Mtsl: A Molecular Link Between the Cytoskeleton and Breast Tumor Metastasis
Abstract
The objectives of this grant are: (1) examine how mtsl expression alters directed cellular motility in vitro; (2) generate myosin-IIA antibodies that mimic mtsl binding to examine how the regulation of myosin-IIA affects directed motility in vitro; and (3) utilize an intravital imaging system to evaluate the impact of mtsl expression on metastasis in live animal models. We have established high expressing MTC-GFP-mtsl and MTLn3-GFP-mts1 cell lines and are now poised to begin our intravital imaging studies, which will visualize the motile behavior of mtsl expressing tumor cells within the primary tumor, during intravasation and extravastion in situ. Importantly, these analyses will allow us to determine how mtsl expression impacts on the motile processes associated with invasion and metastasis in vivo and identify those steps in the metastatic cascade affected by mts1 expression. These cell lines will also be used in an in vitro assay to evaluate the effects of mts1 expression on chemoattractant-stimulated motility. This will allow us to obtain comprehensive behavioral phenotype and will identify which aspects of directed motility are sensitive to the expression of mtsl. We have established a quantitative glutathione-Sepharose pull-down assay for mapping the mts1 binding site on the myosin-IIA rod. Our analysis with a GST fusion of residues 1900-1961 indicates that the entire mts1 binding site is contained within the C-terminal 62 residues of the myosin-IIA heavy chain and further suggests that mtsl binds a linear sequence as previously proposed by us. This assay will be used to further narrow and define the mtsl binding domain for the production of a myosin-IIA antibody that mimics the effects of mtsl binding on myosin-II assembly and activity.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2002
- Accession Number
- ADA408099
Entities
People
- Anne R Bresnick
Organizations
- Albert Einstein College of Medicine