Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia - PubMed (original) (raw)
Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia
Marie Schoumacher et al. J Cell Biol. 2010.
Abstract
Invasive cancer cells are believed to breach the basement membrane (BM) using specialized protrusions called invadopodia. We found that the crossing of a native BM is a three-stage process: invadopodia indeed form and perforate the BM, elongate into mature invadopodia, and then guide the cell toward the stromal compartment. We studied the remodeling of cytoskeleton networks during invadopodia formation and elongation using ultrastructural analysis, spatial distribution of molecular markers, and RNA interference silencing of protein expression. We show that formation of invadopodia requires only the actin cytoskeleton and filopodia- and lamellipodia-associated proteins. In contrast, elongation of invadopodia is mostly dependent on filopodial actin machinery. Moreover, intact microtubules and vimentin intermediate filament networks are required for further growth. We propose that invadopodia form by assembly of dendritic/diagonal and bundled actin networks and then mature by elongation of actin bundles, followed by the entry of microtubules and vimentin filaments. These findings provide a link between the epithelial to mesenchymal transition and BM transmigration.
Figures
Figure 1.
Stages of BM breakage by invasive cancer cells. (A) Mesothelial BM. (left panels) x–y images showing the top of the BM immunostained for laminin (left) or collagen IV (right). (right panels) x–z sections of the corresponding x–y projection revealing the two BM layers present in the rat peritoneum. (B and C) Cells cultured atop of peritoneal BM stained with phalloidin–Alexa Fluor 488 (green) and DAPI (blue). BM was detected by laminin staining (red). Merged images are shown in the right column. HT29 (B) and HCT116 (C) cells are shown. (top) Stage 1, early stage of invasion characterized by degradation of the BM and formation of short invasive protrusions (invadopodia). (middle) Stage 2, intermediate stage of invasion and formation of long invasive protrusions (mature invadopodia). (bottom) Stage 3, late stage of invasion and infiltration of the cell on the other side of the membrane. Asterisks indicate sites of degradation and localization of invasive protrusions. (D) Time requirement for BM penetration by HCT116 cells. Black bars show the percentage of cells in stage 1. Light gray bars show the percentage of cells in stage 2. Dark gray bars show the percentage of cells in stage 3. Bars: (A and C) 5 µm; (B) 10 µm.
Figure 2.
Invasive cancer cells form invadopodia on the native BM. (A–C) Localization of invadopodia markers in invasive protrusions: cortactin (A), MT1-MMP (B), and c-src (C). From left to right, columns show actin revealed by phalloidin-Cy3 (red), invadopodia markers as indicated (green), BM detected by immunostaining for laminin and collagen IV (gray), and merged images. Asterisks indicate invasive protrusions. Bars, 5 µm.
Figure 3.
Invadopodia elongation and chemoinvasion assay. (A) Schematic diagram of the chemoinvasion assay. Blue lines correspond to the x–y planes shown in B. (B) HCT116 cells in the chemoinvasion assay. Fluorescent Matrigel, actin stained with phalloidin-Cy3, cortactin, and a merged image of actin and Matrigel are shown. (top) x–y projections presenting the cell on the top of the filter. (middle) x–y projections presenting the cell below the focal plane of the filter. Arrows and arrowheads indicate a short and long protrusion, respectively. On the merged picture, the white rectangular corresponds to the longitudinal cut shown in the bottom panels. (bottom) x–z projections showing longitudinal cut through the cell at the level of the protrusions. (C) Effect of the metalloprotease inhibitor GM6001 on the formation of invadopodia. (left) Gelatin assay shows the percentage of cells that degraded the gelatin. (right) Chemoinvasion assay shows the percentage of cells that formed mature invadopodia. The results were normalized to the cells treated with DMSO. Light gray bars correspond to the cells treated with 10 µM GM6001. Dark gray bars correspond to control DMSO-treated cells. Error bars indicate SEM. *, P < 0.001; paired t test. Bars, 5 µm.
Figure 4.
Lamellipodial and filopodial markers are present in invadopodia. (A and B) Immunofluorescence analysis in the gelatin degradation assay. (top left) Fluorescently labeled gelatin. (top right) Actin revealed by phalloidin-Cy3. (bottom left) ABP as indicated. (bottom right) Merged image of ABP (green) and actin. Insets show higher magnification images of the boxed regions. (A) ABPs associated with lamellipodia revealed by immunostaining (p34 subunit of Arp2/3 complex) or by expression of GFP fusion proteins (VASP and α-actinin) in MDA-MB-231 cells. (B) ABPs associated with filopodia visualized by expression of GFP fusion proteins (T-fimbrin in MDA-MB-231 cells; fascin and myosinX in MDA-MB-435 cells). Bars, 5 µm.
Figure 5.
The formation of invadopodia relies on lamellipodia and filopodial machineries. (A) Immunoblot analysis after siRNA treatment. Scrambled siRNA served as a control for the nonspecific cell response (si-control). Tubulin served as a loading control. (B and C) Quantification of the gelatin degradation of MDA-MB-231 cells treated with the indicated siRNA and normalized to the cells treated with a scrambled siRNA (si-control). (B) Percentage of cells that degraded the gelatin. (C) Degradation index was calculated as described in Materials and methods. (B and C) Error bars indicate SEM. *, P < 0.001; Kruskal-Wallis analysis of variance, Holm-Sidak method. (D) Time-lapse analysis of invadopodia lifetime. Each graph shows the distribution of invadopodia lifetimes in each of the conditions as indicated. Statistical differences were observed between si-control and si-fascin and si-control and si-p34 but not between si-control and si-myosinX (P < 0.001; Kruskal-Wallis analysis of variance, Dunn’s method).
Figure 6.
The elongation of invadopodia relies on lamellipodial and filopodial machinery. (A and B) Immunofluorescence analysis of HCT116 cells in the chemoinvasion assay: spatial distribution of lamellipodial and filopodial markers in mature invadopodia. (left) x–y projections of the cell at the focal plane of the filter. The image is merged, showing actin revealed by phalloidin-Cy3 and the specified ABPs (green). Insets show the x–y projections of the ventral surface of the cell at the focal planes below the filter. Arrows indicate invasive protrusions. (right) z projections of the indicated protrusion. A, actin (red); ABP, ABP as specified in the left panels (green); M, merge. (A) ABPs associated with lamellipodia revealed by immunostaining (cortactin and p34 subunit of Arp2/3 complex) or by expression of GFP fusion proteins (VASP and α-actinin). (B) ABPs associated with filopodia visualized by immunostaining (myosinX) or by expression of GFP fusion proteins (fascin, T-fimbrin, and mDia2). For the visualization of fascin, the nonphosphorylatable mutant (S39A) was used. (C and D) Effect of the depletion of lamellipodial and filopodial machinery on the elongation of invadopodia in MDA-MB-231 cells in the chemoinvasion assay. (C, left) Immunoblot analysis after siRNA treatment. Scrambled siRNA served as a control for the nonspecific cell response (si-control). Tubulin served as a loading control. (right) mRNA expression levels of mDia2 in the cells transfected with two independent siRNA against mDia2 and control siRNA. β-Actin mRNA levels were used as a loading control. (D) Repartition of the protrusions according to their length in cells transfected with siRNA as indicated. Light gray bars show short protrusions (<5 µm). Black bars show long protrusions (>5 µm). The dashed line (50%) is shown to compare the distribution of protrusion lengths between control and siRNA-treated cells. *, P < 0.001; Kruskal-Wallis analysis of variance, Holm-Sidak method. (E) Effect of the depletion of lamellipodial and filopodial machinery on the elongation of invadopodia in HCT116 cells in the native BM model. Repartition of the protrusions according to their length transfected with siRNAs is shown. Light gray bars show short protrusions (<2.5 µm). Black bars show long protrusions (>2.5 µm). *, P < 0.001; Kruskal-Wallis analysis of variance, Dunn’s method. (D and E) Error bars indicate SEM. Fas, fascin; myoX, myosinX; T-Fimb, T-fimbrin. Bars: (A and B, left) 5 µm; (A and B, right) 1 µm.
Figure 7.
Electron micrographs of invadopodia. (A) Mature invadopodia in MDA-MB-231. (A′) The same invadopodia in which the cytoskeleton networks were color coded: microtubules in orange, intermediate filaments in blue, and actin in light/dark yellow. The majority of the actin filaments are randomly arranged so that only short pieces or cross sections are seen in a thin section (light yellow). Bifurcation of actin bundle at the tip of the invadopodia (dark yellow) is shown. Images on the right are a higher magnification of the corresponding boxed regions in A′. Red indicates the base/central part of mature invadopodia; green indicates the central/tip part of mature invadopodia. c, cell; f, filter; m, Matrigel. Bars: (A and A′ [left]) 0.5 µm; (A′, right) 0.25 µm.
Figure 8.
Localization and role of microtubules during invadopodia formation and elongation. (A) Gelatin degradation assay and localization of microtubules in invadopodia of MDA-MB-231 cells. (top left) Fluorescently labeled gelatin. (top right) Actin (phalloidin-Cy3). (bottom left) The Arp2/3 complex (immunostaining for the p34 subunit). (bottom right) Microtubules (immunostaining for tubulin). Insets show higher magnification images of the boxed regions. (B) Chemoinvasion assay and localization of microtubules in mature invadopodia of HCT116 cells. (left) x–y projections of the cell above the focal plane of the filter. (top left) Actin (phalloidin-Cy3). (top right) Microtubules (immunostaining for tubulin). (bottom left) Tyr-tubulin. (bottom right) Merged image of tubulin and tyr-tubulin (Tyr). Insets show x–y projections of the cell below the focal plane of the filter. Arrows and asterisks indicate protrusions shorter and longer than 5 µm, respectively. (middle) z projections of the indicated protrusions. A, actin; B, tubulin; C, tyr-tubulin; A/B and B/C, merged images as indicated. (right) Presence or absence of microtubules in invasive protrusions according to their length. (C) Immunofluorescence analysis after disruption of the microtubule network by nocodazole in MDA-MB-231 cells. Cells were treated with 5 µM DMSO or 5 µM nocodazole for 5 h. Actin revealed by phalloidin-Cy3, microtubules revealed by immunostaining for tubulin, and merge pictures are shown. (D) Quantification of the gelatin degradation of MDA-MB-231 cells treated with the specified reagent and normalized to their respective controls. Cells treated with 5 µM nocodazole were compared with DMSO-treated cells. The results of DMSO- versus nocodazole-treated cells were not statistically different (P = 0.1 and P = 1; Mann-Whitney rank sum test). (E) Repartition of the protrusions according to their length in MDA-MB-231 cells treated with 2 µM DMSO or nocodazole. Light gray bars show short protrusions (<5 µm). Black bars show long protrusions (>5 µm). *, P < 0.001; Mann-Whitney rank sum test. (B, D, and E) Error bars indicate SEM. Bars: (A and B [left]) 5 µm; (B, middle) 1 µm; (C) 20 µm.
Figure 9.
Localization and role of intermediate filaments during invadopodia formation and elongation. (A) Gelatin degradation assay using MDA-MB-231 cells. (top) Localization of cytokeratins. From left to right, fluorescently labeled gelatin, actin (phalloidin-Cy3), cortactin, and cytokeratin filaments are shown. (bottom) Localization of vimentin. From left to right, fluorescently labeled gelatin, actin (phalloidin-Cy3), the Arp2/3 complex, and vimentin filaments are shown. Insets show higher magnification images of the boxed regions. (B) Chemoinvasion assay and localization of intermediate filaments in mature invadopodia in HCT116 cells. (top) Cytokeratin filaments. (bottom) Vimentin filaments. (left) x–y projections of the cell above the focal plane of the filter. Merged images of actin and intermediate filaments (green) are shown. Insets show x–y projections of the cell below the focal plane of the filter. Arrows and asterisks indicate protrusions shorter and longer than 5 µm, respectively. (middle) z projections of the indicated protrusions. A, actin (red); B, intermediate filaments (green); M, merged image. (right) Presence or absence of intermediate filaments in invasive protrusions according to their length. (C, top) Immunofluorescence analysis after depletion of vimentin filaments by siRNA in MDA-MB-231 cells. DAPI (left), vimentin (middle), and a merged image (right) are shown. si-control indicates cells treated with a scrambled siRNA; si-vim1 and si-vim2 indicate cells treated with siRNAs against vimentin. (bottom) Immunoblot analysis after siRNA treatment. Scrambled siRNA served as a control for the nonspecific cell response (si-control). Tubulin served as a loading control. (D) Repartition of the protrusions according to their length in MDA-MB-231 cells treated with the indicated siRNA. Light gray bars show short protrusions (<5 µm). Black bars show long protrusions (>5 µm). *, P < 0.001; Kruskal-Wallis analysis of variance, Holm-Sidak method. (E) Immunofluorescence analysis of MDA-MB-231 cells transfected with pEGFP (gfp) or pEGFP-vim1A (vim1A) plasmids. Actin revealed by phalloidin-Cy3 (left), GFP (middle), and vimentin filaments revealed by immunostaining for vimentin (right) are shown. (F) Repartition of the protrusions according to their length in MDA-MB-231 cells transfected with pEGFP or pEGFP-vim1A. Light gray bars show short protrusions (<5 µm). Black bars show long protrusions (>5 µm). *, P < 0.001; Kruskal-Wallis analysis of variance, Dunn’s method. (B, D, and F) Error bars indicate SEM. Bars: (A, B [left], and E) 5 µm; (B, middle) 1 µm; (C) 10 µm.
Figure 10.
Model for invadopodia formation and maturation. (A) Stages of BM breaching by invasive cancer cells. Noninvasive tumor cells do not degrade the BM (stage 0). Once cancer cells become invasive, invadopodia form and degrade the BM (stage 1). Further, invadopodia elongate (stage 2) and lead the cell to infiltrate into the stromal compartment (stage 3). (B) Cytoskeleton organization in invasive protrusions. Formation of invadopodia requires the assembly of dendritic and bundled actin networks (1a). Elongation of invadopodia is achieved by growth of actin bundles, which is sustained by the dendritic actin network (1b and 2a). Microtubules and intermediate filaments penetrate mature invadopodia while actin bundles are replaced by the expansion of the dendritic actin network (2b).
References
- Artym V.V., Zhang Y., Seillier-Moiseiwitsch F., Yamada K.M., Mueller S.C. 2006. Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function. Cancer Res. 66:3034–3043 10.1158/0008-5472.CAN-05-2177 - DOI - PubMed
- Artym V.V., Yamada K.M., Mueller S.C. 2009. ECM degradation assays for analyzing local cell invasion. Methods Mol. Biol. 522:211–219 - PubMed
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