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In vivo proplatelet formation
referred as cPPT) and in vivo generated native PPTs (referred as nPPT).
While the mechanisms governing cPPT extension in vitro have been well documented, no study has clearly evaluat- ed the in vivo cytoskeleton-based mechanisms regulating the extension of nPPT. Based on in vitro experiments, sever- al cytoskeletal elements have been identified as playing a major role.22,23 Inside cPPT, the microtubules are organized as linear bundles of mixed polarity running along the shaft and ending as a coil in the cPPT bud, prefiguring the platelet marginal band.2,17,24 Incubating MK in vitro in the presence of microtubule depolymerizing drugs prevented de novo cPPT extension17,21,25,26 and retracted already formed cPPT.16,17,21 More recent work suggests that the slid- ing property of microtubules, rather than their proper poly- merization, is the primary driver of cPPT extension.11,24 On the other hand, actin has been proposed to play a role in the branching process since F-actin depolymerization reduces the number of bifurcations,17 while myosin activity decreases the extension of cPPT and has no impact on their morphology except on the bud size.26,27
In the present study we examined whether the mecha- nisms previously described in vitro also apply to the nPPT by genetically and pharmacologically manipulating cytoskeletal key components. We show that in the bone marrow of living mice, the previously in vitro described mechanism governing nPPT extension differs from the previously one described in vitro.
Methods
For details see the Online Supplementary Appendix. This study was approved by the Local Ethical Committee and experiments were performed according to the Agreement for Experimentation released by the French government (Agreement numbers: 2016090911005304 and 2018061211274514).
Intravital imaging
Intravital imaging was performed with either mT/mG;Pf4-cre mice28 or following MK and nPPT staining by intravenous injec- tion of an AF488-conjugated anti-GPIX antibody derivative.29 Two- photon microscopy was performed by observation of skull bone marrow as described.29 Anesthetized mice where observed for a maximum of 3 hours (h), during which time one to four nPPT could be recorded.
In vitro proplatelet formation
Bone marrow experiments were performed as described.26 In
vitro liquid culture of Lin- mouse progenitors was performed as described previously.30
Immunofluorescence and confocal observations
Bone marrow experiments were performed as described.26 In vitro liquid culture of Lin– mouse progenitors was performed as described previously.30
Results
Distinct morphologies between in vivo native proplatelets and in vitro cultured proplatelets
As already shown by others,4,7,9,10,31,32 nPPT extending into bone marrow sinusoids are unbranched and elongat- ed protrusions that appear mostly larger than cPPT. nPPT
extensions can be observed in situ in fixed tissues by GPIbb immunolabeling or SEM (Figure 1Ai-iii, see also Figure 4C), clearly showing the slightly bulbous aspect of their ends (Figure 1A, ii and iii inset). As also previously shown by others, two-photon microscopy observations in living animal confirmed this elongated morphology, sometimes irregular with constriction zones, which extend over long distances (Figure 1Bi-iii; Online Supplementary Video S1-3; Online Supplementary Figure S1A). nPPT were rarely found to segment at their extrem- ities, but rather broke off as long fragments subsequently further remodeled into individual platelets (Online Supplementary Figure S1B).
In contrast, the morphology of cPPT appears highly dif- ferent. Using various microscopy techniques, cPPT observed in vitro, either from cultured MK or cultured mar- row explants, present a regular thin shaft terminated by a bud (Figure 1Ci-iii). The cPPT shaft diameter is four-times smaller than the shafts measured on nPPT (Figure 1D), notwithstanding the different microscopy techniques used for their observations. The cPPT bud diameter is twice the size of the cPPT shaft (Figure 1D, left panel). Furthermore, the cPPT buds are already discoid as clearly visible by SEM (Figure 1Cii, arrows), prefiguring the future platelet, con- trary to the nPPT ends (Figure 1Aii-iii).
These data that essentially confirm previous observa- tions by others are presented for comparison purposes as these important PPT morphological differences between in vitro and in vivo observations suggest that the underlying mechanisms might be different. We therefore evaluated the role of the cytoskeleton in the dynamics of nPPT for- mation.
In vivo native proplatelet elongation dynamics are regulated by myosin IIA that opposes driving forces
As observed in vivo by two-photon microscopy, nPPT elongation is a dynamic and irregular process which pro- ceeds through elongation periods interspersed with pause and retraction phases as exemplified in Figure 2A (red and blue traces) (see also the Online Supplementary Figure S2B for more tracings), resulting in high variability in elongation speed (Online Supplementary Figure S2A). We hypothesized that this irregular behavior resulted from opposing forces exerted by the cytoskeleton and that the myosin contractile cytoskeleton was a likely contributor. We previously showed that Myh9-/- mice had a quantitative defect in cPPT formation in the explant marrow model, with fewer MK extending PPT which were also less complex compared to WT mice.26 Here using intravital microscopy, we were nevertheless able to find Myh9-/- extensions within sinusoids (Figure 2B). However, in contrast to WT nPPT, myosin-deficient cytoplasmic processes elongation occurred without any pause or retraction phases as exemplified in Figure 2C (Online Supplementary Figure S2C). Furthermore, the elon- gation speed was twice as high as that seen in WT nPPT, which might be explained by the absence of pauses and retractions (Figure 2D). Interestingly, Myh9-/- cPPT were longer and thinner (Figure 2 E-F). Their mean length was increased by 49% in Myh9-/-compared to WT nPPT and the shafts were 28% thinner. These findings suggest that myosin IIA, by increasing intracellular tension, renders the cytoplasmic extensions less stretchable and partici- pates in the pauses and retractions observed under nor- mal conditions.
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