Bone resorption is performed by the mature osteoclast. To resorb bone, osteoclasts require both the presence of dynamic actin adhesion structures in the sealing zone (podosomes) and very efficient endocytosis in the ruffled-border domain. Data generated in our laboratory within the last 5 years has clearly established that two signaling enzyme activities are directly involved in the regulation of both bone resorption and actin dynamics in the osteoclast. On the one hand we have shown that both Src tyrosine kinase activity and the binding of Src to Pyk2 are required, for bone resorption (1) and for the dynamic assembly and disassembly of podosomes (2, see Preliminary Results). On the other hand, we have also shown that dynamin 2 and its GTPase activity are involved in the dynamic assembly and disassembly of podosomes (3, 4) and are required for bone resorption by osteoclasts (4). Furthermore, we have shown that dynamin, itself a substrate of Src, interacts with Pyk2 and with Cbl. We therefore propose to explore the role of dynamin in the integrin-dependent signaling cascade that regulates podosome assembly and disassembly and, we propose, the initiation of endocytosis at the ruffled border, and to understand how dynamin 2 GTPase activity and the Pyk2 and Src tyrosine kinase activities regulate bone resorption in a coordinated manner.
Aim 1 will focus on the early integrin signaling events involving dynamin/Pyk2 interaction and taking place upstream of Cbl, their regulation and their link to actin dynamics and/or endocytosis in osteoclasts.
Aim 2 will explore the signaling events involving the dynamin/Cbl interaction that take place downstream of Cbl, their regulation and their link to both actin dynamics and endocytosis.
Aim 3 will focus on determining the role of dynamin in genetic experiments in vitro and in vivo: analyzing the skeletal and osteoclastic effects of dynamin knockouts/knockdowns and transgenic expression of mutants, selected from the results obtained in Aim 1 and 2. This project proposes to explore a novel concept, based on strong (and partially published) preliminary data, which we believe will help further understand the mechanisms that regulate bone resorption.
|Biosse Duplan, Martin; Zalli, Detina; Stephens, Sebastien et al. (2014) Microtubule dynamic instability controls podosome patterning in osteoclasts through EB1, cortactin, and Src. Mol Cell Biol 34:16-29|
|Shin, Nah-Young; Choi, Hyewon; Neff, Lynn et al. (2014) Dynamin and endocytosis are required for the fusion of osteoclasts and myoblasts. J Cell Biol 207:73-89|
|Coury, Fabienne; Zenger, Serhan; Stewart, Andrew K et al. (2013) SLC4A2-mediated Cl-/HCO3- exchange activity is essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts. Proc Natl Acad Sci U S A 110:2163-8|
|Liu, Shizhou; Ho, Chu Kwen; Ouyang, Jian et al. (2013) Nek1 kinase associates with ATR-ATRIP and primes ATR for efficient DNA damage signaling. Proc Natl Acad Sci U S A 110:2175-80|
|Bruzzaniti, Angela; Neff, Lynn; Sandoval, Amanda et al. (2009) Dynamin reduces Pyk2 Y402 phosphorylation and SRC binding in osteoclasts. Mol Cell Biol 29:3644-56|
|Destaing, Olivier; Sanjay, Archana; Itzstein, Cecile et al. (2008) The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts. Mol Biol Cell 19:394-404|