SIGNAL TRANSDUCTION AND ACTIN DURING AMOEBOID CHEMOTAXIS

Project: Research project

Project Details

Description

Amoeboid cells of Dictyostelium discoideum are chemotactic to a variety of
agents including extracellular cAMP. As in other chemotactic amoeboid
cells, such as leukocytes and metastatic tumor cells, stimulation of
Dictyostelium amoebae with chemoattractant elicits a burst of actin
polymerization. The location and timing of this polymerization response
helps determine subsequent cell polarity, and creates part of the force
for pseudopod extension. In the previous funding period we identified and
purified a protein, aginactin, that may be involved in regulating
chemoattractant induced actin polymerization in cells by uncapping the
barbed ends of cytoskeleton associated actin filaments.

In this renewal we propose to characterize aginactin at the molecular
level. Aginactin is a cytosolic isoform of the HSC-70 family of proteins.
We will identity which members of the HSC-70 family have aginactin-like
capping activity through the use of antibodies that specifically
crossreact with the subset of HSC-70s that have capping activity and by
protein biochemistry. We will analyze the domain structure of aginactin to
identity phosphorylation, actin binding and capping domains in the protein
to gain insights into the mechanism of capping and its regulation. The
localization of aginactin in situ will be determined relative to F-actin
and the free barbed ends of actin filaments particularly immediately
before and after chemotactic stimulation that causes uncapping of barbed
ends.

To determine the function of aginactin in vivo we will prepare gene
disruption, replacement and/or antisense constructs that can be used to
disrupt or lower the expression of, or overexpress aginactin in vivo.
Phenotypes of transformants prepared by these methods will be analyzed
using established biochemical assays, fluorescence microscopy and computer
assisted video microscopy to detect even subtle differences compared to
wild type and control cells.

The combined approaches of protein chemistry, bacterial expression and
molecular genetics in Dictyostelium will provide a definitive analysis of
the structure and function of aginactin.
StatusFinished
Effective start/end date9/30/938/31/94

Funding

  • National Institute of General Medical Sciences

ASJC

  • Cell Biology

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