KINESIN AND INTERMEDIATE FILAMENT DYNAMICS

Vimentin intermediate filaments (IFs) were recently shown to associate with a specific subset of microtubules (MTs). A posttranslational modification, detyrosination, occurs on a subset of stable MTs. Detyrosination removes the C-terminal tyrosine exposing a glutamate residue. Until the finding that vimentin IFs coalign with detyrosinated MTs, not function had been identified for this post-translational modification. The focus of my project will be to further characterized the association of IFs with MTs and determine if detyrosinated MTs play a role in the dynamic movements of IFs. Part of this project will include the isolation of a vimentin-kinesin that was recently identified in the laboratory. I will devise a protocol to purify the vimentin- kinesin for use in biochemical studies. I will also use the purified vimentin-kinesin to obtain microsequence analysis and antibodies, which will be used to further characterize the association of IFs with MTs. To study the dynamic behavior of IFs, and the requirement for MTs, I will use light microscopy techniques. In vivo studies will include time-lapse recordings of cells that express GFP-vimentin and have been microinjected with rhodamine-tubulin. Further studies using this system will determine if the movement of IFs requires detyrosinated MTs. I will also reconstruct the movement of IFs on MTs in vitro using bacterially expresses GFP-vimentin, rhodamine-tubulin and the vimentin- kinesin. The in vitro reconstitution of IF movement on MTs will be the first where system where the movement of biologically relevant cargo is studied.