Direct Real-time Observation of Actin Filament Branching Mediated by Arp2/3 Complex Using Total Internal Reflection Fluorescence Microscopy

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2001 Dec 14

PMID 11742068

Citations 123

Authors

K J Amann

T D Pollard

Affiliations

Soon will be listed here.

Abstract

Existing methods for studying actin filament dynamics have allowed analysis only of bulk samples or individual filaments after treatment with the drug phalloidin, which perturbs filament dynamics. Total internal reflection fluorescence microscopy with rhodamine-labeled actin allowed us to observe polymerization in real time, without phalloidin. Direct measurements of filament growth confirmed the rate constants measured by electron microscopy and established that rhodamine actin is a kinetically inactive tracer for imaging. In the presence of activated Arp2/3 complex, growing actin filaments form branches at random sites along their sides, rather than preferentially from their barbed ends.

Citing Articles

Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein.

Pimm M, Haarer B, Nobles A, Haney L, Marcin A, Alcaide Eligio M J Cell Biol. 2024; 223(9).

PMID: 38787349 PMC: 11117073. DOI: 10.1083/jcb.202305065.

Cooperative actin filament nucleation by the Arp2/3 complex and formins maintains the homeostatic cortical array in Arabidopsis epidermal cells.

Xu L, Cao L, Li J, Staiger C Plant Cell. 2023; 36(3):764-789.

PMID: 38057163 PMC: 10896301. DOI: 10.1093/plcell/koad301.

Inferring local molecular dynamics from the global actin network structure: A case study of 2D synthetic branching actin networks.

Rostami M, Bannish B, Gasior K, Pinals R, Copos C, Dawes A J Theor Biol. 2023; 575:111613.

PMID: 37774939 PMC: 10842209. DOI: 10.1016/j.jtbi.2023.111613.

Crowding alters F-actin secondary structure and hydration.

Chen X, Roeters S, Cavanna F, Alvarado J, Baiz C Commun Biol. 2023; 6(1):900.

PMID: 37660224 PMC: 10475093. DOI: 10.1038/s42003-023-05274-3.

Differential sensitivity of ADF isovariants to a pH gradient promotes pollen tube growth.

Wang J, Shen J, Xu Y, Jiang Y, Qu X, Zhao W J Cell Biol. 2023; 222(11).

PMID: 37610419 PMC: 10445753. DOI: 10.1083/jcb.202206074.

References

Mullins R, Heuser J, Pollard T . The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc Natl Acad Sci U S A. 1998; 95(11):6181-6. PMC: 27619. DOI: 10.1073/pnas.95.11.6181. View

Pantaloni D, Boujemaa R, Didry D, Gounon P, Carlier M . The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins. Nat Cell Biol. 2000; 2(7):385-91. DOI: 10.1038/35017011. View

Robinson R, Turbedsky K, Kaiser D, Marchand J, Higgs H, Choe S . Crystal structure of Arp2/3 complex. Science. 2001; 294(5547):1679-84. DOI: 10.1126/science.1066333. View

Ichetovkin I, Han J, Pang K, Knecht D, Condeelis J . Actin filaments are severed by both native and recombinant dictyostelium cofilin but to different extents. Cell Motil Cytoskeleton. 2000; 45(4):293-306. DOI: 10.1002/(SICI)1097-0169(200004)45:4<293::AID-CM5>3.0.CO;2-1. View

Dabiri G, Sanger J, Portnoy D, Southwick F . Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc Natl Acad Sci U S A. 1990; 87(16):6068-72. PMC: 54473. DOI: 10.1073/pnas.87.16.6068. View

Gouin E, Hansen G, Samarin S, Le Clainche C, Didry D, Dehoux P . Listeria protein ActA mimics WASp family proteins: it activates filament barbed end branching by Arp2/3 complex. Biochemistry. 2001; 40(38):11390-404. DOI: 10.1021/bi010486b. View

Volkmann N, Amann K, Stoilova-McPhie S, Egile C, Winter D, Hazelwood L . Structure of Arp2/3 complex in its activated state and in actin filament branch junctions. Science. 2001; 293(5539):2456-9. DOI: 10.1126/science.1063025. View

Pollard T, Mooseker M . Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores. J Cell Biol. 1981; 88(3):654-9. PMC: 2112767. DOI: 10.1083/jcb.88.3.654. View

Amann K, Pollard T . The Arp2/3 complex nucleates actin filament branches from the sides of pre-existing filaments. Nat Cell Biol. 2001; 3(3):306-10. DOI: 10.1038/35060104. View

10.

Andrianantoandro E, Blanchoin L, Sept D, McCammon J, Pollard T . Kinetic mechanism of end-to-end annealing of actin filaments. J Mol Biol. 2001; 312(4):721-30. DOI: 10.1006/jmbi.2001.5005. View

11.

Boldogh I, Yang H, Nowakowski W, Karmon S, Hays L, Yates 3rd J . Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast. Proc Natl Acad Sci U S A. 2001; 98(6):3162-7. PMC: 30624. DOI: 10.1073/pnas.051494698. View

12.

Spudich J, Watt S . The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971; 246(15):4866-71. View

13.

Bearer E . Direct observation of actin filament severing by gelsolin and binding by gCap39 and CapZ. J Cell Biol. 1991; 115(6):1629-38. PMC: 2289206. DOI: 10.1083/jcb.115.6.1629. View

14.

Blanchoin L, Amann K, Higgs H, Marchand J, Kaiser D, Pollard T . Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins. Nature. 2000; 404(6781):1007-11. DOI: 10.1038/35010008. View

15.

Maciver S, Zot H, Pollard T . Characterization of actin filament severing by actophorin from Acanthamoeba castellanii. J Cell Biol. 1991; 115(6):1611-20. PMC: 2289216. DOI: 10.1083/jcb.115.6.1611. View

16.

Bonder E, Fishkind D, Mooseker M . Direct measurement of critical concentrations and assembly rate constants at the two ends of an actin filament. Cell. 1983; 34(2):491-501. DOI: 10.1016/0092-8674(83)90382-3. View

17.

Kaiser D, Sato M, Ebert R, Pollard T . Purification and characterization of two isoforms of Acanthamoeba profilin. J Cell Biol. 1986; 102(1):221-6. PMC: 2114039. DOI: 10.1083/jcb.102.1.221. View

18.

Higgs H, Blanchoin L, Pollard T . Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization. Biochemistry. 1999; 38(46):15212-22. DOI: 10.1021/bi991843+. View

19.

Pollard T, Blanchoin L, Mullins R . Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Struct. 2000; 29:545-76. DOI: 10.1146/annurev.biophys.29.1.545. View

20.

Pollard T . Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J Cell Biol. 1986; 103(6 Pt 2):2747-54. PMC: 2114620. DOI: 10.1083/jcb.103.6.2747. View