Mapping Cell Membrane Fluctuations Reveals Their Active Regulation and Transient Heterogeneities.

Shape fluctuations of the plasma membrane occur in all cells, are incessant, and are proposed to affect membrane functioning. Although studies show how membrane fluctuations are affected by cellular activity in adherent cells, their spatial regulation and the corresponding change in membrane mechanics remain unclear. In this article, we study how ATP-driven activities and actomyosin cytoskeleton impact basal membrane fluctuations in adherent cells. Using interference imaging, we map height fluctuations within single cells and compare the temporal spectra with existing theoretical models to gain insights about the underlying membrane mechanics. We find that ATP-dependent activities enhance the nanoscale z fluctuations but stretch out the membrane laterally. Although actin polymerization or myosin-II activity individually enhances fluctuations, the cortex in unperturbed cells stretches out the membrane and dampens fluctuations. Fitting with models suggest this dampening to be due to confinement by the cortex. However, reduced fluctuations on mitosis or on ATP-depletion/stabilization of cortex correlate with increased tension. Both maps of fluctuations and local temporal autocorrelation functions reveal ATP-dependent transient short-range (<2 µm) heterogeneities. Together, our results show how various ATP-driven processes differently affect membrane mechanics and hence fluctuations, while creating distinct local environments whose functional role needs future investigation.

The structural flexibility of MAD1 facilitates the assembly of the Mitotic Checkpoint Complex.

The spindle assembly checkpoint (SAC) safeguards the genome during cell division by generating an effector molecule known as the Mitotic Checkpoint Complex (MCC). The MCC comprises two subcomplexes: BUBR1:BUB3 and CDC20:MAD2, and the formation of CDC20:MAD2 is the rate-limiting step during MCC assembly. Recent studies show that the rate of CDC20:MAD2 formation is significantly accelerated by the cooperative binding of CDC20 to the SAC proteins MAD1 and BUB1. However, the molecular basis for this acceleration is not fully understood. Here, we demonstrate that the structural flexibility of MAD1 at a conserved hinge near the C-terminus is essential for catalytic MCC assembly. This MAD1 hinge enables the MAD1:MAD2 complex to assume a folded conformation in vivo. Importantly, truncating the hinge reduces the rate of MCC assembly in vitro and SAC signaling in vivo. Conversely, mutations that preserve hinge flexibility retain SAC signaling, indicating that the structural flexibility of the hinge, rather than a specific amino acid sequence, is important for SAC signaling. We summarize these observations as the 'knitting model' that explains how the folded conformation of MAD1:MAD2 promotes CDC20:MAD2 assembly.

CDC20 assists its catalytic incorporation in the mitotic checkpoint complex.

Open (O) and closed (C) topologies of HORMA-domain proteins are respectively associated with inactive and active states of fundamental cellular pathways. The HORMA protein O-MAD2 converts to C-MAD2 upon binding CDC20. This is rate limiting for assembly of the mitotic checkpoint complex (MCC), the effector of a checkpoint required for mitotic fidelity. A catalyst assembled at kinetochores accelerates MAD2:CDC20 association through a poorly understood mechanism. Using a reconstituted SAC system, we discovered that CDC20 is an impervious substrate for which access to MAD2 requires simultaneous docking on several sites of the catalytic complex. Our analysis indicates that the checkpoint catalyst is substrate assisted and promotes MCC assembly through spatially and temporally coordinated conformational changes in both MAD2 and CDC20. This may define a paradigm for other HORMA-controlled systems.

Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology.

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.