Class-I myosin responds to change in membrane tension during clathrin-mediated endocytosis in human induced pluripotent stem cells

Abstract

Clathrin-mediated endocytosis (CME) is an essential cellular process that needs to operate efficiently across a wide range of conditions. Internalization of the endocytic site involves forces generated by membrane-bound proteins and Arp2/3-mediated branched actin filament assembly to bend the plasma membrane (PM) from flat to omega-shaped. In mammalian CME, the requirement for a branched actin filament network varies depending on cell type and differences in membrane tension. However, how the actin network adapts to changes in load in order to ensure robustness of this process over a range of membrane tensions is not understood. Here, we combine live-cell imaging and super-resolution microscopy of genome-edited human induced pluripotent stem cells (hiPSCs) to investigate the role of the mammalian class-I myosin, Myosin1E (Myo1E), in load adaptation. Under normal conditions, sites that recruit Myo1E are rare and exhibit slow CME dynamics. However, as membrane tension increases and CME dynamics are slowed globally, Myo1E is recruited to more sites, likely to assemble more branched actin, resulting in increased force generation to rescue stalled sites and promote internalization. Loss of Myo1E results in increased Arp2/3 complex lifetime at CME sites under normal conditions, and at high membrane tension, these sites fail to recruit as many Arp2/3 molecules. We propose that Myo1E is recruited to CME sites that have stalled due to increased membrane tension, where it helps build a more effective branched actin network by generating force through motor activity and recruiting additional Arp2/3 complexes to rescue stalled sites. SignificanceFor mammalian cells to internalize extracellular cargo via clathrin-mediated endocytosis (CME), specific regions of the plasma membrane (PM) must bend from flat to inwardly curved, a process that requires force-generating proteins. One key component in generating this force during CME is the branched actin network, in which actin filaments polymerize against the plasma membrane. When PM tension increases, more force is required to generate curvature, prompting the assembly of actin and actin associated proteins to aid the process. We demonstrate that the class-I myosin motor, Myosin1E (Myo1E), becomes increasingly crucial as membrane tension rises, presumably to build a more effective branched actin network to facilitate internalization of slowed sites.