CLIP-170 and CLIP-115 are cytoplasmic linker proteins that associate specifically with the ends of growing microtubules and may act as anti-catastrophe factors. Here, we have isolated two CLIP-associated proteins (CLASPs), which are homologous to the Drosophila Orbit/Mast microtubule-associated protein. CLASPs bind CLIPs and microtubules, colocalize with the CLIPs at microtubule distal ends, and have microtubule-stabilizing effects in transfected cells. After serum induction, CLASPs relocalize to distal segments of microtubules at the leading edge of motile fibroblasts. We provide evidence that this asymmetric CLASP distribution is mediated by PI3-kinase and GSK-3 beta. Antibody injections suggest that CLASP2 is required for the orientation of stabilized microtubules toward the leading edge. We propose that CLASPs are involved in the local regulation of microtubule dynamics in response to positional cues.

Megakaryocytes are terminally differentiated cells that, in their final hours, convert their cytoplasm into long, branched proplatelets, which remodel into blood platelets. Proplatelets elongate at an average rate of 0.85 m/min in a microtubule-dependent process. Addition of rhodamine-tubulin to permeabilized proplatelets, immunofluorescence microscopy of the microtubule plusend marker end-binding protein 3 (EB3), and fluorescence time-lapse microscopy of EB3-green fluorescent protein (GFP)-expressing megakaryocytes reveal that microtubules, organized as bipolar arrays, continuously polymerize throughout the proplatelet. In immature megakaryocytes lacking proplatelets, microtubule plus-ends initiate and grow by centrosomal nucleation at rates of 8.9 to 12.3 m/min. In contrast, plus-end growth rates of microtubules within proplatelets are highly variable (1.5-23.5 m/ min) and are both slower and faster than those seen in immature cells. Despite the continuous assembly of microtubules, proplatelets continue to elongate when net microtubule assembly is arrested. One alternative mechanism for force generation is microtubule sliding. Triton X-100-permeabilized proplatelets containing dynein and its regulatory complex, dynactin, but not kinesin, elongate with the addition of adenosine triphosphate (ATP) at a rate of 0.65 m/min. Retroviral expression in megakaryocytes of dynamitin (p50), which disrupts dynactindynein function, inhibits proplatelet elongation. We conclude that while continuous polymerization of microtubules is necessary to support the enlarging proplatelet mass, the sliding of overlapping microtubules is a IntroductionBlood platelets, tiny cells shed by megakaryocytes, circulate throughout blood vessels and survey the integrity of the vascular system. In response to traumatic injuries in which blood vessel continuity is interrupted, platelets bind to exposed collagen, change shape, secrete granule contents, and aggregate with neutrophils to form a hemostatic plug to seal off the damaged blood vessel. The mechanisms by which blood platelets are formed and released from giant precursor cells, called megakaryocytes, in situ remain to be defined. However, the development of megakaryocyte culture systems that produce platelets has provided a means to study the intermediate structures called "proplatelets," long (up to several millimeters), thin extensions of the megakaryocyte cytoplasm that contain multiple platelet-sized beads along their length. [1][2][3][4][5][6][7] Based on multiple lines of evidence, we have speculated that platelets are not preassembled in the megakaryocyte cytoplasm but instead are constructed de novo, predominantly at the ends of the proplatelets. 8 As predicted, megakaryocytes go to great lengths to amplify the number of proplatelet ends, taking the shaft of each proplatelet and bending it multiple times. Each bend yields a bifurcation in the shaft, generating a new end.The formation of proplatelets is highly dependent upon a complex network of protein filaments that extends throug...