This application is intended to provide additional training and protected time for the principal investigator to develop a new area of research in parallel to current NIAID funded research. Our efforts to describe the activation of hematopoietic integrin receptor activation has progressed rapidly and, along with other recent reports, now suggests that activated beta-3 integrin complexes orchestrate the assembly of actin into stress fibers. This novel hypothesis requires the use of a recently developed in vitro assay of actin assembly and the application of advanced two-color live digital microscopy. Formal advanced training in fluorescent microscopy and significant investigator time will be required for the completion of these experiments. The addition of live cell imaging and fluorescent microscopy will be necessary to appropriately continue the investigators long-term study of hematopoietic cell integrin-mediated adhesion and motility. Current theory of cell motility and adhesion proposes localized actin assembly leading to membrane deformation. This would be followed by integrin interaction first with matrix ligand and then with cytosolic F-actin. Recently, actin nucleating proteins were shown to associate with integrin complexes. We have developed an in vitro assay for actin assembly by purified hematopoietic integrins. This assay reflects deficiencies in cell adhesion through quantitative and qualitative assessment of actin polymerization and was developed to investigate the failure of a Tyr747Phe mutant ?3 integrin to support cell adhesion. Current NIAID funding supports continuing investigation into the signaling mechanisms and physiologic relevance of ?3 tyrosine phosphorylation, but does not provide support for determining integrin control of cytoskeletal assembly. We hypothesize that integrin activation sequesters actin nucleating proteins and that the activated integrin complex serves as the origin of actin fiber assembly during stress fiber formation. If true, this will join integrin signaling and actin assembly fields in a unique approach to understanding cell adhesion and motility. Utilizing integrins purified from hemaotpoietic cells, that exhibit an inactive basal state and applying technology from the actin biochemistry field we will establish in vitro actin stress fiber assembly as an integrin function and use it to understand integrin regulation of cell adhesion and motility.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Scientist Development Award - Research (K02)
Project #
5K02AI057384-05
Application #
7373569
Study Section
Allergy & Clinical Immunology-1 (AITC)
Program Officer
Prograis, Lawrence J
Project Start
2004-06-01
Project End
2010-02-28
Budget Start
2008-03-01
Budget End
2010-02-28
Support Year
5
Fiscal Year
2008
Total Cost
$113,652
Indirect Cost
Name
Upstate Medical University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210
Butler, Boyd; Gao, Chunlei; Mersich, Akos T et al. (2006) Purified integrin adhesion complexes exhibit actin-polymerization activity. Curr Biol 16:242-51
Butler, Boyd; Blystone, Scott D (2005) Tyrosine phosphorylation of beta3 integrin provides a binding site for Pyk2. J Biol Chem 280:14556-62