Blood platelets are specialized anucleate cells that play an essential role in hemostasis, angiogenesis, wound healing, immunity, and inflammation. Abnormal platelet counts result in clinical complications and medical conditions that increasingly require expensive transfusions. As the demand for platelet transfusions rises, the need for an improved understanding of their mechanistic formation has urgently increased. Our investigations will target megakaryocytes (MKs), the precursor cells that generate platelets by remodeling their cytoplasm into beaded proplatelet processes which function as the assembly lines for platelet production. We know that cytoskeletal mechanics power platelet production, but many questions about platelet biogenesis remain unanswered. While microtubule-based forces are critical for proplatelet elongation, there is a surprising lack of understanding of the mechanisms that trigger MKs to undergo the cytoskeletal rearrangements needed to initiate proplatelet production. Aim 1 will test a new hypothesis that centrosome clustering and subsequent monospindle formation cause mature MKs to initiate proplatelet formation. We will interrogate the role of the molecular motor KIFC1 in centrosome clustering and proplatelet induction. Although the mechanism by which sliding of cortical microtubules powers proplatelet extension is unknown, our recent observation that inhibition of the actin assembly process paralyzes proplatelet elongation suggests a role for actin. This exciting new data indicates that actin may function as a molecular clutch and promote microtubule sliding and/or anchoring of microtubules to the membrane, which will be tested in Aim 2. Finally, we know that proplatelet protrusions extend from bone marrow, breach the endothelial barrier, and deposit platelets into the blood, but we do not know how. Aim 3 will employ an engineered, endothelialized, microfluidic bone marrow on-a-chip to test the idea that actin-driven megakaryocyte podosomes provide a mechanism to penetrate the endothelium. Biologically inspired engineering will be used to study how podosomes transition to proplatelet production and to test the role of monospindle formation in proplatelet polarization into the bloodstream. We expect that findings from this investigation will 1) improve the understanding of the molecular mechanisms that regulate platelet formation in health and disease, 2) lay the foundation for novel therapeutic approaches to accelerate platelet production in patients with thrombocytopenia, and 3) enable the future creation of in vitro platelets.