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Characterization of the Role of Myosin II Isoforms in Cell Spreading
John Ganz1 , Robert B. Wysolmerski 2 and Karen H. Martin2
1Biology
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
Abstract
RNAi Knockdown
Myosin IIB Stain
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.
Myosin IIA
Wild Type
K.O. IIB
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.
1Hr
Wild Type
10 hours
5 Hr
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.
Actin
.
Isoform
Composite
20x Objective
Myosin IIB
KO IIB
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.
Actin
Time Course of Cell Spreading
10 Hr
Time Course of Cell Spreading
40x Objective
0.5 hour
Myosin IIB Knock Out
Wild Type
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.
Z-Stack
KO IIB
Cell Spreading Inverse to Myosin
Myosin IIA
Myosin II A KD
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.
Myosin IIB
Wild Type
Actin
KO II B Rescue
Myosin IIB
Myosin IIA Knock Down
1.5 hour
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.