1. Cell Spreading Inverse to Myosin Actin is a 42 KD protein present in most eukaryote cells that polymerizes to form filamentous structures called stress fibers. Actin filaments form cellular structure which supports many cellular processes including cell spreading and cell migration. The actin filaments interact with myosin motors, proteins composed of two sets of heavy chain and four sets of light chains. Myosin II polymers form bipolar filaments that interact with actin stress fibers. Myosin II molecular motors generate cellular tension, thus detaching the cell from a substrate. Protrusive forces cause cell spreading maximizing contact of cell membrane with substrate. Through the regulation of contractile and protrusive forces, a cell achieves a balance appropriate for specific function and physiological behavior. The three isoforms of nonmuscle Myosin II are Myosin IIA, IIB, and IIC, each of which has a slightly different location in the cell suggesting that they have unique functions. The purpose of the project was to determine the specific roles of the myosin IIA and IIB isoforms in cell spreading.. Myosin II colocalizes with actin stress fibers in fibroblasts isolated from day 14.2 embryos from wild type or myosin IIB knock out mice. Actin was stained using rhodamine phalloidin, and myosin II B was stained using rabbit polyclonal affinity purified antibodies specific to the c-terminal end myosin molecule. Abstract Myosin IIA Knock Down Previous experiments with a specific myosin light chain kinase inhibitor KT5926 and a generalized contractile inhibitor Staurosporine indicated that cell spreading is inversely proportional to myosin activity. These investigators hypothesized that myosin binding to actin increases the stiffness of the cell and consequently impedes cell spreading. John Ganz 1, Robert B. Wysolmerski 2 and Karen H. Martin 2 1 Biology Department, Class of 2009 Eckerd College, St. Petersburg, Florida 2 The Mary Babb Randolph Cancer Center and the Department of Neurobiology and Anatomy, West Virginia University, Morgantown, West Virginia Funding provided by the Spurlock, Reed, McClung and Marconi Endowment to the Mary Babb Randolph Cancer Center Myosin IIB Knock Out Z-Stack Time Course of Cell Spreading Characterization of the Role of Myosin II Isoforms in Cell Spreading Wild Type RNAi Knockdown Knock down of myosin II A was achieved using RNAi, a procedure that blocks the transcription and/or translation of proteins from genes. RNAi uses double stranded RNA which is broken down by an enzyme called dicer. The now unzipped single stranded RNA binds to complementary sequences of the messenger RNA. The newly paired strand is incorporated into RNA induced silencing complex where the paired mRNA will be dissociated. The level of expression for the gene of interest will be decreased. Myosin II A KD Myosin IIB StainTime Course of Cell Spreading 20x Objective 1.5 hour KO IIB KO II B Rescue Actin Myosin IIB Wild Type 0.5 hour Wild Type KO IIB 10 hours Cells exist in three dimensional environment and in effort to model cells actual environment; the cells were grown in a 3-D collagen matrix instead of on a plastic dish. The fibroblasts were then stained for actin and images collected at varying focal planes. In compiling the layers of images, a 3-D projection could be made. The picture below was compiled from the 90 1 micron optical sections taken from different layers of a collagen gel. ActinMyosin IIAMyosin IIB Actin Myosin IIA IsoformComposite K.O. IIBWild Type 10 Hr 5 Hr 1Hr 40x Objective