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. 2000 Sep 4;150(5):1001–1012. doi: 10.1083/jcb.150.5.1001

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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PLP treatment inhibits induction of nucleation sites and actin polymerization by Cdc42. Control (Sepharose bead–treated) and PLP (PLP-bead–treated) supernatants were incubated with buffer or 1 μM Cdc42 for 5 min. (a) To measure filament number, the supernatants were diluted into 1.5 μM pyrenylactin and the initial rate of increase in pyrenylactin fluorescence was determined as a measure of nucleation sites (see Materials and Methods). Data from several experiments were normalized by setting the Cdc42-induced increase in polymerization rate in the control supernatant as 100%. PLP treatment decreased the Cdc42-induced change in initial rate to 10.0 ± 12.0% (mean ± SD, n = 7) of control. (b) To measure F-actin, the supernatants were diluted into TRITC–phalloidin, which stains F-actin. The F-actin was pelleted and the TRITC–phalloidin extracted and its fluorescence used as a measure of F-actin (see Materials and Methods). The data from different experiments were normalized by setting the increase in F-actin induced by Cdc42 in control supernatant as 100%. PLP treatment decreased F-actin induced to 11.2±4.8% of control (mean ± SD, n = 12). (c) Time course of polymerization. PLP-treated (▴) or control (Sepharose bead–treated, ○) supernatants were incubated for 5 min without (open symbols) or with (filled symbols) 1 μM Cdc42 before dilution into TRITC–phalloidin. The amount of TRITC–phalloidin bound was measured as in Fig. 3 b. (d and e) Re-addition of profilin restores ability of Cdc42 to induce actin nucleation sites and F-actin in PLP-treated supernatant. Various concentrations of wild-type (WT) or mutant (H133S and R74E) profilins were added to PLP-treated supernatant, which was then incubated for 5 min with Cdc42 before assaying for nucleation sites (d, as described in Fig. 3 a) or F-actin (e, as described in Fig. 3 b). Data are expressed as the percentage of Cdc42–induced nucleation sites in control (Sepharose bead–treated) supernatant; addition of 1.5–2 μM WT profilin restored the nucleation response to ∼60% of control level (d) and F-actin to ∼50% (e) of control level. Little or no restoration was seen with either H133S or R74E profilin.

PLP treatment inhibits induction of nucleation sites and actin polymerization by Cdc42. Control (Sepharose bead–treated) and PLP (PLP-bead–treated) supernatants were incubated with buffer or 1 μM Cdc42 for 5 min. (a) To measure filament number, the supernatants were diluted into 1.5 μM pyrenylactin and the initial rate of increase in pyrenylactin fluorescence was determined as a measure of nucleation sites (see Materials and Methods). Data from several experiments were normalized by setting the Cdc42-induced increase in polymerization rate in the control supernatant as 100%. PLP treatment decreased the Cdc42-induced change in initial rate to 10.0 ± 12.0% (mean ± SD, n = 7) of control. (b) To measure F-actin, the supernatants were diluted into TRITC–phalloidin, which stains F-actin. The F-actin was pelleted and the TRITC–phalloidin extracted and its fluorescence used as a measure of F-actin (see Materials and Methods). The data from different experiments were normalized by setting the increase in F-actin induced by Cdc42 in control supernatant as 100%. PLP treatment decreased F-actin induced to 11.2±4.8% of control (mean ± SD, n = 12). (c) Time course of polymerization. PLP-treated (▴) or control (Sepharose bead–treated, ○) supernatants were incubated for 5 min without (open symbols) or with (filled symbols) 1 μM Cdc42 before dilution into TRITC–phalloidin. The amount of TRITC–phalloidin bound was measured as in Fig. 3 b. (d and e) Re-addition of profilin restores ability of Cdc42 to induce actin nucleation sites and F-actin in PLP-treated supernatant. Various concentrations of wild-type (WT) or mutant (H133S and R74E) profilins were added to PLP-treated supernatant, which was then incubated for 5 min with Cdc42 before assaying for nucleation sites (d, as described in Fig. 3 a) or F-actin (e, as described in Fig. 3 b). Data are expressed as the percentage of Cdc42–induced nucleation sites in control (Sepharose bead–treated) supernatant; addition of 1.5–2 μM WT profilin restored the nucleation response to ∼60% of control level (d) and F-actin to ∼50% (e) of control level. Little or no restoration was seen with either H133S or R74E profilin.