title: FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets.
abstract: Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation.
authors.affiliation: Division of Hematology, Brigham and Women's Hospital, Boston, MA; Department of Medicine, Harvard Medical School, Boston, MA;
authors.forename: Antonija Jurak
authors.initials: AJ
authors.lastname: Begonja
authors.affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada;
authors.forename: Fred G
authors.identifier: http://orcid.org/0000-0002-0451-5840
authors.initials: FG
authors.lastname: Pluthero
authors.affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA;
authors.forename: Worawit
authors.initials: W
authors.lastname: Suphamungmee
authors.affiliation: Division of Hematology, Brigham and Women's Hospital, Boston, MA; Department of Medicine, Harvard Medical School, Boston, MA;
authors.forename: Silvia
authors.initials: S
authors.lastname: Giannini
authors.affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada;
authors.forename: Hilary
authors.initials: H
authors.lastname: Christensen
authors.affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada;
authors.forename: Richard
authors.initials: R
authors.lastname: Leung
authors.affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada; Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, ON, Canada; and.
authors.forename: Richard W
authors.identifier: http://orcid.org/0000-0001-5325-5590
authors.initials: RW
authors.lastname: Lo
authors.affiliation: Division of Hematology, Brigham and Women's Hospital, Boston, MA; Department of Medicine, Harvard Medical School, Boston, MA;
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Nakamura
authors.affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA;
authors.forename: William
authors.identifier: http://orcid.org/0000-0002-9430-9521
authors.initials: W
authors.lastname: Lehman
authors.affiliation: Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Plomann
authors.affiliation: Division of Hematology, Brigham and Women's Hospital, Boston, MA; Department of Medicine, Harvard Medical School, Boston, MA;
authors.forename: Karin M
authors.initials: KM
authors.lastname: Hoffmeister
authors.affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada; Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, ON, Canada; and.
authors.forename: Walter H A
authors.identifier: http://orcid.org/0000-0002-2832-7158
authors.initials: WH
authors.lastname: Kahr
authors.affiliation: Division of Hematology, Brigham and Women's Hospital, Boston, MA; Department of Medicine, Harvard Medical School, Boston, MA;
authors.forename: John H
authors.initials: JH
authors.lastname: Hartwig
authors.affiliation: Division of Hematology, Brigham and Women's Hospital, Boston, MA; Department of Medicine, Harvard Medical School, Boston, MA;
authors.forename: Hervé
authors.identifier: http://orcid.org/0000-0003-0788-9204
authors.initials: H
authors.lastname: Falet
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism / Q000648:ultrastructure; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D002478:Cells, Cultured; D064448:Filamins / Q000378:metabolism* / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D008533:Megakaryocytes / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D011485:Protein Binding / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D011554:Pseudopodia / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C578663:FLNA protein, human; D064448:Filamins; C120714:PACSIN2 protein, human
references.citation: Biophys J. 2003 Apr;84(4):2646-54
references.pmid: 12668473
references.citation: J Cell Biol. 2000 Mar 6;148(5):1047-62
references.pmid: 10704453
references.citation: Science. 2004 Jan 23;303(5657):495-9
references.pmid: 14645856
references.citation: J Virol. 1996 Aug;70(8):5701-5
references.pmid: 8764092
references.citation: FEBS Lett. 1999 Jul 9;454(3):356-62
references.pmid: 10431838
references.citation: Mol Biol Cell. 2005 Aug;16(8):3642-58
references.pmid: 15930129
references.citation: Dev Cell. 2005 Dec;9(6):791-804
references.pmid: 16326391
references.citation: Mol Cell. 2006 Feb 3;21(3):337-47
references.pmid: 16455489
references.citation: Blood. 2006 Mar 1;107(5):1925-32
references.pmid: 16293600
references.citation: J Cell Sci. 2006 Apr 15;119(Pt 8):1504-16
references.pmid: 16551695
references.citation: Blood. 2006 May 15;107(10):3868-75
references.pmid: 16434494
references.citation: Blood. 2006 Jul 1;108(1):134-40
references.pmid: 16522820
references.citation: Biochim Biophys Acta. 2006 Aug;1761(8):897-912
references.pmid: 16938488
references.citation: Cell. 2007 May 18;129(4):761-72
references.pmid: 17512409
references.citation: Structure. 2007 Jul;15(7):839-52
references.pmid: 17540576
references.citation: EMBO J. 2007 Sep 5;26(17):3993-4004
references.pmid: 17690686
references.citation: J Cell Biol. 2007 Dec 3;179(5):1011-25
references.pmid: 18056414
references.citation: Cell. 2008 Mar 7;132(5):807-17
references.pmid: 18329367
references.citation: Dev Biol. 2008 Jul 1;319(1):86-99
references.pmid: 18495106
references.citation: Blood. 2011 Nov 24;118(22):5928-37
references.pmid: 21960593
references.citation: Blood. 2011 Oct 6;118(14):e101-11
references.pmid: 21596849
references.citation: Proc Natl Acad Sci U S A. 2009 Aug 4;106(31):12700-5
references.pmid: 19549836
references.citation: Proc Natl Acad Sci U S A. 2012 Mar 6;109(10):3760-5
references.pmid: 22355135
references.citation: Annu Rev Biophys. 2012;41:227-46
references.pmid: 22404683
references.citation: Cardiovasc Res. 2012 Jun 1;94(3):526-36
references.pmid: 22298643
references.citation: J Biol Chem. 2012 Jun 22;287(26):22387-96
references.pmid: 22573331
references.citation: Histochem Cell Biol. 2012 Aug;138(2):215-30
references.pmid: 22718246
references.citation: Blood. 2012 Oct 11;120(15):e73-82
references.pmid: 22869793
references.citation: Cell Signal. 2013 Jan;25(1):236-46
references.pmid: 22975681
references.citation: Arterioscler Thromb Vasc Biol. 2013 Jan;33(1):e11-8
references.pmid: 23117662
references.citation: J Biol Chem. 2012 Dec 21;287(52):43438-53
references.pmid: 23129763
references.citation: Platelets. 2013;24(1):1-5
references.pmid: 22372530
references.citation: Arterioscler Thromb Vasc Biol. 2013 Jul;33(7):1538-43
references.pmid: 23619296
references.citation: Blood. 2013 Sep 5;122(10):1695-706
references.pmid: 23881916
references.citation: Blood. 2013 Nov 7;122(19):3349-58
references.pmid: 23861251
references.citation: Blood. 2014 Feb 6;123(6):921-30
references.pmid: 24152908
references.citation: Cell Signal. 2014 Sep;26(9):1975-84
references.pmid: 24703939
references.citation: Blood. 2014 Oct 16;124(16):2554-63
references.pmid: 25061177
references.citation: J Biol Chem. 2009 Sep 11;284(37):25450-8
references.pmid: 19622754
references.citation: Blood. 2009 Nov 19;114(21):4729-37
references.pmid: 19692704
references.citation: FEBS Lett. 2010 Mar 19;584(6):1111-8
references.pmid: 20188097
references.citation: Proc Natl Acad Sci U S A. 2010 May 4;107(18):8213-8
references.pmid: 20404169
references.citation: J Mol Biol. 2010 Jul 9;400(2):129-36
references.pmid: 20471395
references.citation: J Cell Mol Med. 2010 Jun;14(6B):1569-93
references.pmid: 19656240
references.citation: Blood. 2010 Aug 5;116(5):793-800
references.pmid: 20457868
references.citation: Blood. 2010 Aug 19;116(7):1147-56
references.pmid: 20439620
references.citation: J Exp Med. 2010 Aug 30;207(9):1967-79
references.pmid: 20713593
references.citation: J Cell Sci. 2011 Jun 15;124(Pt 12):2032-40
references.pmid: 21610094
references.citation: J Cell Sci. 2011 Jul 15;124(Pt 14):2375-88
references.pmid: 21693584
references.citation: J Cell Sci. 2011 Aug 15;124(Pt 16):2777-85
references.pmid: 21807942
references.citation: Blood. 2011 Aug 25;118(8):2285-95
references.pmid: 21652675
references.citation: EMBO J. 2011 Aug 31;30(17):3501-15
references.pmid: 21878992
references.citation: J Cell Sci. 2000 Dec;113 Pt 24:4511-21
references.pmid: 11082044
references.citation: J Biol Chem. 2002 Mar 29;277(13):11432-40
references.pmid: 11790794
references.citation: Blood. 2002 Sep 15;100(6):2113-22
references.pmid: 12200375
references.citation: EMBO J. 2002 Nov 15;21(22):6083-94
references.pmid: 12426380
references.citation: Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16782-7
references.pmid: 12464680
references.citation: Blood. 2015 Feb 5;125(6):1014-24
references.pmid: 25468568
references.citation: Protein Cell. 2013 Sep;4(9):695-701
references.pmid: 23888307
references.citation: J Thromb Haemost. 2003 May;1(5):1062-70
references.pmid: 12871378