title: Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis.
abstract: To produce blood platelets, megakaryocytes elaborate proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. The invaginated demarcation membrane system (DMS), a hallmark of mature cells, has been proposed as the source of proplatelet membranes. By direct visualization of labeled DMS, we demonstrate that this is indeed the case. Late in megakaryocyte ontogeny, the DMS gets loaded with PI-4,5-P(2), a phospholipid that is confined to plasma membranes in other cells. Appearance of PI-4,5-P(2) in the DMS occurs in proximity to PI-5-P-4-kinase alpha (PIP4Kalpha), and short hairpin (sh) RNA-mediated loss of PIP4Kalpha impairs both DMS development and expansion of megakaryocyte size. Thus, PI-4,5-P(2) is a marker and possibly essential component of internal membranes. PI-4,5-P(2) is known to promote actin polymerization by activating Rho-like GTPases and Wiskott-Aldrich syndrome (WASp) family proteins. Indeed, PI-4,5-P(2) in the megakaryocyte DMS associates with filamentous actin. Expression of a dominant-negative N-WASp fragment or pharmacologic inhibition of actin polymerization causes similar arrests in proplatelet formation, acting at a step beyond expansion of the DMS and cell mass. These observations collectively suggest a signaling pathway wherein PI-4,5-P(2) might facilitate DMS development and local assembly of actin fibers in preparation for platelet biogenesis.
authors.affiliation: Dana-Farber Cancer Institute, One Jimmy Fund Way, Boston, MA 02115, USA.
authors.forename: Harald
authors.initials: H
authors.lastname: Schulze
authors.forename: Manav
authors.initials: M
authors.lastname: Korpal
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hurov
authors.forename: Sang-We
authors.initials: SW
authors.lastname: Kim
authors.forename: Jinghang
authors.initials: J
authors.lastname: Zhang
authors.forename: Lewis C
authors.initials: LC
authors.lastname: Cantley
authors.forename: Thomas
authors.initials: T
authors.lastname: Graf
authors.forename: Ramesh A
authors.initials: RA
authors.lastname: Shivdasani
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002462:Cell Membrane / Q000502:physiology; D005434:Flow Cytometry; D008164:Luminescent Proteins / Q000235:genetics; D008533:Megakaryocytes / Q000166:cytology / Q000502:physiology* / Q000821:virology; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D010957:Plasmids; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics; D010980:Platelet Membrane Glycoproteins / Q000235:genetics; D012190:Retroviridae / Q000302:isolation & purification; D034061:Thrombopoiesis
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; C032193:glycoprotein receptor GPIb-IX; C054562:yellow fluorescent protein, Bacteria