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Human CAP1 is a key factor in the recycling of cofilin and actin for rapid actin turnover

Kenji Moriyama* and Ichiro Yahara{ddagger}

Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
{ddagger} Present address: Medical & Biological Laboratories Co., Ltd., 1063-103, Ohara, Terasawaoka, Ina-city, 396-0002, Japan



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  		Fig. 1. Identification of human cellular proteins associated with the cofilin-actin complex. (A) HEK293 cells were transfected with either a plasmid that drove the expression of Cof-His6 (lane +), or a control vector (lane -). Cell lysate (3 mg protein equivalent) was incubated with Ni2+-NTA resin to adsorb Cof-His6 and associated proteins. Bound material was eluted with step-wise increments in the concentration of NaCl. The eluate was electrophoresed on a 10-20% polyacrylamide gradient gel and silver stained. The lanes of the 300 mM NaCl eluates ares shown. The arrowheads point to bands that were recovered specifically from Cof-His6-producing cells. A small amount of Cof-His6 leaked out of the resin as a result of NaCl washing (arrow). (B) HA-tagged actin-binding proteins (p57WD, fascin, CAP1 and EF1{alpha}) were expressed separately in HEK293 cells with (+) or without (-) co-expression of Cof-His6. Cof-His6-associated protein fractions were prepared as above. Crude lysates (lysate) and 500 mM NaCl eluates (bound) were subjected to western blotting with an anti-HA MAb (clone 12CA5). The size of the bands is around 55kDa. (C) Anti-CAP1 blotting of p55. Crude lysate (lane 1) and 300 mM NaCl eluates from Ni2+-resin (lanes 2 and 3) were subjected to western blotting with an affinity-purified rat IgG to CAP1. The NaCl eluate from Cof-His6-producing cells is clearly positive for the CAP1 signal (lane 3), in contrast to the eluate from non-expressing cells (lane 2).

 


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  		Fig. 2. Purification of His6-CAP1. HEK293 cells were transfected with pUSH-CAP1. The cell lysate was prepared (lane 2), and His6-CAP1 was successively enriched upon a Ni2+-NTA- (lane 3) and a SP-Sepharose column (lane 4). Although gel filtration through a Superdex 200 column could not separate bound G-actin from His6-CAP1 (lane 5), bound actin was liberated by treating the Ni2+-column-bound His6-CAP1-actin complex with urea. Pure His6-CAP1 was then eluted with imidazole after the urea was removed (lane 6). Molecular size standards were run on lane 1.

 


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  		Fig. 3. Effects of human CAP1 on actin dynamics. (A) His6-CAP1 promotes the turnover of F-actin in the presence of cofilin. 6 µM 1,N6-etheno ATP ({epsilon}ATP)-actin was polymerized with or without cofilin and/or His6-CAP1. The intensity of {epsilon}ATP fluorescence is higher when it is bound to actin (Wang and Taylor, 1981Go). After a steady state was reached, excess unlabeled ATP was added to chase actin-bound {epsilon}ATP, and the decline in fluorescence was recorded. Although binding of cofilin to {epsilon}ATP-F-actin was observed to increase the basal intensity of fluorescence, it was possible to semi-quantitatively evaluate CAP1 activity. The legends of each graph state the protein concentration in µM. `Cof' designates bacterially produced porcine cofilin without a His-tag. (B) Effect of His6-CAP1 on the rate of actin depolymerization. Gelsolin-capped actin filaments (10%-pyrene labeled) were diluted 20-fold in the presence or absence of His6-CAP1 and/or cofilin. The fluorescence intensity was recorded to monitor gradual depolymerization. Pyrene-labeled actin is more fluorescent when it is in F-actin than in its unpolymerized state (Kouyama and Mihashi, 1981Go). (C) Subunit exchange assay using F-actin seeds with free barbed ends. Unlabeled F-actin was mixed with cofilin and/or His6-CAP1, then a small amount of pyrene-labeled Mg-G-actin was immediately added. The incorporation of pyrene-actin into unlabeled F-actin was monitored by the change in fluorescence. Final concentrations of pyrene-actin and unlabeled actin were 0.2 µM and 2.0 µM, respectively.

 


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  		Fig. 5. Effects of the N- and C-terminal domains of human CAP1 on actin dynamics. (A) Effect of the CAP1 domains on the turnover of F-actin. {epsilon}ATP-actin was polymerized as in Fig. 3A. After a steady state was reached, unlabeled ATP was added and the decline in fluorescence was recorded. 2.4 CT and 2.4 NT represent 2.4 µM CAP1-CT and 2.4 µM CAP1-NT, respectively. (B) Effect of the CAP1 domains on the rate of actin depolymerization. Gelsolincapped actin filaments (10%-pyrene labeled) were diluted in a solution containing the CAP1 domains with or without 0.1 µM of cofilin. When both CAP1 domains were simultaneously used, CAP1-NT was added at a final concentration of 1 µM (triangles, plots c and e). The gradual depolymerization was monitored as in Fig. 3B. The apparent rate constant (kapp) of each curve was calculated and plotted as a function of the amount of the respective CAP1-domains. 6 µM vitamin-D-binding protein (DBP) was used as a control actin-sequestering protein, which does not affect the depolymerization rate constant at the pointed end (Weber et al., 1994Go). (C,D) Effect of CAP1 domains on the rate of nucleotide exchange on G-actin. Mg-ADP-actin was mixed with cofilin or its solvent for 3 minutes. Then, {epsilon}ATP and CAP1 domains were simultaneously added and the exchange of actin-bound ADP to {epsilon}ATP was monitored as fluorescence increased. Final concentrations of actin, cofilin, CAP1-NT and {epsilon}ATP were 1 µM, 1.5 µM, 2 µM and 50 µM, respectively. The exponential curve was fitted for each trace and drawn in the graph C. The apparent rate constants (kapp) of exchange reactions were calculated and plotted as a function of the amount of CAP1 or CAP1-CT (D). (E) Subunit exchange assay using F-actin seeds with free barbed ends. Unlabeled F-actin was mixed with cofilin and/or the CAP1 domains, then a small amount of pyrene-labeled Mg-G-actin was immediately added. The incorporation of pyrene-actin into unlabeled F-actin was monitored as in Fig. 3C. The maximal rate (near initial rate) of each fluorescence trace was derived, normalized and plotted as a function of the amount of CAP1 or CAP1-CT. The results with the use of 1 µM cofilin were plotted by filled symbols, and those with the use of 1 µM CAP1-NT were shown by triangles.

 


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  		Fig. 4. The N-terminal domain of CAP1 is responsible for its cofilin-dependent interaction with unpolymerized actin. (A) The cofilin-actin complex interacts with CAP1-NT but not with CAP1-CT. G-actin (29 µM) was converted to the Mg-ATP-bound form by a 5 minute incubation in 40 µM MgCl2 plus 0.5 mM EGTA. 15 µM of each His6-tagged CAP1 domain was mixed with 7.2 µM of G-actin, 15 µM cofilin, or both, in a physiological salt buffer (lanes 1-6) or in a low salt buffer (lanes 7-12) at 2°C. Then, His6-tagged proteins and associated molecule(s) were adsorbed to a Ni2+-resin. The resin was washed four times with 10 mM Tris-HCl, 1 mM MgCl2, 50 mM KCl, 0.01% Triton X-100, 0.1 mM ATP, 0.1 mM DTT (pH 7.5), then boiled in SDS sample buffer. The bound and unbound fractions were analyzed by SDS-PAGE. NT-His designates the His6-tagged N-terminal domain (amino acids 1-229) of CAP1, and HSE-CT designates the C-terminal domain (amino acids 255-475) carrying both His6- and S-tags. (B,C) Co-sedimentation of the CAP1 domains with F-actin. F-actin (7.2 µM for B and 6 µM for C) was reacted with various amounts of CAP1-CT (B) or CAP1-NT (C) in the presence (+ cof) or absence (- cof) of cofilin (4 µM cofilin for B and 6 µM for C). After 6 hours, the samples were centrifuged at 190,000 g for 20 minutes with a Beckman TLA100 rotor. The supernatant and precipitate were subjected to SDS-PAGE. The amount of sedimented protein was quantified with a densitometer and plotted as a function of the amount of the respective CAP1 domains. Both of the CAP domains did not sediment in the absence of F-actin (data not shown).

 


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  		Fig. 6. Colocalization of CAP1 with cofilin, and analysis of factors that determine its localization.

(A,B) Spreading mouse C3H-2K fibroblasts were fixed and incubated with a rat anti-CAP1 IgG and a rabbit antibody to actin (A) or cofilin (B), followed by labelling with a Cy3-labeled anti-rat IgG and a fluorescein-labeled anti-rabbit IgG. The lamellipodia are indicated by arrowheads, whereas the ruffling areas are indicated by arrows.

(C) HA-tagged CAP1-NT was transiently expressed in C3H-2K cells (panels a-d) by lipofection. HA-tagged CAP1-CT (panels e and f) or HA-tagged CAP2-NT (panels g and h) were expressed as well. Transfected cells were split onto coverslips, and the spreading cells were fixed and reacted with a mouse anti-HA and a rabbit antibody to actin (panels a, b, e and f) or to cofilin (panels c, d, g and h), after which they were stained with a Cy3-labeled anti-mouse IgG and a fluorescein-labeled anti rabbit IgG. The lamellipodia are indicated by arrowheads (for CAP-NTs) or arrows (for CAP1-CT). The white bar represents 10 µm.

 


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  		Fig. 7. A schematic model of co-operation between CAP1 and cofilin in promotion of actin dynamics. The working steps of CAP1 are indicated by open arrowheads. (1) CAP1 facilitates the addition of Mg-ATP-actin monomer onto the barbed end of actin filament. Cofilin-induced severing also contributes to this step by increasing the number of barbed ends. (2) CAP1 accelerates subunit release at the pointed end and enhances the more potent, analogous effect of cofilin. (3) CAP1 relieves the inhibitory effect of cofilin on nucleotide exchange of ADP-actin. (4) CAP1 accelerates nucleotide exchange on G-actin.

 




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