Biology subjects

Piano, V.

Publications and source records attributed to Piano, V..

3 recordsLinked to original sources

bioRxiv2024

Interplay of kinetochores and catalysts drives rapid assembly of the mitotic checkpoint complex

The spindle assembly checkpoint (SAC) makes mitotic exit contingent on completion of sister chromatid biorientation, but how this coordination is achieved in practice remains poorly understood. Kinetochores, megadalton chromosome attachment sites to spindle microtubules, contribute to SAC signaling. However, it is unclear whether kinetochores are mere docking sites for SAC proteins, or further contribute to co-orientation of SAC catalysts, including MAD1:MAD2 and BUB1:BUB3, to facilitate SAC signaling. Here, we combined biochemical reconstitutions of kinetochores and the SAC to address this question in vitro. We engineered recombinant kinetochore particles that recruit most SAC components and activate SAC signaling after induction with Rapamycin, and challenged them with a battery of impairing mutants. At approximately physiological concentrations of SAC catalysts, kinetochores were crucially required for rapid assembly of the mitotic checkpoint complex (MCC), the SAC effector. Our observations depict kinetochores as a cradle that catalyzes rapid MCC assembly by concentrating and co-orienting distinct SAC catalysts.

biochemistry↗

bioRxiv2022

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, and during its assembly, formation of the CDC20:MAD2 subcomplex is the rate-limiting step. Recent studies show that the rate of CDC20:MAD2 formation is significantly accelerated by the cooperative binding of CDC20 to 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 in a "knitting" model that explains how the folded conformation of MAD1:MAD2 promotes CDC20:MAD2 assembly.

cell biology↗

bioRxiv2019

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 kinetochores, a spatially confined and well-studied subcellular structures. After electroporation in human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes displayed 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 reconstituted robust kinetochore localization of Spindly. Thus, electroporation is uniquely versatile for delivering synthetic and, as required, chemically modified functional mimics of endogenous proteins, and is therefore a promising tool for chemical and synthetic biology.

cell biology↗