Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis

Abstract

words)Force generation due to actin assembly is a fundamental aspect of membrane sculpting for many essential processes. In this work, we use a multiscale computational model constrained by experimental measurements to show that a minimal branched actin network is sufficient to internalize endocytic pits against physiological membrane tension. A parameter sweep identified the number of Arp2/3 complexes as particularly important for robust internalization, which prompted the development of a molecule-counting method in live mammalian cells. Using this method, we found that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Our simulations also revealed that actin networks self-organize in a radial branched array with barbed filament ends oriented to grow toward the base of the pit, and that the distribution of linker proteins around the endocytic pit is critical for this organization. Surprisingly, our model predicted that long actin filaments bend from their attachment sites in the coat to the base of the pit and store elastic energy that can be harnessed to drive endocytosis. This prediction was validated using cryo-electron tomography on cells, which revealed the presence of bent actin filaments along the endocytic site. Furthermore, we predict that under elevated membrane tension, the self-organized actin network directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, our study reveals that spatially constrained actin filament assembly utilizes an adaptive mechanism that enables endocytosis under varying physical constraints.

Induced Arp2/3 complex depletion increases FMNL2/3 formin expression and filopodia formation

“…In recent years, the Arp2/3 complex turned out to be of fundamental importance for numerous cellular processes, including actin-based cell edge protrusion during migration and various additional actin assembly processes at both plasma and intracellular membranes (Rotty et al, 2013;Molinie and Gautreau, 2018), as well as specific interactions of viral or bacterial pathogens with their host cells (Welch and Way, 2013). Interestingly, many of these processes, in particular those occurring at the plasma membrane, involve adaptive responses orchestrated by Arp2/3 complex to mechanical forces (Mueller et al, 2017;Akamatsu et al, 2020), as recently emphasized (Papalazarou and Machesky, 2020). Although the canonical complex is comprised of seven subunits throughout eukaryotic cell evolution, expression of functionally diverse or incomplete complexes - even within the same cell type – have also been described (Chorev et al, 2014;Abella et al, 2016).…”

The cell pushes back: The Arp2/3 complex is a key orchestrator of cellular responses to environmental forces

“…Akamatsu et al. [ 21 ] and Sun et al [ 22 ] recently modelled how Arp2/3-generated actin networks assist CME events, using agent-based modelling and quantitative experimental studies. They found that endocytic actin networks adapted to the changing landscape of membrane tension during the various stages of deformation and endocytic uptake of clathrin-coated endocytic vesicles ( Figure 1 ).…”

Force requirements of endocytic vesicle formation

“…Many theoretical studies have attempted to estimate the forces required for endocytic membrane remodelling, proposing values over a range of two orders of magnitude (tens to thousands of pN; Akamatsu et al, 2020; reviewed in Carlsson, 2018; Lacy et al, 2018). Here, we have measured the force required for endocytic vesicle formation in yeast under normal and different genetically and environmentally perturbed conditions.…”