Lab Overview
My laboratory’s research focuses on the tropomodulin family of actin
pointed end capping proteins and their roles in membrane and cytoskeleton organization,
cell migration and adhesion, with a present focus on cardiac development, red
blood cell membrane biogenesis and stability, and epithelial cell polarity
and morphology. Our approaches include biochemical and biophysical studies
with purified proteins, genetic approaches in mouse models, immunofluorescence
microscopy of proteins in cells and tissues, over-expression and siRNA knockdown
experiments in cultured cells, and microscopy of fluorescent-tagged protein
dynamics and cell motility in living cells.
Highlight
Tmod3 binds to actin monomers via a unique interface.
Robert S. Fischer1, E. Yarmola2, K.L. Weber1, K.D. Speicher3, D.W. Speicher3,
M.R. Bubb2 and V.M. Fowler1. 1The Scripps Research Institute, 2University
of Florida and 3The Wistar Institute.
Regulation of the actin cytoskeleton by actin binding proteins is critical
to myriad dynamic cellular functions. The ability of these proteins to bind
both filaments as well as monomers is often central to their cellular functions,
and contributes to regulation of the monomer pool. The ubiquitous pointed end
capping protein Tmod3 acts as a negative regulator of cell migration, yet mechanisms
behind its cellular functions are not understood. Analysis of Tmod3 effects
on kinetics of actin polymerization and steady state monomer levels reveals
that Tmod3, unlike other Tmods, sequesters actin monomers with an affinity
similar to its affinity for capping pointed ends. To map the Tmod3-actin interface,
we performed zero-length cross-linking to produce a Tmod3-actin complex, and
tested the ability of other actin monomer binding proteins to inhibit this
interaction. These experiments, together with analysis of the Tmod3-actin complex
by tryptic digestion and LC-MS/MS reveal a unique binding interface on actin,
distinct from other actin monomer binding proteins, and two binding sites in
Tmod3. We also demonstrate that Tmod3 binds to monomers in vivo, and that in
motile cells with increased expression of Tmod3, there is a decrease in apparent
free actin concentration, consistent with monomer sequestration by Tmod3. We
conclude that Tmod3 binds to actin monomers via a unique binding interface
in vitro, and in vivo in the context of other monomer binding proteins. We
hypothesize that monomer binding may reduce the effective Tmod3 concentration
available to cap filament pointed ends in cells, providing an additional level
of actin dynamics regulation in cell motility.
2006 Publications
Fischer, R.S., E. G. Yarmola, K.L. Weber, K.D. Speicher, D.W. Speicher, M.R.
Bubb, and V.M. Fowler. (2006) Tropomodulin3 binds to actin monomers. J. Biol.
Chem. 281(47):36454-65.
Kalfia, T.A., S. Pushkaran, N. Mohandas, J.H. Hartwig, V.M. Fowler, J.F. Johnson,
C.H. Joiner, D.A. Williams and Y. Zheng. (2006) Rac GTPases regulate the morphology
and deformability of the erythrocyte cytoskeleton. Blood 108(12):3637-45.
Fowler, V.M., C.R. McKeown and R.S. Fischer. (2006) Nebulin: Does it measure
up as a ruler? Current Biology 16(1):R18-R20. Dispatch.
Fowler, V.M. and R.S. Fischer. (2006) A new Rac-ket in red cells. Blood 108:3633-34
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