Vimentin is involved in regulation of mitochondrial motility and membrane potential by Rac1.

In this study we show that binding of mitochondria to vimentin intermediate filaments (VIF) is regulated by GTPase Rac1. The activation of Rac1 leads to a redoubling of mitochondrial motility in murine fibroblasts. Using double-mutants Rac1(G12V, F37L) and Rac1(G12V, Y40H) that are capable to activate different effectors of Rac1, we show that mitochondrial movements are regulated through PAK1 kinase. The involvement of PAK1 kinase is also confirmed by the fact that expression of its auto inhibitory domain (PID) blocks the effect of activated Rac1 on mitochondrial motility. The observed effect of Rac1 and PAK1 kinase on mitochondria depends on phosphorylation of the Ser-55 of vimentin. Besides the effect on motility Rac1 activation also decreases the mitochondrial membrane potential (MMP) which is detected by ∼20% drop of the fluorescence intensity of mitochondria stained with the potential sensitive dye TMRM. One of important consequences of the discovered regulation of MMP by Rac1 and PAK1 is a spatial differentiation of mitochondria in polarized fibroblasts: at the front of the cell they are less energized (by ∼25%) than at the rear part.

Mitochondrial membrane potential is regulated by vimentin intermediate filaments.

This study demonstrates that the association of mitochondria with vimentin intermediate filaments (VIFs) measurably increases their membrane potential. This increase is detected by quantitatively comparing the fluorescence intensity of mitochondria stained with the membrane potential-sensitive dye tetramethylrhodamine-ethyl ester (TMRE) in murine vimentin-null fibroblasts with that in the same cells expressing human vimentin (∼35% rise). When vimentin expression is silenced by small hairpin RNA (shRNA) to reduce vimentin by 90%, the fluorescence intensity of mitochondria decreases by 20%. The increase in membrane potential is caused by specific interactions between a subdomain of the non-α-helical N terminus (residues 40 to 93) of vimentin and mitochondria. In rho 0 cells lacking mitochondrial DNA (mtDNA) and consequently missing several key proteins in the mitochondrial respiratory chain (ρ(0) cells), the membrane potential generated by an alternative anaerobic process is insensitive to the interactions between mitochondria and VIF. The results of our studies show that the close association between mitochondria and VIF is important both for determining their position in cells and their physiologic activity.

Vimentin intermediate filaments modulate the motility of mitochondria.

Interactions with vimentin intermediate filaments (VimIFs) affect the motility, distribution, and anchorage of mitochondria. In cells lacking VimIFs or in which VimIF organization is disrupted, the motility of mitochondria is increased relative to control cells that express normal VimIF networks. Expression of wild-type VimIF in vimentin-null cells causes mitochondrial motility to return to normal (slower) rates. In contrast, expressing vimentin with mutations in the mid-region of the N-terminal non-α-helical domain (deletions of residues 41-96 or 45-70, or substitution of Pro-57 with Arg) did not inhibit mitochondrial motility even though these mutants retain their ability to assemble into VimIFs in vivo. It was also found that a vimentin peptide consisting of residues 41-94 localizes to mitochondria. Taken together, these data suggest that VimIFs bind directly or indirectly to mitochondria and anchor them within the cytoplasm.

Dynein is a motor for nuclear rotation while vimentin IFs is a "brake".

The positioning of the nucleus is achieved by two interconnected processes, anchoring and migration, both of which are controlled by cytoskeleton structures. Rotation is a special type of nuclear motility in many cell types, but its significance remains unclear. We used a vimentin-null cell line, MFT-16, which shows extensive nuclear rotation to study the phenomenon in detail. By selective disruption of cytoskeletal structures and video-microscopic analysis, nuclear rotation was a microtubule-dependent process that F-actin partially impedes. The dynein-dynactin complex is responsible and inhibiting this motor by expression of a dominant negative mutant of its component P-150 completely stops it. Nuclear rotation is powered by dynein associated with the nuclear envelope along stationary microtubules, centrosomes remaining immobile. We confirmed that vimentin IFs inhibit nuclear rotation, and variant proteins of the mutated wild type gene for vimentin that lacked considerable fragments of the N- and C-terminal domains restored nuclear anchoring. Immunochemical analysis showed that these mutated IFs also bound plectin, arguing for a key role of this cytolinker protein in nuclear anchoring. It is proposed that this versatile machinery guarantees not only rotation and the correct location of a nucleus, but also its orientation in a cell.