Load adaptation by endocytic actin networks

Abstract

Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly-mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feed back on assembly. By manipulating membrane tension and Arp2/3 complex activity we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Endocytosis at Extremes: Formation and Internalization of Giant Clathrin-coated Pits Under Elevated Membrane Tension

“…It was previously shown that the inward movement of endocytic CCPs during their internalization is dependent on actin polymerization (Ferguson et al ., 2017). Moreover, it has been shown that actin-driven displacement of clathrin coats (away from the cell surface) gets longer with increasing membrane tension (Ferguson et al ., 2017), in accord with models suggesting that actin polymerization pushing the clathrin coats increases with the increasing mechanical load (Kaplan et al ., 2022).…”

“…Moreover, it has been shown that actin-driven displacement of clathrin coats (away from the cell surface) gets longer with increasing membrane tension (Ferguson et al, 2017), in accord with models suggesting that actin polymerization pushing the clathrin coats increases with the increasing mechanical load (Kaplan et al, 2022).…”

“…Actin dynamics provide the energy required for internalization of clathrin-coated vesicles under mechanically stringent conditions that slow down completion of clathrin coats (Boulant et al ., 2012; Ferguson et al ., 2017; Kaplan et al ., 2022). For instance, uptake of viral particles larger than the canonical clathrin pits take longer than the time required for internalization of smaller cargo molecules, and necessitate a “push” provided by actin polymerization that moves the clathrin coat away from the plasma membrane (Cureton et al ., 2010).…”

“…Increased membrane tension is another factor that renders internalization of clathrin coats dependent on the forces generated by actin polymerization (Boulant et al ., 2012; Kaur et al ., 2014; Kaplan et al ., 2022). When the plasma membrane tension is increased by squeezing cells using a soft polymer cushion, dynamic CCPs are replaced by bright fluorescent puncta corresponding to large and long-lived clathrin-coated structures as imaged under diffraction-limited fluorescence microscopy (Ferguson et al ., 2017).…”

“…Cholesterol depletion increases the adhesion of actin cytoskeleton to the plasma membrane (Khatibzadeh, Nima, Gupta, 2012), which may negatively impact actin dynamics. Whereas osmotic swelling increases the in-plane membrane tension without affecting the function of actin machinery (Dai et al ., 1998; Boulant et al ., 2012; Ferguson et al ., 2017; Kaplan et al ., 2022). Considering the fact that actin machinery becomes indispensable for internalization of clathrin-coated vesicles under high mechanical load (Boulant et al ., 2011; Ferguson et al ., 2017), we propose that splitting and internalization of GCPs under high membrane tension is also dependent on the forces provided by actin polymerization.…”