Actin cytoskeleton at different stages of the bleb life cycle. Correlative video and PREM of blebbing HT1080 cells treated with 200 µM CK-666. (A–G) Cell #1. (A) Last phase-contrast image before extraction at t = 0. (B) PREM of the same cell. (C) Overlay of boxed regions from A (blue) and B (gray). (D and E) Time-lapse images from the framed region in C showing formation of a nascent bleb (arrow) and two retracting blebs (arrowheads). Both retracting blebs emerged at t = −64 s and finished expansion at t = −20 s, but bleb 1 began retracting earlier (at t = −12 s) than bleb 2 (at t = −4 s). (F) Overlay of the framed region in E (blue) and corresponding PREM image (gray). (G) PREM of the framed region in F; dashed line marks the cell contour from the phase-contrast image. (H–J) Cell #2. (H) Time-lapse images showing an expanding bleb (arrowheads); asterisks mark an adjacent filopodium. (I) Overlay of the last video frame (blue) and the corresponding PREM image (gray). (J) PREM of the framed region in I. Dashed line indicates the contour of the bleb labeled in H and I. (K–M) Cell #3. (K) Time-lapse images of a bleb (blue arc) fixed at a stationary/early retraction phase. The bleb emerged at t = −22 s, reached maximal size at t = −18 s, and underwent minor retraction between t = −6 and −2 s. (L) Overlay of the last video frame (blue) and the corresponding PREM image (gray). (M) PREM of the framed region in L. Scale bars: 5 µm (A and B), 2 µm (C, H, I, K, and L), 1 µm (D–F), and 500 nm (G, J, and M). Time in seconds before extraction. See also Fig. S3 and Video 3.