Characterization Of Coronin 2A And Its Role In Regulating Cofilin Activity

Thomas W. Marshall: Characterization of Coronin 2A and its role in regulating Cofilin activity (under the direction of Dr. James E. Bear) Coronins are conserved F-actin binding proteins that are important for motility and actin dynamics. Mammalian Coronin proteins can be broken down into three subtypes: Three Type I Coronins (consisting of Coronin 1A, 1B and 1C), two Type II Coronins (Coronin 2A and 2B), and one Type III Coronins (POD/Coronin 7). Each of these types has distinct localization patterns in the cell. Type I Coronins primarily localize to lamellapodial F-actin structures found at the leading edge of cells. Unlike type I Coronins, the type II Coronin 2A is excluded from the leading edge and localizes to stress fibers and focal adhesions. Studies on POD suggest that it primarily localizes to the Golgi apparatus. Depletion of Coronin 2A in MTLn3 cells decreases cell motility and focal adhesion turnover. Surprisingly, none of the pathways known to regulate focal adhesion turnover are affected by Coronin 2A depletion. Depletion of Coronin 2A does however increase phospho-Cofilin suggesting that misregulation of Cofilin may affect adhesion dynamics. Slingshot-1L, a Cofilin-activating phosphatase, localizes to focal adhesions and interacts with Coronin 2A. Depletion of Coronin 2A reduces Cofilin activity at focal adhesions as measured by barbed end density and actin turnover. Consistent with this idea, expression of an active mutant of Cofilin iii bypasses the defects in cell motility and focal adhesion disassembly seen upon Coronin 2A depletion. These results implicate both Coronin 2A and Cofilin as new factors that can regulate focal adhesion turnover. Enforced expression of Coronin 2A induces the formation of structures that are similar to Cofilin-Actin rods. These are ordered aggregates that form in certain cells in response to stress, such as ATP-depletion. Like Cofilin-Actin rods, Coronin 2A rods also contain Cofilin and Actin, but unlike Cofilin-Actin rods, Coronin 2A rods stain positively with phalloidin. These data suggest that Coronin 2A may form submit Cofilin-Actin rod intermediates that require further investigation. iv Dedication This work is dedicated to my grandma Marie Schwacke who turned 100 years old this year. Her unbelievable longevity showed me that what I have been able to accomplish over the last six years is just a drop in the bucket of what I will hopefully be able to in my lifetime. Also to my family, friends, and Gwendolyn, the love of my life. Without them, my work would be …