terminus (mCherry-Rab3D) (Figure 4.8A). 75 mCherry- Rab3D expressed m LGACs showed similar subapical distribu tion to endogenous Ra b3D (Figure 4.8B) (Evans et a!., 2008), but the morphology of the membrane compartment labeled by mCherry-Ra b3D seemed to be slig htly different fr om that labeled by endogenous Rab3D. Such a diff erence might be due to the overexpression of mCherry- Rab3D, since morphological changes in various membrane compa rtments caused by overexpression of other Rab proteins have been described previously (Bucci et a!., 1992; Wilson et a!., 1994; Wilcke et a!., 2000; Mesa et a!., 2001; Holtta-Vuori et a!., 2002). Previous work in our lab showed a direct interaction between Rab3D and the cytoplasmic domain of plgR (Evans et a!., 2008). Co-immunoprecipitation likewise demonstrated a direct interaction between mCherry- Rab3D and hplgR-EGFP (Figure 4.8C), suggesting that mCherry- Rab3D is functionally compara ble with endogenous Rab3D. This result is also consistent with the extensive coloca lization of subapical hplgR-EGFP with mCherry- Rab3D (Figure 4.9A). 76 Figure 4.9 77 Figure 4.9 The distribution of hplgR-EGFP in the regulated secretory pathway and the transcytotic pathway. Live LGACs co-expressing fluorescent fusion proteins were imaged by confocal fluorescence microscopy to show localization patterns of various markers. Cellular ou tlines were obtained by comparing the respective image with the DIC image . A) hpigR-EGFP was colocalized with mCherr y-Ra b3D. B) The compartment enriched in hpigR-EGFP was distinct fr om YFP-Rab27b-enriched secretory vesicles. C) YFP-Rab27b-enriched secr etory vesicles were distinct fr om mCherry -Rab3D-enriched secretory vesicles. D) EGFP-myosin V c was enriched in the mCherry-Rab 3D-enriched secretory vesicles. E) In cells that co-expressed both proteins, EGFP-myosin V c tail was colocalized with the membrane compa rtment labeled by mCherr y-Rab3D. F) hpigR-EGFP was partially colocalized with mCherr y-myosin Vb tail. The high magnification image displays a region of 12.5 x 12.5 11m (A,B,D,E,F) or 6.25 x 6.25fl m (C) fr om the yellow boxed image to the left. White arrows, colocalization between red and green; *, lumena; scale bar: 5 11m. Interestingly, hpigR-EGFP did not colocalize extensively with YFP-Rab27b (Figure 4.9B), which is another marker of mature secretory vesicles in LGACs (Chiang et a!., 2011) and in other exocrine secr etory cells (Chen et a!., 2004; Imai et a!., 2004). Moreover, mCherry- Rab3D and YFP-R ab27b labeled two different pools of secretory vesicles that did not extensively overlap (Figure 4.9C). Syncollin has been identif ied as a marker of the regulated secretory pathway in LGACs (Jerdeva et a!., 2005) and other secretory cells (Wasle et a!., 2004; Wasle et a!., 2005), and Syncollin-GFP expressed using Adenoviral vector transduction is present in YFP-Rab27b-enriched secretory vesicles (Chiang et a!., 2011 ). Confocal fluorescence microscopy showed that syncollin-GFP was not enriched in mCherry-Ra b3D-labeled secr etory vesicles (Figure 4.10, Figure 4.11). Moreover, treatment with 100 mM CCh substan tially accelerated the 78 release of syncollin-GFP-emiched vesicles at the apical membrane compared with the resting stage, which occurred with different dynamics than the modest changes observed in the movements of mCherry-Rab3D-emiched secretory vesicles (Figure 4.10, Figure 4.11). These observations suggest that Rab3D and Rab27b may label two partially distinct subsets of mature secretory vesicles in LGACs. In addition, plgR is emiched in the Rab3D-emiched membrane compartm ent, but not the Rab27b-emiched membrane compartment; while syncollin-GFP is emiched in the Rab27b-emiched membrane compartm ent, but not the Rab3D-emiched membrane compartment. Figure 4.10 Figure 4.10 Syncollin-GFP-enriched vesicles and mCherry-Rab3D-enriched vesicles 79 in resting LGACs. LGACs grown on a 35-mrn glass-bottomed plate were co-transduced with Ad constructs on the second day of culture for co-expression of syncollin-GFP and mCherr y-Rab3D. After 0.1 Jlg/mL dox ycycline induction overnight, LGACs were mounted in a 37°C incubation chamber and imaged. *, lumena; scale bar: 5 Jlff i. Figure 4.11 Figure 4.11 Syncollin-GFP-enriched vesicles and mCherry-Rab3D-enriched vesicles in LGACs stimulated with CCh. LGACs grown on a 35-mm glass-bottomed plate were co-transduced with Ad constmcts on the second day of culture for co-expression of syncollin-GFP and mCherr y-Rab3D. After 0.1 Jlg/mL doxycycline induction overnight, LGACs were mounted in a 37°C incubation chamber and imaged immediately after application of 100 JlM CCh. Red arrow: mCherry -Rab3D-enriched vesicle; green atTow, vesicles containing syncollin -GFP. *, lumena; scale bar: 5 Jlff i. Myo sin Vc is associated with Rab3D-enriched mature secreto ry vesicles: Our previous study showed the associa tion of the myosin Vc motor protein with the 80 Rab3D-enriched secr etory vesicles in LGACs (Marc helle tta et a!., 2008). Here, live cell imaging showed the co-loca lization of EGFP-full-length-myosin V c with the membrane-compartment enriched in mCherr y-Rab3D (Figure 4.9D), consis tent with the coloca lization by indirect immunofluore scence seen previously. Moreover, the overexpression of EGFP-f ull-l ength-myosin V c significa ntly altered the morphology of overexpressed mCherry- Rab3D compared to the LGACs without overexpressed myosin V c (Figure 4.9 A, C, D). Specifically, in the cells expressmg both the EGFP-full-length-myosin V c and mCherry-Ra b3D, the mCherry-Ra b3D that was recruited to distinct mature secr etory vesicles was clearly more unif ormly distributed on the enlarged mature secretory vesicles, and it was completely co localized with myosin V c. These obser vations suggest a functional interaction between myosin V c and the Rab3D-regulated secretory pathway; further, the expression level of my osin V c may be rate limiting either in recruitment of Rab3D to vesicles or in fo rmation or stabilization of vesicles capable of recruiting Rab3D. However, it should be noted that biochemical assays fa iled to detect any evidence for direct assoc iation of myosin V c and Rab3D in LGACs (data not shown). An Ad construct encoding myosm V c tail fused to EGFP on the N' -terminus (EGFP-myosin V c tail) is known to compete with endogenous myosin V c for the binding site on vesicles, but does not have the motor function, thus it acts as the dominant 81 negative mutant of myosm V c (Rodriguez and Cheney, 2002). Similar to EGFP-full-length-myosin V c, EGFP-myosin V c tail was also highly co localized with the mCherry- Rab3D-enriched membrane compartment (Figure 4.9E), and, compara bly to EGFP-full-length-myosin V c, elicited more associa tion of overexpressed mCherry-ra b3D with secretory vesicles. Interestingly, EGFP-myosin V c tail was also extensively co localized with YFP-Rab27b on large secretory vesicles (Figure 4.12A), and this is consistent with the observed coloca lization described in previous studies (Jacobs et a!., 2009; Chiang et a!., 2012). These observations indicate that the myosin V c motor participates in the traffi cking of both the Ra b3D-enriched and the Rab27b-enriched membrane compartments, but that these compartments are largely distinct from each other. Different Rab proteins, ra ther than motor proteins, seem to be the key delinea tor for marking vesicles with possi bly distinct cargo in the regulated secretory pathway. plgR that enters the transcvtotic pathway remains distinct fr om the regulated secretory pathway: Though both the Rablla-regulated transcytotic pathway and the Rab3D-regulated secretory pathway participate in the traffi cking of plgR (Evans et a!., 2008; Xu et a!., 201 1), whether these two pathways are independently distinct or exchange cargo has not 82 been studied. Therefore, I aimed to interrogate whether these two pathways exchange plgR as their cargo. 83 Figure 4.12 A 8 c D Rab3D [:] ,___. 5�m kD 250 150 100 MyoVb tail * [:] * ,___. 5�m myoVc myoVb (a) tail tail Control .9:1ie rla� ' <.� , ·· � -- �- ,___. 5�m High Mag ' :1 \ .. ., .... "-· f. kD (b) 250 myoVc myoVb 150 100 tail tail Control .. 84 Figure 4.12 Characterization of over -expressed mCherry-myosin Vb tail and EGFP-myosin Vc tail in LGACs. A) Live LGACs co-expressing EGFP-myosin Vc tail and YFP-Rab27b were observed by confocal fluorescence microscopy. Cellular outlines were obtained by comparing the respective image with the diff erential interference contrast (DIC) image. B) Live LGACs co-expressing hplgR-EGFP and mCherr y-myosin Vb tail were observed by confocal fluorescence microscopy. Cellular outl ines were obtained by comparing the respective image with the diff erential interference contrast (DIC) image. C) LGACs expressing mCherr y-myosin Vb tail were fixed, permea bilized and labeled with primary rabbit anti-Rab3D serum, seco ndary Alexa Fluor ®-488- conjugated donkey anti-rabbit antibody and Alexa Fluor®-647-conjugated phalloidin. Actin stained with Alexa Fluor®-647- conjugated phalloidin is displayed in purp le in the overlay image. High magnification image displays a region of 10 x 10 11m (A) or 12.5 x 12.5 11m (B, C) from the yellow boxed image to the left. *, lumena; scale bar: 5 11m. D) The LGACs expressing EGFP-myosin V c tail, mCherry- myosin Vb tail, or LifeAct- TagRFP (control) were ly zed with 4x SDS-P AGE loading buffer and resolved by 7.5% SDS-P AGE gels, which were subseq uently used for further western blotting (a) or commassie blue staining (b). Primary rabbit anti-myosin Vc tail and goa t anti-myosin Vb antibodies, seco ndary IRDye®800- conjugated goat anti-rabbit and IRDye®700-conjugated donkey anti-goat antibodies were used for western blotting. Green arrow, EGFP-myosin V c tail; red arrow, mCherr y-myosin Vb tail. I also used Ad mCherr y-myosin Vb tail that acts as a dominant negative construct of myosin Vb to study the effect of motor proteins on the traff icking of plgR. As demonstrated in Figure 3.9, overexpression of mCherr y-myosin Vb tail disrupted the traffi cking of Rablla-enriched vesicles in LGACs, suggesting that their motility depend on the myosin Vb motor protein. Our previous study also showed that endogenous plgR is co localized with the membrane compartment labeled by the EGFP-myosin V c tail (Marchelletta et a!., 2008). Here, confocal fluorescence microscopy of live LGACs showed that a subset of hplgR-EGFP was localized to the membrane compartment 85 labeled by mCherr y-myosin Vb tail (Figure 4.9F). The mCherry-myo sin Vb tail did not colocalize with either the EGFP-myosin V c tail (Figure 4.12B) or Rab3D (Fignre 4.12C), markers of the regulated secretory pathway. These data indicate that myosin Vb and V c motors are assoc ia ted distinctly and independently with the transcytotic pathway and the regulated secr etory pathway, respectively. Based on these observations, I hypot hesized that, since plgR is distributed into the transcytotic pathway and the Ra b3D-regulated secretory pathway, the overexpression of mCherr y-myosin Vb tail will trap the plgR that enters the transcytotic pathway, but will not interfere with the traffi cking of the plgR present in the Rab3D-enriched secr etory pathway. On the other hand, the overexpression of EGFP-myosin V c tail will trap the plgR sorted into the regulated secr etory pathway, but will not interfere with the traffi cking of the plgR that enters the transcytotic pathway. 86 Figure 4.13 Surf ace labeling of endogenous plgR A 30 mi 60 mi B 30 mi 60 mi 30 min c M: o o Anti-SC ovetJ .. High Mag * * [] * * * * * * * * .......... .......... .......... 5�m 5�m 5�m MyoVc tail Anti-SC Overlay High Mag - \ * * * 60 min cr. [:; J Q� � � · I , . , . * * .......... .......... .......... 5�m 5�m 5�m 87 Figure 4.13 plgR endocytosed from the basolateral membrane enters the transcytotic pathway but does not intersect with the regulated secretory pathway. A) LGACs expressing mCherr y-myosin Vb tail were incubated with sheep anti-rabbit SC antiser um for 1 hour at 4 ,C, rinsed and incubated at 3 TC. Cells were fixed at time points indicated, permea bilized, and labeled with seco ndary Alexa Fluor ®-488-co njugated donkey anti-sheep antibody, and Alexa Fluor®-647-conjugated phalloidin. B) LGACs expressing EGFP-myosin V c tail were incuba ted with sheep anti-rabbit SC antiserum for 1 hour at 4 ,C , rinsed and incubated at 3TC. Cells were fixed at time points indicated, permea bilized, and labeled with seco ndary Alexa Fluor ®-568-conjugated donkey anti-sheep antibody, and Alexa-Fluor -647-con jugated phalloidin. Actin stained with Alexa Fluor®-647-conjugated phalloidin is displayed in purple in the overlay image. The high magnification image displays a region of 12.5 x 12.5 11m fr om the yellow boxed image to the left. White arrows, co localization between red and green; *, lumena; scale bar: 5 11m. To test this hypot hesis, I tracked the cohort of plgR labeled by anti-SC antibody and endocytosed fr om the BLM. In LGACs expressing either mCherry- myosin Vb tail or EGFP-myosin V c tail, the endocytosis of plgR fr om the BLM was not visibly affe cted (data not shown). However, plgR endocytosed in LGACs expressing mCherry-myosin Vb tail accumulated in the membrane compartment labeled by mCherry- myosin Vb tail (Figure 4.13B), and did not show the same extent of subapical distribution as in non-transduced LGACs (Figure 4.13A). In contrast, EGFP-myosin V c tail did not appear to alter the basal-to-apical transport of basola terally endocytosed plgR. Basolaterally endocytosed plgR accumulated subapically, but not in the membrane compartment labeled by EGFP-myosin V c tail (Figure 4.13C). This indicated that basola terally endocytosed plgR is apparently not sorted into the regulated secr etory 88 pathway to any signi ficant extent, and that the transcytotic and the regulated secretory pathways are distinctively regulated by different myosin motor proteins. The transcvtotic pathway and the regulated secr etory pathway distinctively mediate apical ly-targeted trafficking of pigR. In order to distinguish the roles of the tra nscytotic pathway and the regulated secretory pathway in the traffi cking of pigR, I transduced LGACs with Ad hpigR-EGFP and co-transduced with Ad mCherr y-myosin Vb tail, Ad EGFP-myosin V c tail or rAV cMv Lif eAct-TagRFP. rAV cMv LifeAct-TagRFP expresses RFP-tagged Lif eAct, which labels the F -actin network but does not interfere with actin dyna mics in vitro and in vivo (Riedl et a!., 2008; Riedl et a!., 2010 ), and is thus used as a control for co-transduction, relative to the myosin tail domain constructs. The use of control Ad provides the reference for comparison of protein secretion, indicating the process of Ad transduction does not account for the change in protein secretion. mCherr y-myosin Vb tail and EGFP-myosin V c tail were expr essed at compara ble levels in transduced LGACs (Figure 4.12D). Apically-released proteins including SC, can be recovered fr om culture medium since the medium is contiguous with that within the luminal structur es fo rmed within these reconstituted acinar cultures (Jerdeva et a!., 2005; Chiang et a!., 2011 ). And this was also show in Chapter III. Theref ore, hSC cleaved fr om hpigR-EGFP and 89 released into the culture superna tant was quantif ied by western blotting using anti-hSC antibody that does not cross-react with endogenous rabbit plgR or SC (Figure 4.2A,B). 90 Figure 4.14 A 8 30 min 30 min + 30 min resting 30 min + 30 min CCh Relative recovery of hSC 100% +----------- 80% +----------- 60% +------------ * 40% +---t-------1--- 0% LifeAct, Vc tail, Vb tail LifeAct, Vc tail, Vb tail CCh resting resting resting CCh CCh Surface labeling of endogenous plgR Anti-SC * :::1 -· ,___. 5�m · Anti�sc [;] ·'· . . . * ,___. * 5�m Anti-SC . , * • , * 0 * * ,___. 5�m Rab1 1 D * - ,___. 5 �m Rab1 1 Q ,___. * 5�m Rab11 0 ,___. 5�m * * * Over l ay - - -�. '-. � � : / * \ _ . J.: � j, - · �Iii ,___. 5�m o :y e'r: Jay-� , _. A � t . . 't, : ." . . � • l � -: ' . .! ,___. " * ) 5�m Overlay � 0 * ** '· . . ,___. 5�m 1� 12.5 m -1 n1gn Mag _1 lj • -� i : � . .. High Mag ' • f • e � . ,. . ;! · !] ,. High Mag * * 91 Figure 4.14 CCh accelerates the SC release on the apical membrane of LGAC acini. A) Quan tification ofhSC released into culture superna tant. LGACs co-transduced with Ad hplgR-EGFP, Adeno-X Tet -On® and rAV cMV LifeAct- Tag RFP, Ad EGFP-myosin V c tail or Ad mCherr y-myosin Vb tail were cultured overnight with doxycycline. The net release of SC into the culture superna tant during 30-mi nute treatment was quan tified by western blotting, as described in Materials and Methods. n�5; #, p<0.05, one-sample Wilcoxon signed rank test; *, p<0.05 (student t-test) B) Non-trans duced LGACs were incubated with sheep anti-rabbit SC antiser um for 1 hour at 4 'C, rinsed and incubated at 3TC for 30 minutes. Cells were then treated with or without 100 11M CCh fo r additional 30 minu tes. Cells were fixed at time points indicated, permea bilized, and labeled with primary mouse anti-Rabll antibody, secondary Alexa Fluor® -488- conjugated donkey anti-sheep and Alexa Fluor ®-568 goat anti-mouse antibodies, as well as Alexa Fluor®-647- conjugated phalloidin. Actin labeled with Alexa Fluor®-647-conjugated phalloidin is displayed in purple in the overlay image. The high magnification image displays a region of 12.5 x 12.5 11m fr om the yellow boxed image to the left. White arrows, co localization; *, lumena; scale bar: 5 11m. As a cholinergic agonist, CCh stimulates the release of SC into the culture superna tant by LGACs (Evans et a!., 2008), simulating the neural stimulatory response of the lacrimal gland. I investigated the contribution of the tra nscytotic and the regulated secretory pathways to the acu te-phase release of hSC under the treatment of 100 11M CCh. Either at the resting stage or with CCh treatment for 30 minu tes, the LGACs over-expressing mCherry-myo sin Vb tail released signi ficantly less hSC than the LGACs expressmg Life Act-Tag RFP (control) (Figure 4.14A), indicating that the mCherr y-myosin Vb tail inhibits the constitutive transcytosis as well as the CCh-stimulated transcytosis. However, LGACs over-expressing mCherr y-myosin Vb tail 92 released signi ficantly more hSC under CCh treatment than at the resting stage (Figure 4.14A), suggesting that either the myosin V c-regulated secr etory pathway was not inhibited by over-expressed mCherr y-myosin Vb tail and could still be stimulated by CCh, or CCh stimulation could overcome the inhibitory effects of the myosin Vb tail. With CCh treatment, LGACs over-expressing EGFP-myosin V c tail released significa ntly less hSC than the control, indicating the inhibition of SC release fr om the regulated secretory pathway. However, LGACs over-expressing EGFP-myosin V c tail released signi ficantly more hSC under CCh treatment than at the resting stage (Figure 4.14A), suggesting that either the transcytotic pathway was not inhibited by over-expressed EGFP-myosin V c tail and could be accelerated by CCh, or CCh stimula tion could overcome the inhibitory effects of EGFP-myosin V c tail. These results indicate that although the two distinct pathways are regulated by different myosin motors and different Rab pro teins, CCh appears to accel erate plgR traffi cking and hSC release through both the transcytotic pathway and the regulated secretory pathway. The conclusions above are also consi stent with the confocal fluorescence microscopy analysis of basola terally endocytosed plgR. At the 30-minute time point, basola terally endocytosed plgR accumulated in the subapical domain beneath the lumen; when an additional 30-minute incubation at the resting stage was applied, endocytosed plgR continued to accumulate in the subapical domain (Figure 4.14B). However, when an 93 additional 30-minute CCh treatment was applied, most of the endocytosed plgR in the subapical domain was depleted (Figure 4.14B). This obser vation suggested that CCh accelerates the transcytotic pathway, possibly by accel erating the terminal steps. This is also the first study to reveal the CCh-inducible terminal release of plgR at the APM of polarized exocrine cells, through either the transcytotic pathway or the regulated secretory pathway. 94 Figure 4.15 A CCh CCh + B CCh CCh + c - . CCh 30 • I • Anti . :SC ' ' * * - · t] .......... 5 �m Anti-SC B * l . ' J ............. S. � m EGFP-myoVc tail · · · o . * . • .. ............. 5� m ta il / • -� �. .. . EGFP-m Q . . - ' . * ' \ ............. 5 �m I I I I I • I I I • MyoVb tail * * - 8 ............. 5� m M yo D * ............. 5� m Anti-SC - · · o * - ............. S� m Anti-SC • Q • • ............. 5� m Ove.r.la y • I t . · � ' " l . . _, ... .. � -- [B • f ........,, J 5� � Over� � . - · * ' -� . * r •/ � ... . . , ( Ov e rla y - ' *: .... , ?JC * � "' - ' . . -,. '!1 1· ............. S� m O v e � T S l \ JJ � . . ; . . . .. \.. � 1 � •r * ·· ' \ ' \ ............. S� m High Mag (3 . ·- · - . , . · ... High Mag • ' - · ·:tc • - . 1- 12.5 IJm-1 High Mag � ' ., _ ,;._ :.J {- .. . High Mag - J. -.. - ( " * .., \ 95 Figure 4.15 Apically-targeted traffi cking of plgR is regulated by the transcytotic pathway and the regulated secretory pathway distinctively. A) LGACs expressing mCherr y-myosin Vb tail were incubated with sheep anti-rabbit SC antiserum for 1 hour at 4 ,C, rinsed and incubated at 3T Cfor 30 minutes. Cells were then treated with or without 100 11M CCh for additional 30 minutes. Cells were fixed, permea bilized, and labeled with secondary Alexa Fluor ®-488-c onjugated donkey anti-sheep antibody, as well as Alexa Fluor®-647-conjuga ted phalloidin. B) LGACs expressing EGFP-myosin V c tail were incubated with sheep anti-rabbit SC antiserum for 1 hour at 4 ,C , rinsed and incubated at 3T Cfor 30 minutes. Cells were then treated with or without 100 11M CCh for additional 30 minutes. Cells were fixed, permea bilized, and labeled with secondary Alexa Fluor ®-568-conjugated donkey anti-sheep antibody, as well as Alexa Fluor®-647- conjugated phalloidin. Actin stained with Alexa Fluor®-647-conjugated phalloidin is displayed in purp le in the overlay image. C) LGACs expressing EGFP-myosin V c tail was treated with 100 11M CCh or not for 30 minu tes. Cells were fixed, permea bilized and labeled with primary sheep anti-SC serum, secondary Alexa Fluor ®-568-conjugated donkey anti-sheep antibody and Alexa-Fluor-647-conjugated phalloidin. Actin stained with Alexa Fluor®-647- conjugated phalloidin is displayed in purp le in the overlay image . High magnification image displays a region of 12.5 x 12.5 11m fr om the yellow boxed image to the left. White arrows, colocalization between red and green; *, lumena; scale bar: 5 11m. On the other hand, CCh treatment was not able to deplete the endocytosed plgR "trapped" in the membrane compartment labeled by mCherr y-myosin Vb tail (Figure 4.15A). This indicated that the CCh-induc ed accelerated release of SC via the transcytotic pathway is dependent on the myosin Vb motor, and suggested that the additional SC released under CCh stimulation by LGACs expressing mCherry- myosin Vb tail was likely secreted via the regulated secretory pathway. However, the over-expression of EGFP-myosin V c tail did not inhibit the depletion of endocytosed plgR in the subapical region upon CCh treatment (Figure 4.15B), further indicating that the transcytotic 96 pathway is not regula ted by the myosin V c motor. At the resting stage, a subset of endogenous plgR was enriched in the membrane compartment labeled by EGFP-myosin V c tail, and 30-minute CCh treatment was not able to deplete endogenous plgR fr om this compartment (Figure 4.15C). This finding is again consistent with the inhibition of the regulated secretory pathway and accounts for the overall reduction of hSC released into the culture superna tant under CCh treatment (Figure 4.14A). PKC-s is the common regulator for apical release of plgR through both the Rablla-regulated transcvtotic pathway and the Rab3D-regulated secr etory pathway. Previous study revealed that PKC-s promotes the transcytosis of plgR and dlgA in MDCK cells. In LGACs, PKC-s is a well-cha racterized downstream effector of CCh and regulator ofF-a ctin reorganization (Zoukhri eta!., 1997; Jerdeva eta!., 2005). Since CCh treatment seem to accel erate both the transcytotic pathway and the regulated secretory pathway, it is reasonable to hypothe size that PKC-s is involved in the process of apical release of plgR in LGACs (Roj as and Apodaca, 2002a). In this study, I interrogated the role of PKC-s in the transcytotic pathway and the regulated secretory pathway. 97 Figure 4.16 A 8 30 min 30 min + 30 min Control 30 min + 30 min CCh GFP co ,___... 5�m Relative recovery of SC • • A • • • •• A Anti-SC Actin co . co ,___... ,___... 5�m 5�m • • . - o,verl ay !Cfu .. . - ) �, . * ' - . .. ... . . -�· � . .- 98 Figure 4.16 Inhibition of basal-to-apical transcytosis of endogenous plgR by over- expression of PKC-s DN A) Quantification of SC released into culture supe rnatant. LGACs co-transduced with Ad hplgR-EGFP, regulatory virus and rAV cMv LifeAct-TagRFP, Ad PKC-s DN/GFP were cultured overnight with doxycycline. The net release of SC into the culture superna tant during 30-mi nute treatment was quan tified by western blotting, as described in Materials and Methods. n�5; *, p<0.05; **, p<O.OOl B) LGACs expressing PKC-s DN and GFP were incubated with sheep anti-SC serum, rinsed and incubated at 3T C. After 30 minu tes, cells were treated with or without 100 11M CCh for additional 30 minutes. Cells were fixed at time points, permea bilized, and labeled with secondary Alexa Fluor ®-568-conjugated donkey anti-sheep antibody, as well as Alexa Fluor®-647- conjugated phalloidin. Yellow rectangle, lumen and subapical region; white arrow, depletion of subapical plgR in non-transduced LGACs; blue arrow, retention of subapical plgR in LGACs expressing PKC-s DN and GFP; *, lumena; scale bar: 5 11m. I uti lized an Ad construct encoding domina nt negative (DN) PKC-s that inhibits the reorganization of the F-actin network as a tool for investigating the apically-targeted traffi cking of plgR. LGACs were co-transduced with Ad hplgR-EGFP, the AdenoX Te t-on® regulatory virus and Ad PKC-s DN/GFP, whereas rAV cMv Lif eAct-TagRFP was used as control for co-transduction. PKC-s DN almost completely inhibited the release of SC in both the resting and CCh-stimulated stages (Figure 4.16A). 99 Figure 4.17 A Rest ing CCh 30 min GFP * ........... S�m GFP ........... S�m ;�t- ¥ ,... • • . • 8 Rest ing CCh 30 min Anti-SC Actin � • * � * ........... ........... S�m S�m Anti-SC Actin � * � """' . ........... ........... S�m S� m Actin ........... S�m Actin * ........... S �m Omloy ,. t/1' * � l\ · . • to'"\ * � ,.. . · * . ... .. � . \ � . ,.-. - ·� . , ........... \ " S�m .... . Overla� * 1�- � . .. � .. � · r:>.. """' . I '-' '\' • ' -;! ........... . \ S�m Overlay � � � ,. Q �' � ........... '- S�m Overlay �- k- ,.. . )· � ... ........... ·s �m -· Figure 4. 17 Inhibition of apical release of endogenous plgR by PKC-E DN. LGACs expressing PKC-s DN and GFP (A) or non-transduced LGACs (B) were treated with 100 11M CCh or not (resting stage) fo r 30 minutes. LGACs were fixed, permeabilized, and labeled with primary sheep anti-rabbit SC serum, secon dary Alexa-Fluor-568-conjugated donkey anti-sheep antibody, as well as Alexa-Fluor-647-conjugated phalloidin. Star, lumena; scale bar: 5 11m; arrow, accumulation of pigR in the subapical domain 100 Interestingly, the CCh-stimulated release of SC by LGACs over-expressing PKC-e DN was signif icantly less than LGACs over-expressing mCherry-myosin Vb tail or EGFP-myosin Vc tail (Figure 4. 14A). These observations indica te that PKC-e regulates the release of SC through either or both the Rablla-regulated transcytotic pathway and the Rab3D-regula ted secretory pathway. Confocal microscopy showed that over-expression of PKC-e DN did not inhibit the accumulation of endocytosed plgR in the subapical region; however, the treatment of CCh was not able to trigger the depletion of basola terally endocytosed plgR in the subapical region (Figure 4.16A), suggesting the inhibiton of apical release of plgR through the transcytotic pathway. Figure 4.18 A GFP "Jt· Rab3D Actin Overlay � * * * * * ,., .. * -I --< ' "" J .... '\ " .............. * .............. * 1--C . * i .............. (} 5�m 5�m 5�m ; J s�m 8 GFP Rab1 1 Actin Overlay � ... ,.. .... . -4 ·P * * * . \ ' * * * .. • :·-· ., 't . .............. .............. .............. .............. 5 �m 5�m 5�m 5�m Figure 4.18 Expression of PKC-s DN does not alter the localiz ation of Rab3D or Rab11. A) LGACs expressing PKC-e DN and GFP were fixed, permeabilized, and labeled with primary rabbit anti-Ra b3D serum, secondary Alexa-Fluor®- 568-conjugated 101 donkey anti-rabbit antibody, as well as Alexa-Fluor®-647-con jugated phalloidin. B) LGACs expressing PKC-s DN and GFP were fixed, permea bilized, and labeled with primary mouse anti-Rabll antibody, secondary Alexa-Fluor ®-568-conjugated goat anti-mouse antibody, as well as Alexa-Fluor®-647-conjugated phalloidin. Star, lumena; scale bar: 5 11m When all endogenous plgR rather than a single cohort is labeled with anti -SC serum, immunofluore scence confocal microscopy showed accumulation of endogenous plgR in the subapical domain of non-transduced LGACs (Figure 4.17B). The subapical pool of plgR in Figure 4.17A, B represents plgR in both the Rablla-regulated transcytotic pathway and the Rab3D-reg ulated secr etory pathway. The subapical pool of plgR in non-transduced LGACs could be depleted by the treatment of CCh (Figure 4.17B); while over-expression of PKC-s DN inhibited the release of endogenous plgR in the subapical region by the treatment of CCh (Figure 4.17 A). This is consistent with the quan tification ofhSC released by LGACs over-expressing PKC-s DN (Figure 4.16A). Moreover, confocal microscopy showed that PKC-s DN did not substantially alter the localization of Rabll or Rab3D (Figure 4.18), suggesting that PKC-s DN is unlikely to inhibit the Rablla-regulated transcytotic pathway and Rab3D-regulated secretory pathway by directly disrupting Rab localization or functionality. This study identif ied that PKC-s is a crucial regulator for the apical release of SC through either the Rab lla-regulated transcytotic pathway or the Rab3D-reg ulated secr etory pathwa ys. 102 Chapter V. Discussion: The mechanisms of plgR traff icking in LGACs I have used the rabbit LGAC model system to chara cterize the traff icking of pigR and secretion of SC through tandem apically-directed secretory pathwa ys: regulated exocytosis and transcytosis. The observations reported here extend the molecular characterization of the regulation of the transcytotic and secretory pathways in LGAC. Using a combination of unique tools that allowed the tracking of cargo sepa ra tely along the regulated secretory and tra nscytotic pathways, I demonstra ted a total of three pathways for apically-directed traff icking in LGAC: the Rablla-regulated transcytotic pathway, the Rab3D-reg ulated secr etory pathway and the Rab27b-regulated secretory pathway. Moreover, I showed that apically-directed pigR in the Rablla-regulated transcytotic pathway was sorted independently of either of the 2 regulated secr etory pathw ays, i.e., the pathways did not appear to in tersect. In addition, I showed that, the pigR appeared to occupy exclusively Rab3D-enriched secr etory vesicles, but not Rab27b-enriched secretory vesicles. Many previous studies that aimed to iden tify the role of Rablla in transcytotic and other traffi cking pathways have used cells that are not specialized for transcytosis or secrete the proteins under study as model system s. Rablla was shown to participate in 103 the exocytosis of exogenously expressed human growth hormone in bladder umbrella cells (Khandelwal et a!., 2008) and in the basal-to-apical transcytosis of digA mediated by exogenous pigR in MDCK cells (Casanova et a!., 1999; Wang et a!., 2000). These studies have provided valuable insig hts into general traff icking mechanisms of Rab1 1a. I wanted to interrogate the role of Rablla in transcytotic traffi cking of pigR and digA in LGACs, which engage robustly in both constitutive transcytosis and regulated apical exocytosis. Confocal fluorescence microscopy confirmed that basola terally endocytosed pigR is sorted into the Rablla-enriched membrane compa rtment. The sorting of endocytosed materials into Rab 11 a-enriched membrane compartment seems to be cargo-spec ific, because the fluid phase marker TMR-dextran did not accumulate m EGFP-Rablla-enriched membrane compartment. Over-expre ssion of GDP-locked EGFP-Rablla DN inhibited the basal-to-apical traff icking of basola terally endocytosed pigR while inhibiting the transcytosis of digA, which suggests basal-to-apical transcytosis of pigR and digA requires Rablla's functionality, which depends on its cycling between GTP-bound and GDP-bound form s. I also charact erized traff icking mechanism of Rab lla-enriched vesicles associa ted with the cytoskeleton. Localization and traff icking of Rablla vesicles is dependent on both the MT and the F-actin networks, and regulated by a minus-end-directed MT -based 104 motor usmg the dynactin complex and by myosm Vb, respec tively. My study characteriz ed the actin- and MT dependent traffi cking mechanisms of Rablla-enriched vesicles in the constitutive phys iological transcytotic pathway regulated by Rablla . Our data suggest that a minus -end-directed MT -based motor using the dynactin complex and myosin Vb are both recruited onto Rablla-enriched vesicles. The sorting of dlgA and /or plgR into the Rablla compartment is, however, independent of myosin Vb, because in LGACs overexpressing mCherr y-myosin Vb tail, basola terally endocytosed plgR or endocytosed dlgA still accumulates in the compa rtment labeled by myosin Vb tail and Rablla (Figure 3.10 B,D; Figure 4.13B). Rablla participates in regulated exocytosis in other specialized epithelial cells, such as urinary bladder umbrella cells (Khandelwal et a!., 2008) and HCl-sec reting gastric parietal cells (Duman et a!., 1999). In bladder umbrella cells and gastric parietal cells, regulated exocytosis of membrane vesicles with the apical membrane functions to increase apical membrane surf ace area and the number of membrane transporters at the APM, respectively; and this process is inhibited by expression of Rablla DN. However, my data fr om confocal fluorescence microscopy and biochemical analysis of protein and �-hexosaminidase secretion clearly showed that LGACs do not rely upon Rablla to drive regulated secretory vesicle exocytosis, as expression of EGFP-Rablla DN does not inhibit the regulated secretion. Thus, it is possible that the role of Rab lla in regulated 105 exocytosis within bladder umbrella and gastric parietal cells, but not acinar cells, reflects the very different functions of secretory vesicles in these epithelial cells. With respect to pigR transcytosis, I transduced LGAC with a novel Ad hpigR-EGFP construct that appeared faithf ully to reca pitulate our previous findings with the endogenous pigR. In particula r, basola terally endocytosed pigR accumulated in subapical Rablla-enriched vesicles, and this transcytotic pathway ofpigR was dependent upon functional Rablla . The pigR endocytosed fr om the basolateral membrane did not enter the regulated secr etory pathway, and the transcytosis pathway can also accele rated by the cholinergic agonist CCh. While the Rablla-regulated transcytotic pathway 1s responsible for the basal-to-apical transport of pigR endocytosed fr om the basolateral membrane, the Rab3D-regulated secretory pathway contains a subset of pigR that likely originates directly fr om the biosy nthetic pathway. This finding is consi stent with previous studies suggesting the participation of both transcytotic and secr etory pathways in the traffi cking ofpigR and secretion of SC by LGACs (Evans et a!., 2008). I also further characteriz ed the regulated secretory pathway in LGAC with respect to the Ra b3D- and Rab27b-enriched compartments. Rab3D and Rab27b are both present in pancreatic acinar cells (Ohnishi et a!., 1996; Valentijn et a!., 1996; Chen et a!., 2004), parotid acinar cells (R affan iello et a!., 1999; Nguyen et a!., 2003; Imai et a!., 2004; Imai 106 et a!., 2009), and LGACs (Evans et a!., 2008; Chiang et a!., 2011 ). In rat parotid acinar cells, Rab27b regulates the release of amylase (Imai et a!., 2004), and Rab3D is localized to the zymogen granule membrane (Raffa niello et a!., 1999). However, these previous studies did not charact erize the interrelation between the membrane compartments labeled by Rab3D and Rab27b in the same cell. Indirect immunoflu orescence showed that Rab3D and Rab27b show partial coloca lization in LGACs (Chiang et a!., 2011) , but the studies in fixed cells may not have been able to resolve clearly individual pools of Rab3D- and Rab27b-enriched compartments. The Ad mCherr y-Rab3D construct exhibited plgR interaction and secr etory vesicle associa tion comparably to the endogenous Rab3D protein. Co-expression of mCherry-Ra b3D and YFP-R ab27b showed that these two markers label two largely distinct pools of secr etory vesicles in LGACs: Rab3D regulates the apical release of plgR, but not syncollin; while Rab27b regulates the apical release of syncollin, but not plgR. This result suggests that LGACs may utilize different Rabs to regulate apically-targeted traff icking of different secretory cargo proteins, revealing diff erentiation and comple xity of the regulated secretory pathway in exocrine cells. Although not previously investigated in live cell studies for these two particular Rab isof orms (Rab3D and Rab27b) in acinar cells, the relationship between other Rab3 and Rab27 isof orms have been previously investigated in other cells. The enrichment of 107 GTP-bound Ra b27 in sperm enhanced recruitment of active Rab3 to the vesicles that media te acrosome fusion, suggesting a temporal regulation and relationship between the two Rab isof orms (Bustos et a!., 2012). Another study in PC 12 cells has suggested that Rab27a is associated more stably with exocytotic gran ules upon format ion, and that Rab3A associates with these same granules more dyna mically and preferentially with newly fo rmed rather than aged granul es (Handley et a!., 2007). Still other stud ies suggest the temporal and seq uential activation of Rab27, Rab3 and Rab8 in apical membrane traff icking in epithelial development (Galvez-Santisteban et a!., 2012). Work in LGACs has long recognized that individual acinar cells display secr etory vesicles of significa ntly different composition; EM analys is reveals serous and mucous granu les with clea rly distinct content (Hann et a!., 1989; Edman et a!., 2010); the nature of the molecular machinery that resides on each type of granu le has not been identif ied due to complexities associated with biochemical isolation of these large (0.5 11M-1 11M) and fr agile vesicle popula tions. Collec tively this work suggests that secretory Rab assoc iation with secr etory vesicles may be quite complex, dependent upon the local signaling environment, the age of the secretory vesicle, the contents of the vesicle and the second messengers involved. Myosin Vb and V c motors are members of the myosin V fam ily. This work, to our knowledge, constitutes the first research to study these two motors in paral lel in the same 108 cell model, and distinguishes their fun ctional differentia tion: Myosin Vb motor is associated with Rablla-enriched vesicles, and myosin Vc motor with Rab3D- and Rab27b-enriched vesicles. Myosin Vb and myosin Vc motors do not seem to have fun ctional overlap: over-expression of mCherr y-myosin Vb did not alter the regulated secr etory pathway labeled by YFP-Rab27b or Rab3D in LGACs; whereas it induced substantial relocation and complete traff icking inhibition of EGFP-Rablla-enriched vesicles, as well as the inhibition of basal-to-apical transcytosis of basola terally endocytosed plgR sorted into Rablla-enriched vesicles. These obser vations clea rly indicate the differentiation in the functions of myosin V motor isof orms in specialized secr etory acinar cells that express all 3 known class V myosin isof orms (Marchelletta et a!., 2008), and further demonstrates that Rablla regulates a pathway distinct fr om the Ra b3D-regulated secretory pathway and the Rab27b-regulated secretory pathway in exocrine acinar cells. PKC-s is demonstra ted to be a crucial regulator for apical release of secr etory pro teins. Our previous study showed that PKC-s DN signif icantly decreased the syncollin-GFP released into the culture superna tant under CCh treatment (Jerdeva et a!., 2005). In this study, quantitative western blots and confocal immunofluore scence microscopy showed that PKC-s DN also significantly decreased the hSC released into the culture superna tant, either at the resting stage or under CCh treatment. Syncollin-GFP is 109 released at the apical membrane through the Ra b27b-regulated secr etory pathway, while hSC is released at the apical membrane through either the Rab3D-regulated secr etory pathway or the Rablla-regulated transcytotic pathway. Our obser vations indicate that PKC-s is a common regulator of the terminal release of secr etory proteins at the apical membrane through Rab27b-regulated secretory pathway, Rab3D-reg ulated secretory pathway and the Rablla-regulated transcytotic pathway. In other words, these 3 apically-targeted pathways do have intersections at their terminal-release step. PKC-s has been shown to regulate multip le downstream signaling pathways, including the MEK/ERK and the NFKB pathways that regulate gene expression, the p38/MAPK pathway assoc iated with apoptosis, etc (Akita, 2002). PKC-s also interacts with RACK2 and RACK! scaffolding pro teins, which are involved in Golgi vesicular traffi cking and cell ad hesion, respectively (Akita, 2002). PKC-s has an acti n-binding motif in its regulatory domain, and this motif is ess ential for inducing neurite outgrowth during neuronal differentiation by PKC-s (Zeidman et a!., 2002). PKC-s is involved in many intracellular signaling processes, and yet this study does not provide direct evidence showing by aff ecting which signaling process can PKC-s DN inhibit the apical release of cargoes via the Rablla-regulated transcytotic pathway, the Rab3D-regulated secretory pathway and the Rab27b-regulated secretory pathway. However, these 3 pathways involve either myosin Vb or myosin V c motor, suggesting their terminal release 110 of cargo at the apical membrane may be dependent on the F -actin network. Our previous research showed that PKC-s DN inhibits the reorganiza tion of the F -actin network in the subapical domain (Jerdeva et a!., 2005), and PKC-s is capable of directly interacting with F-actin (Prekeris et a!., 1996). Therefore, it is reasonable to hypot hesize that PKC-s DN disrupts the Ra blla-regulated transcytotic pathway, the Rab3D-regulated secr etory pathway and the Rab27b-regulated secr etory pathway likely by inhibiting the reorganization of F -actin in the subapical domain. lll Figure 5.1 � plgR Q Early Hypo. endosome ('{ secretory , '/' Dynein vesicle I J y sc Q Rab11a vesicle •: Syncollin � F-actin -V- dlgA 0 Rab3D vesicle - MT VM yosin Vb -\tl- slgA Rab27b • MTOC � Myosin Vc vesicle Figure 5.1 Working model of the trafficking of plgR and dlgA in LGACs. Newly synthesized plgR is segregated into two subsets in the trans-Golgi network (TGN). One subset is transported onto the basola teral membrane fo r the transcytotic pathway. At the basolateral membrane, plgR with or without bound dlgA is endocytosed and transported into early endosomes, and then sorted into Rab lla-enriched vesicles. These are then transported on microtubules towards the minus ends anchored on MTOCs located in the subapical region by cytoplasmic dynein. During the process of basola teral-to-apical transport, Rablla-enriched vesicles recruit myosin Vb, which enables their movement on the subapical F-actin network. When Rab lla-enriched vesicles that contain plgR and dlgA are released at the apical surface, the extracellular domain of the plgR is proteolytically cleaved, and slgA as well as fr ee SC are released into mucosal secretions. 112 The other subset is sorted to Rab3D-enriched secr etory vesicles, which are not accessible to the basola terally endocytosed plgR that is sorted into the Rablla-regulated secretory pathway. The terminal release of plgR through the Rab3D-regulated secr etory pathway is myosin V c. Rab27b labels a different pool of mature secr etory vesicles than Rab3D-enriched secr etory vesicles. Rab27b- enriched secr etory vesicles are regulated by myosin V c, and they are responsible for the apical release of syncollin, but not plgR. APM, apical membrane; BLM, basal membrane ; SC, secretory component; dlgA, dimeric IgA; slgA, secretory IgA; MT, microtu bule; MTOC, microtu bule-o rga nizing center. 113 On the basis of these observations, I propose a model for the traffi cking of pigR in LGACs (Fignre 9). A subset of newly synthe sized pigR is transported to the baso lateral membrane. Bound to its ligand or not, basola teral pigR is rapidly endocytosed and transported into the early endosomes labeled by EEA- 1 and Ra b5a. Thereafter, they are sorted into Rablla-enriched vesicles, which are transported on MTs, towards the apical region, where they form a subapical pool for the further release of sigA into the lumen. Due to the enrichment of the p150 subunit of the dynactin complex with Rablla , combined with the known pola rity of MTs in LGACs with minus -ends apical ( da Costa et a!., 1998), the cytoplasmic dyn ein motor is highly likely responsible for driving Rablla-enriched vesicles towards subapical MTOCs. During the process of basal-to-apical transport, the Rab11 a-enriched vesicles experience a motor switch step by recruiting myosin Vb, which regulates the movement of these vesicles on the cortical F-actin network towards the apical plasma membrane. The traffi cking mechanism of Rablla-enriched vesicles based on the coordination of MT-based and F-actin-based motors in this model is consistent with other proposed coordination between MT -based and actin-based motors that fac il itate transport processes such as regulated exocytosis of secretory granules in melanocytes (Barra! and Seabra, 2004; Ross et a!., 2008). In this model, Rablla-enriched subapical vesicle dissoc iation fr om the MT network should, in the presence of active myosin Vb motor, result in their redistribution to the 114 subapical F-actin network; whereas their dissocia tion fr om the F-actin network in LGACs that over-express the mCherr y-myosin Vb-tail should, in the presence of active cytoplasmic dynein, cause these vesicles to accumulate on MTs and at the MTOC. These are all consi stent with the obser vations in this study. Rablla is known to participate in the apical recycling of dlgA in MDCK cells that express exogenous plgR (Casanova et a!., 1999). Therefore, it is reasonable to hypothe size that Rablla also participates in the apical recycling of specific cargoes in LGACs, which is similar to the apical recycling mechanism in MDCK cells. The subapical pool of Rablla vesicles might represent a compa rtment engaged both in basolateral-to-apical transcytosis as well as apical recycling. Another subset of newly synt hesized plgR is directly sorted into Rab3D-enriched secretory vesicles in the subapical domain. Myosin V c is required for the apically-targeted traffi cking of SC m Rab3D-enriched vesicles. Although Rab27b-enriched secretory vesicles also recruit my osm V c, some Rab27b-enriched secretory vesicles consti tute a clea rly distinct membrane compartment fr om Rab3D-enriched secr etory vesicles as evidenced by their ability to fuse and release syncollin-GFP without any apparent assoc iation or recruitment of overexpressed mCherry- Rab3D. Additional vesicle populations labeled by both Rab27b and Rab3D are hypotheti cally shown that may carry distinct cargo proteins and/or represent vesicles at a 115 different matura tional or regulatory sta tes. The mechanism that disting uishes the basola terally endocytosed plgR fr om that sorted into the Ra b3D-se cretory pathway requires further study. However, a reasonable expla nation is that phosphor ylation of serine 664 or 726 on the cytoplasmic domain of plgR provides the crucial signal for internalization and tra nscytosis (Hirt et a!., 1993; Okamoto et a!., 1994) may also serve as a sorting signal. This may differentiate the endocytosed plgR fr om the newly synt hesized, but perhaps non-phosph oryla ted plgR sorted into the Rab3D-reg ulated secr etory pathway. Moreover, the direct binding between plgR and Rab3D demonstrated in previous study (Evans et a!., 2008) as well as in this study may explain the sequestration of a subset of newly synthe sized plgR by Rab3D into the membrane compartment of secr etory vesicles. My model also suggests the phys iological functions of different traff icking pathways of plgR: The transcytotic pathway provides a constitutive mechanism for transporting dimeric IgA fr om the serum and interstitial space to the tears. This is ess ential for maintaining the normal functions of the mucosal immune system. However, the regulated secr etory pathway may play the role of reinforcing mucosal innate immunity under conditions of ocular surf ace challenge assoc iated with physiological reflex and triggering of tear flow through regulated secretion by LGACs. Free SC is known to provide protective function against certain pathogens (Giugliano et a!., 1995; 116 Hammerschmidt et a!., 1997; Dallas and Rolfe, 1998; de Araujo and Giugliano, 2001; de Oliveira et a!., 2001; Perrier et a!., 2006). The rapid release of SC may immediately increase the SC concentration in tears, to saturate and neutralize path ogens and/or allergens, the sources of eye irrita tion. LGACs may deliberately sequester plgR and SC in a specialized population of Rab3D-enriched secr etory vesicles, which may be mobilized and released under specific stimu li, particularly for this pur pose. In summ ary, this study shows that Rablla regulate the cargo-speci fic basal-to-apical transcytotic pathway of plgR and/or dlgA in LGACs, polarized epithelial cells specialized physiologically for transcytosis and apical exocytosis. This compa rtment is distinct fr om the lysosomal pathway, the Ra b3D-reg ulated secretory pathway and the Rab27b-regulated secr etory pathway. The traffi cking of the Rablla-enriched vesicles occurs in assoc iation with the MT and the F -actin networks, regulated by cytoplasmic dynein and myosin Vb, respectively. A subset of plgR and its cleaved SC domain appear segregated in Ra b3D-enriched secretory vesicles (lacking Rab27b) and regulated by myosin V c. In addition, the exocytosis of plgR fr om both the Rablla-regulated transcytotic pathway and the Rab3D-regulated secretory pathway can be accele rated with cholinergic agonists . 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Am J Physio/ 272, C263-269. 125 Appendices Protein sequences of EGFP-Rablla constructs EGFP-Rablla WT: MVSKGEELFT GVVPILVELDGDVNG HKFSVS GEGEGDATYGKLTLKFICTTGKLP VPWP TLVTTLTY GVQCFSRYPDHMKQHDFFKS AMPEGYVQERTIFFKDDGNYKT RAEVKFEGDT LVNRIELKGIDFKEDG NILGHKLEYNYN SHNVYIMADKQKNGIK VNFKIRHNIEDGSVQL ADHYQQNTPIGDGPVLLP DNHYLSTQSALSKDPNEKRDH MVLLEF VTAAGI TLGMDEL YKSGRTQISSSSFEFDHMGTRDDEYDYLFKVVLIGD SGVGKSNLLSRFTRNEFNLESKS TIGVEF ATR SIQVDGKTIKAQIWDT AGQERYRA ITSA YYRGAVGALL VYDIAKHLTYENVER WLKELRDHADSNIVIMLVGNKSDLRH LRA VPTDE ARAFAE KNGLSFIETSALDST NVEAAFQTIL TEIYRIVSQKQMS DRRE NDMSP SNNVVPIHVPPTTENKPKVQCCQNI Theoretical pi!Mw: 5.74 I 52983.71 53kD EGFP-Rablla DN (S25N): MVSKGEELFT GVVPILVELDGDVNG HKFSVS GEGEGDATYGKLTLKFICTTGKLP VPWP TLVTTLTY GVQCFSRYPDHMKQHDFFKS AMPEGYVQERTIFFKDDGNYKT RAEVKFEGDT LVNRIELKGIDFKEDG NILGHKLEYNYN SHNVYIMADKQKNGIK VNFKIRHNIEDGSVQL ADHYQQNTPIGDGPVLLP DNHYLSTQSALSKDPNEKRDH MVLLEF VTAAGI TLGMDEL YKSGRTQIS SSSFEFDHMG TRDDEYDYLFKVVLIGD SGVGKNNLLSRFTR NEFNLESKS TIGVEFA TRSIQVDGKTIKAQIWDTAGQERYRA ITSA YYRGAVGALL VYDIAKHLTYENVER WLKELRDHADSNIVIMLVGNKSDLRH LRA VPTDE ARAFAE KNGLSFIETSALDST NVEAAFQTIL TEIYRIVSQKQMS DRRE NDMSP SNNVVPIHVPPTTENKPKVQCCQNI EGFP-Rablla CA (S20V): MVSKGEELFT GVVPILVELD GDVNGHK FSV SGEGE GDATY GKLTLKFICT TGKL PVPWP TL VTTL TYGVQ CFSRYPDHMK QHDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTLVNRIELKGID FKEDGNILGH KLEYNYNSHN VYIMADKQKN GIKVNFKIRH NIEDGSVQL ADHYQQNTPIG DGPVLLPDNH YLSTQSALSK DPNEKRDHMV LLEFVT AAGI TLGMDELYKSGRT QISSSSF EFHMGTRDDE YDYLFKVVLI GDVGVGK SNL LSRFTRNEFN LESKS TIGVE FATR SIQVDG KTIKAQIWDT AGQERYRAIT SAYYRGAVGA LL VYDIAKHL TYENVERWLK ELRDHADSNI VIMLVGNKSD LRHLRA VPTD EARAFAEKNG LSFIETSALD STNVEAAFQTILTEIYRIVS QKQMSDR REN DMSPSNNVVP IHVPPTTENK PKVQCCQNI Theoretical pi!Mw: 5.82 I 52880.68 126 Protein sequence of hplgR-EGFP MLLF VLTCLLA VFP AISTKSPI FGPEEVNSVEGNSVS ITCYYPPTSVNRHTRKYWCR QGARGGCIT LISSEGYVSSKY AGRANLT NFPENGTFVVNIAQLSQDDS GRYKCGL GINSRGLSFDVSLEVS QGPGLL NDTKVYTVDLGR TVTINCPFKTENAQKRK SL YK QIGL YPVL VIDSSGYVNPNYTGRIRLDIQGTGQ LLFSVVINQLRLSDAGQYLCQAG DDSNSNKKNADLQVL KPEPELVYEDLRGSVTFHCALGPEV ANVAKFLCRQSSGE NCDVVVNTLGKRAPAFEGRILL NPQDKDGSFS VVITGLRKEDAGRYLCGAHSDG QLQEGSPIQAWQLFVNEESTIPRSPTV VKGVAGGSVAVLCPYNR KESK SIKYWCL WEGAQNGRCPLLVDSEGWVKAQYEGRLSLLEE PGNGTFTVILNQL TSRDAGFY WCLTNGDTLWR TTVEIKIIEGEPNLKVPG NVT AVLGETL KVPCHFPCKFSSYEKY WCKWNNTGCQALP SQDEGPS KAFVNCDENSRL VSL TLNL VTRADEGWYWCGV KQGHFYGETAAVYVAVEERKAAGSRDVSLAKADAAPDEKVLDSGFREIENKAIQ DPRLFAEEKA VADTR DQADGSRAS VDSGSSE EQGGSS RALVSTL VPLGL VLAVGA VAVGV ARARHRKN VDR VSIRSYRT DISMSDFENSR EFGANDNMGAS SITQETSLG GKEEFV A TTESTTETKEPKKAKRSS KEEAEMAYKDFLLQ SSTV AAEAQDGPQEA Y LELKLRILQSTVPRARDPPV ATMVSKGE ELFTGVVPILVELDGDVNG HKFSVSGE GEGDATYGKLTLK FICTTGKLP VPWPT LVTTL TYGVQCFSR YPDHMKQHDFFKSA MPEGYVQER TIFFKDDGNYKTRAEVKFEG DTLV NRIELKGIDFKEDGNILGHKLE YNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLP DNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK Protein sequence of mCherrv-Ra b3D MVS KGEEDNMAIIKEFMRFKVHMEGSVNGHEFEI EGEGEGRPYEGTQTAKLKVT KGGPLPFA WDILSPQFMYGS KA YVKHP ADIPDYLKLSFPEGFKWER VMNFEDGG VVTVTQDSS LQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGA LKGEIKQRLKLKDGGHYDAE VKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTI VEQYERAEGRHSTG GMDELYKL DGGSGGGSGGGSGMASAGDPPAGPRDAADQ NFDYMFKILIIG NSSVGKTSFL FRYADDSFTPA FVST VGIDFKVKTVYRHDKRIKL QIWDT AGQER YRTITTA YYRGAMGFLLVYDIANQES FNAVQDWATQIKTYSWDN AQVILVGNKCDLEDERA VPAEDGRRLADDLGEYEQTEAKTLNPRPTFERL VDSIC EKMNESLEP SSSPGSNG KGPALGDAPPPQPSSC GC Theoretical pi!Mw: 5.13 I 517 06.88 127

Abstract (if available)

Abstract This study characterizes the intracellular trafficking pathway of polymeric immunoglobulin receptor (pIgR) that mediates the basal-to-apical transcytosis of dimeric immunoglobulin A (dIgA) in lacrimal gland acinar cells (LGACs). This study demonstrates that the trafficking of pIgR to the apical plasma membrane in LGACs involves two distinct pathways: the transcytotic pathway and the regulated secretory pathway. Rab3D and myosin Vc facilitate the release of a distinct pool of pIgR from the regulated secretory pathway to the apical plasma membrane in a process stimulated by the muscarinic cholinergic agonist carbachol (CCh)

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Asset Metadata

Creator Xu, Shi (Ben) (author)

Core Title The characterization of Rab11a and trafficking mechanisms of polymeric immunoglobulin receptor (pIgR) in lacrimal gland acinar cells (LGACs)

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 02/20/2013

Defense Date 01/25/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag lacrimal gland,myosin Vb,myosin Vc,OAI-PMH Harvest,polymeric immunoglobulin receptor,Rab11a,secretory pathway,transcytosis

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hamm-Alvarez, Sarah F. (committee chair), Garner, Judy A. (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email jqrshdsy@gmail.com,jqrshdsy@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-221919

Unique identifier UC11292644

Identifier usctheses-c3-221919 (legacy record id)

Legacy Identifier etd-XuShiBen-1444.pdf

Dmrecord 221919

Document Type Dissertation

Rights Xu, Shi (Ben)

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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