Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy

Mohri

Journal of Biological Chemistry

2004

Actin-depolymerizing factor (ADF)/cofilin and gelsolin are the two major factors to enhance actin filament disassembly. Actin-interacting protein 1 (AIP1) enhances fragmentation of ADF/cofilin-bound filaments and caps the barbed ends. However, the mechanism by which AIP1 disassembles ADF/cofilin-bound filaments is not clearly understood. Here, we directly observed the effects of these proteins on filamentous actin by fluorescence microscopy and gained novel insight into the function of ADF/cofilin and AIP1. ADF/cofilin severed filaments and AIP1 strongly enhanced disassembly at nanomolar concentrations. However, gelsolin, gelsolinactin complex, or cytochalasin D did not enhance disassembly by ADF/cofilin, suggesting that the strong activity of AIP1 cannot be explained by simple barbed end capping. Barbed end capping by ADF/cofilin and AIP1 was weak and allowed filament elongation, whereas gelsolin or gelsolin-actin complex strongly capped and inhibited elongation. These results suggest that AIP has an active role in filament severing or depolymerization and that ADF/cofilin and AIP1 are distinct from gelsolin in modulating filament elongation.

Section: Discussionmentioning

confidence: 82%

Microscopic Evidence That Actin-interacting Protein 1 Actively Disassembles Actin-depolymerizing Factor/Cofilin-bound Actin Filaments

Mohri

Journal of Biological Chemistry

2004

“…Actin was labeled on Cys-374 with pyrene iodoacetamide (Pollard, 1984) and Oregon-green 488 iodoacetamide (Amann and Pollard, 2001). AFH1 FH1FH2 was purified according to Michelot et al (2005), and Arabidopsis profilin 4 was purified roughly according to the purification procedure of maize (Zea mays) recombinant profilin (Gibbon et al, 1997).…”

Section: Protein Productionmentioning

confidence: 99%

“…The flow cell was made as described previously (Amann and Pollard, 2001). The slide was coated with 100 nM NEM-Myosin in the absence or presence of AFH3 FH1FH2 and AFH3 FH2 for 2 to 4 min and was subsequently equilibrated with 1% BSA.…”

Section: Direct Visualization Of Actin Assembly By Tirfmmentioning

confidence: 99%

ArabidopsisFormin3 Directs the Formation of Actin Cables and Polarized Growth in Pollen Tubes

et al. 2009

Cytoplasmic actin cables are the most prominent actin structures in plant cells, but the molecular mechanism underlying their formation is unknown. The function of these actin cables, which are proposed to modulate cytoplasmic streaming and intracellular movement of many organelles in plants, has not been studied by genetic means. Here, we show that Arabidopsis thaliana formin3 (AFH3) is an actin nucleation factor responsible for the formation of longitudinal actin cables in pollen tubes. The Arabidopsis AFH3 gene encodes a 785-amino acid polypeptide, which contains a formin homology 1 (FH1) and a FH2 domain. In vitro analysis revealed that the AFH3 FH1FH2 domains interact with the barbed end of actin filaments and have actin nucleation activity in the presence of G-actin or G actin-profilin. Overexpression of AFH3 in tobacco (Nicotiana tabacum) pollen tubes induced excessive actin cables, which extended into the tubes' apices.

Journal of Biological Chemistry

“…Frozen stocks were 2-4-fold less active than freshly prepared MmCP. Myosin from rabbit skeletal muscle (19) was treated with N-ethylmaleimide (Sigma) (20) and stored at Ϫ20°C in 10 mM imidazole, pH 7, 0.5 M KCl, 1 mM DTT, and 50% glycerol.…”

Section: Proteinsmentioning

confidence: 99%

Single Molecule Kinetic Analysis of Actin Filament Capping

Kuhn

Pollard

2007

Self Cite

We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy. The barbed end rate constants for mouse capping protein (CP) association of 2.6 ؋ 10 6 M ؊1 s ؊1 and dissociation of 0.0003 s ؊1 agree with published values measured in bulk assays. The polyphosphoinositides (PPIs), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ), PI(4,5)P 2 , and PI(3,4,5)P 3 , prevent CP from binding to barbed ends, but three different assays showed that none of these lipids dissociate CP from filaments at concentrations that block CP binding to barbed ends. The affinity of fission yeast CP for barbed ends is a thousandfold less than mouse CP, because of a slower association rate constant (1.1 ؋ 10 5 M ؊1 s ؊1 ) and a faster dissociation rate constant (0.004 s ؊1 ). PPIs do not inhibit binding of fission yeast CP to filament ends. Comparison of homology models revealed that fission yeast CP lacks a large patch of basic residues along the actin-binding surface on mouse CP. PPIs binding to this site might interfere sterically with capping, but this site would be inaccessible when CP is bound to the end of a filament.The motility of animal cells depends on the coordinated assembly of actin filaments at the leading edge. Arp2/3 complex promotes actin polymerization by nucleating filaments that grow as branches from a mother filament (1, 2). Capping protein (CP, 3 called CapZ in muscle) appears to play an important role in generating the zone of short, highly branched actin filaments at the leading edge. CP is a heterodimer of structurally similar ␣ and ␤ subunits that form a mushroom-like structure with one flexible and one immobile COOH-terminal "tentacle" that bind the two terminal subunits of an actin filament (3). CP binds the barbed end of actin filaments tightly with a K d of 0.1-1 nM. Given an association rate constant of 3-4 M Ϫ1 s Ϫ1 and a cytoplasmic concentration of 1 M (4), CP should terminate the growth of actin filaments with a half-time of about 0.2 s after their nucleation. Terminating growth quickly keeps the filaments short, so that they do not buckle under the force of polymerization as they push the membrane forward.Vertebrate CP dissociates from barbed ends with a half-time of 30 min (4), far too slow for simple, spontaneous dissociation to be involved with the conversion of the network of short, branched filaments at the leading edge to longer, unbranched filaments toward the cell body. As CP prevents both dissociation of subunits from the barbed end and end-to-end filament annealing, some mechanism must accelerate the removal of CP from filament ends.Both lipids and proteins, such as Ena/VASP (5-7) and CARMIL (8), can inhibit the interaction of CP with actin filaments. Polyphosphatidylinositides (PPIs) bind CP and block its interaction with filaments both in vivo (9) and in vitro (10). Addition of PIP 2 to mixtures of capped filaments, CP, and actin monomers in vi...