The octameric exocyst complex is associated with the junctional complex and recycling endosomes, and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. I observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by the expression of the C-terminus of Sec15A or downregulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multi-purpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells, and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated chloride channel that is a member of the ATP-binding cassette (ABC) transporter family [1]. It is involved in the movement of chloride ions across epithelial membranes in the airways, sweat glands, intestine and pancreas [2]. Mutations in CFTR that result in a loss of channel function result in the disease cystic fibrosis, affecting nearly 1 in 2500 people in northern Europe and the United States [3]. As a member of the ABC transporter family, CFTR shares the structural features of these proteins. Unique to CFTR is the presence of a cytoplasmic R-domain, that contains multiple phosphorylation sites. Phosphorylation of the R domain is required for CFTR channel gating, and cAMP/PKA simulation can also elicit insertion of CFTR into the plasma membrane from intracellular compartments [4]. We evaluated the structural basis of regulated CFTR trafficking by determining agonist-evoked increases in plasma membrane capacitance (Cm) of Xenopus oocytes expressing CFTR deletion mutants. Expression of CFTR as a split construct that omitted the R-domain (´aa 635-834) produced a channel with elevated basal current (Im) and no ´Im or trafficking response (´Cm) upon cAMP/PKA stimulation, indicating that the structure(s) required for regulated CFTR trafficking are contained within the R domain. Additional deletions showed that removal of amino acids 817-838 produced a channel with regulated gating that lacked the agonist-induced increase in CFTR trafficking. This 22aa region exhibits helical structure, bears a net negative charge of -9, is highly conserved among species, and has been termed NEG2 [5, 6]. Injection of NEG2 peptide into oocytes expressing split ´NEG2 CFTR prior to stimulation restored the agonist-evoked ´Cm, consistent with the concept that this sequence mediates regulated CFTR trafficking. Further modifications of NEG2 suggest that the trafficking phenotype depends primarily on its helical structure. These observations suggest that the NEG2 region at the C-terminus of the R domain allows CFTR to enter a regulated intracellular compartment from which it traffics to the plasma membrane in response to cAMP/PKA-stimulation.

Lysosomal storage diseases (LSDs) are a group of inherited disorders that are caused by the defective activity of lysosomal proteins, resulting in the intracellular accumulation of undigested lysosomal metabolites. Mucolipidosis type IV (MLIV) is a neurodegenerative, recessive disease that results in the accumulation of undigested material in most tissue types. MLIV is caused by mutations in the MCOLN1 gene, encoding the transient receptor potential (TRP) cation channel family member mucolipin-1 (TRP-ML1). While previous work has provided insight into the role of this protein in progression of MLIV, a detailed knowledge of TRP-ML1 function and subsequent role in MLIV pathogenesis remains unclear. The aim of this project was to gain a better understanding of TRP-ML1 function and to provide further insight into the molecular mechanisms behind MLIV pathogenesis. Since TRP-ML1 is lysosomally localized and is subject to proteolysis, I wanted to first examine the trafficking and characterize the processing of this protein. TRP-ML1 undergoes cleavage within its first extracellular loop during its biosynthetic delivery to lysosomes. The lysosomal delivery of TRP-ML1 is impaired by depletion of the Adaptor Protein (AP) complex AP-1, while proteolysis remains unaffected in cells lacking AP-1, indicating that cleavage may occur in a pre-lysosomal compartment. Current models of MLIV pathogenesis suggest that TRP-ML1 directly regulates the postendocytic membrane trafficking by mediating interactions between late endosomes and lysosomes. Acute down-regulation of TRP-ML1 reveals that the lysosomal delivery and degradation of various markers is unperturbed, arguing against this model for MLIV pathogenesis. However, LDL-derived cholesterol ester hydrolysis in MLIV fibroblasts was found to be selectively impaired, suggesting that loss of TRP-ML1 alters lysosomal hydrolytic activity and has a cumulative effect on lysosome function. My findings support a role for TRP-ML1 in maintenance of the ionic balance that is critical for proper lysosome function. This work is significant because it shifts focus away from the concept that TRP-ML1 only functions to regulate specific membrane trafficking events along the endocytic pathway. These results contribute to a more complete understanding of the physiological role of TRP-ML1 and broaden current understanding of MLIV progression, providing the basis for potential therapeutic treatment strategies.

Polarized epithelial cell function relies on the proper sorting and distribution of newly synthesized proteins to either the cells apical or basolateral domains. If trafficking is altered by disruptions either the fidelity or efficiency of this sorting then disease can result. There is an increasing appreciation for the role of phosphatidylinositol metabolism in membrane traffic, including the sorting and delivery of newly synthesized proteins. I have studied how biosynthetic delivery pathways are regulated by the expression of phosphatidylinositol-metabolizing enzymes. The phosphatidylinositol 4,5-bisphosphate (PIP2)-synthesizing enzyme, murine phosphatidylinositol 4-phosphate 5-kinase I alpha (PI5KIá) localizes to the apical pole of Madin-Darby canine kidney (MDCK) cells and increases cellular PIP2 concentrations over control cell levels. Interestingly, expression of exogenous PI5KIá stimulated the rate of surface delivery of a subset apical proteins that associate with lipid rafts, including influenza hemagglutinin (HA). Conversely, overexpression of the PIP2-5-phosphatase OCRL (oculocerebrorenal syndrome of Lowe), which is defective or absent in patients with Lowe syndrome, decreased cellular PIP2 levels and inhibited the rate of HA delivery. The observation that increases in PIP2 stimulate apical delivery of HA suggests the possibility that depletion of OCRL may have a similar effect to overexpression of PI5K. I used siRNA to knock down OCRL in MDCK cells and human proximal tubule (HK2) cells and examined the consequence on HA surface delivery. Knockdown of OCRL slightly increased cellular PIP2 levels but did not stimulate HA delivery. PI5K-mediated increases in PIP2 results in activation of neuronal Wiskott-Aldrich syndrome protein (N-WASP) leading to downstream actin cytoskeleton rearrangements including the formation of actin comets. To examine the potential role of N-WASP in HA delivery I expressed a dominant negative inhibitor of N-WASP function, the WA domain from a WASP family member, WAVE1. Expression of the WA domain significantly and selectively inhibited the rate of HA surface delivery. siRNA-mediated knockdown of N-WASP also inhibited HA delivery, confirming a role for N-WASP in biosynthetic traffic. Consistent with this, PI5KIá and HA (but not p75) were visualized on actin comets in formaldehyde-fixed MDCK cells. In summary, my data support a role for PI5K-stimulated actin comet formation in apical delivery of a subset of newly synthesized proteins.

Hutchinson-Gilford Progeria Syndrome (HGPS) is an autosomal dominant disorder caused by de novo mutations in the gene (LMNA) encoding lamin A that results in premature aging and early death. HGPS belongs to a group of disorders collectively referred to as, segmental progeroid syndromes, because multiple organs and tissues exhibit premature degenerative phenotypes consistent with physiological aging. The results presented here indicate that HGPS cells exhibit an elevated steady-state level of DNA double-stranded breaks (DSBs) and impaired repair of ionizing radiation (IR)-induced DSBs, both of which correlate strongly with the nuclear structural irregularities observed in a fraction of HGPS cells. These DNA damage-associated defects are due to the presence of Progerin in a dominant gain of function manner. Accordingly, reduction of Progerin levels by treatment with a farnesyl transferase inhibitor (FTI) improves repair of IR-induced DSBs. Interestingly, MRN (MRE11/Rad50/Nbs1) repair complex factors, involved in DSB repair, exhibit delayed localization to IR-induced DSBs, which may contribute to the repair defect. Furthermore, many segmental progeroid syndromes including HGPS exhibit phenotypes consistent with pre-natal developmental defects. These developmental defects are significant in their own right, but also because they may contribute to the post-natal progressive degeneration observed in HGPS. To begin investigating whether the HGPS mutation has adverse effects during pre-natal development, the expression of nuclear lamins was characterized in mouse (mESCs) and human (hESCs) embryonic stem cells which have been shown to be representative of pluripotent inner mass cells of blastocyst-stage embryos. This study shows that lamin A is not expressed in undifferentiated mESCs and hESCs, but becomes expressed quickly during in vitro hESC differentiation. Theses results suggest that Progerin is absent during early pre-natal development, but becomes expressed at later stages when the LMNA gene is activated, perhaps adversely affecting the developmental process. Altogether, theses studies identify impaired DNA DSB repair as a molecular mechanism that may contribute to the progressive degeneration phenotype and suggest that at least some of the pathophysiology may begin before birth.

Mouse bronchiolar stem cells have been identified in vivo based on functional characteristics including naphthalene resistance, long-term retention of labeled DNA precursors, and dual expression of markers for airway (CCSP) and alveolar (pro-SPC) epithelium. Further characterization would benefit from establishment of rigorous enrichment strategies allowing analysis of their behavior in vitro and following transplantation, and the establishment of a defining gene expression signature. We have determined that Epithelial Cell Adhesion Molecule (EpCAM) and Integrin α6 are expressed on the cell surface of both alveolar and bronchiolar epithelial cells and that low levels of Sca-1 expression characterize the bronchiolar epithelium. Within the Sca-1low EpCAMpos Integrinα6pos population of bronchiolar epithelial cells, autofluorescence (AF) levels distinguish the facultative transit-amplifying population which is AFhi from bronchiolar stem cells which are AFlow. Use of transgenic animal models allowing expansion or depletion of the stem cell compartment and use of lineage tracing strategies have allowed us to determine the identity of cells isolated based on their cell surface phenotype and autofluorescence characteristics. Injury models associated with depletion of terminally differentiated ciliated cells (ozone) or facultative transit amplifying population (naphthalene) were used to validate the functional characteristics of the two fractions of bronchiolar progenitors. In conclusion, we have developed and validated a fractionation approach for the generation of highly purified preparations of bronchiolar stem and Clara cells from the mouse lung. These data enable establishment of robust in vitro and transplantation assays to further validate the functional behavior of stem and facultative TA (Clara) cells and allows analysis of gene expression profile of the two populations towards a better understanding of unique characteristics of the bronchiolar stem cell compartment.

Sertoli cells provide nutrients and growth factors for developing germ cells. Each Sertoli cell can support a finite number of developing germ cells. During development of the testis, cessation of Sertoli cell proliferation and the onset of differentiation establishes the final number of Sertoli cells and hence the number of sperm that can be produced. Studies in this dissertation explore the hypothesis that the transition from proliferation to differentiation is facilitated by E-box transcription factors that induce the expression of differentiation-promoting genes. The relative activities of E-box proteins were studied in primary Sertoli cells isolated from 5, 11, and 20 day-old rats, representing proliferating, differentiating, and differentiated cells, respectively. DNA binding by E-box proteins is nearly undetectable at 5 days after birth but peaks with initiation of differentiation at 11 days after birth and remains elevated. Upstream Stimulatory Factors 1 and 2 (USF1, USF2) were the predominant E-box proteins present within DNA-protein complexes formed after incubating E-box containing probes with nuclear extracts from developing Sertoli cells. Increased USF binding activity corresponded with elevated Usf1 mRNA and USF1 protein levels 11 days after birth. The potentiator of Sertoli cell differentiation, thyroxine, induces USF DNA binding in Sertoli cells prior to differentiation. Decreased nuclear expression of ID proteins may permit increased USF DNA binding during Sertoli cell differentiation. Several genes required for Sertoli cell differentiation and differentiated Sertoli cell functions have USF binding sites within their promoters. Two potential USF target genes, Nr5a1 and Shbg, were induced in 11 day-old Sertoli cells when compared with 5 day-old Sertoli cells. DNA binding studies of Nr5a1 and Shbg promoter sequences determined that USF1 and USF2 binding to E-box motifs increased during differentiation. In vivo binding assays confirmed that USF1 and USF2 occupy the E-box within the Nr5a1 promoter. These data support the ideas that increased USF protein expression induces differentiation-associated gene expression and that USF-mediated alterations in gene transcription are responsible for the onset of differentiation in developing Sertoli cells. These USF-mediated processes would then determine the final number of Sertoli cells present within the testes and the upper limit of fertility.

The Role of Gap Junctions in Cellular Migration Bado Hewa DeFranco, MS. University of Pittsburgh, 2009 The body of work presented here focuses on characterizing the role that gap junction intercellular channels play in regulating cellular migration. Cell migration is a ubiquitous process that is required for embryonic development and for maintaining the integrity of tissues and organs. Yet, the status of gap junction channels, with regard to structure and function, in migrating cells is not completely understood. We hypothesized that, Gap junction channels, as mediators of intercellular communication, play a role in cell migration, and have investigated and characterized gap junctions in migrating cells. Accordingly, the aims of this dissertation were: (i) to characterize gap junctions and their function during migration, (ii) to determine the effect of altering the status of gap junction expression on cell migration and to (iii) characterize the mechanism of gap junction internalization in migrating cells. With combined molecular and imaging approaches we have demonstrated that in cells migrating as a sheet, gap junction plaque structures are retained on the plasma membrane surface; that gap junction communication is continuous in migrating cells and that interrupting this communication with connexin 43 specific mimetic peptides reduces migration. We have established a system in a human adrenal cortical cell line (SW13) wherein we significantly reduce gap junction protein expression with siRNA and show that cellular migration is inhibited. We have also demonstrated that gap junction plaque size and orientation are modified during migration. We also discovered that sometimes, migrating cells will spontaneously detach from one another at cellular processes and that during this event the gap junction plaques are internalized. Analysis of gap junction plaque internalization in migrating cells revealed that clathrin and several adaptor proteins associate with surface gap junctions and cytoplasmic annular gap junction structures and possibly regulate the unique mechanism of gap junction plaque removal from the plasma membrane. Within the field of gap junction research this work expands our understanding of gap junctions in living cells as dynamic structures that may play a key role in coordinating the migration of entire cell populations.

The regulated recycling of endocytosed membrane proteins at the plasma membrane is an important intracellular trafficking process that can regulate the copy number of proteins at the membrane and thereby modulate their function and the physiology of the tissue as a whole. The Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes rapid and efficient recycling at the apical plasma membrane in polarized epithelial cells. The cellular mechanisms that facilitate CFTR recycling are understood poorly, especially in polarized cell systems, yet this process ensures the proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Using a physiologically relevant model that recapitulates the in vivo secretory functions of the intestinal epithelia, I tested the hypothesis that distinct members of the Rab family of GTPases mediate the recycling of CFTR at the apical plasma membrane and that this process facilitates transepithelial anion secretion by maintaining a physiologically functional surface density of CFTR channels. The role of the Rab11 isoforms in CFTR recycling was investigated using the colonic epithelial cell line, T84, and rat intestine for in vivo studies. Immuno-isolation of Rab11 vesicles revealed endogenous CFTR within both the Rab11a and Rab11b compartments; however, only perturbing Rab11b expression or function attenuated the CFTR-mediated, cAMP-activated anion efflux. In polarized epithelial monolayers, mutant Rab11b inhibited the forskolin-stimulated transepithelial anion secretion by reducing the apical membrane density of CFTR. Biotin protection assays revealed a dependence of CFTR recycling on functional Rab11b, not Rab11a, demonstrating the selective requirement for the Rab11b isoform. Therefore, apical CFTR copy number is regulated by Rab11b-mediated recycling, which facilitates the agonist-stimulated, transepithelial anion response in polarized intestinal epithelial cells. The identification of Rab11b as a mediator of recycling reveals an apical recycling itinerary unidentified previously and emphasizes the importance of elucidating the membrane recycling itineraries in polarized epithelial cell models, which mimic the normal physiology of tissue. Understanding these processes within relevant models have direct implications for understanding normal cell biology and tissue physiology and form a foundation for future clinical therapies designed to correct disease processes arising from improper membrane recycling.

Wounding disrupts the primary function of an epithelium, which is to provide a barrier to the outside environment. The longer the epithelial defect remains unhealed, the greater the risks of morbidity and mortality from infection, loss of tissue homeostasis, and fibrosis. Normally, epithelial cells restore barrier function by becoming highly motile and migrating to cover the defect, but in many situations cells do not move fast enough to prevent tissue malfunction. This is especially true in the cornea, where even minor wounds can impair vision. Therefore, there is considerable therapeutic interest in identifying signals that induce epithelial migration. Activation of the epidermal growth factor receptor (EGFR) is a key signaling event that promotes cells to move and cover wounds in many epithelia. The broad goal of this dissertation research was to identify mechanisms of wound-induced EGFR activation so that therapies may be developed to improve normal and pathological healing. I hypothesized that mechanisms of EGFR activation may differ with respect to distance from the wound, so I developed wounding models to analyze signaling specifically in cells near to or far from wounds in a human corneal epithelial cell line. I have examined the involvement of extracellular ATP, phospholipase D, Src-family kinases (SFKs), and the focal adhesion kinase Pyk2, all of which are signals that have been hypothesized to be stimulated by environmental cues related to wounding and to activate the EGFR. I have found that the proximal mechanism of EGFR activation is the proteolytic release of membrane-bound ligands, which is regulated by activation of SFKs. After wounding, multiple pathways converge on SFKs to regulate EGFR activation and cell motility. In one pathway, extracellular ATP transactivates the EGFR through phospholipase D2. In a distinct pathway that functions specifically near the wound edge, Pyk2 triggers SFK and EGFR activation. Finally, my data suggest the presence of a third distinct pathway that promotes SFK and EGFR activation in response to a physically unconstrained edge. By delineating signaling pathways that stimulate EGFR activation, I have identified potential therapeutic targets for modulating EGFR signaling and cell motility in wound healing and other pathologies.