PROJECT SUMMARY Platelets are specialized anucleate cells that circulate in the blood and serve to prevent bleeding and minimize blood vessel injury. In addition to their hemostatic functions, platelets participate in wound healing, angiogenesis, inflammation, and immunity, and are therefore central players in maintaining normal physiology and in disease pathogenesis. Platelets are derived from their precursor cells, megakaryocytes (MKs), that reside principally in the bone marrow. During maturation, MKs undergo an altered cell cycle called endomitosis in which they replicate their DNA but avoid cell division, resulting in polyploid MKs with amplified microtubule (MT)-organizing centers called centrosomes. Once fully mature, MKs extend long cytoplasmic protrusions called proplatelets via MT-dependent forces into sinusoidal blood vessels, eventually giving rise to circulating platelets. Despite progress in elucidating key steps of platelet production, there is a conspicuous lack of understanding of what triggers mature, polyploid MKs undergo the MT rearrangements required for proplatelet production. Using live cell imaging of mouse fetal liver-derived MKs expressing fluorescent tubulin, we found that MKs cluster amplified centrosomes into monospindles containing an enlarged array of MTs oriented towards the cell cortex. We also observed monospindles in mouse bone marrow-derived and human cord blood MKs, suggesting that centrosome clustering is a general phenomenon in MKs. KIFC1 is a microtubule- based mitotic motor protein that facilitates centrosome clustering in cancer cells. Interestingly, we detected increased levels of KIFC1 in more mature MKs preceding proplatelet formation. Of note, small molecule inhibition of KIFC1 severely impaired both monospindle formation and proplatelet production. This proposal will test the hypothesis that KIFC1-mediated centrosome clustering and subsequent monospindle formation cause mature MKs to terminate endomitotic progression and initiate proplatelet formation. In Aim 1, we will determine whether elevated KIFC1 levels drive centrosome clustering and cause mature MKs to cease endomitosis. In Aim 2, we will determine whether KIFC1-mediated centrosome clustering triggers proplatelet production in vitro, ex vivo, and in mice by reorienting MTs towards the cell cortex. Elucidating the mechanisms by which mature MKs initiate proplatelet formation will yield a coherent, molecular understanding of how polyploidization is linked with the MT rearrangements necessary for proplatelet production. In addition, these data will help inform basic cell biology, as there are important parallels between centrosome clustering in MKs and cancer cells. Finally, results from the proposed experiments may yield novel therapeutic strategies for treating patients with thrombocytopenia by directly stimulating platelet production from mature MKs residing in the bone marrow.