Analysis of focal adhesions and cytoskeleton by custom microarray.
Focal adhesions and the cell cytoskeleton (intermediate filaments, microfilaments, microtubules) are involved in mechanotransduction-both direct (transduction of mechanical forces to the nucleus) and indirect (transduction of chemical signaling cascades to the nucleus). Thus, observation of changes in focal adhesion and cytoskeletal organization can be invaluable in research such as drug treatments and medical material testing in vitro. Here we describe how to stain human fibroblasts for vinculin (located to focal adhesions), actin (microfilaments), tubulin (microtubules), and vimentin (intermediate filaments) and how to perform custom microarray experiments. Comparative analysis of the immunofluorescence and array data should allow the researcher to build up a global picture of changes to both direct and indirect mechanotransduction through the cytoskeleton from focal adhesions.
Dalby
Matthew J
MJ
Center for Cell Engineering, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK.
Yarwood
Stephen J
SJ
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D016000:Cluster Analysis; D003599:Cytoskeleton / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D022001:Focal Adhesions / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D007382:Intermediate Filaments / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D014746:Vimentin / Q000235:genetics / Q000378:metabolism; D016596:Vinculin / Q000235:genetics / Q000378:metabolism
D000199:Actins; D014404:Tubulin; D014746:Vimentin; D016596:Vinculin