Journal of Thrombosis and Haemostasis, 13: 851–859

DOI: 10.1111/jth.12887

BRIEF REPORT

Distinct localizations and roles of non-muscle myosin II during proplatelet formation and platelet release I. BADIROU,*†‡ J. PAN,*†‡ S. SOUQUERE,†‡§ C. LEGRAND,*†‡ G. PIERRON,†‡§ A. WANG,¶ A . E C K L Y , * * † † ‡ ‡ § § A . R O Y , * † ‡ C . G A C H E T , * * † † ‡ ‡ § § W . V A I N C H E N K E R , * † ‡ Y . C H A N G * † ‡ and C . L EO N * * † † ‡ ‡ § §

*Institut National de la Sante et de la Recherche Medicale, Villejuif; †Universite Paris-Sud, Le Kremlin-Bic^etre; ‡Institut Gustave Roussy;

§Centre National de la Recherche Scientifique, Villejuif, France; ¶Laboratory of Molecular Cardiology, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA; **Institut National de la Sante et de la Recherche Medicale; ††Etablissement Francßais du Sang-Alsace; ‡‡Faculte de Medecine, Universite de Strasbourg; and §§Federation de Medecine Translationnelle, Strasbourg, France

To cite this article: Badirou I, Pan J, Souquere S, Legrand C, Pierron G, Wang A, Eckly A, Roy A, Gachet C, Vainchenker W, Chang Y, on C. Distinct localizations and roles of non-muscle myosin II during proplatelet formation and platelet release. J Thromb Haemost 2015; 13: Le 851–9.

Introduction Summary. Background: At the end of maturation, megakaryocytes (MKs) form long cytoplasmic extensions called proplatelets (PPT). Enormous changes in cytoskeletal structures cause PPT to extend further, to re-localize organelles such as mitochondria and to fragment, leading to platelet release. Two non-muscle myosin IIs (NMIIs) are expressed in MKs; however, only NMII-A (MYH9), but not NMII-B (MYH10), is expressed in mature MKs and is implicated in PPT formation. Objectives: To provide in vivo evidence on the specific role of NMII-A and IIB in MK PPT formation. Methods: We studied two transgenic mouse models in which non-muscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of IIB. Results and Conclusions: This work demonstrates that the kinetic properties of NM-IIA, depending on the Nterminal domain, render NMII-A the better NMII candidate to control PPT formation. Furthermore, the carboxyl-terminal domain determines myosin II localization in the constriction region of PPT and is responsible for the specific role of NMII in platelet release. Keywords: megakaryocyte; Non-muscle myosin type IIA; Non-muscle myosin type IIB; platelet; stem cell.

Correspondence: Yunhua Chang, Institut National de la Sante et de la Recherche Medicale, U1009, Villejuif, France. Tel.: +33 1 42 11 42 33; fax: +33 1 42 11 52 40. E-mail: [email protected] Received 24 October 2014 Manuscript handled by: Y. Ozaki Final decision: P. H. Reitsma, 24 February 2015 © 2015 International Society on Thrombosis and Haemostasis

For a long time, NMII-A has been considered as the only non-muscle myosin II heavy chain expressed in megakaryocytes (MKs). Our recent results show that NMII-B is expressed in immature MKs and silenced during MK maturation [1,2]. This discovery revealed that different myosin II isoforms play specific roles at different steps of MK differentiation: NMII-B is involved in cytokinesis and ploidization and IIA in migration and proplatelet (PPT) formation (PPF). Interestingly, dysfunction or deregulated expression of the two isoforms (NMII-A and – B) is associated with several platelet disorders. NMII-A (MYH9) is the only myosin isoform present in platelets. Its mutations are responsible for the MYH9-related platelet disorders (MYH9-RD) characterized by a macrothrombocytopenia [3,4]. Furthermore, NMII-A expression and NMII-B silencing during normal MK differentiation are both regulated by RUNX1 [2,5]. Constitutional or acquired RUNX1 gene alterations are implicated in various acquired and inherited hemopathies. Patients with loss of function mutations of RUNX1 or FLI1 (which forms a complex with RUNX1) present NMII-B persistence in platelets and a decrease of NMII-A in mature MKs [5,6]. This deregulation of MYH9 and MYH10 expression may lead to defects both in ploidization and PPF in these patients. Some specific myosin II functions are determined by their distinct cellular localizations, which are controlled by the carboxyl (C)-terminal domain, whereas others are reliant on the motor activity of their amino (N)-terminal domain [7–11]. In this work, we tried to better understand the distinct and redundant roles of non-muscle myosin II isoforms and functional domains during the last phases of MK maturation: proplatelet formation and platelet release. We have studied two mouse lines [8] in which the

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Non-muscle myosin II and platelet production 853 Fig. 1. Proplatelet formation and platelet number in heterozygous A+/Ab* and A+/Aab mice. (A) NMII-A (red) and NMII-B (green) are present at the periphery of mature heterozygous A+/Ab* megakaryocytes (MKs). Bars indicate 10 lm. (B) Proplatelet formation is increased significantly in MKs from heterozygous A+/Ab* mice compared with MKs from A+/A+ wild-type (WT) mice. (C) Representative micrographs of MKs and PPT from heterozygous A+/Ab* mice or from A+/A+ WT mice. Bars represent 50 lm. (D) NMII-A (red) and chimeric NMII (hNMHCII-AB) (green) all presented around the periphery of mature heterozygous A+/Aab MKs. Bars indicate 10 lm. (E) Proplatelet formation is not changed in MKs from heterozygous A+/Aab mice compared with MKs from A+/A+ WT mice. (F) and (G) The proportion of megakaryocytes extending proplatelets was determined 3 and 6 h after bone marrow explant culture set-up. (F) A+/Ab* mice and their controls, n = 6 animals, representing a total number of 1547 A+/A+ and 2232 A+/Ab* megakaryocytes observed. (G) A+/Aab mice and their controls, n = 5 animals, representing a total number of 1073 A+/A+ and 1116 A+/Aab megakaryocytes observed. Results are mean  SEM. (H) Heterozygous A+/Ab* mice present a normal platelet number compared with A+/A+ WT mice. (I) Heterozygous A+/Aab mice present a thrombocytopenia compared with A+/A+ WT mice.

murine Myh9 first coding exon was disrupted either by a complementary DNA (cDNA) encoding a green fluorescent protein (GFP) human NMII-B (Ab*/Ab* mice) or by a cDNA encoding a chimeric GFP human non-muscle myosin heavy chain (hNMHC) II-AB (the N-terminal domain of NMII-A fused to the C-terminal II-B domain) (Aab/Aab mice). Complete substitution of NMII-A by IIB or chimeric hNMHCII-AB is lethal. However, the heterozygous A+/Ab* and A+/Aab mice, which present a haplo-insufficiency of NMII-A and expression of NMII-B or chimeric NMII during MK differentiation, provide some good models to study the different roles of the two NMIIs during PPF and platelet release. Materials and methods

proplatelets was quantified as described [14]. For ex vivo PPF, MKs visualized at the periphery of bone marrow slices after 3 and 6-h incubations were quantified by phase contrast microscopy [15]. Platelet collection and function assay

Mouse blood was collected by cardiac puncture into plastic tubes containing 1.138 M trisodium citrate (nine volumes/one volume). To obtain platelet-rich plasma (PRP), blood samples were centrifuged at 59 9 g for 10 min at room temperature. PRP was then centrifuged at 835 9 g for 10 min at room temperature to obtain isolated platelets. A clot retraction assay was performed on PRP while platelet aggregation and adhesion were performed on washed platelets, as described [16].

Mice

Ab*/Ab*and Aab/Aab mouse lines were generated in Adelstein’s laboratory [8]. The mice phenotypes and their genotyping have been reported [8,12]. The mice were maintained in a mixed C57BL/6 and 129S6SvEv background. Whenever possible, comparisons were made among same sex mice from the same litter. In vitro murine megakaryocyte culture

To obtain MKs in vitro, Lin- cells were isolated with the Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and subsequently cultured in serumfree medium supplemented with recombinant murine TPO (50 ng mL 1, Peprotech, Rocky Hill, NJ, USA) and hirudin (50 U mL 1, Sigma, St. Louis, MO, USA) as described [12,13]. After 24-h culture, cells were labeled with antiCD41-PE after pre-incubation with anti-CD16/CD32 Fc (III/II) (eBioscience, San Diego, CA, USA). CD41+ MK were sorted by an Influx flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) using a 100-lM nozzle. PPF analysis

For in vitro assay, sorted CD41+ MKs were seeded at 20 9 103 cells mL 1 in serum-free liquid medium with murine TPO and hirudin. The percentage of MKs bearing © 2015 International Society on Thrombosis and Haemostasis

Electron microscopy analysis

Platelets and bone marrow samples were fixed in 1.5% glutaraldehyde (Fluka) for 1 h and washed three times in 0.1 M phosphate buffer, pH 7.2, post-fixed in 1% osmic acid, dehydrated in ethanol, and embedded in Epon by standard methods. Samples were counterstained and observed with an FEI Technai Spirit transmission electron microscope (Hillsboro, OR, USA). Western blot

Western blot analysis was performed on platelet lysate as previously described [2,14]. Antibodies were: rabbit antiNMYII-B (Cell Signaling, Danvers, MA, USA), rabbit anti-NMII-A (Cell Signaling) and rat anti-HSC70 (Stressgen, San Diego, CA, USA). Immunofluorescence

The sorted CD41+ MKs were cultivated until PPF (day 4 culture) and immunofluorescence were performed as previously described [14]. Cells were examined under a Zeiss laser scanning microscope (LSM 510, Carl Zeiss, Aalen, Baden-W€ urttemberg, Germany) with a 63 9 1.4 NA oil objective. Antibodies were: rabbit anti-NMII-B (Cell Signaling), mouse anti-NMII-A (Sigma) and mouse

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Fig. 2. NMII-A, NMII-B and hNMHCII-AB localization in heterozygous A+/Ab* and A+/Aab proplatelets. (A) NMII-A (red), but not NMII-B, is present in the PPT formed by A+/A+ wild-type (WT) megakaryocytes (MKs). 3D: three dimensional graph. (B) Both NMII-A (red) and NMII-B (green) are present in PPT formed by heterozygous A+/ Ab* MKs. NMII-A is well distributed along all PPT, even in the constricted region. However, NMII-B does not accumulate in the constricted region, and cannot even be detected (see the arrows). (C) Both NMII-A (red) and hNMHCII-AB (green) are present in PPT formed by heterozygous A+/Aab MKs, but hNMHCII-AB does not accumulate in the constricted region, and cannot even be detected.

anti-a-tubulin (Sigma). TOTO-3 Iodide (Molecular Probes, Eugene, OR, USA) was applied for nucleus staining. Flow cytometry analysis of bone marrow MK number

Mouse bone marrow was flushed from femurs and tibia and dispersed with DMEM medium (10% FBS) by a 1-

mL syringe attached to a 23G 0.6 9 25 mm needle. The bone marrow suspension was incubated with Anti-CD41 APC and Anti-CD42 PE (BD Biosciences, Franklin Lakes, NJ, USA) for 30 min at 4 °C after pre-incubation with Anti–CD16/CD32 Fc (III/II). The CD41+/CD42+ MK populations were counted per 106 single events by an influx flow cytometer. © 2015 International Society on Thrombosis and Haemostasis

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Fig. 3. Platelet length is increased in heterozygous A+/Ab* mice. (A) No modification of the ultrastructure of mature A+/ Ab* megakaryocytes (MKs) in bone marrow was observed. Bars represent 5 lm. (B) NMII-A, but not NMII-B, is detected in the platelets formed by A+/ A+ wild-type (WT) MKs. However, both NMII-A and NMII-B are present in platelets from A+/ Ab* mice. (C) Platelets isolated from heterozygous A+/ Ab* mice have a longer size than those from WT A+/A+ mice. Bars represent 5 lm. (D) 100 platelets from three heterozygous A+/ Ab* and three WT A+/A+ mice are measured for length and width. Statistical significance was determined by T-test (unpaired t-test with Welch’s correction). (E) The number of platelets with a size longer than 3.5 lm was counted in three heterozygous A+/ Ab* and three WT A+/A+ mice. For each 1000 lm [2], heterozygous A+/ Ab* mice have higher numbers of platelets longer than 3.5 lm compared with WT A+/A+ mice. © 2015 International Society on Thrombosis and Haemostasis

856 I. Badirou et al Fig. 4. Platelet length is increased in heterozygous A+/Aab mice. (A) No modification of the ultrastructure of mature A+/Aab megakaryocytes (MKs) in bone marrow was observed. Bars represent 10 lm. (B) Platelets isolated from heterozygous A+/Aab mice presented a longer size than those from wild-type (WT) A+/A+ mice. Bars represent 5 lm. (C) One hundred and fifty platelets from three heterozygous A+/Aab and three WT A+/A+ mice were measured for length and width. Statistical significance was determined by T-test (unpaired t-test with Welch’s correction). (D) Platelets isolated from heterozygous MK-restricted Myh9 KO A+/A- mice showed the same size as those from WT A+/A+ mice. Bars represent 5 lm. (E) One hundred platelets from three heterozygous A+/A- mice and three WT A+/A+ mice were measured for length and width.

Statistics

Data are presented as means ( SEM). Statistical significance was determined by Student’s t-test. A P value < 0.05 was considered as statistically significant. Results and discussion The N-terminal of NMII-A and NMII-IIB determined their distinct role in proplatelet formation (PPF)

It has been shown that in in vitro liquid MK cultures, PPF is enhanced in MK derived from megakaryocyterestricted Myh9 inactivated mice (Myh9 KO) [17]. Previous results revealed also that inhibition of NMII-A activity or myosin light chain MLC2 phosphorylation, which controls NMII-A activation, also increased PPF [14,18,19]. All these data suggest that NMII-A may somehow control PPT extension. To check if NMII-B expression could play a similar role, the heterozygous A+/Ab* mice, which present a NMII-A haplo-insufficiency and a persistence of NMII-B during late MK differentiation, were analyzed for PPF after MK differentiation in vitro. As shown in Fig. 1(A), though NMII-A and NMII-B are all present at the periphery of mature heterozygous A+/Ab* MK, PPF was increased significantly in MKs from heterozygous A+/Ab* mice compared with MKs from A+/A+ WT (wild-type) mice (Fig. 1B,C). The heterozygous A+/Aab mice, which present an NMII-A haplo-insufficiency, but also an expression of a chimeric NMII (hNMHCII-AB) with the head of NMIIA and the tail of II-B, had myosin II localization similar to A+/Ab* (Fig. 1D). Interestingly, in vitro they had similar PPF efficiency to A+/A+ WT (Fig. 1E). As NMIIB and hNMHCII-AB have the same C-terminus but different N-terminus, their different effects on PPT formation should be related to their N-terminal domains. Although both of them catalyze adenosine triphosphate hydrolysis (ATP), the N-terminal domain of NMII-A appears more efficient in restraining membrane protrusion and controlling more accurately PPF. The kinetic properties of NMII-A, which is dependent on its N-terminal, render NMII-A the better NMII candidate for controlling PPF. In ex vivo fresh bone marrow, the proportion of MKs forming proplatelets was determined 3 and 6 h after explant culture set-up. The heterozygous A+/Aab mice

and the control show the same PPF ratio as in in vitro culture. By contrast, the heterozygous A+/Ab* mice show a trend towards a decrease in PPF at 6 h compared with the control (Fig. 1F,G). This is similar, though to a far lesser extent, to what was observed with Myh9 KO mice: in this mouse line, PPF is also increased in vitro, but decreased strongly in ex vivo bone marrow explant experiments [15,17]. Similarly to Myh9 KO, A+/Ab* also exhibited an increased bone marrow MK number, as measured by flow cytometry. The average MK number per 106 cells was increased by 29.9% in A+/Ab* mice (2557) compared with WT mice (1969) (n = 8, P = 0.02). In contrast, such an increase was less evident for A+/Aab mice and did not reach significance (1833 MK 10 6 cells in A+/Aab mice compared with 1559 for their control, n = 8, P = 0.11). Nevertheless, contrary to Myh9 KO mice [15], in both mouse lines, no evidence for in situ MK death was observed, as shown by immunohistology (Fig. S1) or electron microscopy (Fig. 3A and 4A). Remarkably, a thrombocytopenia was observed in heterozygous A+/Aab mice, but not in heterozygous A+/Ab* mice (Fig. 1H,I). As our previous work has shown that both mouse lines presented a decreased MK polyploidisation [12], and because the lower ploidy cannot be compensated for by an increased MK number in A+/Aab mice, it is expected that A+/Aab mice present a slight thrombocytopenia. This is in contrast to A+/Ab* mice, where the increased MK number most probably counteracts the lower ploidy. The C-terminal domain determined the different localizations of NMII-A and NMII-B in proplatelets

NMII-A and NMII-B show the same localization in mature heterozygous A+/Ab* MKs; however, some subtle differences were observed in their localization in PPT. In A+/A+ WT mice (Fig. 2A), as in heterozygous A+/Ab* (Fig. 2B) or A+/Aab mice (Fig. 2C), NMII-A was well distributed all along the PPT extension, even in the constricted regions, where platelets will be presumably cut and released. However, NMII-B or hNMHCII-AB in the constricted region was at the limit of detection or even undetected (Fig. 2B,C). Because hNMII-B and hNMHCII-AB possess the C-terminus of NMII-B, this result suggests that the C-terminal domain of NMII-A is responsible for NMII localization in the constricted region and consequently for the platelet release function © 2015 International Society on Thrombosis and Haemostasis

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of NMII. As NMII-A is the only myosin expressed in mature MKs and platelets, this specific localization of NMII-A in the constricted region should be very important for platelet release: in the MYH9 syndrome, MYH9 mutations could perturb this function and lead to production of macroplatelets. NMII-B or hNMHC II-AB expression leads to platelets with an abnormal length but normal function

NMII-B expression did not modify the ultrastructure (membranes and granules) of mature MKs in bone marrow (Fig. 3A) and platelets isolated from blood (Fig. 3B, C). However, some abnormality in platelet morphology, particularly regarding their length, was observed. In fact, when 100 platelets from three heterozygous A+/ Ab* and three WT A+/A+ mice were measured for their length and width, A+/Ab* platelets presented a longer size than wild-type platelets (Fig. 3D,E), suggesting a defect in platelet release. Expression of hNMHCII-AB likewise did not modify the ultrastructure of mature MKs in bone marrow, but increased platelet length (Fig. 4A,C). The platelet size abnormality in heterozygous A+/Aab and A+/Ab* mice could be due to either the presence of NMII-B/hNMHCII-AB or a NMII-A haplo-insufficiency or both. However, platelets from the heterozygous mice with MK-restricted Myh9 knockout (A+/A- mice) showed no difference in either their length or width (Fig. 4D,E) [17]. This suggests that NMII-A haplo-insufficiency per se does not affect platelet morphology. Thus the longer platelets in heterozygous A+/Ab* and A+/Aab mice are probably linked to NMII-B persistence in PPT. As NMII-B and hNMHCII-AB do not accumulate in the PPT restriction region, they cannot replace the function of NMII-A; in addition, their expression may increase the tension along the PPT, which could make proplatelet fragmentation more difficult compared with heterozygous A+/A- mice and produce longer platelets. Interestingly, blood platelets from patients with constitutional RUNX1 mutations, which presented a decreased expression of NMII-A and MYL9 (MLC2 or myosin light chain 9, regulatory) as well as a persistence of NM-IIB [5], also presented a remarkable heterogeneity in platelet size, suggesting the important role of myosin II in the control of platelet size. Finally, we checked whether the presence of NMII-B or hNMHCII-AB affects platelet functions. Platelet aggregation (Fig. S2) and clot retraction (Fig. S3), as well as platelet shape change and static adhesion on fibrinogen (data not shown), were performed. No difference was observed between the A+/Ab*, A+/Aab mice and their proper control. Thus, the slightly increased platelet length does not seem to affect the platelet function. Because the clot retraction was delayed in heterozygous A+/A- mice [16] but not modified in A+/Ab* and A+/Aab mice, expression of NMII-B and hNMHCII-AB therefore compensates for the

haplo-insufficiency of MYH9 in platelet function and thus may have similar functions to NMII-A in platelets. Addendum I. Badirou, J. Pan, S. Souquere, C. Legrand, G. Pierron, A. Eckly and A. Roy performed research; A. Wang generated the Ab*/Ab*and Aab/Aab mouse lines; C. Leon and C. Gachet generated the MK-restricted Myh9 knockout mice and performed research. Y. Chang designed and performed research; Y. Chang wrote the paper with the help of W. Vainchenker, C. Gachet, C. Leon and I. Badirou. Acknowledgements This work was supported by INSERM, by the Agence Nationale de la Recherche (ANR Jeune chercheur) (Y. Chang), and by grants from la Ligue Nationale Contre le Cancer (Equipe labellisee 2012). I. Badirou was supported by the ANR, and J. Pan by the China Scholarship Council and la Societe Francßaise d’Hematologie. A. Roy is supported by a grant from la Fondation de la Recherche Medicale (FRM). We thank L. Lodier, J. Weber and P. Laeuffer for their technical help. Disclosure of Conflict of Interests The authors state that they have no conflict of interests. Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1 Sections of A+/A+ and A+/Ab* bone marrow samples were stained with hematoxyline and von Willebrand factor, which marked megakaryocytes in bone marrow (Brown) (n=3). Fig. S2 Aggregation of washed mouse platelets. Fig. S3 Clot retraction. References 1 Lordier L, Jalil A, Aurade F, Larbret F, Larghero J, Debili N, Vainchenker W, Chang Y. Megakaryocyte endomitosis is a failure of late cytokinesis related to defects in the contractile ring and Rho/Rock signaling. Blood 2008; 112: 3164–74. 2 Lordier L, Bluteau D, Jalil A, Legrand C, Pan J, Rameau P, Jouni D, Bluteau O, Mercher T, Leon C, Gachet C, Debili N, Vainchenker W, Raslova H, Chang Y. RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization. Nat Commun 2012; 3: 717. 3 Balduini CL, Pecci A, Savoia A. Recent advances in the understanding and management of MYH9-related inherited thrombocytopenias. Br J Haematol 2011; 154: 161–74. 4 Chen Z, Shivdasani RA. Regulation of platelet biogenesis: insights from the May-Hegglin anomaly and other MYH9related disorders. J Thromb Haemost 2009; 7 (Suppl. 1): 272–6.

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Non-muscle myosin II and platelet production 859 5 Bluteau D, Glembotsky AC, Raimbault A, Balayn N, Gilles L, Rameau P, Nurden P, Alessi MC, Debili N, Vainchenker W, Heller PG, Favier R, Raslova H. Dysmegakaryopoiesis of FPD/ AML pedigrees with constitutional RUNX1 mutations is linked to myosin II deregulated expression. Blood 2012; 120: 2708–18. 6 Antony-Debre I, Bluteau D, Itzykson R, Baccini V, Renneville A, Boehlen F, Morabito M, Droin N, Deswarte C, Chang Y, Leverger G, Solary E, Vainchenker W, Favier R, Raslova H. MYH10 protein expression in platelets as a biomarker of RUNX1 and FLI1 alterations. Blood 2012; 120: 2719–22. 7 Bao J, Ma X, Liu C, Adelstein RS. Replacement of nonmuscle myosin II-B with II-A rescues brain but not cardiac defects in mice. J Biol Chem 2007; 282: 22102–11. 8 Wang A, Ma X, Conti MA, Liu C, Kawamoto S, Adelstein RS. Nonmuscle myosin II isoform and domain specificity during early mouse development. Proc Natl Acad Sci USA 2010; 107: 14645–50. 9 Wang A, Ma X, Conti MA, Adelstein RS. Distinct and redundant roles of the non-muscle myosin II isoforms and functional domains. Biochem Soc Trans 2011; 39: 1131–5. 10 Sandquist JC, Means AR. The C-terminal tail region of nonmuscle myosin II directs isoformspecific distribution in migrating cells. Mol Biol Cell 2008; 19: 5156–67. 11 Sato MK, Takahashi M, Yazawa M. Two regions of the tail are necessary for the isoform-specific functions of nonmuscle myosin IIB. Mol Biol Cell 2007; 18: 1009–17. 12 Badirou I, Pan J, Legrand C, Wang A, Lordier L, Boukour S, Roy A, Vainchenker W, Chang Y. Transgenic mouse models demonstrate the Carboxyl-terminal depended recruitment of nonmuscle myosin II to megakaryocyte contractile ring during polyploidization. Blood 2014; 124: 2564–8.

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Distinct localizations and roles of non-muscle myosin II during proplatelet formation and platelet release.

At the end of maturation, megakaryocytes (MKs) form long cytoplasmic extensions called proplatelets (PPT). Enormous changes in cytoskeletal structures...
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