Actins R62D, G13R, VP.C, and G150P can be quantitatively removed from cells by detergent extraction. NIH3T3 cells were transiently transfected with expression plasmids encoding FLAG-actin or mutant derivatives as in Figure 1, but the cells were subjected to brief extraction with 0.5% Triton X-100 before fixation and staining. Left, transfected actins (anti-FLAG; green). Right, merge of transfected actins (anti-FLAG; green), total F-actin (rhodamine-phalloidin; red) and transfection marker (anti-LexA; blue). Bar, 50 μm.

Actins R62D, G13R, VP.C, and G150P do not colocalize with cellular F-actin structures. NIH3T3 cells were transiently transfected with expression plasmids encoding FLAG-actin or mutant derivatives together with the nuclear-localized LexA derivative NLexA as transfection marker. Transfected cells were formaldehyde fixed and stained as described in MATERIALS AND METHODS. Left, transfected actins (anti-FLAG; green). Right, merge of transfected actins (anti-FLAG; green), total F-actin (rhodamine-phalloidin; red) and transfection marker (anti-LexA; blue). Bar, 50 μm.

SRF activation does not require CRM1. (A) Leptomycin B does not inhibit serum activation of SRF. NIH3T3 cells transfected with 3D.ALuc were serum starved and restimulated in the presence or absence of 5 nM leptomycin B (LMB). (B) Leptomycin B does not inhibit SRF transcriptional shutdown after serum stimulation. Total cell RNA was prepared from 3D.A-fosHA cells, which contain a stably integrated SRF reporter gene, at various times after serum stimulation after pretreatment with 10 nM LMB as indicated. Transcripts of the 3D.A-fosHA reporter, endogenous c-fos, and control GAPDH were analyzed by RNase protection. An identical result was obtained using 50 nM LMB. (C) Quantification of RNase protection assay. The experiment in B was analyzed using the PhosphorImager. Charts show reporter and c-fos transcript levels relative to GAPDH control.

Nonpolymerizable actin mutants inhibit SRF activation. (A) Actins G13R and R62D block serum-induced SRF activation. NIH3T3 cells were transfected with 0.2, 0.5, 1.0, or 2.0 μg of expression plasmid encoding FLAG-tagged wild-type actin, actin G13R, actin R62D, or empty vector, together with the SRF reporter 3D.ALuc. After serum stimulation, luciferase activities were measured and expressed relative to activation by SRF-VP16, taken as 100. Inset shows a FLAG immunoblot of cell extracts. Data show mean of at least three independent experiments with SEM indicated by the bars. (B) Actin/VP-16 fusion proteins inhibit SRF activation. NIH3T3 cells were transfected with expression plasmids encoding FLAG-tagged wild-type actin, NLS-actin, actin VP.N, actin NLS-VP.N (0.5 μg each), or actin VP.C (2 μg), or empty vector, together with the SRF reporter gene 3D.ALuc, and reporter activity was analyzed as in A. (C) CCT-binding mutant actin G150P does not inhibit SRF activation. Cells were transfected with the indicated mutants and 3D.ALuc as in A, except that activities were normalized to a cotransfected tk-luc reference plasmid rather than total recovered protein to minimize variations arising from the toxicity of actin G150P. (D) Nonpolymerizable actins inhibit SRF activation by cytochalasin D. Cells were transfected with actin expression plasmids (0.5, 1.0, or 2.0 μg) and 3D.ALuc and analyzed as in C, with stimulation by 10 μM cytochalasin D before analysis of reporter gene activity.

Actins G13R, R62D, and VP.C are soluble and do not enter the F-actin pool. (A) Solubility. Cells transfected with the indicated FLAG-tagged actin mutants were either left untreated (lanes 1 and 2) or treated with the actin-stabilizing drug jasplakinolide (0.3 μM; lanes 3 and 4) for 90 min before lysis and separation into 100,000-g supernatant (S) and pellet (P) fractions. Equal amounts were separated by SDS-PAGE, and proteins in each fraction were detected by immunoblotting by using anti-actin antibodies (top) or anti-FLAG antibodies (bottom). (B) G13R and R62D expression increase cellular F-actin substantially less than wild-type. Cells were transfected with the indicated FLAG-tagged actins; 36 h later they were fixed and stained for FLAG epitope and F-actin; F-actin levels in the transfected population were then quantitated relative to the untransfected population by using FACS, as described in MATERIALS AND METHODS. The relative increase in mean F-actin content is shown (mean of at least three independent experiments ± SEM). Asterisks indicate statistical significance at p < 0.01 according to the unpaired Student's t test.

Actins V159N and S14C stabilize F-actin and activate SRF. (A) Actins V159N and S14C colocalize with endogenous F-actin structures. NIH3T3 cells were transiently transfected with expression plasmids encoding FLAG-actin or mutant derivatives and subjected to brief extraction with 0.5% Triton X-100 before fixing and staining as in Figure 2. The Figure shows a merge of transfected actins (anti-FLAG; green), total F-actin (rhodamine-phalloidin; red), and transfection marker (anti-LexA; blue). Bar, 50 μm. (B) Actins V159N and S14C accumulate preferentially in the Triton X-100–insoluble fraction. Extracts of cells expressing the indicated FLAG-tagged actin mutants were prepared and separated into 100,000-g supernatant (S) and pellet (P) fractions as described in MATERIALS AND METHODS; equal amounts were separated by SDS-PAGE and FLAG-actin in each fraction was detected by immunoblotting with anti-FLAG antibodies. (C) Actins V159N and S14C copolymerize with wild-type actin and decrease its detergent solubility. Extracts were prepared from cells expressing the indicated actins, or the catalytic domain of LIMK1, together with HA-tagged wild-type actin and fractionated as in B before detection of HA-actin in each fraction by immunoblotting with anti-HA antibodies. (D) Expression of actins V159N and S14C increases F:G-actin ratio. Cells were transfected with expression plasmids encoding the indicated actins and fixed and stained for the FLAG epitope and either F-actin, with phalloidin, or G-actin, with DNase I. The levels of F-actin or G-actin in the transfected population were then quantitated relative to the untransfected population by using FACS, as described in MATERIALS AND METHODS. Bars represent the increase in mean relative F-actin (±SEM, n = 4) or G-actin (± half range, n = 2) contents. (E) Expression of actins V159N or S14C activates SRF in the absence of extracellular signals. NIH3T3 cells were transfected with expression plasmids encoding the indicated FLAG-tagged actins together with the SRF reporter gene 3D.ALuc as described in MATERIALS AND METHODS. After serum stimulation, luciferase activities were measured and expressed relative to activation by SRF-VP16, taken as 100. Data show mean of at least four independent experiments with SEM indicated by the bars.