Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair

Abstract

Efficient cell membrane repair mechanisms are essential for maintaining membrane integrity and thus for cell life. Here we show that the Ca2+- and phospholipid-binding proteins annexin A4 and A6 are involved in plasma membrane repair and needed for rapid closure of micron-size holes. We demonstrate that annexin A4 binds to artificial membranes and generates curvature force initiated from free edges, whereas annexin A6 induces constriction force. In cells, plasma membrane injury and Ca2+ influx recruit annexin A4 to the vicinity of membrane wound edges where its homo-trimerization leads to membrane curvature near the edges. We propose that curvature force is utilized together with annexin A6-mediated constriction force to pull the wound edges together for eventual fusion. We show that annexin A4 can counteract various plasma membrane disruptions including holes of several micrometers indicating that induction of curvature force around wound edges is an early key event in cell membrane repair.

Cellular mechanisms and signals that coordinate plasma membrane repair

“…Calcium-dependent Annexin A4 also binds preferentially to PS-rich membrane regions and curves the wounded plasma membrane edges, which is subsequently constricted inward by Annexin A6 to aid in closing the wound. This proposed mechanism seems to fit with the wound stabilizing role of Annexin A5, which is recruited to the injury site within seconds [8], while later arrival of Annexins A4 and A6 facilitates wound closure [52]. Annexin A6 has been reported to localize to the extracellular face of the plasma membrane in skeletal muscle [51,11], which is consistent with the role of Annexin A6 in constricting the damaged free edges of the wound site.…”

“…While annexins with lower calcium sensitivity respond later, and can involve binding partners such as S100 proteins [16]. The difference in response to calcium signaling may determine how largely similar annexin proteins including Annexin A1, A2, A4, A5 and A6 can accumulate and potentially facilitate different aspects of plasma membrane repair (Figure 2a) [51,52]. However, annexin accumulation at the injury site does not necessarily indicate a role in repair.…”

“…In addition, Annexin A5 – like other annexins – binds phosphatidylserine (PS) enriched at the site near injury (see discussion on lipid patterning below). In contrast to the structural lattice formed by Annexin A5, the Annexins A4 and A6 play complimentary roles in membrane curvature and constriction after injury [52]. Calcium-dependent Annexin A4 also binds preferentially to PS-rich membrane regions and curves the wounded plasma membrane edges, which is subsequently constricted inward by Annexin A6 to aid in closing the wound.…”

“…This model, in which PS binding proteins may regulate PS translocation, also invokes a potential role of annexin and MG53 localization in PS translocation. PS patterning at the wound edge helps annexins to localize and stabilize the wound edge and induce folding of the wounded membrane edge that aids in repair [52]. …”

Imaging Membrane Repair in Single Cells Using Correlative Light and Electron Microscopy

“…Cell imaging is the most suitable approach for studying plasma membrane repair. Fluorescence microscopy, whether confocal or super‐resolution techniques (Lek et al., ; Middel et al., ), electron microscopy (Carmeille et al., ; Miyake & McNeil, ) and atomic force microscopy (Boye et al., ; Miyagi, Chipot, Rangl, & Scheuring, ) have been successfully used in this context. Cellular structures that are involved in membrane resealing and have to be spatially resolved are plasma membrane (5‐ to 10‐nm thick), cytoplasmic vesicles (diameter range from 50 to 500 nm), organelles (µm‐size), microfilaments (6 nm in diameter) and microtubules (20 to 25 nm in diameter).…”