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Molecular requirements for actin-based lamella formation in Drosophila S2 cells.

Rogers SL, Wiedemann U, Stuurman N, Vale RD - J. Cell Biol. (2003)

Bottom Line: Cell migration occurs through the protrusion of the actin-enriched lamella.Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells.Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.

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RNAi-mediated inhibition of actin regulatory proteins disrupts normal cellular morphology in S2 cells on con A. Untreated cells are shown in Fig. 1 (c and d). Cells were treated with dsRNA against the p20 subunit of the Arp2/3 complex (a), profilin/chickadee (b), cofilin/twinstar (c), slingshot (d), capping protein β (e), Cdc42 (f), Rho1 (g), and myosin II/zipper (h) for 7 d and then plated on con A and stained with rhodamine-phalloidin (red) and DAPI (blue) to visualize filamentous actin and DNA, respectively. (i) Immunoblots demonstrating the effectiveness of RNAi on the levels of 13 different proteins: cofilin/twinstar, capping protein β (CPB), SCAR, Rho1, diaphanous (Dia), enabled (Ena), myosin VI (MVI), Nck/dreadlocks (Dock), Pod1, fascin/singed, lethal giant larvae (LGL), and Trio. Exactly 10 μg of total cellular protein was loaded for each lane. Bars, 5 μm.
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fig3: RNAi-mediated inhibition of actin regulatory proteins disrupts normal cellular morphology in S2 cells on con A. Untreated cells are shown in Fig. 1 (c and d). Cells were treated with dsRNA against the p20 subunit of the Arp2/3 complex (a), profilin/chickadee (b), cofilin/twinstar (c), slingshot (d), capping protein β (e), Cdc42 (f), Rho1 (g), and myosin II/zipper (h) for 7 d and then plated on con A and stained with rhodamine-phalloidin (red) and DAPI (blue) to visualize filamentous actin and DNA, respectively. (i) Immunoblots demonstrating the effectiveness of RNAi on the levels of 13 different proteins: cofilin/twinstar, capping protein β (CPB), SCAR, Rho1, diaphanous (Dia), enabled (Ena), myosin VI (MVI), Nck/dreadlocks (Dock), Pod1, fascin/singed, lethal giant larvae (LGL), and Trio. Exactly 10 μg of total cellular protein was loaded for each lane. Bars, 5 μm.

Mentions: DNA microarray analysis demonstrated that only five genes in this list are not expressed above background levels in S2 cells (Table I; Hollien, J., and J. Weissman, personal communication). As very low expressing genes nevertheless may be important for cell function, we still subjected these genes to RNAi analysis. A 7-d RNAi treatment was used to deplete proteins before assaying the cells for lamella formation on con A–treated coverslips. Filamentous actin was visualized with rhodamine-phalloidin, and DNA was stained with DAPI to screen for multiple nuclei reflecting cytokinesis defects. For every treatment, we examined at least 500 cells. We verified the efficacy of our RNAi treatments by immunoblotting extracts from dsRNA-treated cells using a panel of antibodies to 13 proteins to which we had access (Fig. 3 i). Immunoblotting for those tested revealed that RNAi reduced protein expression by at least 90% of endogenous levels and in many cases was not detectable. This immunoblot analysis included five proteins for which RNAi did not elicit an obvious phenotype (Table I). In the accompanying paper (Goshima and Vale, 2003), we also demonstrate >90% reduction in the levels of 10 motor proteins subjected to RNAi and have yet to encounter a case where RNAi has failed to reduce protein levels. We, therefore, speculate that dsRNAs against proteins that we could not quantitate most likely produced a similar degree of inhibition.


Molecular requirements for actin-based lamella formation in Drosophila S2 cells.

Rogers SL, Wiedemann U, Stuurman N, Vale RD - J. Cell Biol. (2003)

RNAi-mediated inhibition of actin regulatory proteins disrupts normal cellular morphology in S2 cells on con A. Untreated cells are shown in Fig. 1 (c and d). Cells were treated with dsRNA against the p20 subunit of the Arp2/3 complex (a), profilin/chickadee (b), cofilin/twinstar (c), slingshot (d), capping protein β (e), Cdc42 (f), Rho1 (g), and myosin II/zipper (h) for 7 d and then plated on con A and stained with rhodamine-phalloidin (red) and DAPI (blue) to visualize filamentous actin and DNA, respectively. (i) Immunoblots demonstrating the effectiveness of RNAi on the levels of 13 different proteins: cofilin/twinstar, capping protein β (CPB), SCAR, Rho1, diaphanous (Dia), enabled (Ena), myosin VI (MVI), Nck/dreadlocks (Dock), Pod1, fascin/singed, lethal giant larvae (LGL), and Trio. Exactly 10 μg of total cellular protein was loaded for each lane. Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172842&req=5

fig3: RNAi-mediated inhibition of actin regulatory proteins disrupts normal cellular morphology in S2 cells on con A. Untreated cells are shown in Fig. 1 (c and d). Cells were treated with dsRNA against the p20 subunit of the Arp2/3 complex (a), profilin/chickadee (b), cofilin/twinstar (c), slingshot (d), capping protein β (e), Cdc42 (f), Rho1 (g), and myosin II/zipper (h) for 7 d and then plated on con A and stained with rhodamine-phalloidin (red) and DAPI (blue) to visualize filamentous actin and DNA, respectively. (i) Immunoblots demonstrating the effectiveness of RNAi on the levels of 13 different proteins: cofilin/twinstar, capping protein β (CPB), SCAR, Rho1, diaphanous (Dia), enabled (Ena), myosin VI (MVI), Nck/dreadlocks (Dock), Pod1, fascin/singed, lethal giant larvae (LGL), and Trio. Exactly 10 μg of total cellular protein was loaded for each lane. Bars, 5 μm.
Mentions: DNA microarray analysis demonstrated that only five genes in this list are not expressed above background levels in S2 cells (Table I; Hollien, J., and J. Weissman, personal communication). As very low expressing genes nevertheless may be important for cell function, we still subjected these genes to RNAi analysis. A 7-d RNAi treatment was used to deplete proteins before assaying the cells for lamella formation on con A–treated coverslips. Filamentous actin was visualized with rhodamine-phalloidin, and DNA was stained with DAPI to screen for multiple nuclei reflecting cytokinesis defects. For every treatment, we examined at least 500 cells. We verified the efficacy of our RNAi treatments by immunoblotting extracts from dsRNA-treated cells using a panel of antibodies to 13 proteins to which we had access (Fig. 3 i). Immunoblotting for those tested revealed that RNAi reduced protein expression by at least 90% of endogenous levels and in many cases was not detectable. This immunoblot analysis included five proteins for which RNAi did not elicit an obvious phenotype (Table I). In the accompanying paper (Goshima and Vale, 2003), we also demonstrate >90% reduction in the levels of 10 motor proteins subjected to RNAi and have yet to encounter a case where RNAi has failed to reduce protein levels. We, therefore, speculate that dsRNAs against proteins that we could not quantitate most likely produced a similar degree of inhibition.

Bottom Line: Cell migration occurs through the protrusion of the actin-enriched lamella.Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells.Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.

Show MeSH
Related in: MedlinePlus