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A. Bornert et al.
(L) and the apparent viscosity η of the blood in microves- sels varying from 2-4x10-3 Pa.s,34-36 the Stokes’ force is approximatly 30-60 pN on the bud. This value can be con- sidered as the minimal force as the Stokes’ force is also applied all along the nPPT shaft. It is known from the lit- erature that a force around 20-50 pN is usually required to extend membrane nanotubes in cells including blood cells such as neutrophils or erythrocytes.37-39 Hence the force exerted by the blood flow on the whole nPPT would be high enough to substantially contribute to nPPT exten- sion.
Discussion
In this study we evaluated the mechanisms of nPPT for- mation as it occurs in vivo inside bone marrow sinusoid vessels. We found that the mechanisms differ from those taking place in order to produce cPPTs under in vitro condi- tions, especially with regards to the relative implication of two main cytoskeletal components, i.e., microtubules and myosin. We show that the non-continuous nPPT elonga- tion process resulting from pause and retraction phases resulted from myosin IIA. MK are known to activate myosin IIA in response to local increase in shear.40,41 Blood flow within sinusoid vessels presents heterogeneous shear stresses ranging from zero to 10 dyn/cm2,42,43 generating forces that are sufficient to trigger cellular mechanotrans- duction in endothelial cells.44 Hence, depending on the flow forces, transient myosin activation could increase membrane tension to preserve its integrity. Conversely, a decreased myosin IIA activity would increase membrane compliance and stretching, promoting thinner and longer protrusions such as observed in Myh9-/- mice. Another hypothesis could be that myosin-promoted pauses serve to slow down the extension process in order for DMS to properly enter the nPPT. In favor of this hypothesis, we observed in situ that myosin-deficient nPPT contain very few DMS membranes compared to WT ones (Online Supplementary Figure S8), which could also explain the thinner morphology of these Myh9-/- nPPT.
Unexpectedly, the microtubule behavior was found to differ between nPPT and cPPT. This was first revealed in Tubb1-/- mice in vivo. Although the number of MK extend- ing nPPT was decreased, in agreement with the moderate thrombocytopenia, their morphology and elongation speed were fully normal. This was in stark contrast to the almost total inability of Tubb1-/- MK to form cPPT in vitro. These findings are a further indication that the defects of Tubb1-/- mice are exacerbated in vitro and they are clear evi- dence of mechanistic differences between the two envi- ronments.
Given these observations, we hypothesize that MK extensions generated in vivo are less dependent on micro- tubules than anticipated from cPPT produced in vitro. We observed that microtubules were present in the nascent nPPT, although not organized in bundles, as also previous- ly mentioned in an earlier work.8 Their presence however suggested that they might still play a role in nPPT initia- tion, in agreement with the decreased number of Tubb1-/- nPPT observed in situ. In vivo, the precise intracellular mechanisms controlling the initiation and transmural pas- sage of nPPTs are still unknown. The first step of nPPT extension, which initiates in the marrow stroma and in the absence of blood forces, might require higher protru-
sive forces to push against the endothelial barrier com- pared to liquid culture. It is most probable that both F-actin and microtubule cytoskeletons jointly play a role as we also observed strong F-actin accumulation in MK at the site where the nascent nPPT cross the vessel wall (Figure 4B). This F-actin accumulated in structures resem- bling shoulder-like structures which could correspond to the fibrillary-rich collars previously observed by Behnke and Forer and could be an anchorage point for facilitating the initial protrusion.31,4 At this stage, whether micro- tubules directly contribute together with F-actin to pro- mote the initial pushing force for the transmural passage, or whether they are indirectly required to organize vesi- cle/organelle transport to bring essential components or play a role as information carriers for F-actin is not known.45
Upon subsequent nPPT growth inside sinusoids, we observed a non-uniform distribution of microtubules inside elongated nPPT which clearly differs from the microtubule bundles uniformly lining the cPPT shafts and ending as coils observed in vitro. Although we were beyond the resolution limit to see microtubule arrange- ment in areas of strongest nPPT constriction in situ, these were clearly observed as unbundled in larger areas, in agreement with early observations by Behnke mentioning random arrangement in large clumps of MK cytoplasm released in sinusoids in situ.8 Radley and Scurfield also observed in situ that microtubules were aligned in constric- tion zones but splayed out on either side of the constric- tion.46 These results confirm and extend those recently published by Brown et al. showing by tomography that in situ, microtubules were individual and randomly distrib- uted in MK protrusions.4 Hence all the above data point to a different mechanism depending on whether MK extend protrusions in vitro or in vivo. However, microtubules are clearly important for maintaining the elongated nPPT structure in WT mice. This was evidenced here after inducing in vivo microtubule depolymerization on pre- existing nPPT.
A much unexpected observation in Myh9-/- mice was the continuous growth of MK protrusions within sinusoids even when microtubules were depolymerized by vin- cristine. This indicated that under conditions where con- tractile forces are weakened, abrogating nPPT retraction, elongation of protrusions is still occurring. Hence, inside the blood vessels, microtubules would be less crucial for nPPT elongation. Isolated microtubules have a low push- ing force in the range of 3-4 pN, while it has been demon- strated that organization in bundles increases their force- generating capacity in an additive fashion.47-49 While in vitro microtubule bundles could conceivably be the primary driving force for elongation, this is different in vivo where the essential role of microtubules would be to act as a backbone to transport constituents and to prevent and counteract myosin-mediated nPPT retraction. Of note, Tubb1-/- platelets do not present abnormalities in their granule distribution (see the Online Supplementary Figure S3C) contrary to knockout mice having actomyosin impairments,32,50 showing that partial microtubule content is sufficient to promote normal organelle transport into the maturing MK and the nPPT.
Our finding then raised the question of the force pro- moting MK fragment elongation in vivo. Our data suggest that the main motor for nPPT elongation may come from hemodynamic forces. The importance of blood flow was
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haematologica | 2021; 106(5)