Organization of contractile elements in cardiomyocytes and during cardiac repair after injury depends on the coordinated functioning of microtubules (MTs), microfilaments (MFs) and intermediate filaments (IFs). Mutations in IF genes cause several types of familial cardiomyopathies. IF distribution is determined by their interaction with MTs and MFs via linkers and motors, and is regulated by phosphorylation. However, the molecular machinery that mediates and regulates IF interaction with MTs and MFs and IF transport remains elusive. This work will combine live cell imaging with biochemical techniques and siRNA-mediated knockdown to study regulation of IF movement by motors along MTs and MFs. As a model for IF transport, we will use cell lines stably expressing a GFP-tagged Y117L vimentin mutant which blocks VIF assembly beyond the unit-length filament (ULF) but preserves vimentin interaction with other components of the cytoskeleton. Since VIFs form a dense network, this well-defined short oligomer is easier to track in a cell and to isolate biochemically. In addition, we will use wild-type vimentin tagged with a photoactivatable version of GFP to observe the dynamics of individual filaments in the VIF network. Our work has two specific aims, and our preliminary data show both of them are feasible. AIM 1. To identify motors and adapters responsible for ULF transport and VIF alignment along MTs and MFs. To identify candidates, we will use GFP-ULF as bait to pull-down any adapters or motors that could connect vimentin to MTs or MFs. The requirement of individual adapters and motors for ULF transport and VIF distribution will be determined using siRNA-mediated knockdown. AIM 2. To study the regulation of ULFs transport and VIFs distribution by vimentin phosphorylation. Chemical inhibitors and siRNA-knock down will determine the contribution of specific kinases to 1) ULF transport, 2) VIF distribution, and 3) the recruitment of adapters and motors. Vimentin phospho-residues affected by the kinases inhibition treatments will be identified by mass spectrometry. We will determine whether phosphomimetic and non-phosphorylatable mutants of the identified residues can bind to motors and adapters, move along MTs and actin and affect VIF function on cell migration. This work will contribute to the analysis of the regulation of the cytoskeletal interactions defining organization of many types of eukaryotic cells, including cardiomyocytes and endothelial cells.
|Effective start/end date||7/1/13 → 6/30/15|
- American Heart Association Midwest Affiliate (13POST16210010)