Microtubule-dependent transport and dynamics of vimentin intermediate filaments

Abstract

We studied two aspects of vimentin intermediate filament dynamics-transport of filaments and subunit exchange. We observed transport of long filaments in the periphery of cells using live-cell structured illumination microscopy. We studied filament transport elsewhere in cells using a photoconvertible-vimentin probe and total internal reflection microscopy. We found that filaments were rapidly transported along linear tracks in both anterograde and retrograde directions. Filament transport was microtubule dependent but independent of microtubule polymerization and/or an interaction with the plus end-binding protein APC. We also studied subunit exchange in filaments by long-term imaging after photoconversion. We found that converted vimentin remained in small clusters along the length of filaments rather than redistributing uniformly throughout the network, even in cells that divided after photoconversion. These data show that vimentin filaments do not depolymerize into individual subunits; they recompose by severing and reannealing. Together these results show that vimentin filaments are very dynamic and that their transport is required for network maintenance.

FIGURE 1:

Rapid motility of mature filaments at cell periphery revealed by TIRF-SIM. (A) Edge of Emerald-vimentin–expressing RPE cell. (B) Time-lapse at 1-s intervals shows rapid transport of filaments in the boxed region. Arrow points to the end of a filament that could be followed between frames. Scale bar, 1 μm. Corresponds to Supplemental Video S1, first sequence.

FIGURE 2:

Photoconvertible-vimentin reveals the rapid transport of mature vimentin intermediate filament (IF) in multiple cell types. (A) Left, Eos3.2-vimentin RPE cell immediately after a circular region was exposed to UV light. All converted filaments were initially confined to the region of exposure (see inset). Right, same cell after 3 min. Many filaments had moved outside of the initial region of conversion, including long filaments (inset). Scale bars, 5 μm. Corresponds to Supplemental Video S2, first sequence. (B) Vimentin IF transport occurred in many cell types. Several filaments had moved outside the region of photoconversion, marked with a circle, within 3 min in (from left to right) NIH3T3, SW13, and primary MEF cells. Scale bars, 5 μm. Corresponds to Supplemental Video S3.

FIGURE 3:

Vimentin IF transport depends on microtubules. (A) Two-­dimensional TIRF-SIM at the cell periphery of an emerald-vimentin–expressing RPE cell treated with nocodazole (top) shows that filaments remained stationary without microtubules (time lapse; bottom). Scale bar, 1 μm. Corresponds to Supplemental Video S1, second sequence. (B) Control (left) and nocodazole-treated (right) cells immediately after conversion and at 1 min, 20s and 3 min. Scale bar, 5 μm. Corresponds to Supplemental Video S2, second sequence. (C) Quantification of filament motility in control (n = 23) vs. nocodazole-treated (n = 19) cells. The 95% confidence interval is represented by error bars. (D) mtagRFPt-cells under control conditions (top) and after nocodazole treatment (bottom). Scale bar, 5 μm.

FIGURE 4:

Method to quantify vimentin filament motility. (A) Control cell 0 and 3 min after photoconversion. (B) Filaments detected using custom software to detect linear segments. (C) Enlargement of boxed regions in A and B. (D) The overlay of boxed regions. Scale bars, 5 μm. (E) Plot of filament spreading from cell represented in A–D.

FIGURE 5:

Blocking microtubule dynamics does not affect vimentin IF transport. (A) mtagRFPT-EB3–labeled growing microtubule plus ends. Frames from time-lapse sequences were individually pseudocolored and superimposed. Differences in frames result in the appearance of rainbows; where frames overlap, colors merge and appear white. Comets can be seen in control (left; see also first sequence of Supplemental Video S4) but not in the presence of 10 nM vinblastine (right; see also second sequence of Supplemental Video S4). Scale bar, 10 μm; color scale, 16 s. (B) Microtubule network is indistinguishable between control (left) and the presence of 10 nM vinblastine (right). Scale bar, 5 μm. (C) Examples of photoconverted Eos3.2-vimentin RPE cells after 3 min in the absence (left) and presence (right) of 10 nM vinblastine shows filament motility in both conditions. (D) Quantification of filament motility in control (n = 16) vs. vinblastine-treated (n = 13) cells. The 95% confidence interval is represented by error bars.

FIGURE 6:

Vimentin filament transport along microtubules revealed by live-cell, two-color TIRF-SIM. Frames from time-lapse imaging show vimentin filaments (left) moving in the periphery of a cell. Arrowheads indicate the ends of two filaments in each frame. The microtubule network was also captured at each time point (middle). The merged images show that vimentin filaments are translocating along microtubules (right). Note that the filaments marked with yellow and blue arrowheads are traveling in opposing directions along the same microtubule. Scale bar, 1 μm.

FIGURE 7:

Long-term imaging after photoconversion reveals extensive severing and reannealing of filaments. Eos3.2-vimentin–expressing SW13 cell immediately after photoconversion and 6 and 17 h later. (A) The 488 channel shows that nonconverted and incompletely converted labeling of filaments was uniform throughout the network. (B) The 561 channel shows filaments distributed in segments throughout the network. Even at 17 h, 561 labeling was restricted to patches along filaments (inset, bottom right). Scale bars, 5 μm. Corresponds to Supplemental Video S7.

FIGURE 8:

Filaments remain in polymerized form throughout cell division in SW13 cells. Eos3.2-vimentin–expressing SW13 cell immediately after photoconversion (left), rounding and lifting from the coverslip in preparation for division at 4 h (middle), and after division (right). (A) Green (nonconverted and incompletely converted) labeling of filaments is uniform. (B) Converted, red filaments remain in segmented form throughout the network and in daughter cells. Scale bar, 5 μm. Corresponds to Supplemental Video S8.

FIGURE 9:

Model of vimentin filament dynamics. (A) Filament dynamics can be followed by producing fiduciary marks on them using photoconversion. (B) Within only minutes, filaments can be seen to be transported outside the region of conversion, likely mediated by microtubule motors kinesin (purple) and dynein (orange). (C) After several hours, converted filaments appear in a patch-like pattern as a result of filament severing and reannealing.