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A. Bornert et al.
labeling, we found that the number of MK was increased by around 75% compared to the WT mice (Online Supplementary Figure S3A), probably as a compensatory response to the thrombocytopenia (Online Supplementary Figure S3B). Tubb1-/- mouse MK were able to extend protru- sions in marrow sinusoids, although we found a 45% decrease in the number of nPPT extensions compared to WT nPPT, in agreement with the decreased number of cir- culating platelets (Figure 3B; Online Supplementary Figure S3B). Tubb1-/- nPPT exhibited a fully normal morphology (Figure 3C) with a surprisingly normal elongation speed (Figure 3D) and a fully normal mean length and width (Figure 3E-F). These results indicate that while the absence of an essential tubulin isoform almost totally abrogates PPT formation in vitro, it partially affects the number of
PPT in vivo but not their elongation speed. Overall, this points to different mechanisms contributing to PPT exten- sion when considering the in vivo situation with a less cru- cial importance of microtubules compared to in vitro.
Microtubules are non-uniformly distributed in proplatelets in vivo
Another difference between in vivo and in vitro was observed by looking at the cytoplasmic distribution of microtubules. Within cPPT obtained in vitro, microtubules are organized into a bundle running along the shaft as pre- viously described17 and as illustrated in Figure 4A. We then explored their organization in situ under conditions that preserved microtubules as denoted by the intact marginal band of platelets and mitotic spindles in marrow cells (see
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Figure 5. Vincristine infusion leads to shrinkage of preformed wild-type native proplatelets. A) Representative z-projection time-lapse images showing an native pro- platelets (nPPT) before and after vincristine administration (1 mg/kg). (B) Length of individual nPPT plotted as a function of time before and after 1 mg/kg vincristine administration. Seven mice analyzed. (C) Scatter plot of nPPT length difference during a 10-minute observation showing elongation in the absence of vincristine and retraction following vincristine injection (from 0.5-10.5 min). Seven mice were analyzed for each condition. (D) Measurement of the difference in width close to the base during a 10-minute window showing small positive or negative variation in the absence of vincristine (left) and increasing width following vincristine injection (right). Seven mice were analyzed. (E) Doubling the vincristine dose administration accelerates nPPT shrinkage. Seven mice were analyzed. All data analyzed using Mann-Whitney test. ns: not significant.
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haematologica | 2021; 106(5)