Cell motility contributes to both normal (e.g. wound healing) and pathological (e.g. metastasis) cell behavior. Underlying the migratory abilities of cells are the dynamics of actin at the leading edge, regulated in response to Rho GTPases. One such Rac GTPase-initiated pathway crucial to directed cell migration involves the precise spatial regulation of cofilin-dependent actin depolymerizing/severing activity through a phosphoregulatory cycle. During the past grant period, our laboratory identified and characterized a novel cofilin/ADF phosphatase which we termed chronophin (CIN). Our data show CIN to be an important regulator of cofilin phosphocycling, thereby modulating actin dynamics during cell division and motility. The regulation of CIN activity by upstream signals modulating Rac-dependent actin remodeling and cell motility will be investigated. We will use biochemical and molecular biological approaches, along with microscopy-based cell imaging, to determine the signaling pathways, proteins, and molecular complexes that influence cofilin regulation by CIN. The role(s) of CIN in coordinating Rac-dependent actin dynamics and cell motility will be studied. We will use quantitative fluorescent speckle microscopy (qFSM) to analyze the regulation of actin dynamics through CIN-modulated cofilin phosphocycling at the leading edge. In addition, we have shown for the first time that we can apply FSM to directly investigate growth factor (EGF)- regulated leading edge actin dynamics in the carcinoma cell line MTLn3. Cytoskeletal rearrangements induced by heat shock and/or ischemia/ATP depletion are a major cause of injury to cardiovascular cells, neuronal cells, and renal proximal tubule cells. These cytoskeletal responses may have both adaptive (survival) and injurious consequences. We have found that Hsp90 is an endogenous binding partner of CIN and negatively regulates CIN activity. We will examine the participation of Rho GTPase-Hsp90/CIN-cofilin signaling in ATP-depletion-mediated cytoskeletal remodeling. We will investigate the biochemical role of Hsp90 as a direct regulator of CIN activity under these conditions, and assess how the CIN-dependent cofilin cycle participates in stress-mediated cytoskeletal responses and cell survival. Relevance to public health: Cell motility plays important roles in normal physiology and in numerous disease states. Our studies will investigate a novel regulator of this important process. In addition, this protein is likely to play important roles in ischemic disorders and other stress responses. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM044428-17
Application #
7264097
Study Section
Cellular Signaling and Dynamics Study Section (CSD)
Program Officer
Anderson, Richard A
Project Start
1991-07-01
Project End
2011-06-30
Budget Start
2007-09-01
Budget End
2008-06-30
Support Year
17
Fiscal Year
2007
Total Cost
$293,375
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Delorme-Walker, Violaine; Seo, Ji-Yeon; Gohla, Antje et al. (2015) Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels. Proc Natl Acad Sci U S A 112:E5150-9
Huang, Timothy Y; Michael, Sarah; Xu, Tao et al. (2013) A novel Rac1 GAP splice variant relays poly-Ub accumulation signals to mediate Rac1 inactivation. Mol Biol Cell 24:194-209
Delorme-Walker, Violaine D; Peterson, Jeffrey R; Chernoff, Jonathan et al. (2011) Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration. J Cell Biol 193:1289-303
Kim, Jun-Sub; Huang, Timothy Y; Bokoch, Gary M (2009) Reactive oxygen species regulate a slingshot-cofilin activation pathway. Mol Biol Cell 20:2650-60
Huang, Timothy Y; Minamide, Laurie S; Bamburg, James R et al. (2008) Chronophin mediates an ATP-sensing mechanism for cofilin dephosphorylation and neuronal cofilin-actin rod formation. Dev Cell 15:691-703
Chang, Yuan-Chen; Nalbant, Perihan; Birkenfeld, Jorg et al. (2008) GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA. Mol Biol Cell 19:2147-53
Zoudilova, Maria; Kumar, Puneet; Ge, Lan et al. (2007) Beta-arrestin-dependent regulation of the cofilin pathway downstream of protease-activated receptor-2. J Biol Chem 282:20634-46
Delorme, Violaine; Machacek, Matthias; DerMardirossian, Celine et al. (2007) Cofilin activity downstream of Pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks. Dev Cell 13:646-62
Zhao, Tieming; Bokoch, Gary M (2007) Transduction of proteins into intact neutrophils. Methods Mol Biol 412:115-23
Huang, Timothy Y; DerMardirossian, Celine; Bokoch, Gary M (2006) Cofilin phosphatases and regulation of actin dynamics. Curr Opin Cell Biol 18:26-31

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