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.2000 Sep;11(9):2999-3012.
doi: 10.1091/mbc.11.9.2999.

Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration

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Free PMC article

Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration

C Ballestrem et al. Mol Biol Cell.2000 Sep.
Free PMC article

Abstract

Migrating cells are polarized with a protrusive lamella at the cell front followed by the main cell body and a retractable tail at the rear of the cell. The lamella terminates in ruffling lamellipodia that face the direction of migration. Although the role of actin in the formation of lamellipodia is well established, it remains unclear to what degree microtubules contribute to this process. Herein, we have studied the contribution of microtubules to cell motility by time-lapse video microscopy on green flourescence protein-actin- and tubulin-green fluorescence protein-transfected melanoma cells. Treatment of cells with either the microtubule-disrupting agent nocodazole or with the stabilizing agent taxol showed decreased ruffling and lamellipodium formation. However, this was not due to an intrinsic inability to form ruffles and lamellipodia because both were restored by stimulation of cells with phorbol 12-myristate 13-acetate in a Rac-dependent manner, and by stem cell factor in melanoblasts expressing the receptor tyrosine kinase c-kit. Although ruffling and lamellipodia were formed without microtubules, the microtubular network was needed for advancement of the cell body and the subsequent retraction of the tail. In conclusion, we demonstrate that the formation of lamellipodia can occur via actin polymerization independently of microtubules, but that microtubules are required for cell migration, tail retraction, and modulation of cell adhesion.

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Figures

Figure 1
Figure 1
Dynamics of microtubules in a migrating melanoma cell. (A) Distribution of microtubules in a migrating B16 melanoma cell transfected with β5-tubulin-GFP. Note that the lamella is almost devoid of microtubules. (B) Consecutive fluorescence micrographs of the protruding lamella and lamellipodium were taken at 1-min intervals and show the dynamics of microtubules in the advancing lamella. Microtubules perpendicular to the leading edge barely enter the lamella, whereas microtubules growing parallel to the lamellipodium remain fixed with respect to the substrate. The arrow indicates the increasing distance between microtubules oriented parallel to the lamellipodium and the protruding leading edge. (C) As in B after stop of lamellipodium protrusion. Many microtubules reach the leading edge where they become stabilized (empty arrowhead) and eventually depolymerize from the minus end in the perinuclear region (filled arrowhead). Bar, 15 μm.
Figure 2
Figure 2
Actin dynamics in B16 cells during disruption of microtubules. GFP-actin–transfected cells were plated on FN and treated with 10 μg/ml nocodazole at time 00′00". Images at time 13′27" and 25′50" demonstrate the inhibition of cell edge ruffling, (filled arrowheads) and the formation of new stress fibers and focal contacts (inserts). A′–c′) are high-power images of a–c, respectively, the depicted region is indicated in c. Improved visibility of the newly formed dense actin network was achieved by inverting colors. Bar, 10 μm.
Figure 3
Figure 3
Lamellipodium formation after stimulation with PMA is Rac1 dependent. (A) GFP-actin–transfected B16 cells were plated on FN and treated with 100 ng/ml PMA at time 0′. Cells show intense ruffling and lamellipodium formation 55′ after addition of PMA (arrowheads). Bar, 40 μm. (B) B16 melanoma cells transiently transfected with N17Rac (dominant negative) were plated on FN and stimulated with 100 ng/ml PMA for 30 min. Fixed cells were stained for actin (a) and double labeled for N17Rac expression (b). In contrast to nontransfected cells, the N17Rac-transfected cell does not show stress fibers and lamellipodia (a, b). Bar, 20 μm. B16 cell cotransfected with GFP-actin and L61Rac (constitutively active) plated on FN (c) exhibits stress fibers and a smooth rim around the cell edge. Bar, 20 μm.
Figure 4
Figure 4
PMA-induced lamellipodium formation in cells pretreated with nocodazole. (A) GFP-actin–transfected cells were plated on FN and treated with 10 μg/ml nocodazole for 1 h prior to stimulation with 100 ng/ml PMA (time 00′00"). Time-lapse images show lamellipodia formation resulting in disruption of the cell after PMA stimulation (time 05′00"-21′38"). Bar, 20 μm. (B) Tubulin and actin distribution in cells treated with nocodazole and PMA. GFP-actin–expressing cells were plated on FN and treated subsequently with nocodazole and PMA. Cells were then fixed and stained for tubulin. a, actin distribution; b, tubulin distribution; and c, overlay of tubulin (red) and actin (green) within the cell. Note the cells show lamella and actin-rich lamellipodium (a, c) without any filamentous tubular structures (b, c). The red staining in lamella and lamellipodium of the cell in b and c represents unpolymerized tubulin. Bar, 20 μm
Figure 5
Figure 5
PMA-induced lamellipodium formation in B16 cells pretreated with taxol. (A) Melanoma cells transfected with GFP-actin were plated on FN and treated with 10 μM taxol for 1 h prior to 100 ng/ml PMA at time 0′. Subsequent images show circular actin ruffles and lamellipodium formation after addition of PMA (time 10′ and 23′). Bar, 20 μm. (B) Tubulin and actin distribution in cells treated with taxol and PMA. GFP-actin–expressing cells were plated on FN and treated subsequently with taxol and PMA. Cells were then fixed and stained for tubulin. a, actin distribution; b, tubulin distribution; and c, overlay of tubulin (red) and actin (green) within the cell. Note the cells show lamella and actin-rich lamellipodium (a, c) without any tubular structures (b, c). Bar, 20 μm.
Figure 6
Figure 6
Kymograph analysis. (A) Motility of B16 melanoma cells was analyzed by measuring cell edge movements along regions of interest (insert, white line). Movements at these regions (20-μm line) were recorded in 1-s intervals for a period of 5 min. Pictures were assembled resulting in a stroboscopic image, which displays lamellipodia protrusion (black lines) and ruffle retraction (white lines). Lamellipodia protrusion velocity and ruffle retraction rate in these time/space images are indicated by the ascent (dx/dt, μm/min) of protrusive or retracted structures with notable gray values; frequencies are measured counting the number of lamellipodia or ruffles per minute (1/period, min−1). (B) Stroboscopic images under different conditions. Taxol (+tax) and nocodazole (+noc) treatment for 60 min reduced velocity and frequency of lamellipodia protrusions and ruffle retractions. Protrusions and retractions are indicated by white arrows. Cell edge motility was enhance by PMA (+PMA); stimulation with PMA restored the motility of cells that were pretreated with taxol (+tax +PMA), or nocodazole (+noc +PMA). (C) Quantification of B16 cell motility by kymograph analysis. B16 melanoma cells were left untreated (nt) or were treated with taxol (tax), nocodazole (noc), or/and stimulated with PMA. Cell motility was recorded by SACED. The following cell motility parameters were quantified from stroboscopic images: 1) lamellipodia protrusion velocity, 2) ruffle retraction velocity (retraction rate), 3) lamellipodia frequency, and 4) ruffle frequency. Compared with nt cells, noc and tax decreased cell motility. Stimulation by PMA restored lamellipodia and ruffle velocity and frequency of cells pretreated with taxol (tax +PMA) or nocodazole (noc +PMA). At least 15 cells were analyzed per experimental condition. Error bars indicate SD of mean values, calculated from five independent experiments. p ≤ 0.01, p ≤ 0.001 compared with untreated control B16 cells..
Figure 6
Figure 6
Kymograph analysis. (A) Motility of B16 melanoma cells was analyzed by measuring cell edge movements along regions of interest (insert, white line). Movements at these regions (20-μm line) were recorded in 1-s intervals for a period of 5 min. Pictures were assembled resulting in a stroboscopic image, which displays lamellipodia protrusion (black lines) and ruffle retraction (white lines). Lamellipodia protrusion velocity and ruffle retraction rate in these time/space images are indicated by the ascent (dx/dt, μm/min) of protrusive or retracted structures with notable gray values; frequencies are measured counting the number of lamellipodia or ruffles per minute (1/period, min−1). (B) Stroboscopic images under different conditions. Taxol (+tax) and nocodazole (+noc) treatment for 60 min reduced velocity and frequency of lamellipodia protrusions and ruffle retractions. Protrusions and retractions are indicated by white arrows. Cell edge motility was enhance by PMA (+PMA); stimulation with PMA restored the motility of cells that were pretreated with taxol (+tax +PMA), or nocodazole (+noc +PMA). (C) Quantification of B16 cell motility by kymograph analysis. B16 melanoma cells were left untreated (nt) or were treated with taxol (tax), nocodazole (noc), or/and stimulated with PMA. Cell motility was recorded by SACED. The following cell motility parameters were quantified from stroboscopic images: 1) lamellipodia protrusion velocity, 2) ruffle retraction velocity (retraction rate), 3) lamellipodia frequency, and 4) ruffle frequency. Compared with nt cells, noc and tax decreased cell motility. Stimulation by PMA restored lamellipodia and ruffle velocity and frequency of cells pretreated with taxol (tax +PMA) or nocodazole (noc +PMA). At least 15 cells were analyzed per experimental condition. Error bars indicate SD of mean values, calculated from five independent experiments. p ≤ 0.01, p ≤ 0.001 compared with untreated control B16 cells..
Figure 6
Figure 6
Kymograph analysis. (A) Motility of B16 melanoma cells was analyzed by measuring cell edge movements along regions of interest (insert, white line). Movements at these regions (20-μm line) were recorded in 1-s intervals for a period of 5 min. Pictures were assembled resulting in a stroboscopic image, which displays lamellipodia protrusion (black lines) and ruffle retraction (white lines). Lamellipodia protrusion velocity and ruffle retraction rate in these time/space images are indicated by the ascent (dx/dt, μm/min) of protrusive or retracted structures with notable gray values; frequencies are measured counting the number of lamellipodia or ruffles per minute (1/period, min−1). (B) Stroboscopic images under different conditions. Taxol (+tax) and nocodazole (+noc) treatment for 60 min reduced velocity and frequency of lamellipodia protrusions and ruffle retractions. Protrusions and retractions are indicated by white arrows. Cell edge motility was enhance by PMA (+PMA); stimulation with PMA restored the motility of cells that were pretreated with taxol (+tax +PMA), or nocodazole (+noc +PMA). (C) Quantification of B16 cell motility by kymograph analysis. B16 melanoma cells were left untreated (nt) or were treated with taxol (tax), nocodazole (noc), or/and stimulated with PMA. Cell motility was recorded by SACED. The following cell motility parameters were quantified from stroboscopic images: 1) lamellipodia protrusion velocity, 2) ruffle retraction velocity (retraction rate), 3) lamellipodia frequency, and 4) ruffle frequency. Compared with nt cells, noc and tax decreased cell motility. Stimulation by PMA restored lamellipodia and ruffle velocity and frequency of cells pretreated with taxol (tax +PMA) or nocodazole (noc +PMA). At least 15 cells were analyzed per experimental condition. Error bars indicate SD of mean values, calculated from five independent experiments. p ≤ 0.01, p ≤ 0.001 compared with untreated control B16 cells..
Figure 7
Figure 7
Lamellipodium formation in melanoblasts (melb-a) after c-kit stimulation with SCF. Starved melanoblasts pretreated with nocodazole (A) or taxol (B) were stimulated with 50 ng/ml SCF. Both taxol- and nocodazole-pretreated cells show lamellipodium formation 10 min after addition of SCF (10′).
Figure 8
Figure 8
Tail retraction is inhibited in cells devoid of microtubules. (A) Migration of a melb-a melanoblast after stimulation with SCF. Cell form lamellipodia and advance quickly (arrowhead). (B) In contrast cells treated with nocodazole prior to SCF form lamellipodia but are inhibited in cell migration. The depicted cell advances very slowly and leaves a trace of membrane behind, indicating inhibition of tail retraction (arrowhead).
Figure 9
Figure 9
(A) B16 cell migration is inhibited upon disruption of microtubules. Cells plated on serum-coated plastic dishes were treated with control medium (nt), 100 ng/ml PMA, 10 μg/ml nocodazole, or nocodazole and PMA. The migration distance of at least 50 cells/condition was measured and calculated as average speed of migration per hour (μm/h). The histogram represents one of five independent experiments with similar results. (B) Cell fragment migration. GFP-actin–transfected B16 cells were plated on FN and were subsequently treated with nocodazole and PMA. Time-lapse images show the separation of a part of the cell from the main cell body. The advancing cell fragment leaves a trace of actin-containing membrane behind (arrowhead). Bar, 15 μm. (C) Adhesion of B16 melanoma cells to FN (2.5 μg/ml) is significantly enhanced by addition of nocadozole or the combination of nocadozole with PMA. Stimulation of B16 with PMA alone leads only to little increase in adhesion.
Figure 9
Figure 9
(A) B16 cell migration is inhibited upon disruption of microtubules. Cells plated on serum-coated plastic dishes were treated with control medium (nt), 100 ng/ml PMA, 10 μg/ml nocodazole, or nocodazole and PMA. The migration distance of at least 50 cells/condition was measured and calculated as average speed of migration per hour (μm/h). The histogram represents one of five independent experiments with similar results. (B) Cell fragment migration. GFP-actin–transfected B16 cells were plated on FN and were subsequently treated with nocodazole and PMA. Time-lapse images show the separation of a part of the cell from the main cell body. The advancing cell fragment leaves a trace of actin-containing membrane behind (arrowhead). Bar, 15 μm. (C) Adhesion of B16 melanoma cells to FN (2.5 μg/ml) is significantly enhanced by addition of nocadozole or the combination of nocadozole with PMA. Stimulation of B16 with PMA alone leads only to little increase in adhesion.
Figure 9
Figure 9
(A) B16 cell migration is inhibited upon disruption of microtubules. Cells plated on serum-coated plastic dishes were treated with control medium (nt), 100 ng/ml PMA, 10 μg/ml nocodazole, or nocodazole and PMA. The migration distance of at least 50 cells/condition was measured and calculated as average speed of migration per hour (μm/h). The histogram represents one of five independent experiments with similar results. (B) Cell fragment migration. GFP-actin–transfected B16 cells were plated on FN and were subsequently treated with nocodazole and PMA. Time-lapse images show the separation of a part of the cell from the main cell body. The advancing cell fragment leaves a trace of actin-containing membrane behind (arrowhead). Bar, 15 μm. (C) Adhesion of B16 melanoma cells to FN (2.5 μg/ml) is significantly enhanced by addition of nocadozole or the combination of nocadozole with PMA. Stimulation of B16 with PMA alone leads only to little increase in adhesion.
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