Our current investigations concern the structure and function of podocytes, focusing on regulation of the actin cytoskeleton, the GTPase dynamin, and clathrin-mediated endocytosis. A better understanding of podocyte pathobiology will pave the way for developing a cure for kidney diseases.
What is the role of dynamin in podocyte structure and function?
Podocytes are terminally differentiated cells that form the filtration barrier in the kidney, and podocyte damage or loss is an early symptom of many kidney diseases. Our recent studies suggest that the GTPase dynamin is a critical regulator of actin dynamics in healthy and diseased podocytes. In normal podocytes, dynamin influences actin organization in a GTP-dependent manner. During proteinuric kidney disease, induction of a cytoplasmic form of the protease cathepsin L leads to cleavage of dynamin at a conserved site, resulting in reorganization of the podocyte actin cytoskeleton and proteinuria (elevated protein levels in urine due to defective ultrafiltration). Strikingly, podocyte damage and proteinuria do not occur when cathepsin L-resistant dynamin mutants are delivered to the kidney. Our study identifies dynamin as a critical regulator of renal permselectivity, which is specifically targeted by proteolysis under pathological conditions. We are currently elucidating the mechanisms that lead to the presence of the cathepsin L in the cytoplasm, as well as the mechanisms by which dynamin regulates structure and function of healthy and diseased podocytes. Better understanding of podocyte pathobiology will pave the way for developing a cure for kidney diseases in the future.
How does dynamin regulate actin in podocytes?
The function of podocyte in the ultrafiltration barrier requires a highly dynamic actin cytoskeleton. Our data suggest that dynamin is a master regulator of actin dynamics in podocytes. We are using molecular biology, biochemistry, and mouse models to elucidate the molecular mechanism by which dynamin regulates actin dynamics in podocytes.
What is the role of dynamin in clathrin mediated endocytosis?
Clathrin-mediated endocytosis is the process by which cells internalize receptors, transmembrane channels, transporters, and extracellular ligands such as hormones, growth factors and nutrients. In neurons, endocytosis is critical to allow rapid synaptic vesicle regeneration. In addition, endocytosis of ligand-activated receptors is essential for the proper attenuation of a variety of signal transduction processes, as well as for co-localization of activated receptors with downstream signaling molecules. Thus, defective regulation of this process can cause many aberrations of normal cellular function, including neoplastic transformation. In contrast to the classical view that dynamin acts as a mechanochemical enzyme or "pinchase" that serves vesicles from the plasma membrane, our work suggests an alternative model in which dynamin is a regulatory GTPase that orchestrates formation of clathrin-coated vesicles. In this view, dynamin recruits other proteins that execute vesicle budding. In support of our model, we have identified Hsc70 and it's co-chaperone auxilin as downstream effectors of dynamin activity (Newner et al., 2003 Sever et al., 2006). These observations suggest that dynamin instructs the chaperone machinery to induce conformational changes within the clathrin coat that drive vesicle constriction and fission. We are now examining the mechanism by which dynamin regulates the chaperone machinery. Ultimately, we would like to identify the minimal molecular machinery that executes the fission reaction.
Sever S, Muhlberg AB, Schmid SL. Impairment of dynamin's GAP domain stimulates receptor-mediated endocytosis. 1999. 398: 481-486.
Sever S, Damke H, Schmid SL. Dynamin: GTP controls formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis. J Cell Biol. 150: 1137-1147.
Sever S, Damke H, Schmid SL. Garrotes, springs, ratchets and whips: putting dynamin models to the test. 2000. 5: 385-392.
Damke H, Muhlberg AB, Sever S, Sholly S, Warnock DE, Schmid SL.Expression, purification and functional assays for self-association of dynamin-1. Meth Enz. 329: 447-457.
Sever S. Dynamin and endocytosis. Curr Opinion in Cell Biol. 14: 463-467.
Newmyer S, Christensen A, and Sever S. Auxilin-dynamin interactions link the uncoating ATPase chaperone machinery with vesicle formation. Developmental Cell. 4: 929-920.
Sever S, Newmyer S, Skoch J, Ko D, McKee M, Bouley R, Ausiello D, Hyman BT and Backskai BJ. Physical and functional connection between auxilin and dynamin during endocytosis. EMBO J. 2006; 25: 4163-74.
Sever S, Altintas MM, Nankoe SR, Möller CC, Ko D, Wei C, Henderson J, del Re EC, Hsing L, Erickson A, Cohen CD, Kretzler M, Kerjaschki D, Rudensky A, Nikolic B, Reiser J. Proteolytic processing of dynamin by cytoplasmic cathepsin L is a mechanism for proteinuric kidney disease. J Clin Invest. 2007; 117: 2095-104.
Gu C, Yaddanapudi S, Weins A, Osborn T, Reiser J, Pollak M, Hartwig J, Sever S. Direct dynamin-actin interactions regulate the actin cytoskeleton. EMBO J. 2010; 29: 3593-606.
Yaddanapudi S, Altintas MM, Kistler AD, Fernandez I, Möller CC, Wei C, Peev V, Flesche JB, Forst AL, Li J, Patrakka J, Xiao Z, Grahammer F, Schiffer M, Lohmüller T, Reinheckel T, Gu C, Huber TB, Ju W, Bitzer M, Rastaldi MP, Ruiz P, Tryggvason K, Shaw AS, Faul C, Sever S, Reiser J. CD2AP in mouse and human podocytes controls a proteolytic program that regulates cytoskeletal structure and cellular survival. J Clin Invest. 2011; 121: 3965-80.
Schiffer M, Teng B, Gu C, Shchedrina VA, Kasaikina M, Pham VA, Hanke N, Rong S, Gueler F, Schroder P, Tossidou I, Park JK, Staggs L, Haller H, Erschow S, Hilfiker-Kleiner D, Wei C, Chen C, Tardi N, Hakroush S, Selig MK, Vasilyev A, Merscher S, Reiser J, Sever S. Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models. Nat Med. 2015; 21: 601-9.
Hayek SS, Sever S, Ko YA, Trachtman H, Awad M, Wadhwani S, Altintas MM, Wei C, Hotton AL, French AL, Sperling LS, Lerakis S, Quyyumi AA, Reiser J. Soluble Urokinase Receptor and Chronic Kidney Disease. N Engl J Med. 2015; 373: 1916-25.