Molecular density-accelerated binding-site maturation underlies CENP-T-dependent kinetochore assembly.

Formation of macromolecular cellular structures relies on recruitment of multiple proteins, requiring the precisely controlled pairwise binding interactions. At human kinetochores, our recent work found that the high molecular density environment enables strong bonding between the Ndc80 complex and its two binding sites at the CENP-T receptor. However, the mechanistic basis for this unusual density-dependent facilitation remains unknown. Here, using quantitative single-molecule approaches, we reveal two distinct mechanisms that drive preferential recruitment of the Ndc80 complex to higher-order structures of CENP-T, as opposed to CENP-T monomers. First, the Ndc80 binding sites within the disordered tail of the CENP-T mature over time, leading to a stronger grip on the Spc24/25 heads of the Ndc80 complexes. Second, the maturation of Ndc80 binding sites is accelerated when CENP-T molecules are clustered in close proximity. The rates of the clustering-induced maturation are remarkably different for two binding sites within CENP-T, correlating with different interfaces formed by the corresponding CENP-T sequences as they wrap around the Spc24/25 heads. The differential clustering-dependent regulation of these sites is preserved in dividing human cells, suggesting a distinct regulatory entry point to control kinetochore-microtubule interactions. The tunable acceleration of slowly maturing binding sites by a high molecular-density environment may represent a fundamental physicochemical mechanism to assist the assembly of mitotic kinetochores and other macromolecular structures.

CLASP2 recognizes tubulins exposed at the microtubule plus-end in a nucleotide state-sensitive manner.

CLASPs (cytoplasmic linker-associated proteins) are ubiquitous stabilizers of microtubule dynamics, but their molecular targets at the microtubule plus-end are not understood. Using DNA origami-based reconstructions, we show that clusters of human CLASP2 form a load-bearing bond with terminal non-GTP tubulins at the stabilized microtubule tip. This activity relies on the unconventional TOG2 domain of CLASP2, which releases its high-affinity bond with non-GTP dimers upon their conversion into polymerization-competent GTP-tubulins. The ability of CLASP2 to recognize nucleotide-specific tubulin conformation and stabilize the catastrophe-promoting non-GTP tubulins intertwines with the previously underappreciated exchange between GDP and GTP at terminal tubulins. We propose that TOG2-dependent stabilization of sporadically occurring non-GTP tubulins represents a distinct molecular mechanism to suppress catastrophe at the freely assembling microtubule ends and to promote persistent tubulin assembly at the load-bearing tethered ends, such as at the kinetochores in dividing cells.

Effects of bacterial lipopolysaccharides on platelet function: inhibition of weak platelet activation.

Platelets are anucleate blood cells with reported roles in hemostasis and immune responses, which possess a functional receptor for bacterial lipopolysaccharides (LPSs), the well-known inducers of inflammation. However, LPSs effects on platelets are contradictory. Here we aim to investigate mechanisms of platelet functioning in the presence of LPS and to find the cause of the discrepancy in the previously published data. Cell activity was analyzed by flow cytometry, western blotting, and aggregometry. Thrombus growth was assessed by fluorescent microscopy. LPS' activity was checked by their capability to induce PMN activation. However, LPSs did not substantially affect either thrombus growth in flow chambers, irreversible platelet aggregation, or platelet responses to strong activation. Platelet aggregation in response to 1 µM of ADP was significantly inhibited by LPSs. Flow cytometry analysis revealed that platelet activation responses to weak stimulation were also diminished by LPSs, while VASP phosphorylation was weakly increased. Additionally, LPSs were capable of inhibition of ADP-induced P2-receptor desensitization. Incubation of platelets with a pan-PDE inhibitor IBMX significantly enhanced the LPSs-induced platelet inhibition, implying cAMP/cGMP dependent mechanism. The discrepancy in the previously published data could be explained by LPS-induced weak inhibition of platelet activation and the prevention of platelet desensitization.