Load adaptation of endocytic actin networks

Abstract

Clathrin-mediated endocytosis (CME) remains robust despite variations in plasma membrane tension. Actin assembly-mediated force generation becomes essential for CME under high membrane tension, but the underlying mechanisms are not understood. We investigated actin network ultrastructure at each stage of CME by super-resolution imaging. Actin and N-WASP spatial organization indicate that polymerization initiates at the base of clathrin-coated pits and that the actin network then grows away from the plasma membrane. Actin network organization is not tightly coupled to endocytic clathrin coat growth and deformation. Membrane tension-dependent changes in actin organization explain this uncoupling. Under elevated membrane tension, CME dynamics slow down and the actin network grows higher, resulting in greater coverage of the clathrin coat. This adaptive mechanism is especially crucial during the initial membrane curvature-generating stages of CME. Our findings reveal that adaptive force generation by the actin network ensures robust CME progression despite changes in plasma membrane tension.Highlights-Clathrin coat surface area and actin ultra-structure adapt to elevated membrane tension.-The actin network is nucleated at the base of the clathrin-coated pit and grows upward.-Actin ultra-structural organization is not tightly coupled to CME progression.-Actin force generation is required earlier in CME progression under elevated membrane tension.SummaryKaplan et al. revealed that actin assembly compensates for changes in plasma membrane tension by an adaptive force generating mechanism to ensure robust endocytosis. Under elevated membrane tension the network grows deeper, even in early endocytic stages, from the base upward.

Actin force generation in vesicle formation: mechanistic insights from cryo-electron tomography

“…Variation in branched actin filament organization and in the number of clusters and filaments between individual CME sites likely reflects the stochastic nature of filament assembly as well as local adaptive responses to variable conditions. High variability in actin network organization and density from one CME site to the next might result from such factors as variance in mother filament number and orientation, Hip1R localization, active Arp2/3, N-WASP position and differences in plasma membrane tension (this study, (Akamatsu et al, 2020;Clarke and Royle, 2018;Engqvist-Goldstein et al, 2001;Kaplan et al, 2021;Sochacki et al, 2017)).…”

Multiple roles for actin in secretory and endocytic pathways

“…Clathrin-mediated endocytosis in mammals is understood in much less detail, however. There have also been conflicting results on the requirement of actin for mammalian clathrin-mediated endocytosis, but this has been resolved with the finding that actin's role in this process increases in importance with increasing membrane tension 40,41 . Another variable might be the extent and nature of the cortical actin network in specific cellular contexts.…”

“…Platinum replica EM evidence in mammals suggests that barbed ends orient toward the neck of the pit, with pointed ends anchored as branches from other cellular filaments 46 . Super-resolution microscopy studies suggest a more variable orientation of the network, dependent on membrane tension 41 , and EM tomography shows longer filaments that are bent, as if under tension 45 . Given that force is exerted at the barbed ends of a polymerizing dendritic network 1 , the orientation of the network dictates the direction of force and is thus an important parameter to establish.…”

WASP integrates substrate topology and cell polarity to guide neutrophil migration

“…3B). In contrast, others have reported saddle-enrichment of N-WASP (Kaplan et al, 2020) and the yeast orthologue Las17 (Mund et al, 2018) on this size scale using STORM to image CME in fixed cells. Finally, we compared WASP intensities at uniformly coated 200 and 500 nm bead-induced invaginations measured with 3D STED and again found an increased enrichment to beads with higher radii of curvature (Fig.…”