Visualization and Manipulation of the Platelet and Megakaryocyte Cytoskeleton

Driven by the application of immunofluorescence (IF) microscopy and modern molecular biology approaches to cytoskeletal manipulation, the last 5 years have yielded considerable progress to our understanding of the molecular mechanisms governing megakaryocyte development and platelet biogenesis. Such studies have visualized endomitotic spindle dynamics, characterized the maturation of the �demarcation membrane system, delineated the mechanics of organelle transport and microtubule assembly in living megakaryocytes, described the process of platelet production in vivo, and revealed factors contributing to and the mechanisms driving proplatelet production and platelet release. Here, we describe methods to (1) culture megakaryocytes from murine fetal livers, (2) manipulate the tubulin and actin cytoskeleton of both platelets and cultured megakaryocytes, and (3) examine these by live-cell microscopy and fixed-cell immunofluorescence microscopy.