title: Signaling and cytoskeletal requirements in erythroblast enucleation.
abstract: To understand the role of cytoskeleton and membrane signaling molecules in erythroblast enucleation, we developed a novel analysis protocol of multiparameter high-speed cell imaging in flow. This protocol enabled us to observe F-actin and phosphorylated myosin regulatory light chain (pMRLC) assembled into a contractile actomyosin ring (CAR) between nascent reticulocyte and nucleus, in a population of enucleating erythroblasts. CAR formation and subsequent enucleation were not affected in murine erythroblasts with genetic deletion of Rac1 and Rac2 GTPases because of compensation by Rac3. Pharmacologic inhibition or genetic deletion of all Rac GTPases altered the distribution of F-actin and pMRLC and inhibited enucleation. Erythroblasts treated with NSC23766, cytochalasin-D, colchicine, ML7, or filipin that inhibited Rac activity, actin or tubulin polymerization, MRLC phosphorylation, or lipid raft assembly, respectively, exhibited decreased enucleation efficiency, as quantified by flow cytometry. As assessed by high-speed flow-imaging analysis, colchicine inhibited erythroblast polarization, implicating microtubules during the preparatory stage of enucleation, whereas NSC23766 led to absence of lipid raft assembly in the reticulocyte-pyrenocyte border. In conclusion, enucleation is a multistep process that resembles cytokinesis, requiring establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte.
authors.affiliation: Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
authors.forename: Diamantis G
authors.initials: DG
authors.lastname: Konstantinidis
authors.forename: Suvarnamala
authors.initials: S
authors.lastname: Pushkaran
authors.forename: James F
authors.initials: JF
authors.lastname: Johnson
authors.forename: Jose A
authors.initials: JA
authors.lastname: Cancelas
authors.forename: Stefanos
authors.initials: S
authors.lastname: Manganaris
authors.forename: Chad E
authors.initials: CE
authors.lastname: Harris
authors.forename: David A
authors.initials: DA
authors.lastname: Williams
authors.forename: Yi
authors.initials: Y
authors.lastname: Zheng
authors.forename: Theodosia A
authors.initials: TA
authors.lastname: Kalfa
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism* / Q000502:physiology; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D004900:Erythroblasts / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D004920:Erythropoiesis / Q000235:genetics / Q000502:physiology; D021962:Membrane Microdomains / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008870:Microtubules / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012156:Reticulocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology
chemical_list: D000199:Actins; D020744:rac GTP-Binding Proteins