Technical Reference #1716

1716.

Beta- and Gamma-cytoplasmic actins display distinct distribution and functional diversity Vera Dugina1; Ingrid Zwaenepoel2;*; Giulio Gabbiani2; Sophie Clément2 and Christine Chaponnier2, Moscow State University, Journal of Cell Science, 122(1716), (2009)
Link To Paper

Abstract:
Using newly generated monoclonal antibodies we have compared the distribution of - and -cytoplasmic actin in fibroblastic and epithelial cells in which they play crucial roles during various key cellular processes. Whereas -actin is preferentially localized in stress fibers circular bundles and at cell-cell contacts suggesting a role in cell attachment and contraction -actin displays a more versatile organization according to cell activities. In moving cells -actin is mainly organized as a meshwork in cortical and lamellipodial structures suggesting a role in cell motility; in stationary cells -actin is also recruited into stress fibers. -actin-depleted cells become highly spread display broad protrusions and reduce their stress-fiber content; by contrast -actin-depleted cells acquire a contractile phenotype with thick actin bundles and shrinked lamellar and lamellipodial structures. Moreover - and -actin depleted fibroblasts exhibit distinct changes in motility compared with their controls suggesting a specific role for each isoform in cell locomotion. Our results reveal new aspects of - and -actin organization that support their functional diversity.

Keywords:
actin isoforms; actin network; cytoskeleton

Materials & Methods:
Live-cell imaging was performed on an Axiovert 100M microscope (Zeiss) at 37°C with 5% CO2 using a CCD camera (Hamamatsu Photonics Massy France) operated by Openlab software (Improvision Basel Switzerland). HSCFs were transfected with 50 nM siRNA and 50 nM BLOCK-iT Fluorescent Oligo (Invitrogen) to label transfected cells and plated at single-cell density onto glass-bottom dishes (MatTek Ashland MA). Images for kymography analysis were taken with a 40 0.75 Plan- Neofluar objective every 2 seconds for 5 minutes. Kymographs were produced from image sequences as described (Hinz et al. 1999) using MetaMorph software (Universal Imaging West Chester PA).

Microscopic Technique
Confocal Microscopy, Laser Scanning

Cell Type(s)
HSCFs, rat subcutaneous fibroblasts, pig aortic endothelial