![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.](https://cdn.rupress.org/rup/content_public/journal/jcb/189/3/10.1083_jcb.200909113/6/jcb_200909113r_rgb_fig8.jpeg?Expires=1772567138&Signature=MfxG7~esVPAJvKl9vqH2o3q940u-S1DvXcMS6u0T~DHpNT~-6mda7frlE8Z1j7F8oTloNm~~fUmVdcXOZqZxhfi2NM4TdT01sqJz5eq1bCITH3FOE6XP5C~pjOSKLF5loMCLslELTw51fQtnSmuub2an10vpyPpQNijZAfmc6nT0Cwgu3t2S7PVYv~e8nW2uTE9K8hvK7QVAk6ZVE~BW0odCvXbz6uOy3m5v~kSYrK~aKTU0LLxhGooIWvUDP-rrntAwjSm~kSkOC0ghSh4ZUFMzv8dFw5NgSZi6bHoe2rjNnWvptibnBlAFR-PJSjUyUmDcZ6VM1PwfDrxNlbWNkg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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.
