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In vivo proplatelet formation
on Figure 4C yellow arrowheads and arrows). As shown in Figure 4B (left, arrowhead), microtubules were not organ- ized as bundles but aligned roughly parallel in the direction of the extension in nascent nPPT. This different micro- tubule organization suggests a different role in the process of nPPT extension. Of note, strong F-actin labeling was observed laterally at the site of transmigration, indicating also the importance of this cytoskeleton in this first step (Figure 4B, middle). Focusing on later stages in the elongat- ed nPPT, we observed that again, unlike in vitro, the micro- tubules were not arranged in large bundles running along the extension, and microtubules were not longitudinally aligned in parallel but were found in various non-uniform orientations along the nPPT (Figure 4B arrow; Figure 4C-E; especially compare Figure 4Aii with Figure 4B-C which
have the same scale). As seen in Figure 4C showing a three- dimensional (3D) view of three portions of intertwined nPPT, the labelling was not always constant, being some- times decreased (Figure 4C left, orange arrowhead) or even absent (Figure 4C left, orange arrow), suggesting few microtubules and mainly non-polymerized tubulin. Hence the difference between the in vivo and in vitro role of micro- tubules in the process of MK extension is further illustrated by the difference in microtubule organization/distribution within nascent and elongating nPPT compared to cPPT.
Administration of vincristine leads to shrinkage of preexisting wild-type proplatelets
In a second approach, and because Tubb1-/- mice com- pensate the b1 tubulin deficiency by overexpressing b2
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Figure 6. Myh9-/- native proplatelets still elongate after vincristine administration. (A) Representative z-projection time-lapse images showing Myh9-/- protrusions before and after vincristine administration. (B) Measurement of protrusions length in Myh9-/-mice before and after 2 mg/kg vincristine administration plotted as a function of time. Note that some Myh9-/- protrusions could not be recorded after 4.5 minutes since they escaped the observation field. Six mice were analyzed. (C) Measurement of the difference in length during a 4-minute window (0.5–4.5 minutes post 2 mg/kg vincristine injection), showing retraction in wild-type (WT) mice and elongation in Myh9-/- mice. Seven WT mice and six Myh9-/- mice analyzed, using Mann-Whitney statistical analysis. (D) Immunolabeling of bone marrow sections from Myh9-/- mice injected with vincristine (1 mg/kg) 10 minutes prior to the removal of the femurs and fixation of the bone marrow, showing the absence of micro- tubule labeling in a long protrusion (arrow). Left, Magenta labeling showing GPIbb; center, green labeling showing α tubulin; images represent a 3D view. Right panel, 3D representation of the megakaryocytes (MK) extending the nPPT obtained by image segmentation and 3D reconstruction with AMIRA software.
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