Figure 10.
IST1 structures are recruited to sites of membrane damage. (A) STRING protein network of consensus adhesion proteins (Horton et al., 2015) identified by DAVID gene ontology that were depleted in response to 100 μM latrunculin B (Lat B) treatment. (B) Confocal micrographs of moDCs pre-incubated with or without Lat B. After washing away the Lat B containing medium, cells were seeded and incubated for 1 h followed by gentle washing with PBS to remove non-adherent cells. Left graph: average number of adhering moDCs. Right graph: average number of IST1 structures per cell. (n = 5 donors; >80 cells per donors; paired 2-sided t test). (C) Confocal micrographs of moDCs cultured with and without serum and in the presence of 0.5 μg/ml DAPI. Graph shows fluorescent intensities of DAPI. Data points show individual donors (>50 cells per condition; paired two-sided t test; *: P < 0.05; **: P < 0.01; ****: P < 0.0001; NS: not significant). (D) Confocal micrographs showing recruitment of ESCRT structures to plasma membrane contact sites (arrow heads) with membrane-disrupting silica crystals. (E) Model scheme with proposed mechanism. Integrins and other known extracellular cargo proteins (CD63, GPI-anchored proteins, ubiquitinated proteins) are enriched in membrane domains surrounded by ESCRT structures. The cortical F-actin cytoskeleton is disassembled at these clusters. The integrin clusters tightly adhere to the extracellular substrate, making the surrounding membrane vulnerable to damage. This results in formation of the ESCRT structures. ESCRT repairs the membrane by shedding of damaged plasma membrane regions. For the statistical analysis of B and C, data distribution was assumed to be normal, but this was not formally tested. Scale bars: 10 μm.

IST1 structures are recruited to sites of membrane damage. (A) STRING protein network of consensus adhesion proteins (Horton et al., 2015) identified by DAVID gene ontology that were depleted in response to 100 μM latrunculin B (Lat B) treatment. (B) Confocal micrographs of moDCs pre-incubated with or without Lat B. After washing away the Lat B containing medium, cells were seeded and incubated for 1 h followed by gentle washing with PBS to remove non-adherent cells. Left graph: average number of adhering moDCs. Right graph: average number of IST1 structures per cell. (n = 5 donors; >80 cells per donors; paired 2-sided t test). (C) Confocal micrographs of moDCs cultured with and without serum and in the presence of 0.5 μg/ml DAPI. Graph shows fluorescent intensities of DAPI. Data points show individual donors (>50 cells per condition; paired two-sided t test; *: P < 0.05; **: P < 0.01; ****: P < 0.0001; NS: not significant). (D) Confocal micrographs showing recruitment of ESCRT structures to plasma membrane contact sites (arrow heads) with membrane-disrupting silica crystals. (E) Model scheme with proposed mechanism. Integrins and other known extracellular cargo proteins (CD63, GPI-anchored proteins, ubiquitinated proteins) are enriched in membrane domains surrounded by ESCRT structures. The cortical F-actin cytoskeleton is disassembled at these clusters. The integrin clusters tightly adhere to the extracellular substrate, making the surrounding membrane vulnerable to damage. This results in formation of the ESCRT structures. ESCRT repairs the membrane by shedding of damaged plasma membrane regions. For the statistical analysis of B and C, data distribution was assumed to be normal, but this was not formally tested. Scale bars: 10 μm.

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