Project Summary—Project 2 (Gelfand) The goal of this proposal is to study dynamic interactions of vimentin intermediate filaments with two other components of the cytoskeleton, microtubules and actin microfilaments, as well as interactions of VIF with membrane organelles. We will study how intermediate filaments move along microtubules and identify microtubule motors that power this movement. We will determine biochemical basis of vimentin interactions with microtubule motors and identify molecular mechanisms involved in the regulation of these interactions, such as posttranslational modifications of vimentin. We will also find molecular mechanisms of vimentin interaction with actin microfilaments and their bundles and with components of focal adhesions. We will test if interactions of VIF with focal adhesions affect stability of cell-substrate interactions and how vimentin in focal adhesions modulates cell motility. We will identify molecular linkers between actin and intermediate filaments and study functional implications of this association. We will also find how intermediate filaments invade focal adhesions, which focal adhesion proteins link them in place and how intermediate filaments regulate adhesion turnover. Finally, the role of vimentin filaments in transport of membrane organelles will be investigated using two model systems, mitochondria and pigment organelles (melanosomes) in melanophores, because both of these organelles contain bound PKA and PKA-dependent phosphorylation is likely regulated in concert with intermediate filament dynamics and organelle transport.