Motile properties of vimentin intermediate filament networks in living cells - PubMed (original) (raw)

Motile properties of vimentin intermediate filament networks in living cells

M Yoon et al. J Cell Biol. 1998.

Abstract

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.

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Figures

Figure 1

GFP-vimentin IF networks in interphase cells 72 h after transfection. (A and B) Phase-contrast (A) and fluorescence (B) images of a live BHK cell show that GFP-vimentin IF networks extend from the perinuclear region to the cell periphery. (C and D) In PtK2 cells that contain vimentin and keratin IF networks, GFP-vimentin is incorporated into the vimentin IF network, as indicated by fixation and double indirect immunofluorescence. GFP-vimentin-myc (C) was visualized with monoclonal anti-human c-myc antibody and keratin (D) with polyclonal anti-bovine tongue keratin antibody. (E and F) Phase-contrast (E) and GFP (F) images of a live BHK cell after treatment with 600 nM nocodazole for 5 h. The majority of the vimentin IF network is reorganized into a perinuclear cap. Bar, 5 μm.

Figure 6

Continuity of vimentin fibrils across bleach zones following photobleaching. A GFP-vimentin-myc expressing BHK-21 cell was fixed at ∼1 min after photobleaching, and was processed for indirect immunofluorescence. The continuous vimentin-staining pattern across the bleach zones indicates that photobleaching does not damage vimentin fibrils. (A–C) This series depicts a live cell in phase-contrast (A) and GFP before (B) and immediately after (C) photobleaching. (D–F) Phase-contrast image (D). GFP-vimentin (E) is visualized with c-myc antibody and vimentin (F) with a polyclonal BHK IF antibody (F) in the same cell after fixation and processing for double indirect immunofluorescence. Bar, 10 μm.

Figure 5

Time-lapse observations of GFP-vimentin fibrils in a live CPAE cell. Phase-contrast (A) and GFP images (B–I) indicate that GFP-vimentin fibrils extend into a region lacking IF at the edge of a cell. Intervals between frames are 1 min. Bar, 5 μm.

Figure 2

Dynamic properties of GFP-vimentin in live cells during mitosis. (A–F) Mitotic BHK-21 cells. BHK cell in prometaphase showing chromosomes stained with Hoechst-33258 (A) and GFP-vimentin nonfilamentous aggregates (D). Phase-contrast (B) and fluorescence (E) images in anaphase. Phase-contrast (C) and GFP (F) images show reassembly into juxtanuclear caps in two daughter cells. (G–L). Phase-contrast (G, H, and I) and fluorescent (J, K, and L) images of PtK2 cells showing the cage-like structure of vimentin fibrils during pro-metaphase (G and J), telophase (H and K) and in late cytokinesis (I and L). Note the loss of fluorescence in the region of the midbody in L. L is the same cell as K, taken 10 min later. Bar, 5 μm.

Figure 3

Analysis of an IF- enriched cytoskeletal preparation of BHK-21 cells 72 h after transfection with GFP-vimentin- myc. SDS-PAGE analysis (A) reveals the presence of vimentin/desmin (*) and other proteins that coisolate with IF such as the nuclear lamins (NL) as well as 82-kD GFP-vimentin- myc, which is detected by immunoblotting with c-myc antibody (B). When these preparations were solubilized and immunoprecipitated with the c-myc antibody, GFP-vimentin-myc as well as endogenous vimentin were found in the immunoprecipitates. C is an immunoblot of the immunoprecipitate using the c-myc antibody showing a single 82-kD band; and D shows a blot of the same preparation using a monoclonal antibody directed against vimentin (V9; Sigma Chemical Co.) showing both the 82-kD GFP-vimentin and 55-kD untagged vimentin.

Figure 4

Time-lapse observations of GFP-vimentin IF networks in live BHK-21 cells. (A–C) GFP-vimentin fibrils often interconnect bright foci (arrows), and in this case, the foci move towards each other while the fibrils interconnecting these foci appear to shorten. (D–F) Vimentin fibrils can change their shapes, as indicated by bending (arrows). (G–H) GFP-vimentin fibrils often appear in the focal plane over relatively short time intervals (arrows). Lapsed time (min:sec) is indicated at lower left. Bar, 5 μm.

Figure 7

Time-lapse observations of FRAP in GFP-vimentin fibrils in a BHK-21 cell. (A–D) The bleached GFP-vimentin fibrils completely recover their fluorescence within 20 min. (E–H) Frequently the bleach zones on individual fibrils move at different rates during fluorescence recovery, as indicated by the transformation of the straight bleach zone into a wavy line. Lapsed time (min:sec) is indicated at the lower left of each image. Bar, 10 μm.

Figure 8

Fluorescence intensity measurements along a photobleached GFP-vimentin fibril. Gray-scale pixel values were determined along a bleached vimentin fibril every 2 min with the Metamorph image analysis program. In this case, complete recovery took place in 14 min with a recovery half-time (t1/2) of ∼6 min.

Figure 9

Vimentin squiggles in live BHK-21 cells. (A–C) Phase-contrast (A) and fluorescence (B and C) images showing that squiggles are present in the peripheral region of the same cell. (D–F) Time-lapse observations demonstrating that the majority of vimentin squiggles are motile. The squiggle indicated by the top arrow shows a bending movement and a change of direction at the cell surface. Time intervals between frames are 30 s. Bar, 5 μm.

Figure 10

GFP-vimentin in live BHK-21 cells treated with nocodazole, cytochalasin B, and metabolic inhibitors. (A) Cell treated with 600 nM nocodazole for 5 h. Note that the majority of vimentin fibrils are reorganized into a perinuclear cap, but that some fibrils remain associated with the cell surface. (B and C) Nocodazole-insensitive fibrils seen in A at higher magnification to show that shape changes continue in the absence of microtubules. (D) Phase-contrast image of the edge of a cell 30 min after the addition of 20 μM cytochalasin B. Cell margins have retracted and the overall morphology becomes arborized. (E and F) GFP-vimentin squiggles are present in the same region of this cell. Two squiggles (arrows) showing translocations in the 30-s interval between these two images. (G and H) Motility of GFP-vimentin fibrils is completely arrested 15 min after adding 50 mM 2-deoxy-

-glucose and 0.05% sodium azide. This is most obvious when observing squiggles at the edge of a cell (see arrows). Lapsed time (min:sec) is indicated at lower left of figures. Bar, 5 μm.

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Motile properties of vimentin intermediate filament networks in living cells - PubMed (original) (raw)