Restructuring of the plasma membrane upon damage by LC3-associated macropinocytosis

Abstract

The plasma membrane shapes and protects the eukaryotic cell from its surroundings and is crucial for cell life. Although initial repair mechanisms to reseal injured membranes are well established, less is known about how cells restructure damaged membranes in the aftermath to restore homeostasis. Here, we show that cells respond to plasma membrane injury by activating proteins associated with macropinocytosis specifically at the damaged membrane. Subsequent to membrane resealing, cells form large macropinosomes originating from the repair site, which eventually become positive for autophagy-related LC3B protein. This process occurs independent of ULK1, ATG13, and WIPI2 but dependent on ATG7, p62, and Rubicon. Internalized macropinosomes shrink in the cytoplasm, likely by osmotic draining, and eventually fuse with lysosomes. We propose that a form of macropinocytosis coupled to noncanonical autophagy, which we term LC3-associated macropinocytosis (LAM) functions to remove damaged material from the plasma membrane and restore membrane integrity upon injury.

Spatiotemporal Patterns of Substance P-Bound MRGPRX2 Reveal a Novel Connection Between Macropinosome Resolution and Secretory Granule Regeneration in Mast Cells

“…However, our results, showing that inhibition of endocytosis by pitstop2 does not prevent MRGPRX2 targeting to the SGs ( Figure 2C ), strongly suggested that MRGPRX2 can reach the SGs also directly from macropinosomes. Recently, it was demonstrated that cells respond to plasma membrane injury by forming large macropinosomes at the repair site ( 37 ). These structures eventually become positive for the autophagy-related LC3B protein, followed by their shrinkage and fusion with lysosomes ( 37 ).…”

“…Recently, it was demonstrated that cells respond to plasma membrane injury by forming large macropinosomes at the repair site ( 37 ). These structures eventually become positive for the autophagy-related LC3B protein, followed by their shrinkage and fusion with lysosomes ( 37 ). Given the lysosomal nature of the MC SGs, the previous reports that documented the presence of LC3 on MC SGs ( 38 , 39 ), and our recent findings on inhibition of MRGPRX2-stimulated secretion by autophagy targeting drugs ( 21 ), we were motivated to examine if LC3 plays a role in MRGPRX2 delivery from macropinosomes to the SGs.…”

“…Motivated to delineate such pathway, we hypothesized that macropinosome resolution may serve as a mechanism of regenerating SGs. We based our hypothesis on parallel mechanisms that exist during phagocytosis, where phagosome fragmentation leads to lysosome reformation ( 44 ), or repair of plasma membrane injury, during which LC3-mediated resolution of macropinosomes is linked with fusion with lysosomes ( 37 ). Indeed, our results reveal a novel link between macropinosomes, LC3 and the SGs.…”

The V-ATPases in cancer and cell death

“…The molecular basis for the formation and maturation of macropinocytosis involves multiple signals and pathways. V-ATPases play a crucial role in macropinocytosis by keeping lysosomes acidified [ 113 ]. V-ATPases containing a3 localize to plasma membrane, which is necessary for the distribution of regulatory proteins of macropinocytosis, such as RAC1 [ 114 ].…”

Atg8ylation as a general membrane stress and remodeling response

“…The plasma membrane is by its boundary nature under continuous risk of being damaged by exogenous agents, and conversely is used for programmed permeabilization events associated with cell death pathways. Recent studies [124][125][126] suggest that Atg8ylation-dependent processes and other ATG proteins engaged in non-canonical functions contribute to the protection of plasma membrane against physical damage (modeled by laser damage or detergent treatment), programmed permeabilization by gasderminpores during pyroptosis or MLKL during necroptosis, by MLKL-like permeabilization function of SARS-CoV-2 ORF3a, bacterial pore forming toxins (pneumolysin, lysteriolysin and streptolysin O), and harsh components of the cell walls of microbes such as Mycobacterium tuberculosis [124,126]. Whereas ATG9A engages ESCRT proteins to seal plasma membrane holes immediately upon permeabilization [126], Atg8ylation results in blebbing due to lysosomal exocytosis [124] or endocytic processes termed LC3-associated micropinocytosis which appears to be a Lapoid process [125].…”