title: Malaria parasite actin polymerization and filament structure.
abstract: A novel form of acto-myosin regulation has been proposed in which polymerization of new actin filaments regulates motility of parasites of the apicomplexan class of protozoa. In vivo and in vitro parasite F-actin is very short and unstable, but the structural basis and details of filament dynamics remain unknown. Here, we show that long actin filaments can be obtained by polymerizing unlabeled rabbit skeletal actin (RS-actin) onto both ends of the short rhodamine-phalloidin-stabilized Plasmodium falciparum actin I (Pf-actin) filaments. Following annealing, hybrid filaments of micron length and "zebra-striped" appearance are observed by fluorescence microscopy that are stable enough to move over myosin class II motors in a gliding filament assay. Using negative stain electron microscopy we find that pure Pf-actin stabilized by jasplakinolide (JAS) also forms long filaments, indistinguishable in length from RS-actin filaments, and long enough to be characterized structurally. To compare structures in near physiological conditions in aqueous solution we imaged Pf-actin and RS-actin filaments by atomic force microscopy (AFM). We found the monomer stacking to be distinctly different for Pf-actin compared with RS-actin, such that the pitch of the double helix of Pf-actin filaments was 10% larger. Our results can be explained by a rotational angle between subunits that is larger in the parasite compared with RS-actin. Modeling of the AFM data using high-resolution actin filament models supports our interpretation of the data. The structural differences reported here may be a consequence of weaker inter- and intra-strand contacts, and may be critical for differences in filament dynamics and for regulation of parasite motility.
authors.affiliation: Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
authors.forename: Stephan
authors.initials: S
authors.lastname: Schmitz
authors.forename: Iwan A T
authors.initials: IA
authors.lastname: Schaap
authors.forename: Jens
authors.initials: J
authors.lastname: Kleinjung
authors.forename: Simone
authors.initials: S
authors.lastname: Harder
authors.forename: Munira
authors.initials: M
authors.lastname: Grainger
authors.forename: Lesley
authors.initials: L
authors.lastname: Calder
authors.forename: Peter B
authors.initials: PB
authors.lastname: Rosenthal
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Holder
authors.forename: Claudia
authors.initials: C
authors.lastname: Veigel
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000648:ultrastructure; D000199:Actins / Q000737:chemistry* / Q000648:ultrastructure; D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000737:chemistry* / Q000648:ultrastructure; D016778:Malaria, Falciparum / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D010590:Phalloidine / Q000031:analogs & derivatives / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000378:metabolism* / Q000648:ultrastructure; D011817:Rabbits; D012235:Rhodamines / Q000494:pharmacology
chemical_list: D000199:Actins; D012235:Rhodamines; C504731:rhodamine-phalloidin; D010590:Phalloidine
references.citation: J Cell Biol. 1997 Aug 25;138(4):771-81
references.pmid: 9265645
references.citation: Microbes Infect. 1999 Jul;1(9):653-62
references.pmid: 10611742
references.citation: J Cell Biol. 1987 Apr;104(4):1005-17
references.pmid: 3558475
references.citation: Proc Natl Acad Sci U S A. 2007 Nov 6;104(45):17656-61
references.pmid: 17978193
references.citation: Nat Rev Microbiol. 2006 Aug;4(8):621-8
references.pmid: 16845432
references.citation: Biochemistry. 1975 Jul 15;14(14):3250-6
references.pmid: 1148203
references.citation: Mol Biochem Parasitol. 1988 Aug;30(2):143-53
references.pmid: 2459617
references.citation: Trends Parasitol. 2006 Apr;22(4):146-7
references.pmid: 16500149
references.citation: Mol Biol Cell. 2000 Jan;11(1):355-68
references.pmid: 10637313
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: J Struct Biol. 1999 Dec 1;128(1):82-97
references.pmid: 10600563
references.citation: J Biol Chem. 1992 Oct 5;267(28):20339-45
references.pmid: 1400353
references.citation: Parasitol Res. 2002 Sep;88(9):844-8
references.pmid: 12172817
references.citation: FEBS Lett. 2005 Jan 31;579(3):655-60
references.pmid: 15670824
references.citation: Science. 2003 Sep 12;301(5639):1503-8
references.pmid: 12893887
references.citation: Traffic. 2006 Nov;7(11):1433-9
references.pmid: 17010119
references.citation: Mol Biol Cell. 2005 Sep;16(9):4013-23
references.pmid: 15975905
references.citation: Methods Enzymol. 1982;85 Pt B:164-81
references.pmid: 7121269
references.citation: Methods. 2003 Feb;29(2):142-52
references.pmid: 12606220
references.citation: Biophys J. 1989 Feb;55(2):293-8
references.pmid: 2713442
references.citation: Biochim Biophys Acta. 2001 Oct 18;1549(2):137-47
references.pmid: 11690650
references.citation: J Biol Chem. 2007 Dec 14;282(50):36133-7
references.pmid: 17965017
references.citation: Cell. 1996 Mar 22;84(6):933-9
references.pmid: 8601316
references.citation: Biophys J. 1998 Sep;75(3):1424-38
references.pmid: 9726944
references.citation: J Mol Biol. 1993 Dec 5;234(3):826-36
references.pmid: 8254675
references.citation: Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):29-34
references.pmid: 9874766
references.citation: J Cell Biol. 1984 Sep;99(3):769-77
references.pmid: 6540783
references.citation: Cell Host Microbe. 2008 Mar 13;3(3):188-98
references.pmid: 18329618
references.citation: Cell Motil Cytoskeleton. 1997;37(3):253-62
references.pmid: 9227855
references.citation: Proc Natl Acad Sci U S A. 2007 May 22;104(21):8827-32
references.pmid: 17517656
references.citation: J Biol Chem. 1988 Nov 25;263(33):17429-36
references.pmid: 3182855
references.citation: Biophys J. 2001 Feb;80(2):841-51
references.pmid: 11159451
references.citation: Curr Biol. 2002 Apr 2;12(7):570-5
references.pmid: 11937026
references.citation: Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9095-9
references.pmid: 10430901
references.citation: Rev Sci Instrum. 2007 Jan;78(1):013705
references.pmid: 17503926
references.citation: Mol Microbiol. 1997 Oct;26(1):163-73
references.pmid: 9383198
references.citation: Proc Natl Acad Sci U S A. 1986 Sep;83(17):6272-6
references.pmid: 3462694
references.citation: FEBS Lett. 2005 Jan 3;579(1):6-10
references.pmid: 15620683
references.citation: J Biol Chem. 1994 May 27;269(21):14869-71
references.pmid: 8195116
references.citation: Biochim Biophys Acta. 1990 Aug 17;1035(2):197-200
references.pmid: 2393669
references.citation: J Res Natl Inst Stand Technol. 1997 Jul-Aug;102(4):425-454
references.pmid: 27805154
references.citation: Mol Biol Cell. 2003 Feb;14(2):396-406
references.pmid: 12589042
references.citation: Mol Biol Cell. 2006 Feb;17(2):895-906
references.pmid: 16319175
references.citation: Nature. 2004 Sep 2;431(7004):104-7
references.pmid: 15343340
references.citation: J Struct Biol. 1996 Jan-Feb;116(1):190-9
references.pmid: 8742743
references.citation: J Cell Sci. 2003 Jan 1;116(Pt 1):39-49
references.pmid: 12456714
references.citation: Biophys J. 2007 Mar 15;92(6):2199-211
references.pmid: 17208981
references.citation: Biochemistry. 1991 Feb 19;30(7):1973-80
references.pmid: 1899622
references.citation: BMC Genomics. 2005 Dec 12;6:179
references.pmid: 16343347
references.citation: Protein Profile. 1995;2(1):1-103
references.pmid: 8548558
references.citation: Methods Enzymol. 1982;85 Pt B:55-71
references.pmid: 6214692
references.citation: Eur Biophys J. 2004 Aug;33(5):462-7
references.pmid: 14762705
references.citation: Mol Biochem Parasitol. 1988 Jan 15;27(2-3):313-20
references.pmid: 3278231
references.citation: Science. 2007 Mar 2;315(5816):1270-4
references.pmid: 17332412
references.citation: Nature. 1982 Jul 8;298(5870):131-5
references.pmid: 7201078
references.citation: Biochemistry. 1988 Oct 4;27(20):7766-72
references.pmid: 3207708
references.citation: J Mol Biol. 2005 May 27;349(1):113-25
references.pmid: 15876372
references.citation: Biophys J. 2000 Apr;78(4):2180-5
references.pmid: 10733996
references.citation: Biochemistry. 1996 Nov 12;35(45):14054-61
references.pmid: 8916890
references.citation: Adv Parasitol. 2004;56:213-63
references.pmid: 14710998
references.citation: CRC Crit Rev Biochem. 1978 Dec;5(3):185-260
references.pmid: 363352