Regulation of CapZ, an actin capping protein of chicken muscle, by anionic phospholipids

Heiss, Steven G.; Cooper, John A.

doi:10.1021/bi00100a006

Cited by 99 publications

(78 citation statements)

References 38 publications

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“…We confirmed earlier observations that PI(3,4)P 2 and PI(4,5)P 2 inhibit capping in vitro (4,10) at the single filament level, but unlike Schafer (4), we also found a substantial inhibition of capping by PI(3,4,5)P 3 . Assuming a bimolecular reaction of MmCP with PPI and that only free MmCP participates in capping, the dissociation equilibrium constants for MmCP binding PIP 2 and PIP 3 are in the range of 5-10 M in 1 mM MgCl 2 and about 10-fold lower in 0.1 mM MgCl 2 .…”

Section: Discussionsupporting

confidence: 92%

“…Under conditions where the polymerization rate depended on the concentration of ends, we confirmed (Fig. 4A) that addition of PIP 2 to bulk samples of capped actin filaments increased the concentration of ends (10,36). Polymerization was faster in PIP 2 and PIP 3 than in PI or control capped filaments (Fig.…”

Section: Effects Of Polyphosphoinositides On Capping and Uncapping Bysupporting

confidence: 61%

“…Capping proteins were dialyzed into Ca 2ϩ / Mg 2ϩ -free CP buffer (10 mM imidazole, pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mM EGTA, 2 mM NaN 3 ) containing 50% glycerol, flash-frozen in liquid N 2 , and stored at Ϫ80°C. We determined the activity of a frozen MmCP stock by equilibrating a range of MmCP concentrations with actin seeds and then measuring the rate of pyrene actin assembly from capped seeds (10). Frozen stocks were 2-4-fold less active than freshly prepared MmCP.…”

Section: Proteinsmentioning

confidence: 99%

“…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 vitro increases the rate of polymerization (4).…”

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confidence: 99%

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Single Molecule Kinetic Analysis of Actin Filament Capping

Kuhn

Pollard

2007

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 92%

Section: Effects Of Polyphosphoinositides On Capping and Uncapping Bysupporting

confidence: 61%

Section: Proteinsmentioning

confidence: 99%

mentioning

confidence: 99%

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Single Molecule Kinetic Analysis of Actin Filament Capping

Kuhn

Pollard

2007

Journal of Biological Chemistry

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“…Phosphoinositides inhibit the actinbinding activity of CP in vitro (Heiss and Cooper, 1991;, which is a poten-tial mechanism for coupling cell surface signals to changes in actin assembly and cell motility. CP forms a complex with hsc70 in Dictyostelium, which may affect its actin-binding activity (Eddy et al, 1993;Haus et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Mutational analysis of capping protein function in Saccharomyces cerevisiae.

Sizonenko

Karpova

Gattermeir

et al. 1996

MBoC

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To investigate physiologic functions and structural correlates for actin capping protein (CP), we analyzed site-directed mutations in CAP1 and CAP2, which encode the alpha and beta subunits of CP in Saccharomyces cerevisiae. Mutations in four different regions caused a loss of CP function in vivo despite the presence of mutant protein in the cells. Mutations in three regions caused a complete loss of all aspects of function, including the actin distribution, viability with sac6, and localization of CP to actin cortical patches. Mutation of the fourth region led to partial loss of only one function-formation of actin cables. Some mutations retained function and exhibited the complete wild-type phenotype, and some mutations led to a complete loss of protein and therefore loss of function. The simplest hypothesis that can explain these results is that a single biochemical property is necessary for all in vivo functions. This biochemical property is most likely binding to actin filaments, because the nonfunctional mutant CPs no longer co-localize with actin filaments in vivo and because direct binding of CP to actin filaments has been well established by studies with purified proteins in vitro. More complex hypotheses, involving the existence of additional biochemical properties important for function, cannot be excluded by this analysis.

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