The global epidemic of chronic kidney disease is progressing at an alarming rate. In the United States alone, glomerular kidney diseases affect some 20 million people, and this number has roughly doubled in the last two decades. Kidney-related diseases are rapidly eluding current available treatment options and resources. Podocytes are unique cells within the kidney glomerulus, which have a complex organization consisting of a cell body, microtubule-driven membrane extensions (primary processes), and actin-cytoskeleton driven membrane extensions (foot processes). Since the podocyte cytoskeleton is composed of three distinct elements - actin filaments, microtubules, and intermediate filaments - their cytoskeleton must be coordinately regulated for podocytes to fulfill their complex cellular function. Coordination between cytoskeletal elements is achieved through signaling pathways, which involve common regulators such as Rho GTPases as well as the large GTPase dynamin. We have shown that dynamin directly regulates the actin cytoskeleton in podocytes. Our novel findings suggest that dynamin also regulates microtubules ? specifically that dynamin forms helices around microtubules. Dynamin helices exhibit very high rates of GTP hydrolysis, which in turn inhibit microtubule polymerization by lowering the local concentration of GTP. A high GTP concentration is required for efficient tubulin oligomerization since tubulin uses GTP binding to initiate its polymerization into microtubules. Additionally, high rate of GTP hydrolysis by dynamin helices also constricts microtubules to a small radii, thus destabilizing microtubules and initiating microtubule catastrophe. Therefore, these two GTPases, dynamin and tubulin, are regulated by the availability of GTP in their vicinity and dynamin's ability to oligomerize into helices around microtubules. The ability of dynamin to directly affect dynamics of microtubules and actin suggests that dynamin might be one of the proteins in podocytes that these two cytoskeletal proteins use to communicate with each other.
In Specific Aim 1 we use real time single-molecule imaging to decipher mechanistic details of dynamin-microtubule and dynamin-actin interactions.
In Specific Aim 2 we use a panel of diverse dynamin mutants, podocyte cell culture, biochemistry using purified proteins, and electron microscopy to investigate the role that dynamin plays in regulating microtubules in podocytes, and how microtubules affect the formation of focal adhesions and stress fibers.
In Specific Aim 3 we test our hypothesis in animals by using our novel dynamin-specific mouse model, and a mouse model of HIV-associated nephropathy (HIVAN). We test whether a dynamin mutant that has the propensity to oligomerize and is expected to promote microtubule depolymerization can counteract podocyte injury by using microtubule drugs that either promote or inhibit microtubule polymerization. Given the essential role of microtubules in cytokinesis, we will also examine the effect of microtubule drugs on podocyte proliferation observed in HIVAN.
Under this grant application we propose to study the role of the regulatory GTPase dynamin in regulating crosstalk between the actin cytoskeleton and microtubules in podocytes. While podocytes are terminally differentiated cells, some forms of glomerular injury such as HIV-associated nephropathy result in podocyte de- differentiation and cell proliferation, a process driven by crosstalk between actin and microtubules. Under this grant application we study the role of dynamin as a link between microtubules and the actin cytoskeleton in podocytes in health and diseases.
|Sever, Sanja; Schiffer, Mario (2018) Actin dynamics at focal adhesions: a common endpoint and putative therapeutic target for proteinuric kidney diseases. Kidney Int 93:1298-1307|
|Gu, Changkyu; Lee, Ha Won; Garborcauskas, Garrett et al. (2017) Dynamin Autonomously Regulates Podocyte Focal Adhesion Maturation. J Am Soc Nephrol 28:446-451|
|Müller-Deile, Janina; Teng, Beina; Schenk, Heiko et al. (2016) Drugs targeting dynamin can restore cytoskeleton and focal contact alterations of urinary podocytes derived from patients with nephrotic syndrome. Ann Transl Med 4:439|
|Sever, Sanja; Reiser, Jochen (2015) CD2AP, dendrin, and cathepsin L in the kidney. Am J Pathol 185:3129-30|
|Schiffer, Mario; Teng, Beina; Gu, Changkyu et al. (2015) Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models. Nat Med 21:601-9|
|Gu, Changkyu; Chang, Joann; Shchedrina, Valentina A et al. (2014) Regulation of dynamin oligomerization in cells: the role of dynamin-actin interactions and its GTPase activity. Traffic 15:819-38|
|Reiser, Jochen; Sever, Sanja; Faul, Christian (2014) Signal transduction in podocytes--spotlight on receptor tyrosine kinases. Nat Rev Nephrol 10:104-15|
|Sever, Sanja; Chang, Joann; Gu, Changkyu (2013) Dynamin rings: not just for fission. Traffic 14:1194-9|