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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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Immunofluorescence localization of actin regulatory proteins to lamellae of S2 cells. S2 cells were plated on con A for 1 h and then fixed and double stained for actin (red) and Arp3 (a, green), SCAR (b, green), cofilin/twinstar (c, green), profilin (d, green), enabled (e, green), or capping protein (f, green). Bar, 5 μm.
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fig2: Immunofluorescence localization of actin regulatory proteins to lamellae of S2 cells. S2 cells were plated on con A for 1 h and then fixed and double stained for actin (red) and Arp3 (a, green), SCAR (b, green), cofilin/twinstar (c, green), profilin (d, green), enabled (e, green), or capping protein (f, green). Bar, 5 μm.

Mentions: To better understand the organization of actin in S2 cells, we fixed con A–adhered S2 cells expressing GFP–actin or stained them with Texas red X-phalloidin, a probe that selectively binds to filamentous actin. When examined by fluorescence microscopy, most S2 cells (90%) exhibited a highly developed, radially symmetrical actin cytoskeleton that could be divided into three zones: a dense peripheral network at the extreme periphery of the cells (∼1 μm wide), a second central zone (4–6 μm wide) of lower actin density composed of filaments, and a third circular bundle of filaments that surrounded the nucleus (Fig. 2, a and b). Arp3, cofilin, and capping protein were enriched in this first actin-dense zone at the leading edge, especially at membrane ruffles (Fig. 2, a, c, and f). Enabled/VASP was further restricted to the extreme edge of the periphery (<1 μm) (Fig. 2 e). In contrast, immunolocalization of profilin/chickadee revealed puncta that were distributed throughout the cell and particularly abundant in the inner nuclear and organelle-rich domain (Fig. 2 d). These puncta were not associated with adhesion structures, as immunofluorescent staining against phosphotyrosine failed to stain the ventral surface of the cells (unpublished data). The distributions of these well-characterized actin-binding proteins are generally similar to those described in other cell types that form actin-rich lamellae.


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

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

Immunofluorescence localization of actin regulatory proteins to lamellae of S2 cells. S2 cells were plated on con A for 1 h and then fixed and double stained for actin (red) and Arp3 (a, green), SCAR (b, green), cofilin/twinstar (c, green), profilin (d, green), enabled (e, green), or capping protein (f, green). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Immunofluorescence localization of actin regulatory proteins to lamellae of S2 cells. S2 cells were plated on con A for 1 h and then fixed and double stained for actin (red) and Arp3 (a, green), SCAR (b, green), cofilin/twinstar (c, green), profilin (d, green), enabled (e, green), or capping protein (f, green). Bar, 5 μm.
Mentions: To better understand the organization of actin in S2 cells, we fixed con A–adhered S2 cells expressing GFP–actin or stained them with Texas red X-phalloidin, a probe that selectively binds to filamentous actin. When examined by fluorescence microscopy, most S2 cells (90%) exhibited a highly developed, radially symmetrical actin cytoskeleton that could be divided into three zones: a dense peripheral network at the extreme periphery of the cells (∼1 μm wide), a second central zone (4–6 μm wide) of lower actin density composed of filaments, and a third circular bundle of filaments that surrounded the nucleus (Fig. 2, a and b). Arp3, cofilin, and capping protein were enriched in this first actin-dense zone at the leading edge, especially at membrane ruffles (Fig. 2, a, c, and f). Enabled/VASP was further restricted to the extreme edge of the periphery (<1 μm) (Fig. 2 e). In contrast, immunolocalization of profilin/chickadee revealed puncta that were distributed throughout the cell and particularly abundant in the inner nuclear and organelle-rich domain (Fig. 2 d). These puncta were not associated with adhesion structures, as immunofluorescent staining against phosphotyrosine failed to stain the ventral surface of the cells (unpublished data). The distributions of these well-characterized actin-binding proteins are generally similar to those described in other cell types that form actin-rich lamellae.

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