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Cucurbitacin I inhibits cell motility by indirectly interfering with actin dynamics.

Knecht DA, LaFleur RA, Kahsai AW, Argueta CE, Beshir AB, Fenteany G - PLoS ONE (2010)

Bottom Line: However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments.In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration.Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA. david.knecht@uconn.edu

ABSTRACT

Background: Cucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton.

Methodology/principal findings: We show that cucurbitacin I potently inhibits the migration of Madin-Darby canine kidney (MDCK) cell sheets during wound closure, as well as the random motility of B16-F1 mouse melanoma cells, but has no effect on movement of Dictyostelium discoideum amoebae. Upon treatment of MDCK or B16-F1 cells with cucurbitacin I, there is a very rapid cessation of motility and gradual accumulation of filamentous actin aggregates. The cellular effect of the compound is similar to that observed when cells are treated with the actin filament-stabilizing agent jasplakinolide. However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments. In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration. At elevated concentrations, the depolymerization rate was also unaffected, although there was a delay in the initiation of depolymerization. Therefore, cucurbitacin I targets some factor involved in cellular actin dynamics other than actin itself. Two candidate proteins that play roles in actin depolymerization are the actin-severing proteins cofilin and gelsolin. Cucurbitacin I possesses electrophilic reactivity that may lead to chemical modification of its target protein, as suggested by structure-activity relationship data. However, mass spectrometry revealed no evidence for modification of purified cofilin or gelsolin by cucurbitacin I.

Conclusions/significance: Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism. Furthermore, the proximal target of cucurbitacin I relevant to cell migration is unlikely to be the same one involved in activation of the JAK2/STAT3 pathway.

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Actin aggregates persist after removal of cucurbitacin I.B16-F1 cells expressing mCherry-actin were imaged before (A) and after 4 h in the presence of 200 nM cucurbitacin I (B). The compound was then washed out and imaging continued. Images are shown at (C) 24 h and (D) 40 h after washout. Each panel is a maximum-intensity projection of 7 confocal Z sections through a representative cell. (E) After 4 h in cucurbitacin I, one sample of B16-F1 cells was fixed and stained with fluorescein-conjugated phalloidin to visualize F-actin. A single confocal Z slice showing the aggregates stained with fluorescein-phalloidin (left panel), mCherry-actin (center panel) is shown along with a differential interference contrast (DIC) image (right panel).
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pone-0014039-g007: Actin aggregates persist after removal of cucurbitacin I.B16-F1 cells expressing mCherry-actin were imaged before (A) and after 4 h in the presence of 200 nM cucurbitacin I (B). The compound was then washed out and imaging continued. Images are shown at (C) 24 h and (D) 40 h after washout. Each panel is a maximum-intensity projection of 7 confocal Z sections through a representative cell. (E) After 4 h in cucurbitacin I, one sample of B16-F1 cells was fixed and stained with fluorescein-conjugated phalloidin to visualize F-actin. A single confocal Z slice showing the aggregates stained with fluorescein-phalloidin (left panel), mCherry-actin (center panel) is shown along with a differential interference contrast (DIC) image (right panel).

Mentions: Having shown that cucurbitacin I has a rapid and reversible effect on cellular motility in MDCK and B16-F1 cells, changes in the localization of actin-containing structures were examined in compound-treated cells expressing mCherry-actin. During wound closure in MDCK cell monolayers, punctate fluorescent structures appeared in the cytoplasm after addition of cucurbitacin I (Figure 3). In low-density MDCK cell cultures, mCherry-actin produced a diffuse signal throughout the cytoplasm, with more intense localization in areas where rapid actin polymerization was occurring, such as in new protrusions (Figure 6A and Movie S5). Upon addition of 200 nM cucurbitacin I, cells ceased lamellar extension, and small punctate aggregates began to form throughout the cytoplasm (Figure 6A and Movie S5). The aggregates became larger over time and the diffuse cytoplasmic signal decreased, suggesting a shift from actin monomer (globular actin; G-actin) to actin polymer (filamentous actin; F-actin). Similar results were obtained when B16-F1 cells expressing mCherry-actin were treated with the compound (Figure 6B). In cells treated with 200 nM cucurbitacin I for 4 h, aggregates persisted for days after the compound was washed out (Figure 7). To demonstrate that the aggregates are formed from F-actin rather than G-actin, cells treated with cucurbitacin I for 4 h were fixed and stained with FITC-phalloidin, which only binds F-actin; the aggregates that were labeled with mCherry-actin were also stained by FITC-phalloidin (Figure 7E). Cucurbitacin I did not cause actin aggregates to form in Dictyostelium amoebae (Figure 6C).


Cucurbitacin I inhibits cell motility by indirectly interfering with actin dynamics.

Knecht DA, LaFleur RA, Kahsai AW, Argueta CE, Beshir AB, Fenteany G - PLoS ONE (2010)

Actin aggregates persist after removal of cucurbitacin I.B16-F1 cells expressing mCherry-actin were imaged before (A) and after 4 h in the presence of 200 nM cucurbitacin I (B). The compound was then washed out and imaging continued. Images are shown at (C) 24 h and (D) 40 h after washout. Each panel is a maximum-intensity projection of 7 confocal Z sections through a representative cell. (E) After 4 h in cucurbitacin I, one sample of B16-F1 cells was fixed and stained with fluorescein-conjugated phalloidin to visualize F-actin. A single confocal Z slice showing the aggregates stained with fluorescein-phalloidin (left panel), mCherry-actin (center panel) is shown along with a differential interference contrast (DIC) image (right panel).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991314&req=5

pone-0014039-g007: Actin aggregates persist after removal of cucurbitacin I.B16-F1 cells expressing mCherry-actin were imaged before (A) and after 4 h in the presence of 200 nM cucurbitacin I (B). The compound was then washed out and imaging continued. Images are shown at (C) 24 h and (D) 40 h after washout. Each panel is a maximum-intensity projection of 7 confocal Z sections through a representative cell. (E) After 4 h in cucurbitacin I, one sample of B16-F1 cells was fixed and stained with fluorescein-conjugated phalloidin to visualize F-actin. A single confocal Z slice showing the aggregates stained with fluorescein-phalloidin (left panel), mCherry-actin (center panel) is shown along with a differential interference contrast (DIC) image (right panel).
Mentions: Having shown that cucurbitacin I has a rapid and reversible effect on cellular motility in MDCK and B16-F1 cells, changes in the localization of actin-containing structures were examined in compound-treated cells expressing mCherry-actin. During wound closure in MDCK cell monolayers, punctate fluorescent structures appeared in the cytoplasm after addition of cucurbitacin I (Figure 3). In low-density MDCK cell cultures, mCherry-actin produced a diffuse signal throughout the cytoplasm, with more intense localization in areas where rapid actin polymerization was occurring, such as in new protrusions (Figure 6A and Movie S5). Upon addition of 200 nM cucurbitacin I, cells ceased lamellar extension, and small punctate aggregates began to form throughout the cytoplasm (Figure 6A and Movie S5). The aggregates became larger over time and the diffuse cytoplasmic signal decreased, suggesting a shift from actin monomer (globular actin; G-actin) to actin polymer (filamentous actin; F-actin). Similar results were obtained when B16-F1 cells expressing mCherry-actin were treated with the compound (Figure 6B). In cells treated with 200 nM cucurbitacin I for 4 h, aggregates persisted for days after the compound was washed out (Figure 7). To demonstrate that the aggregates are formed from F-actin rather than G-actin, cells treated with cucurbitacin I for 4 h were fixed and stained with FITC-phalloidin, which only binds F-actin; the aggregates that were labeled with mCherry-actin were also stained by FITC-phalloidin (Figure 7E). Cucurbitacin I did not cause actin aggregates to form in Dictyostelium amoebae (Figure 6C).

Bottom Line: However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments.In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration.Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA. david.knecht@uconn.edu

ABSTRACT

Background: Cucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton.

Methodology/principal findings: We show that cucurbitacin I potently inhibits the migration of Madin-Darby canine kidney (MDCK) cell sheets during wound closure, as well as the random motility of B16-F1 mouse melanoma cells, but has no effect on movement of Dictyostelium discoideum amoebae. Upon treatment of MDCK or B16-F1 cells with cucurbitacin I, there is a very rapid cessation of motility and gradual accumulation of filamentous actin aggregates. The cellular effect of the compound is similar to that observed when cells are treated with the actin filament-stabilizing agent jasplakinolide. However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments. In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration. At elevated concentrations, the depolymerization rate was also unaffected, although there was a delay in the initiation of depolymerization. Therefore, cucurbitacin I targets some factor involved in cellular actin dynamics other than actin itself. Two candidate proteins that play roles in actin depolymerization are the actin-severing proteins cofilin and gelsolin. Cucurbitacin I possesses electrophilic reactivity that may lead to chemical modification of its target protein, as suggested by structure-activity relationship data. However, mass spectrometry revealed no evidence for modification of purified cofilin or gelsolin by cucurbitacin I.

Conclusions/significance: Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism. Furthermore, the proximal target of cucurbitacin I relevant to cell migration is unlikely to be the same one involved in activation of the JAK2/STAT3 pathway.

Show MeSH
Related in: MedlinePlus