A Wayne Vogl


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Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

The role of tubulobulbar complex-endoplasmic reticulum contact sites during junction turnover in the seminiferous epithelium (2021)

The focus of this thesis is the characterization of the functions of the membrane contact sites (MCSs) between tubulobulbar complexes (TBCs) and the endoplasmic reticulum (ER). TBCs are double membrane endocytic invaginations that are endemic to Sertoli cells of the mammalian testis. They have been proposed to internalize intact intercellular junctions between late spermatids and Sertoli cells, allowing late spermatids to be released into the seminiferous tubule lumen. However, there is little data showing how TBCs may be regulated temporally and spatially to initiate junction turnover and subsequent spermatid release (spermiation). One of the most striking features of TBCs is the close association of ER to the ‘bulb’ portion, which is present for much of the time a TBC is developing and up until the moment of its vesiculation, all while it is still continuous with the plasma membrane. Although ER contacts are known to help regulate the form and function of other organelles such as endosomes, there have never been an investigation into what the functions of the TBC-ER contact sites are. It is my overall hypothesis that the TBC-ER contact sites are necessary for the normal development of TBCs into mature endocytic structures. Some of the well-established functions of ER contacts are both calcium and lipid exchange. Both processes have been shown to be fundamental to the progression of endosomes through the endosomal-lysosomal system. In this thesis, I begin by using qualitative approaches to establish that proteins known to be involved in both calcium and lipid exchange are present at TBC-ER contacts. I follow-up on my qualitative works with functional studies where I explore the role some of these proteins play in TBC development and in spermiation. Using in vivo knockdown experiments in rat testes to target two of these proteins (IP3R1 and ORP9), I demonstrate that both are necessary for the normal maturation of TBC bulbs and for sperm release to occur. These data support that conclusion that both calcium and lipid exchange are functions of the TBC-ER contact sites and provide evidence that the contact sites are necessary components of the sperm release mechanism.

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Intercellular junctions and tubulobulbar complexes between sertoli cells in primary culture and in vivo (2019)

Tubulobulbar complexes (TBCs) are actin-rich structures that form at intercellular junctions in the seminiferous epithelium of the mammalian testis. Massive intercellular junctions form at the base of the epithelium where Sertoli cells are connected to each other, and at the apex where Sertoli cells are connected to mature spermatids. TBCs are proposed to be responsible for internalizing these intact junctions during spermiation at the apex and during the translocation of spermatocytes from basal to adluminal compartments near the base of the epithelium. A growing body of evidence indicates that apical TBCs internalize tissue specific adhesion junctions at the apex of the epithelium and are involved in sperm release. In comparison, relatively little is known about basal TBC function and spermatocyte translocation. This thesis explores the hypothesis that the presence of spermatogenic cells influences the structure of TBCs at basal junctions between Sertoli cells in vitro. A primary Sertoli-germ cell co-culture system was optimized and used to explore TBC structure in vitro. In the seminiferous epithelium, if basal TBCs are responsible for internalizing junctions, then interfering with TBC structure or formation should lead to delayed translocation of spermatocytes and increased mass of basal intercellular junctions. An in vivo ribonucleic acid interference (RNAi) procedure was optimized to knockdown cortactin, a component of TBCs, and the morphological differences on basal TBCs were observed. This work is a necessary prelude to future work to evaluate the role of basal TBCs during spermatocyte translocation. Finally, this thesis shows that disruption of actin at basal TBCs results in the same altered TBC structure as has been observed at apical sites.

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Study of the Minute Virus of Mice Cell Entry (2014)

The minute virus of mice prototype (MVMp) is a non-enveloped single stranded DNA virus of the family Parvoviridae. MVMp is one of the smallest viruses and shows intriguing abilities to preferentially infect and kill cancer cells (oncotropism/oncolytism), suggesting a potential for MVMp as an anti-cancer agent. Unfortunately, there is a lack of knowledge of the early events of MVMp infection cycle, such as binding to the cell surface and subsequent endocytosis. In an attempt to identify cellular partners of MVMp infection, our lab performed a mass spectrometry analysis of MVMp potential binding partners. Following this analysis, the galactose-binding lectin (galectin) 3 (Gal-3) was identified as binding partner for MVMp. Given the involvement of this extra-cellular matrix protein in the clustering and endocytosis of cell surface receptors, and its up-regulation in various aggressive tumor cells, I hypothesized that Gal-3 could play a role in MVMp cell entry, and potentially in its oncotropism. Using siRNA knockdown of Gal-3 in different cells followed by immunofluorescence microscopy analysis, I found that Gal-3 is necessary for an efficient MVMp cell entry and infection in different cells. Moreover, I discovered that the Golgi enzyme β1,6-acetylglucosaminyltransferase 5 (Mgat5), whose role is the addition of complex N-glycosylation to various cell surface receptors for Gal-3 binding, is required for MVMp infection. I also found that cancer cells with higher Gal-3 expression are more susceptible to MVMp infection than cells with lower Gal-3 levels.Next I used a combination of flow cytometry, immuno-fluorescence and transmission electron microscopy to characterize the early events of MVMp infection in various tissue-culture cell lines. My results show that many crucial parameters of the mesenchymal cell migration process regulate MVMp cellular entry and infection. I found that MVMp relies on cell protrusions to cluster at the leading edge of migrating cells rapidly after binding to the plasma membrane, from where it is subsequently endocytosed. Moreover, transmission electron microscopy analysis revealed that MVMp uses various endocytic pathways, which was confirmed using drug inhibitors of endocytosis. Finally, I found that epithelial-mesenchymal transition, an inducer of cancer cell migration, triggers MVMp infection in highly dividing non-permissive cancer cells.

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Assigning a function to tubulobulbar complexes in testis (2013)

The focus of this thesis is the characterization of apical tubulobulbar complexes in mammalian testis. These double-membrane, actin-based structures form at sites of attachment between germ cells and Sertoli cells. The location, timing and morphology of these complexes have inspired several proposed functions. It has been proposed that tubulobulbar complexes serve as an anchor to prevent early release of spermatids from the epithelium or that they are a device for elimination of excess cytoplasm. I hypothesize that tubulobulbar complexes are subcellular machines responsible for the internalization of intact intercellular junctions thereby contributing to the process of spermatid release from the seminiferous epithelium. A descriptive approach is taken to determine if key components that are present at similar structures in other systems also are present at tubulobulbar complexes, to determine if integral junction molecules are present at tubulobulbar complexes and to determine the fate of internalized junction material. A functional approach is taken to deplete the expression of an actin-based protein that is localized to tubulobulbar complexes to test the prediction that the structures are involved with spermatid release. Dendritic actin components were localized around the cuff of tubulobulbar complexes and clathrin was localized to coated pits at the ends of the structures. Based on these data, a model of tubulobulbar complex formation was proposed that incorporates clathrin-mediated endocytosis and dendritic actin assembly.Junction molecules known to be present at ectoplasmic specializations were found in vesicles at the ends of tubulobulbar complexes that label positively for early endosome markers. Interestingly, junction protein nectin 2 was colocalized with recycling marker Rab11 at newly forming junctions deeper in the epithelium. This suggests that recycling of junctional proteins may be occurring in Sertoli cells.Finally, depletion of cortactin, a key protein at tubulobulbar complexes, resulted in a short phenotype – an indication that the structures were not able to acquire or maintain their normal length after treatment. Significantly, delay in spermatid release was detected. The data presented here supports the junction internalization hypothesis and introduces a new paradigm for junction internalization generally in cells and links the mechanism to a biologically significant event – sperm release.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Implicating calcium signaling at intercellular junctions and structures associated with junction turnover in rat Sertoli cells (2017)

The endoplasmic reticulum (ER) is a prominent organelle in Sertoli cells. It is an integral component of unique adhesion junctions (ectoplasmic specializations - ESs) in this cell type, and is closely associated with structures termed tubulobulbar complexes (TBCs) that internalize intercellular junctions during sperm release and during the translocation of spermatocytes through the blood-testis barrier. A role for the ER in Ca²⁺ regulation at ESs and TBCs has been suspected, but evidence for this function has proved elusive. The focus of this thesis is identification of molecular machinery involved in Ca²⁺ signaling and obtaining functional evidence of Ca²⁺ regulation of the actin networks at TBCs. Functional experiments using EGTA and thapsigargin to lower and raise Ca²⁺ levels did not provide evidence that TBC actin networks are regulated by Ca²⁺. Using immunofluorescence, I demonstrated that Ca²⁺ regulatory machinery is present at the ESs attached to spermatid heads, and at ER-PM contacts. SERCA2 is present at ESs, IP3R is present at ER-PM contacts associated with TBC bulbs, and STIM1, ORAI1 and SERCA2 are present at the ER-PM contacts around the margins of Sertoli cell apical processes. The results support the conclusion that the molecular machinery necessary for ER generated Ca²⁺ fluxes is present in regions and structures directly related to junction remodeling in Sertoli cells, a process necessary for sperm release.

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Tubulobulbar complexes contribute to basal juntion remodeling in the seminiferous epithelium (2013)

Tubulobulbar complexes are cytoskeleton-related membrane structures that develop at sites of intercellular attachment in the mammalian seminiferous epithelium. At apical junctions between Sertoli cells and spermatids the structures internalize adhesion junctions and are a component of the sperm release mechanism. Here I explore the possibility that tubulobulbar complexes that form at the ‘blood-testis barrier’ are sub-cellular machines that internalize basal junction complexes. Electron microscopy reveals that morphologically identifiable tight and gap junctions are present in basal tubulobulbar complexes in rats. In addition, immunological probes for claudin-11 (CLDN11), connexin-43 (GJA1), and nectin-2 (PVRL2) react with linear structures at the light level that I interpret as tubulobulbar complexes, and probes for early endosome antigen 1 (EEA1) and Rab5 (RAB5A) react in similar locations. Significantly, fluorescence staining patterns for actin and claudin-11 indicate that the amount of junction present is dramatically reduced over the time period that tubulobulbar complexes are known to be most prevalent during spermatogenesis. I also demonstrate, using electron microscopy and fluorescence microscopy, that tubulobulbar complexes develop at basal junctions in primary cultures of Sertoli cells. These structures not only morphologically resemble their in vivo counterparts, but they also contain junction proteins. I use this culture system together with transfection techniques to show that junction proteins from one transfected cell project into and are likely internalized by adjacent non-transfected cells as predicted by the junction internalization hypothesis. On the basis of my findings I present a new model for basal junction remodeling as it relates to spermatocyte translocation in the seminiferous epithelium.

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