Doctor of Philosophy in Cell and Developmental Biology (PhD)
Junction Turnover in Testis
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.
No abstract available.
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.
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.