Abstract
Purpose: :
The adverse effect of R120G mutation of B-crystallin on the assembly of muscle desmin intermediate filaments (IF) is well known. Recent study shows that this mutation also affects lens vimentin filaments. Since the lens contains other -crystallins (A-crystallin), we have investigated the effects of A-crystallin and its hetero-oligomers (A/B, A/R120GB, and R116CA/B) on vimentin and beaded filament structures.
Methods: :
We used confocal fluorescence microscopy to visualize assembly of vimentin and beaded filaments (filensin and CP49) with fusion green or red fluorescence protein (GFP or RFP) as probes. GFP-IF and -crystallin genes were cotransfected to HeLa cells and images of living cells were visualized. Protein-protein interactions were determined by FRET using either sensitized emission or photo bleaching method with the donor-acceptor pairs of GFP-IF and RFP--crystallin.
Results: :
The confocal fluorescence images showed that either homo- or hetero-oligomers (A-, B-, or A/B) assist the proper assembly of IFs, but -crystallins containing myopathy-associated mutants (A/R120GB and R116CA/B) disrupt either vimentin assembly or beaded filament bundle structure. FRET analyses indicated that both A/R120GB and R116CA/B increase protein-protein interactions with both vimentin and beaded filaments.
Keywords: crystallins • cytoskeleton • microscopy: confocal/tunneling