The mechanisms responsible for pathfinding are largely unexplored. Pathfinding is defined in two-dimensional culture as the formation of ventral protrusions that are required for focal contact formation. The reason that the relationships between ventral protrusions, lateral protrusions and adhesion formation have not been adequately studied to date is in part due to the inability of current light microscopes to image actin polymerization dynamics at the ventral surface in live cells during stimulated protrusion. In this application we describe how recent advances in optical imaging may be combined with existing biosensors in order to elucidate the choreographed sequence of spatial and temporal events that are involved in ventral protrusions and their relationship to focal contact formation. We will: 1. Extend imaging methods for observation of ventral cell surface dynamics to live cells. a) Develop hardware and software to enable total internal reflection fluorescence (TIRF) imaging using excitation from a pulsed laser. b) Develop hardware and software to enable fluorescence lifetime imaging microscopy (FLIM) using TIRF illumination. c) Develop hardware and software in order to optimize interference microscopy (IRM). 2. Visualize ventral protrusions in live cells and determine the actin compartments on the ventral surface that contribute to ventral protrusion activity and the formation of local contacts.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM076293-04
Application #
7618490
Study Section
Microscopic Imaging Study Section (MI)
Program Officer
Deatherage, James F
Project Start
2006-05-01
Project End
2012-02-29
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
4
Fiscal Year
2010
Total Cost
$279,255
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
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