Mammalian cells integrate mitogen and stress signaling prior to the end of G1 phase to decide whether or not to enter the cell cycle1–4. Before cells can replicate their DNA in S phase, they have to activate cyclin-dependent kinases (CDKs), induce an E2F transcription program, and inactivate an E3 ubiquitin ligase, the anaphase promoting complex/cyclosome (APC/CCdh1). It was recently shown that stress can return cells to quiescence after CDK2 activation and E2F induction but cannot after inactivation of APC/CCdh1, arguing that APC/CCdh1 inactivation is the point-of-no-return for cell cycle entry3. While rapid inactivation of APC/CCdh1 requires early mitotic inhibitor 1 (Emi1)3,5, the molecular mechanism controlling this cell cycle commitment step is unknown. Here we show that cell cycle commitment is mediated by an Emi1-APC/CCdh1 dual-negative feedback switch, in which Emi1 is both a substrate and an inhibitor of APC/CCdh1. The inactivation switch triggers a transition between a state with low Emi1 levels and high APC/CCdh1 activity during G1 to a state with high Emi1 levels and low APC/CCdh1 activity during S and G2. Cell-based analysis, in vitro reconstitution, and modeling data show that the underlying dual-negative feedback is bistable and represents a robust irreversible switch. Together, our study argues that mammalian cells commit to the cell cycle by increasing CDK2 activity and Emi1 mRNA expression to trigger a one-way APC/CCdh1 inactivation switch mediated by Emi1 transitioning from a substrate to an inhibitor of APC/CCdh1.

EPI64 is a TBC domain–containing protein that binds the PDZ domains of EBP50, which binds ezrin, a major actin-binding protein of microvilli. High-resolution light microscopy revealed that ezrin and EBP50 localize exclusively to the membrane-surrounded region of microvilli, whereas EPI64 localizes to variable regions in the structures. Overexpressing EPI64 results in its and EBP50's relocalization to the base of microvilli, including to the actin rootlet devoid of ezrin or plasma membrane. Uncoupling EPI64's binding to EBP50, expression of any construct mislocalizing its TBC domain, or knock down of EBP50 results in loss of microvilli. The TBC domain of EPI64 binds directly to Arf6-GTP. Overexpressing the TBC domain increases Arf6-GTP levels, and expressing dominant-active Arf6 results in microvillar loss. These data reveal that microvilli have distinct cytoskeletal subdomains and that EPI64 regulates microvillar structure.

No abstract

Both Cdc2 and PKC-mediated phosphorylation of EBP50 alter accessibility of its first PDZ domain when its second PDZ domain is occupied to regulate microvilli formation on epithelial cells.

Cell migration is driven by local membrane protrusion through directed polymerization of F-actin at the front. However, F-actin next to the plasma membrane also tethers the membrane and thus resists outgoing protrusions. Here, we developed a fluorescent reporter to monitor changes in the density of membrane-proximal F-actin (MPA) during membrane protrusion and cell migration. Unlike the total F-actin concentration, which was high in the front of migrating cells, MPA density was low in the front and high in the back. Back-to-front MPA density gradients were controlled by higher cofilin-mediated turnover of F-actin in the front. Furthermore, nascent membrane protrusions selectively extended outward from areas where MPA density was reduced. Thus, locally low MPA density directs local membrane protrusions and stabilizes cell polarization during cell migration.