title: Mutations responsible for MYH9-related thrombocytopenia impair SDF-1-driven migration of megakaryoblastic cells.
abstract: MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for the heavy chain of non-muscle myosin-IIA (NMMHC-IIA). Recent  in vitro studies led to the hypothesis that thrombocytopenia of  MYH9-RD derives from an ectopic platelet release by megakaryocytes in the osteoblastic areas of bone marrow (BM), which are enriched in type I collagen, rather than in vascular spaces. SDF-1-driven migration of megakaryocytes within BM to reach the vascular spaces is a key mechanism for platelet biogenesis. Since myosin-IIA is implicated in polarised migration of different cell types, we hypothesised that  MYH9 mutations could interfere with this mechanism. We therefore investigated the SDF-1-driven migration of a megakaryoblastic cell line, Dami cells, on type I collagen or fibrinogen by a modified transwell assay. Inhibition of myosin-IIA ATPase activity suppressed the SDF-1-driven migration of Dami cells, while over-expression of NMMHC-IIA increased the efficiency of chemotaxis, indicating a role for NMMHC-IIA in this mechanism. Transfection of cells with three MYH9 mutations frequently responsible for  MYH9-RD (p.R702C, p.D1424H, or p.R1933X) resulted in a defective SDF-1-driven migration with respect to the wild-type counterpart and in increased cell spreading onto collagen. Analysis of differential localisation of wild-type and mutant proteins suggested that mutant NMMHC-IIAs had an impaired cytoplasmic re-organisation in functional cytoskeletal structures after cell adhesion to collagen. These findings support the hypothesis that a defect of SDF-1-driven migration of megakaryocytes induced by  MYH9 mutations contributes to ectopic platelet release in the BM osteoblastic areas, resulting in ineffective platelet production.
authors.affiliation: Dept. of Internal Medicine, IRCCS Policlinico San Matteo Foundation, University of Pavia, Piazzale Golgi, 27100 Pavia, Italy. alessandro.pecci@unipv.it
authors.forename: Alessandro
authors.initials: A
authors.lastname: Pecci
authors.forename: Valeria
authors.initials: V
authors.lastname: Bozzi
authors.forename: Emanuele
authors.initials: E
authors.lastname: Panza
authors.forename: Serena
authors.initials: S
authors.lastname: Barozzi
authors.forename: Cristian
authors.initials: C
authors.lastname: Gruppi
authors.forename: Marco
authors.initials: M
authors.lastname: Seri
authors.forename: Carlo L
authors.initials: CL
authors.lastname: Balduini
mesh_terms: D002448:Cell Adhesion / Q000235:genetics; D002460:Cell Line; D054377:Chemokine CXCL12 / Q000378:metabolism*; D002633:Chemotaxis / Q000235:genetics; D019457:Chromosome Breakage; D025063:Chromosome Disorders / Q000235:genetics* / Q000473:pathology / Q000503:physiopathology*; D024042:Collagen Type I / Q000378:metabolism; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D055016:Megakaryocyte Progenitor Cells / Q000378:metabolism* / Q000473:pathology; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D013921:Thrombocytopenia / Q000151:congenital* / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D034061:Thrombopoiesis / Q000235:genetics; D019076:Transgenes / Q000235:genetics
chemical_list: C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D024042:Collagen Type I; C103814:MYH9 protein, human; D020409:Molecular Motor Proteins; D018995:Myosin Heavy Chains