Vimentin intermediate filaments modulate the motility of mitochondria

Abstract

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.

FIGURE 1:

Mitochondria colocalize with VimIF network in mouse fibroblasts. Cells (3T3) were stained with MitoTracker Red CMXRos (Invitrogen; see Materials and Methods). Cells were then fixed in methanol (4°C) and stained with anti-vimentin primary antibodies and with secondary anti-rabbit antibodies conjugated with AlexaFluor (Invitrogen). VimIFs, mitochondria, and phase-contrast image of the same cell are shown as overlay (A). Higher magnification images of the two regions shown in A are depicted in B and C; arrows in B show vimentin squiggles. Bar, 10 μm.

FIGURE 2:

Disruption of the VimIF network leads to an altered distribution of mitochondria. Cells (3T3) were transfected with plasmid pEGFP-Vim_(1–138) (A–D) or for control with plasmid pEGFP-Vim (E and F) using FuGene 6 (Roche). Cells at 24 h posttransfection were stained with MitoTracker Deep Red FM (Invitrogen; see Materials and Methods) (B, F pseudocolor) and then were fixed in methanol (–20°C) and stained with antibodies against α-tubulin (Serotech) (C) and anti-vimentin (A), and secondary antibodies conjugated with AlexaFluor 568 and AlexaFluor 488 (Invitrogen), respectively. (D) Overlay of images in A, B, and C. Normal IFs in the cell transfected with the control plasmid in E are visualized by EGFP. Bars, 10 μm.

FIGURE 3:

EGFP-Vim_(1–138) localizes to mitochondria. Cells (3T3) were transfected with plasmid pEGFP-Vim_(1–138) using FuGene 6 (Roche) and 24 h later were stained with MitoTracker Deep Red FM (Invitrogen; see Materials and Methods) (B) and then were fixed in methanol. (A) Epifluorescent image showing GFP localization. (C) Overlay of images in A and B. Bar, 10 μm.

FIGURE 4:

An intact VimIF network alters mitochondrial motility. (A) MFT-6 cells transfected with pmCherry-Mito (left bar) or with pmCherry-Mito and pEGFP-Vim_(1–138) (right bar) using Maxifectin (DiaM). (B) MFT-6 cells (+/+); vimentin-null MFT-16 cells (–/–); vimentin null MFT-16 cotransfected with plasmids pVim and pEGFP-Vim (–/–, +Vim). Cells were also stained with MitoTracker Red CMXRos, and movements of fluorescently labeled mitochondria were recorded by time-lapse microscopy at 4-s intervals. Values are mean percentages of movements exceeding 0.2 μm/s ± SEM; n = number of cells, and, in brackets, the number of movements of mitochondria. Disrupting the intact VimIF network results in faster mitochondrial movements (A), whereas restoring the VimIF network in null cells slows mitochondrial movement (B).

FIGURE 5:

Effects of vimentin mutants on mitochondrial motility. (A) Schematic representation of wild type and mutant vimentin constructs used in this study. WT – full-length vimentin showing N- and C-terminal domains, and central domain formed by four α-helical rich regions; deletion mutants of vimentin: Vim_Δ(26–39), Vim_Δ(41–96), and Vim_Δ(45–70) and full-length vimentin containing P57R point mutation. (B) Western blot analysis of vimentin expression in MFT-6 cells (+/+) using antibody RVIM-AT directed against mouse vimentin or MFT-16 cells before (–/–) or after transfection with plasmids encoding wild type or mutant forms of human vimentin using the V9 antibody. α-tubulin was used as a loading control (DM1a antibody; Sigma). (C) Movements of mitochondria were analyzed in vimentin-null cells cotransfected with plasmids encoding the vimentin mutants and plasmids that encode the same mutants tagged with mCherry and pEGFP-Mito using Maxifectin. Only cells that contained well-formed IF networks (examples are shown in Supplemental Figure S1) were selected for the analysis. Values are mean percentage of movements exceeding 0.2 μm/s ± SEM; n = number of cells (approximately 500 movements in each cell). Statistical analysis (Student's t test) of the data is given in supplemental materials.

FIGURE 6:

Localization of the vimentin N-terminal fragment Vim_(41–94) and its mutant variant expressed in vimentin-null cells. (A) Schematic of the vimentin fragment tagged with the fluorescent protein Dendra2 (green aster); positively charged arginine residues are highlighted in green; R above P-57 represents the substitution of proline for arginine in mutant variant. Cells were transfected with pVim_(41–94)-Dendra2 (B) or pVim_(41–94)-P57R-Dendra2 (D) using Lipofectamine 2000, and mitochondria were stained with MitoTracker Red CMXRos (C and E). Bar, 10 μm.

FIGURE 7:

VimIFs mediate the interaction of mitochondria with F-actin. Cells were transfected with plasmid pEGFP-Mito using Maxifectin. The movements of fluorescently labeled mitochondria in normal and vimentin-null cells were recorded using time-lapse video microscopy before and after incubation with 5 μM LPA for 5 min (A) or before and after incubation with 0.2 μM Lat B for 20 min (B). Values are mean percentage of movements exceeding 0.2 μm/s ± SEM; n = number of cells, and, in brackets, number of mitochondrial movements.

FIGURE 8:

Schematic representation of the association of VimIFs with mitochondria through vimentin N termini exposed on the filament surface and by means of plectin. IF interactions with F-actin bundles (stress fibers) mediated by plectin are also shown.