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The Sever Laboratory Basic Scientific Research With Clinical Relevance HOME RESEARCH PUBLICATIONS LAB MEMBERS OPPORTUNITES LAB FUN CONTACT US HOME Why do we study molecular mechanisms of kidney diseases? Despite hundreds of millions of people losing kidney function world-wide every year, no kidney-specific therapeutics exist. The current treatments for kidney loss, dialysis and transplant, significantly diminish quality of life and life span. The goal of our research is to elucidate the molecular mechanisms of kidney diseases to identify druggable pathways and develop new therapeutics. We primarily focus on the role of the GTPase dynamin in regulating actin cytoskeleton dynamics and clathrin-mediated endocytosis. Disregulation of both of these processes has been implicated in the loss of kidney-specific cells called podocytes that are required for kidney function. Gaining a better understanding of podocyte pathobiology will lay the groundwork to cure kidney disease. Why study dynamin? Dyamin is a founding member of a superfamily of large GTPases that exist in multiple oligomerization states. Dynamin is best known for its role in clathrin-mediated endocytosis. Its ability to self-assemble into helices on lipid templates in vitro has led to the paradigm that dynamin directly executes the fission reaction in which coated pits are freed from the plasma membrane. We have identified regulation of the actin cytoskeleton as an additional and distinct role for dynamin oligomerization in podocytes and other cell types. By using classic cell biology, biochemistry, single molecule imaging, and animal models, we are establishing novel paradigms for dynamin function, the actin cytoskeleton, and endocytosis in healthy and injured kidney cells. Why join the Sever laboratory? The Sever lab is a welcoming and highly interactive environment. Post-doctoral fellows and research technicians share expertise, reagents, and ideas to reach their common goal of understanding how dynamin regulates the actin cytoskeleton in healthy and diseased tissue. We are always looking for energetic and accomplished post-doctoral fellows with expertise in molecular biology, biochemistry, and/or biophysics. RESEARCH The establishment of distinct, cell-type specific, cellular features, including cell polarity, involve signaling cascades, membrane trafficking, and cytoskeletal dynamics, all of which need to be highly coordinated and regulated. We seek to understand the role of the GTPase dynamin as one of the major coordinators of multiple cellular processes including endocytosis, actin cytoskeleton dynamics, as well as microtubule dynamics, in healthy and injured cells. Regulation of the actin cytoskeleton In 2010 we identified direct interactions between dynamin and actin filaments and we have since shown that the dynamin oligomerization cycle plays a direct role in regulating actin polymerization and crosslinking of actin filaments. These interactions have since been implicated in highly diverse cellular processes including endocytosis, the formation of lamellipodia, filopodia invadopodia, and growth cones in neuronal cells. All these processes are driven by the association of distinct actin structures with the plasma membrane. We now seek to identify the molecular mechanisms by which dynamin establishes such diverse cellular processes. Utilizing biochemical and cell biology assays, and total internal reflection fluorescence (TIRF) single-molecule imaging, we plan to elucidate the mechanism by which dynamin regulates actin and microtubule dynamics. We use kidney specific cells named podocytes as an experimental model system to study the role of dynamin with regard to endocytosis, the actin cytoskeleton, and microtubule dynamics. Podocytes are terminally differentiated cells that form the filtration barrier in the kidney. Damage or loss of podocytes is an early symptom of many kidney diseases as structural integrity of the podocyte actin and microtubule cytoskeleton is critical for its proper function. Better understanding podocyte pathobiology has lead to the establishment of novel paradigms with regard to role of dynamin in the cell and has the potential to pave the way for developing a cure for kidney diseases. This photo shows the structure of podocytes in mice expressing dynamin mutant R725A, which increases dynamin’s propensity to oligomerize into higher-order structures such as rings. Dynamin oligomerization has been implicated in clathrin-mediated endocytosis, but here we show for the first time that its oligomerization plays an essential physiological role by directly regulating the actin cytoskeleton. This, in turn, drives the formation of foot processes that are significantly longer than those in wild-type animals as well as those in animals before Doxycycline treatment (used to drive expression of DynR725A). It is very rare to see such a dramatic effect on the length of the foot processes (FPs) since dominant-negative mutations of diverse proteins expressed in podocytes typically result in the loss of FPs. It should be noted that this particular dynamin mutant (DynR725A) has been published by Dr. Sever back in 1999 in Nature (Sever et al, Nature 1999). This mutant suggested that dynamin is a regulatory GTPase and not a pinchase. As you can see, this mutant has a long history. Clathrin Mediated Endocytosis We are also interested in the role of dynamin in clathrin-mediated endocytosis. The classical view of dynamin holds that it acts as a mechanochemical enzyme or pinchase,” severing vesicles from the plasma membrane. Our work suggests an alternative model in which dynamin is a regulatory GTPase, orchestrating formation of clathrin-coated vesicles. In this view unoligomerized dynamin recruits additional proteins that drive formation of fully invaginated coated pits and subsequent budding of free vesicles. Our latest studies suggest that dynamin oligomerization may play an indirect but global role in endocytosis through regulation of actin. This image shows localization of dynamin (labeled with black dots) on clathrin coated vesicles (labeled ‘V’) in foot processes of mice. Molecular mechanisms of kidney diseases Chronic kidney disease, which is loss of kidney function over time, affects hundreds of millions of people worldwide. It is often associated with the appearance of significant amounts of high-molecular-weight plasma proteins such as albumin in the urine (termed proteinuria), a symptom of a compromised glomerular filtration barrier. Chronic kidney disease can occur due to genetic mutations in house keeping genes” such as a-actinin 4, or more often as a secondary effect of diabetes and hypertension. Irrespective of genetic or disease-based causes, podocyte injury underlies loss of kidney function. Podocytes are terminally differentiated cells of the glomerulus, which consist of a cell body, primary microtubule-driven membrane extensions, as well as secondary actin-based membrane extensions called foot processes. Sustained dis-regulation of the actin cytoskeleton in foot processes ultimately leads to podocyte loss. We have shown that pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models. Our study established the first successful targeting of the actin cytoskeleton dynamics in foot processes and in the whole organism. Current research in the lab focuses on interplay between actin, microtubule dynamics, and endocytosis, and the role of dynamin in coordinating these processes in podocytes. We are also examining role of these processes in polarized epithelial cells of renal tubules. Cells of the renal tubules are often injured during anti-cancer therapies, resulting in acute loss of kidney function. The immerging view of kidney disease is that regardless of the injury (diabetes, hypertension, anti-cancer drugs, genetics), the whole organ is somehow affected. Our current challenge is to establish...