Figure 4
From: Annexin A7 is required for ESCRT III-mediated plasma membrane repair
ANXA7 is needed for Chmp4b buildup and ectosome shedding. (a) Representative sequential images of the translocation behavior and shedding of ANXA7-RFP and ALG-2-GFP in MCF7-p95ErbB2 A7-CRISPR upon localized laser injury and corresponding intensity plots of proteins intracellularly (b) or extracellularly (c) measured by Volocity software (blue arrow indicates injury site). (d-f) similar experiment as in a, but with ANXA7 mutant (A7ΔN99) lacking most of the N-terminus. Note, this mutant have decreased Ca2+-sensitivity and shedding activity and fail to localize ALG-2-GFP at the injured membrane. (g) Typical sequential images showing ANXA7-RFP/ALG-2-GFP translocation to injured membrane and subsequently excision and shedding of membrane containing protrusions. (h) Cell shedding frequency in MCF7-p95ErbB2 A7-CRISPR cells expressing Ctrl-RFP, wildtype ANXA7-RFP or A7ΔN99-RFP mutant. Error bars represent SD for at least 15 independent cells per condition. P-values based on Student’s t-test: ***P ≤ 0.001. (i) Number of shedded vesicles as measured from the very edge of the cells membrane upon laser injury of MCF7-p95ErbB2 A7-CRISPR cells expressing wildtype ANXA7-RFP or A7ΔN99-RFP mutant. Cells were injured at the very edge of the cell membrane and shedded vesicles captured by time lapse imaging (Also see Supplementary Movies S3–S5). (j) Average vesicle diameter of shedded vesicles after laser injury. Examples of vesicle diameter indicated in a and c. Data were obtained from time-lapse movies using Volocity software. SD for at least 4 experiments. (k) Typical sequential images of Chmp4b-YFP translocation in MCF7-p95ErbB2 Ctrl-CRISPR or A7-CRISPR cell upon laser injury. (l) Graph showing Chmp4b-YFP translocation kinetic at the site of injury in MCF7-p95ErbB2 Ctrl-CRISPR and A7-CRISPR cells. Error bars represent SEM for at least 8 independent cells per condition. P-values based on Student’s t-test: **P ≤ 0.01.
