Technical Reference #1670

1670.

BPAG1e Maintains Keratinocytes Polarity Through Beta 4 Integrin-mediated Modulation of Rac 1 and Cofilin Activities Kevin J. Hamill; Susan B. Hopkinson; Philip DeBiase and Jonathan C.R. Jones, Northwestern University Medical School, Molecular Biology of the Cell, 20(1670), (2009)
Link To Paper

Abstract:
Integrin a component of hemidesmosomes also plays a role in keratinocyte migration via signaling through Rac1 to the actin-severing protein cofilin. Here we tested the hypothesis that the 4 integrin-associated plakin protein bullous pemphigoid antigen 1e (BPAG1e) functions as a scaffold for Rac1/ cofilin signal transduction. We generated keratinocyte lines exhibiting a stable knockdown in BPAG1e expression. Knockdown of BPAG1e does not affect expression levels of other hemidesmosomal proteins nor the amount of  integrin expressed at the cell surface. However the amount of Rac1 associating with 4 integrin and the activity of both Rac1 and cofilin are significantly lower in BPAG1e deficient cells compared to wild type keratinocytes. In addition keratinocytes deficient in BPAG1e exhibit loss of front-to-rear polarity and display aberrant motility. These defects are rescued by inducing expression of constitutively active Rac1 or active cofilin. These data indicate that the BPAG1e is required for efficient regulation of keratinocyte polarity and migration by determining the activation of Rac1.

Materials & Methods:
Observations of Live Cells Motility Assays Single cell motility was measured as detailed by us previously (Sehgal et al. 2006). Briefly cells were plated onto uncoated 35-mm glass-bottomed culture dishes (MatTek Corp. Ashland MA) 18-24 h prior to cell motility assays. Cells were viewed on a Nikon TE2000 inverted microscope (Nikon Inc. Melville NY). Images were taken at 2-min intervals over 2 h and cell motility behavior was analyzed by a MetaMorph Imaging System (Universal Imaging Corp. Molecular Devices Downingtown PA). Lamellipodia number was quantified by scoring the number of sheet like extensions in phase contrast images of live cells. Cell surface area was measured from the same phase contrast images by tracing the outline of individual cells (including lamellipodia but ignoring filpodia/microspikes and retraction fibers) using MetaMorph Imaging software. Statistical analyses were performed using Microscoft Excel and signficance determined by ANOVA and Student’s T test as appropriate.

Microscopic Technique
Fluorescence Microscopy, Immunofluorescence

Cell Type(s)
HEK-293