M111998–2003 · The regulated bleb, 1995–2003 · strongly supported

ROCK–myosin contraction drives blebbing in dying and in invading cells

Until the late 1990s, the blebbing of stressed and dying cells was widely seen as a non-specific response to injury. Studies in 1998–2003 showed instead that myosin II contraction drives it, that caspase cleavage of Rho-associated kinase I (ROCK I) switches this contraction on in apoptosis, and that the same Rho–ROCK pathway drives a rounded, blebbing mode of tumor-cell invasion. That ROCK-driven myosin contraction powers these blebs is strongly supported, although for invasion the evidence comes from cell lines in vitro.

Before

Hints of an active process existed. Burnham and Williams (1982) saw over-stimulated mouse pancreatic acini form blebs that cytochalasin B largely prevented, and suggested that “Elevated contractility may raise intracellular fluid pressure” (p. 211), without measuring either. Outside apoptosis, the ROCK inhibitor Y-27632 abolished the blebbing of 293T cells stimulated through the receptor EphA3 (Lawrenson et al., 2002). Still unknown were the motor that drives these blebs, how death signaling switches it on, and whether moving cells use the same machinery.

What was done

The first studies separated blebbing from death. In serum-deprived PC12 cells, z-VAD-FMK, an inhibitor of caspases, the proteases that execute programmed cell death, stopped death but not blebbing. Among kinase inhibitors, only those acting on myosin light-chain kinase (MLCK) reduced ongoing blebbing, and phosphorylation of the myosin regulatory light chain was 3–5-fold higher in blebbing cultures (Mills et al., 1998). Rat pancreatic acini, apparently not apoptotic, gave the same answer: they blebbed reversibly in supramaximal cholecystokinin, phosphorylated myosin ringed the bleb necks, and the myosin ATPase inhibitor BDM or the MLCK inhibitor ML-9 blocked blebbing (Torgerson and McNiven, 1998; figure).

How does apoptosis switch myosin on? Caspase cleavage made ROCK I 7.8 ± 0.5-fold more active (s.e.m., 5 experiments; Coleman et al., 2001), and in MCF-7 cells cleavage required caspase-3 (Sebbagh et al., 2001). In Jurkat cells triggered to die through Fas, Y-27632 abolished both light-chain phosphorylation and blebbing, whereas inactivating Rho affected neither (Sebbagh et al., 2001), and truncated ROCK I made cells bleb even with caspases blocked (Coleman et al., 2001; Sebbagh et al., 2001). Caspases thus activate ROCK I without Rho, and ROCK I activates myosin.

To ask whether moving cells use the same machinery, Sahai and Marshall (2003) compared five tumor lines invading Matrigel, a three-dimensional (3D) gel. Inhibiting Rho or ROCK almost abolished invasion by two rounded, blebbing lines but spared elongated SW962 cells (table), and active ROCK I made A375P cells round and blebbing (figure). Protease inhibitors did not reduce invasion by the rounded lines but made elongated BE cells invade with a rounded shape, which Y-27632 then cut sharply. The authors proposed that contractility raises hydrostatic pressure to push out blebs, but did not measure pressure or bleb dynamics.

Quantity Value System Source
Inhibition of ongoing blebbing at 1 h staurosporine 89%, KT5926 39% (text); ML-9 94–99% at 80 µM (mean ± s.e.m., 3 experiments) PC12, caspase-inhibited Mills et al., 1998, Fig. 5
Invasion with Y-27632 (10 µM), depth ≥12 µm A375m2 3.8; LS174T 1.0; WM266.4 34; BE 78; SW962 157 (% of control; mean of 3 experiments, ≥1,000 cells each; no error given) Transwell Matrigel Sahai and Marshall, 2003, Table 1
Invasion, depth ≥8 µm: Y-27632 / protease inhibitors / both A375m2 12 / 150 / 6; LS174T 13 / 250 / 9; BE 81 / 115 / 16 (% of control; 3 experiments) Transwell Matrigel Sahai and Marshall, 2003, Table 3
Later: apoptotic cells with surface blebs, control vs ROCK1−/− clones C1, C2, C3 ≈100 vs ≈4, ≈89, ≈84% (read from plot; s.e.m., 3 experiments) Jurkat, anti-Fas Tixeira et al., 2020, Fig. 2e
Phosphorylated myosin light chain is faint in resting rat acini (A) and rings bleb bases (B; blebs labeled B) after 30 min of supramaximal cholecystokinin; its level rises ≈5–8-fold at 5 min (C, two experiments). BDM or ML-9 reduce blebbed…
Phosphorylated myosin light chain is faint in resting rat acini (A) and rings bleb bases (B; blebs labeled B) after 30 min of supramaximal cholecystokinin; its level rises ≈5–8-fold at 5 min (C, two experiments). BDM or ML-9 reduce blebbed acini from ≈96% to ≈9–13% (D, read from plot). Source: Torgerson and McNiven, 1998, J Cell Sci, Fig. 8A–D; shown with citation for educational, non-profit use.
Rho–ROCK signaling and cell shape in 3D Matrigel. Active RhoA, RhoC or ROCK I round A375P cells, and active ROCK I (ROCK-Δ3) makes them bleb; Rho or ROCK inhibition removes blebs from A375m2 cells (a). Rounding elongated BE cells leaves…
Rho–ROCK signaling and cell shape in 3D Matrigel. Active RhoA, RhoC or ROCK I round A375P cells, and active ROCK I (ROCK-Δ3) makes them bleb; Rho or ROCK inhibition removes blebs from A375m2 cells (a). Rounding elongated BE cells leaves invasion at ≈90–107% of control (c, d; read from plot). Source: Sahai and Marshall, 2003, Nat Cell Biol, Fig. 2; shown with citation for educational, non-profit use.

What it changed

The work turned blebbing into the output of a defined pathway shared by dying and moving cells. Mills et al. (1999) named myosin II, contracting against focally weakened membrane–cortex links, as the force in apoptotic and non-apoptotic blebbing alike, and Fackler and Grosse (2008) treated Rho–ROCK–myosin as the core machinery of both. Genetic evidence was partial: of three ROCK1-knockout Jurkat clones, one almost lost apoptotic blebbing and two lost part of it (Tixeira et al., 2020; table).

Tumor cells also proved able to switch between rounded and elongated movement. In A375M2 cells, ROCK inhibition made rounded cells elongated without slowing them, and depletion of the GTPase Rac left only amoeboid movement (Sanz-Moreno et al., 2008). This plasticity, the mesenchymal–amoeboid transition, became a standard frame for tumor invasion (Lämmermann and Sixt, 2009).

Later work and open questions

In our reading, several apparently contradictory findings reflect differences between cell types and stimuli. Blebbing continued without caspase activity in PC12 cells, where it fell when Rho was inhibited (Mills et al., 1998), and in cisplatin-treated Rat-1 cells, where it needed the stress kinase p38 (Deschesnes et al., 2001). In NIH 3T3 fibroblasts and Jurkat cells, by contrast, it depended on caspases and not on Rho (Coleman et al., 2001; Sebbagh et al., 2001); Sebbagh et al. suggested that adherent cells use Rho as they round up. In endothelial cells treated with combretastatin A-4-phosphate, inhibitors of MLCK and of myosin even increased blebbing (Kanthou and Tozer, 2002).

Invasion, too, depends on the environment. In cross-linked native collagen, tumor cells needed the membrane protease MT1-MMP to invade (Sabeh et al., 2009), so protease-free invasion in Matrigel cannot be assumed in tissue. In rat mammary tumors in mice, ROCK inhibition cut the number of moving tumor cells by about 70% and a broad metalloprotease inhibitor by only about a fifth (no statistics given), but in collagen gels these cells led with an actin-rich front, and blebs are not mentioned (Wyckoff et al., 2006). On flat collagen, unlike on plastic, Y-27632 slowed A375M2 cells (Tozluoğlu et al., 2013).

Two questions remain open. What ends apoptotic blebbing, given that truncated ROCK I sustains blebbing for hours whereas apoptotic blebbing of NIH 3T3 cells lasts about 20–30 min (Coleman et al., 2001)? And do blebs themselves give rounded tumor cells their invasive advantage, or does ROCK-driven contractility act otherwise? No perturbation yet removes blebs selectively (Guzman et al., 2020). See also Dying and injured cells and Migration.

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