The absence of a cell wall affords animal cells diverse functionality at the cost of acute sensitization to plasma membrane (PM) damage. Thus, animal cells tightly monitor and maintain PM integrity to prevent cell death. Genetic loss of PM repair factors is associated with human diseases such as muscular dystrophy. Despite evidence that annexin and endosomal sorting complex required for transport (ESCRT) proteins are required for PM repair, the extent to which their recruitment is coordinated at sites of membrane damage remains unclear. Here, leveraging quantitative organellar proteomics and genome-wide CRISPR interference screens, we identify sorcin as a PM repair factor that couples annexin A11 (ANXA11)-mediated sensing of PM damage to ESCRT-III assembly. We show that sorcin directly binds ANXA11 and ALIX in the presence of Ca2+ via its penta-EF-hand domain and flexible N terminus, respectively, and is required for ESCRT-III recruitment to PM lesions and membrane resealing. Our data support a model in which ANXA11, recruited to the PM upon damage-induced Ca2+ influx, serves as an anchor that facilitates the sequential recruitment of sorcin and ESCRT-III at PM lesions. Together, these findings establish a Ca2+-dependent scaffolding mechanism that couples PM damage sensing to ESCRT-III assembly for PM repair.
A defining feature of animal cells is the lack of a cell wall. Although this absence confers animal cells with distinct functional advantages compared to cells from other taxa, it also renders animal cells susceptible to plasma membrane (PM) damage. PM disruption is common, and a subset of cells, particularly those found in mechanically active tissues, are especially vulnerable to damage (
1). For example, muscle and endothelial cells experience high levels of PM damage due to repeated contractions and fluid shear stress, respectively (
2–
4). If left unresolved, persistent PM damage leads to cell death. Accordingly, animal cells tightly monitor and maintain PM integrity.
Extracellular calcium (Ca
2+) is essential for PM repair (
5). Upon PM damage, Ca
2+ flows from the extracellular space into the cytoplasm and initiates the recruitment of membrane repair factors to the lesion site (
1). Annexins are cytosolic proteins that bind phospholipids in the presence of Ca
2+ and participate in various aspects of PM repair, including membrane patching and tension reduction (
1,
6,
7). Consistent with an important role in PM repair, genetic depletion of annexins in cultured cells impairs membrane resealing (
8–
12), and loss of annexin expression or function in mice causes muscular dystrophy (
13–
15).
In addition to annexins, the endosomal sorting complex required for transport (ESCRT) machinery promotes PM repair (
16). The core ESCRT machinery, consisting of ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III, ALIX, and Vps4, were originally identified for their role in sorting ubiquitylated membrane proteins into the intraluminal vesicles of multivesicular bodies (
17,
18). However, it is now clear that the ESCRT machinery mediates reverse-topology membrane scission in diverse cellular processes including nuclear envelope sealing, autophagosome closure, cytokinesis, viral budding, and lysosome repair (
19–
21). Interestingly, ESCRT I-III are recruited to damaged lysosomes (
22,
23), whereas only ESCRT-III is recruited to PM lesions (
24). Upon PM disruption, annexin A7 (ANXA7) localizes to the membrane lesion and recruits ALG-2, which subsequently recruits ESCRT-III (
25). However, recent studies have suggested that ALG-2 can directly bind membranes in the presence of Ca
2+ (
26,
27). Thus, the extent to which annexin recruitment is coupled to ESCRT-III assembly during PM repair remains unclear.
Mutations in annexin A11 (ANXA11) and CHMP2B (a member of ESCRT-III) have been linked to amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and limb-girdle muscular dystrophy (
28–
31), and a recent report has indicated that these mutations compromise PM repair (
32). Given increasing evidence that PM repair is compromised in neurodegeneration and certain types of muscular dystrophy, we sought to employ complementary biochemical and genetic approaches to identify novel factors required for this essential membrane repair pathway.
