Figure 2. S. rosetta amoeboid cells generate blebs.
(A) Protrusions in eukaryotic crawling cells can either be F-actin-filled pseudopods that form by polymerization of F-actin (pink) reticulated by the Arp2/3 complex (purple, left) or F-actin-free blebs that form through the action of contractile forces in the actomyosin cortex underlying the plasma membrane (right). The cytosol is green in both panels. Modified from Fritz-Laylin et al., 2017a. (B) Formation of dynamic protrusions required F-actin and myosin II activity, but not Arp2/3-mediated F-actin polymerization. Protrusions were abundant in DMSO-treated control cells (N = 32, 34, and 50 cells in the three respective biological replicates) and in cells treated with the Arp2/3 inhibitor CK666 (100 μM, N = 47, 56, and 59 cells in the three respective biological replicates) but virtually absent in cells treated with the F-actin polymerization inhibitor latrunculin B (at both 2 μM [N = 33, 76, and 11 cells in the three respective biological replicates] and 20 μM [N = 32, 49, and 22 cells in the three respective biological replicates]) or the myosin II inhibitor blebbistatin (17 μM, N = 36, 77, and 17 cells in the three respective biological replicates). This suggests that the dynamic protrusions were blebs. All cells were under 1 μm confinement. (C–K) Dynamic protrusions that form under confinement are blebs, as indicated by live imaging of two S. rosetta amoeboid cells expressing an F-actin marker (LifeAct-mCherry, magenta in C/F/I and black in E/H/K) (Video 5). (C, F, I and E, H, K) We observed that expanding blebs (e; defined as blebs that increased in size during the period of observation currently increasing in size) were cytoplasm-filled, but F-actin-free. We found that F-actin subsequently re-invaded blebs that then initiated retraction (r; defined as blebs that decreased in size during the period of observation). F-actin was also present as a cortical layer (cx), as in animal cells (Chugh and Paluch, 2018), and accumulated in cytoplasmic foci (cf). The cells were under 1 μm confinement. (L–S) S. rosetta cells fixed and stained for F-actin (phalloidin, magenta) and cytoplasm (FM 1–43 FX, which distributes to the cytoplasm of S. rosetta following fixation, green) confirmed that the protrusions of amoeboid cells initially lack F-actin and are therefore blebs. (L to N) A flagellate cell showing collar microvilli (black arrowheads) and F-actin cortex (cx). (O) Linescan of F-actin fluorescent intensity along the line of interest in (N), showing cortical actin as two peaks where the lines intersects the cells cortex. (P–R) An amoeboid cell showing both F-actin-free and F-actin-encased protrusions, respectively, interpreted as expanding (e) and retracting (r) blebs. (S) Linescan of F-actin fluorescent intensity along the line of interest in (R), showing cortical actin in the putative retracting bleb but not the putative expanding bleb. In all panels: white arrowheads: blebs, black arrowheads: microvilli, e: expanding blebs, r: retracting blebs, cf: cytoplasmic foci. Time stamps in black boxes shown as min:sec.
Figure 2—figure supplement 1. Latrunculin B and blebbistatin, but not CK666, inhibited the formation of dynamic cellular protrusions under cell confinement.
Figure 2—figure supplement 2. Flow-through chamber for immunostaining of confined cells.
