Limits...
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.

Show MeSH

Related in: MedlinePlus

Control of SCAR degradation and activation. Inhibition of SCAR-associated proteins kette, Sra-1, or Abi by RNAi causes degradation of SCAR itself. S2 cells were treated with dsRNA corresponding to the coding sequence for Sra-1 (a) for 7 d and then plated on con A and stained with phalloidin to visualize actin (red) and DAPI to view DNA (blue). The morphology of these cells closely resembles the defects in lamellae formation produced by Arp2/3 and SCAR RNAi (Fig. 3, b and c). Similar results were observed with RNAi against kette and Abi (not depicted). Bar, 5 μm. (b) Quantitative immunoblotting of cells treated with dsRNA versus Abi, kette, SCAR, and Sra-1 with antibodies against SCAR. Depletion of these proteins by RNAi decreases the amount of SCAR present in S2 cells. Equal protein loading was verified by Bradford assay (not depicted). (c and d) Cells treated with dsRNAi to simultaneously inhibit Rac1, Rac2, Mtl, and Nck show a variety of lamella defects. Among these are a malformed, serrate cell margin (c) and the stellate morphology similar to SCAR RNAi (Fig. 3, b and c). (e) Graph showing the quantitation of morphological defects caused by inhibition of Nck, Rac1/2, and Mtl. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172842&req=5

fig4: Control of SCAR degradation and activation. Inhibition of SCAR-associated proteins kette, Sra-1, or Abi by RNAi causes degradation of SCAR itself. S2 cells were treated with dsRNA corresponding to the coding sequence for Sra-1 (a) for 7 d and then plated on con A and stained with phalloidin to visualize actin (red) and DAPI to view DNA (blue). The morphology of these cells closely resembles the defects in lamellae formation produced by Arp2/3 and SCAR RNAi (Fig. 3, b and c). Similar results were observed with RNAi against kette and Abi (not depicted). Bar, 5 μm. (b) Quantitative immunoblotting of cells treated with dsRNA versus Abi, kette, SCAR, and Sra-1 with antibodies against SCAR. Depletion of these proteins by RNAi decreases the amount of SCAR present in S2 cells. Equal protein loading was verified by Bradford assay (not depicted). (c and d) Cells treated with dsRNAi to simultaneously inhibit Rac1, Rac2, Mtl, and Nck show a variety of lamella defects. Among these are a malformed, serrate cell margin (c) and the stellate morphology similar to SCAR RNAi (Fig. 3, b and c). (e) Graph showing the quantitation of morphological defects caused by inhibition of Nck, Rac1/2, and Mtl. Bars, 5 μm.

Mentions: For complete gene information, see the online supplemental material (available at http://www.jcb.org/cgi/content/full/jcb.200303023/DC1). Depletion of myosin II/zipper also caused impaired lamella spreading and actin disorganization. Inhibition of Rac1/2 also generated an increase in stellate phenotypes in combination with Nck (see Fig. 4).


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

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

Control of SCAR degradation and activation. Inhibition of SCAR-associated proteins kette, Sra-1, or Abi by RNAi causes degradation of SCAR itself. S2 cells were treated with dsRNA corresponding to the coding sequence for Sra-1 (a) for 7 d and then plated on con A and stained with phalloidin to visualize actin (red) and DAPI to view DNA (blue). The morphology of these cells closely resembles the defects in lamellae formation produced by Arp2/3 and SCAR RNAi (Fig. 3, b and c). Similar results were observed with RNAi against kette and Abi (not depicted). Bar, 5 μm. (b) Quantitative immunoblotting of cells treated with dsRNA versus Abi, kette, SCAR, and Sra-1 with antibodies against SCAR. Depletion of these proteins by RNAi decreases the amount of SCAR present in S2 cells. Equal protein loading was verified by Bradford assay (not depicted). (c and d) Cells treated with dsRNAi to simultaneously inhibit Rac1, Rac2, Mtl, and Nck show a variety of lamella defects. Among these are a malformed, serrate cell margin (c) and the stellate morphology similar to SCAR RNAi (Fig. 3, b and c). (e) Graph showing the quantitation of morphological defects caused by inhibition of Nck, Rac1/2, and Mtl. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172842&req=5

fig4: Control of SCAR degradation and activation. Inhibition of SCAR-associated proteins kette, Sra-1, or Abi by RNAi causes degradation of SCAR itself. S2 cells were treated with dsRNA corresponding to the coding sequence for Sra-1 (a) for 7 d and then plated on con A and stained with phalloidin to visualize actin (red) and DAPI to view DNA (blue). The morphology of these cells closely resembles the defects in lamellae formation produced by Arp2/3 and SCAR RNAi (Fig. 3, b and c). Similar results were observed with RNAi against kette and Abi (not depicted). Bar, 5 μm. (b) Quantitative immunoblotting of cells treated with dsRNA versus Abi, kette, SCAR, and Sra-1 with antibodies against SCAR. Depletion of these proteins by RNAi decreases the amount of SCAR present in S2 cells. Equal protein loading was verified by Bradford assay (not depicted). (c and d) Cells treated with dsRNAi to simultaneously inhibit Rac1, Rac2, Mtl, and Nck show a variety of lamella defects. Among these are a malformed, serrate cell margin (c) and the stellate morphology similar to SCAR RNAi (Fig. 3, b and c). (e) Graph showing the quantitation of morphological defects caused by inhibition of Nck, Rac1/2, and Mtl. Bars, 5 μm.
Mentions: For complete gene information, see the online supplemental material (available at http://www.jcb.org/cgi/content/full/jcb.200303023/DC1). Depletion of myosin II/zipper also caused impaired lamella spreading and actin disorganization. Inhibition of Rac1/2 also generated an increase in stellate phenotypes in combination with Nck (see Fig. 4).

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