Endoplasmic spreading requires coalescence of vimentin intermediate filaments at force-bearing adhesions

Abstract

For cells to develop long-range forces and carry materials to the periphery, the microtubule and organelle-rich region at the center of the cell-the endoplasm-needs to extend to near the cell edge. Depletion of the actin cross-linking protein filamin A (FlnA) causes a collapse of the endoplasm into a sphere around the nucleus of fibroblasts and disruption of matrix adhesions, indicating that FlnA is involved in endoplasmic spreading and adhesion growth. Here, we report that treatment with the calpain inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN) restores endoplasmic spreading as well as focal adhesion (FA) growth on fibronectin-coated surfaces in a Fln-depleted background. Addback of calpain-uncleavable talin, not full-length talin, achieves a similar effect in Fln-depleted cells and indicates a crucial role for talin in endoplasmic spreading. Because FA maturation involves the vimentin intermediate filament (vIF) network, we also examined the role of vIFs in endoplasmic spreading. Wild-type cells expressing a vimentin variant incapable of polymerization exhibit deficient endoplasmic spreading as well as defects in FA growth. ALLN treatment restores FA growth despite the lack of vIFs but does not restore endoplasmic spreading, implying that vIFs are essential for endoplasm spreading. Consistent with that hypothesis, vIFs are always displaced from adhesions when the endoplasm does not spread. In Fln-depleted cells, vIFs extend beyond adhesions, nearly to the cell edge. Finally, inhibiting myosin II-mediated contraction blocks endoplasmic spreading and adhesion growth. Thus we propose a model in which myosin II-mediated forces and coalescence of vIFs at mature FAs are required for endoplasmic spreading.

FIGURE 1:

Calpain inhibition rescues endoplasmic spreading in Fln-depleted MEFs. (A) Fln-depleted MEFs were transfected with FL FlnA, (B) treated with DMSO, or (C) treated with 50 μM ALLN for 22 h, plated on 10 μg/ml FN-coated glass for 30 min, fixed, stained for paxillin, and imaged with confocal microscopy. GFP signal was associated with successful FlnA shRNA transfection and expression (scale, 20 μm). (D) Areas of peripheral adhesion plaques were measured manually from a single confocal slice in each condition (Fln-depleted with FL FlnA, 7 cells, 128 adhesions; Fln-depleted with DMSO, 10 cells, 305 adhesions; Fln-depleted with ALLN, 12 cells, 323 adhesions; *p < 0.001). (E) Peripheral adhesion areas were measured at various time points using the same method as in D (with DMSO, 10 min: 16 cells, 139 adhesions; with DMSO, 20 min: 15 cells, 174 adhesions; with DMSO, 30 min: 12 cells, 198 adhesions; with ALLN, 10 min: 16 cells, 295 adhesions; with ALLN, 20 min: 9 cells, 168 adhesions; with ALLN, 30 min: 10 cells, 213 adhesions). (F) Fln-depleted MEFs were transfected with FL FlnA, (G) treated with DMSO, or (H) treated with 50 μM ALLN for 22 h, plated on FN-coated glass for 30 min, and fixed (scale, 20 μm). (I) Endoplasm/whole-cell area was determined by measuring RFP-ER area and whole-cell area in fluorescence and DIC channels, respectively (with DMSO, 40 cells; with ALLN, 41 cells; three separate experiments; *p < 0.001).

FIGURE 2:

Calpain-uncleavable talin expression rescues endoplasmic spreading in Fln-depleted MEFs. (A) Fln-depleted MEFs were cotransfected with GFP-tagged FL talin or (B) calpain-uncleavable talin (NC talin), spread for 30 min, and fixed (scale, 20 μm). (C) Endoplasm/whole-cell area ratios were measured using DIC images for Fln-depleted MEFs cotransfected with GFP-tagged wild-type FlnA, FL talin, or NC talin (at least 33 cells analyzed/experimental condition; *p > 0.1, **p < 0.001). (D) Fln-depleted MEFs, expressing mCherry as an shRNA marker, were cotransfected with GFP-tagged wild-type or (E) calpain-uncleavable talin, spread on FN for 30 min, fixed, and stained for paxillin (scale, 20 μm). (F) Areas of peripheral adhesion plaques were measured manually from a single confocal slice in each condition (Fln-depleted with FL FlnA, 7 cells, 128 adhesions; Fln-depleted, 10 cells, 305 adhesions; Fln-depleted with FL talin, 11 cells, 166 adhesions; Fln-depleted with NC talin, 5 cells, 82 adhesions; *p > 0.1, **p < 0.01). (G) Peripheral adhesion areas were measured at various time points using the same method as in F (75 adhesions measured across 5 cells per time point for each cell type).

FIGURE 3:

Vimentin intermediate filaments are required for endoplasmic spreading. (A) RPTPα^+/+ (control) MEFs were transfected with GFP–FL Vim, (B) transfected with GFP Vim 1-138, or (C) transfected with GFP–Vim 1-138 and treated with ALLN, spread for 30 min on FN-coated glass, and fixed (scale, 20 μm). (D–G) Control MEFs were transfected and spread as in A–C, with subsequent immunostaining for paxillin (scale, 20 μm). (H) Endoplasm/whole-cell ratios were measured as in Figure 2C (control, 30 cells; with FL Vim, 39 cells; with Vim1-138, 30 cells; with Vim 1-138 + ALLN, 45 cells; *p < 0.001, **p > 0.1). (I) Peripheral adhesion areas were measured manually from a single confocal slice in each condition (with FL Vim, 6 cells, 120 adhesions; with Vim 1-138, 6 cells, 120 adhesions; with Vim 1-138 + ALLN, 9 cells, 222 adhesions; *p < 0.001). (J) Peripheral adhesion plaque areas were measured at various time points using the same method as in I (with FL Vim, 10 min: 4 cells, 42 adhesions; 20 min: 6 cells, 114 adhesions; 30 min: 170 adhesions; with Vim1-138, 10, 20, 30 min: 5 cells each, 100 adhesions each).

FIGURE 4:

Endoplasmic spreading requires coalescence of vimentin intermediate filaments at mature focal adhesions. (A) RPTPα^+/+ (control) MEFs were treated with DMSO or (B) 500 nM WFA overnight, spread on FN-coated glass for 30 min, fixed, stained for vimentin, and subsequently subjected to fluorescence confocal imaging (green, anti-vimentin; red, phalloidin). (C) Cells were treated with WFA as in A, fixed, and imaged in DIC. (D) Endoplasm/whole-cell area ratios were significantly lower for cells treated with WFA than controls (30 cells measured for each condition; *p < 0.001). (E) Each cell type was plated on FN-coated glass for 30 min, fixed, and stained for vimentin and paxillin. FA plaques colocalizing with vIF tips were quantified as a percentage of total FA plaques (control, 13 cells; Fln-depleted, 13 cells; control with DMSO, 13 cells; control with WFA, 13 cells; *p < 0.001). (F–I) Sample images used to generate E. Cells were prepared as described in E and subjected to confocal imaging.

FIGURE 5:

VIFs extend closer to the cell edge in Fln-depleted MEFs in a microtubule-dependent manner. (A–F) In each condition, MEFs were plated on FN-coated glass for 30 min, fixed, and stained for vimentin and phalloidin, with GFP signal corresponding with shRNA expression. Cells treated with nocodazole and/or Y-27632 were exposed 30 min before spreading (scale, 20 μm). (G) Distances of vIFs to the cell edge were determined by averaging the distance from the vIF boundary to the cell edge at 10 points in the cell, using at least six cells per cell type (*p < 0.001, **p < 0.05). (H) FlnA^−/− MEFs were transfected with p3xGFP-EMTB and visualized in TIRF. Shown is the initial stage of spreading (∼2 min from initiation) (scale bar, 20 μm). (I) Kymograph generated across white line from F. Yellow dotted line indicates MT boundary. Note that MTs stay confined even as the cell continues to spread after ∼10 min; scale, 20 μm, 5 min.

FIGURE 6:

Endoplasmic spreading requires myosin II–mediated contraction. (A) Control MEFs were treated with 50 μM blebbistatin or an equal volume of DMSO and plated on 10 μg/μl FN for 30 min (scale, 20 μm). (B) Endoplasm/whole-cell ratios show a reduction in endoplasmic spreading upon blebbistatin treatment (50 cells/cell type, n = 2 experiments, *p < 0.01). (C) Cells were treated and spread as in A and then fixed and stained for phalloidin and paxillin (scale, 20 μm).

FIGURE 7:

Endoplasmic spreading is a continuous process dependent on coalescence of vimentin filaments at force-bearing adhesions. A cage-like structure surrounds the endoplasm from the beginning of spreading. As microtubules migrate outward, vimentin fragments are transported out to early, maturing adhesions. Vimentin fragments collect at growing adhesions, subsequently forming vimentin filaments that can interact with the central endoplasmic cage. Myosin II contraction in the endoplasmic cage and associated actomyosin structures causes the endoplasm to pull forward on the vIF-strengthened connection to the growing adhesion, resulting in further growth. Vimentin fragments continue to be transported to the periphery on microtubule tracks toward the next layer of growing adhesions. Vimentin filaments collect at the next round of adhesions, connecting with the spreading endoplasmic cage. The process repeats, allowing the endoplasm to spread in any direction that the cell spreads.