Cell mechanics control rapid transitions between blebs and lamellipodia during migration

Abstract

Protrusion formation is an essential step during cell migration. Cells migrating in three-dimensional environments and in vivo can form a wide variety of protrusion types, including actin polymerization-driven lamellipodia, and contractility-driven blebs. The ability to switch between different protrusions has been proposed to facilitate motility in complex environments and to promote cancer dissemination. However, plasticity in protrusion formation has so far mostly been investigated in the context of transitions between amoeboid and mesenchymal migration modes, which involve substantial changes in overall cell morphology. As a result, the minimal requirements of transitions between blebs and lamellipodia, as well as the time scales on which they occur, remain unknown. To address these questions, we investigated protrusion switching during cell migration at the single cell level. Using cells that can be induced to form either blebs or lamellipodia, we systematically assessed the mechanical requirements, as well as the dynamics, of switching between protrusion types. We demonstrate that shifting the balance between actin protrusivity and actomyosin contractility leads to immediate transitions between blebs and lamellipodia in migrating cells. Switching occurred without changes in global cell shape, polarity, or cell adhesion. Furthermore, rapid transitions between blebs and lamellipodia could also be triggered upon changes in substrate adhesion during migration on micropatterned surfaces. Together, our data reveal that the type of protrusion formed by migrating cells can be dynamically controlled independently of overall cell morphology, suggesting that protrusion formation is an autonomous module in the regulatory network that controls the plasticity of cell migration.

Fig. 1.

Protrusion formation in the sublines of Walker cells. (A) Schematic description of subline selection. (B) Example of DIC and fluorescent images of cells of the two sublines: AdhSL cells form thin, actin-filled lamellipodia (arrow). SuspSL cells show polarized blebbing at their leading edge (arrowheads). Newly formed blebs do not contain F-actin (asterisk). An actin cortex reassembles at the bleb membrane over time (bottom row). [Scale bars, 10 μm (top and middle row), 5 μm (bottom row)]. (C) Quantification of the protrusions formed by adhSL cells on a 2D substrate and by suspSL cells placed under agarose. n: number of cells analyzed in two independent experiments. (D) Time lapses of migrating adhSL and suspSL cells. AdhSL cells migrate on flat 2D substrates (Movie S1), whereas suspSL cells need confined environments (e.g., placed under agarose, Movie S4). Arrows: lamellipodia; arrowhead: bleb. (Scale bars, 10 μm.).

Fig. 2.

Contractility favors bleb formation and limits lamellipodia outgrowth. (A) Cortical tension in the sublines probed by micropipette aspiration. P-Value: Welch’s two-sided T-Test; n: number of cells measured in three independent experiments. (B) Quantification of the response to cortex ablation in suspSL and adhSL cells. n: number of cells ablated in two to four independent experiments. (Scale bars, 2 μm. Images: examples of response to ablation in Walker cells expressing Lifeact-mCherry). (C) DIC and fluorescent images of adhSL cells transfected with pEGFP-ROCK-Δ3. Cells show a rounder morphology and spontaneously form blebs (arrowhead). (Scale bars, 10 μm.). (D) Time lapse of adhSL cells treated with 10 μM Y27632. t = 0 s: drug addition. (Scale bar, 10 μm.). (E) Quantification of the change in lamellipodia area upon Y27632 and blebbistatin treatments. Lamellipodia area was measured for at least three frames before and after drug treatment and the ratio of the mean values were plotted. P-Value: Welch’s two-sided T-Test; n: number of cells measured in two independent experiments.

Fig. 3.

Actin polymerization promotes lamellipodia formation at the expense of blebs. (A) DIC, fluorescent and interference reflection microscopy (IRM) images of adhSL cells treated with DMSO or 100 μM CK-666. Blocking Arp2/3 activity switches the protrusion type formed by adhSL cells from lamellipodia (arrows) to blebs (arrowheads), with no apparent change in cell adhesion (IRM). (B) Quantification of the protrusions formed by adhSL cells upon CK-666 treatment. n: number of cells analyzed in two independent experiments. (C) Time lapse of adhSL cells treated with Y27632 and CK-666. The cells stop forming lamellipodia upon Arp2/3 inhibition but do not bleb. 10 μM Y27632 was added at t = 0 sec and 100 μM CK-666 was added at t = 700 sec. Arrow: small lamellipodia. (D) Rac1 activation in suspSL cells under agarose. Upon activation with a 458 nm laser, cells stopped blebbing (upper box series, Movie S8) or stopped blebbing and formed at least one lamellipodium (middle box series, Movie S9). Lamellipodia formation was also observed on PEG-coated glass (lower box series). Arrows: lamellipodia. (Scale bars, 10 μm.).

Fig. 4.

Changing the balance between polymerization and contractility leads to immediate switching between blebs and lamellipodia. Schematic summary of the effects of various treatments performed (arrows). Red color indicates treatments where immediate transitions could be observed.

Fig. 5.

Adhesion can trigger immediate formation of lamellipodia in blebbing cells. (A) Examples of protrusions formed by suspSL cells placed between agarose and fibronectin coated PDMS 30 min before imaging. Arrows: lamellipodia; arrowheads: blebs. (B) Quantification of protrusions formed by suspSL cells placed between agarose and substrates with varying adhesiveness. FN: fibronectin; F127: nonadhesive coating. n: number of cells analyzed in two to three independent experiments. (C) SuspSL cells migrating under agarose on a PDMS layer with microcontact printed adhesive regions (fibronectin, red) and nonadhesive regions (F127-coated). Asterisk: Cell migrating across boundaries. (D) Quantification of protrusions formed by suspSL cells that crossed boundaries between regions and formed lamellipodia when contacting adhesive areas (9 out of 18 cells, three independent experiments). The other 9 cells displayed continuous blebbing. Frequencies of lamellipodia and blebs formed on FN and F127 normalized to the mean frequency on F127 were determined. Data points corresponding to the same cell on F127 and FN are labeled with the same color. P-Value: Welch’s two-sided, paired T-Test. (E) Examples of protrusions of the cell tracked in box (C) (marks on track correspond to displayed image frames). Arrows: lamellipodia; arrowheads: blebs. (Scale bars, 10 μm.).