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  • pten inhibitor br Materials and Methods br Results br Discus

    2018-10-25


    Materials and Methods
    Results
    Discussion The podocyte, a highly differentiated glomerular epithelial cell, makes up the most distal portion of the kidney filtration barrier. Recent studies show that Rho GTPase plays a key role in maintaining podocyte morphology and function (Wang et al., 2012). Excess activation of RhoA in podocytes induces proteinuria and FSGS (Zhu et al., 2011). Our earlier studies in cultured podocytes demonstrated that INF2 modulates Rho/Dia signaling by binding to and antagonizing the pten inhibitor polymerizing activity of Dia, a major downstream effecter of RhoA. Here we demonstrate that the activity of Dia, as measured by its membrane association, correlates with the severity of the observed INF2-deficient zebrafish phenotype, despite the absence of significant Rho activation. These data suggest that INF2 likely functions though the direct opposition of the Dia protein, rather than through activation of Rho. Ultrastructurally, the podocytes of INF2 morphants lose the normal regularity of podocyte foot processes and normal slit diaphragm structure. Instead, they exhibit jumbled protrusions with prominent microvillus formation and loss of normally oriented slit diaphragms. Microvilli are finger-like membrane protrusions with central actin bundle elongation characterized by Dias localized to the tip of these protrusions and consistent with excess Rho/Dia signaling in these INF2 morphants (Ridley, 2006). Trafficking of nephrin to specific membrane domains of precursor podocytes during development has been shown to initiate foot process budding and intracellular junction formation (slit diaphragms) (George and Holzman, 2012). Mistrafficking of slit diaphragm proteins may result from unbalanced Rho/Dia signaling in podocytes with INF2 deficiency (Sun et al., 2013; Homem and Peifer, 2009; Wallar et al., 2007). In the present studies, we found a significant change in nephrin trafficking in INF2 morphant zebrafish. These findings suggest that uncontrolled Dia activity in the absence of INF2 contributes to the abnormal development of the glomerular filtration barrier, caused at least in part by defective trafficking of slit diaphragm proteins.
    Acknowledgments We thank Eva Plovie and Amy Ronco for the help with zebrafish experiments. We thank Suzanne L. White, Yi Zheng, and Lay-Hong Ang from Histology Core and the Confocal Core at BIDMC for the help with zebrafish tissue processing, sectioning, staining and imaging. We thank Mary McKee for help with zebrafish embryo processing and transmission electron microscopy. This work was support by NIH grants HL109264 to CAM, and DK088826 to MRP. Conflicts of Interest None.
    Introduction Epstein Barr virus (EBV), a γ-herpes virus, is a remarkably successful human pathogen with more than 95% of the entire adult human population being infected (Rickinson et al., 2014). 50years ago, EBV was discovered as the first human tumor virus candidate in tissue samples of Burkitt\'s lymphoma patients (Epstein et al., 1964). Cell-mediated immune control is thought to keep persistent EBV infection in check and prevent virus-associated malignancies. These malignancies harbor latent EBV proteins, which drive cellular proliferation and survival in order for EBV infected B cells to gain access to the memory B cell pool, the virus\' site of persistence (Babcock et al., 1998; Kutok and Wang, 2006). EBV can reactivate from this memory B cell pool into lytic replication, and infectious particle production is initiated upon plasma cell differentiation (Laichalk and Thorley-Lawson, 2005). The resulting virions are similar to those of other members of the herpes virus family. EBV\'s double stranded DNA is encased by an icosahedral nucleocapsid, sheltered by a protein tegument and ultimately pten inhibitor surrounded by a viral envelope decorated with viral glycoproteins (Johannsen et al., 2004). A primary viral envelope is acquired after nucleocapsid assembly via budding through the inner nuclear membrane. This first envelope is shed after fusion with the outer nuclear membrane, thereby releasing the de-enveloped nucleocapsid into the cytosol. This nucleocapsid acquires then its second and final envelope in the cytosol. It is thought that the membrane source of the final herpes virus envelope is perinuclear organelles like the trans-Golgi-network and often requires virus induced organelle reorganization (Henaff et al., 2012). The molecular machinery of macroautophagy can achieve such membrane remodeling, and the second enveloping of EBV is topologically reminiscent of macroautophagy. Indeed, α- and β-herpes virus infections are able to induce macroautophagy (English et al., 2009; McFarlane et al., 2011), and herpes simplex virus (HSV) particles can be found in autophagic vesicles (English et al., 2009). However, no requirement of macroautophagy for the replication of these α- and β-herpes viruses was reported so far.