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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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Drosophila S2 cells attach, spread, and form lamellae when plated on con A. S2 cells expressing EGFP–actin were plated on polylysine (a and b) or con A (c and d) and examined by phase contrast (a and c) or fluorescence microscopy (b and d). Cells on polylysine retain a spherical morphology but form actin-containing membrane ruffles along their surface. When plated on con A, the majority of S2 cells (>90%) spread to form a radially symmetrical actin-based lamellae (c and d). Bar, 5 μm. (e) A single frame from a time-lapse movie of an S2 cell expressing GFP–actin and plated on con A. The yellow line represents the region of the movie used to generate the kymograph shown in f. Bar, 1 μm. (f) This kymograph shows the behavior of actin over time in a lamella. The shark fin shape is indicative of cycles of extension and retraction at the cell margin, while the diagonal lines visualize the retrograde flow of actin at the cell periphery. Bar, 30 s.
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fig1: Drosophila S2 cells attach, spread, and form lamellae when plated on con A. S2 cells expressing EGFP–actin were plated on polylysine (a and b) or con A (c and d) and examined by phase contrast (a and c) or fluorescence microscopy (b and d). Cells on polylysine retain a spherical morphology but form actin-containing membrane ruffles along their surface. When plated on con A, the majority of S2 cells (>90%) spread to form a radially symmetrical actin-based lamellae (c and d). Bar, 5 μm. (e) A single frame from a time-lapse movie of an S2 cell expressing GFP–actin and plated on con A. The yellow line represents the region of the movie used to generate the kymograph shown in f. Bar, 1 μm. (f) This kymograph shows the behavior of actin over time in a lamella. The shark fin shape is indicative of cycles of extension and retraction at the cell margin, while the diagonal lines visualize the retrograde flow of actin at the cell periphery. Bar, 30 s.

Mentions: Under routine culture conditions, S2 cells display a roughly spherical morphology with a diameter of ∼10 μm (Fig. 1, a and b). These cells are not motile and exhibit no obvious morphological polarity, but time-lapse microscopy of cells expressing GFP–actin revealed that their surfaces are dynamic and continuously extend and absorb membrane ruffles (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200303023/DC1). S2 cells may be induced to undergo a dramatic change in their morphology when plated on glass coverslips coated with the lectin concanavalin A (con A) (Rogers et al., 2002). Within 20 to 30 min after plating on this substrate, these cells avidly attach, flatten, and spread to adopt a discoid morphology of approximately double their normal diameter (20 μm). Spread cells resemble a “fried egg” with a domed central region containing the nuclei and majority of organelles surrounded by a thin, organelle-free zone (Fig. 1, c and d; Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200303023/DC1).


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

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

Drosophila S2 cells attach, spread, and form lamellae when plated on con A. S2 cells expressing EGFP–actin were plated on polylysine (a and b) or con A (c and d) and examined by phase contrast (a and c) or fluorescence microscopy (b and d). Cells on polylysine retain a spherical morphology but form actin-containing membrane ruffles along their surface. When plated on con A, the majority of S2 cells (>90%) spread to form a radially symmetrical actin-based lamellae (c and d). Bar, 5 μm. (e) A single frame from a time-lapse movie of an S2 cell expressing GFP–actin and plated on con A. The yellow line represents the region of the movie used to generate the kymograph shown in f. Bar, 1 μm. (f) This kymograph shows the behavior of actin over time in a lamella. The shark fin shape is indicative of cycles of extension and retraction at the cell margin, while the diagonal lines visualize the retrograde flow of actin at the cell periphery. Bar, 30 s.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Drosophila S2 cells attach, spread, and form lamellae when plated on con A. S2 cells expressing EGFP–actin were plated on polylysine (a and b) or con A (c and d) and examined by phase contrast (a and c) or fluorescence microscopy (b and d). Cells on polylysine retain a spherical morphology but form actin-containing membrane ruffles along their surface. When plated on con A, the majority of S2 cells (>90%) spread to form a radially symmetrical actin-based lamellae (c and d). Bar, 5 μm. (e) A single frame from a time-lapse movie of an S2 cell expressing GFP–actin and plated on con A. The yellow line represents the region of the movie used to generate the kymograph shown in f. Bar, 1 μm. (f) This kymograph shows the behavior of actin over time in a lamella. The shark fin shape is indicative of cycles of extension and retraction at the cell margin, while the diagonal lines visualize the retrograde flow of actin at the cell periphery. Bar, 30 s.
Mentions: Under routine culture conditions, S2 cells display a roughly spherical morphology with a diameter of ∼10 μm (Fig. 1, a and b). These cells are not motile and exhibit no obvious morphological polarity, but time-lapse microscopy of cells expressing GFP–actin revealed that their surfaces are dynamic and continuously extend and absorb membrane ruffles (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200303023/DC1). S2 cells may be induced to undergo a dramatic change in their morphology when plated on glass coverslips coated with the lectin concanavalin A (con A) (Rogers et al., 2002). Within 20 to 30 min after plating on this substrate, these cells avidly attach, flatten, and spread to adopt a discoid morphology of approximately double their normal diameter (20 μm). Spread cells resemble a “fried egg” with a domed central region containing the nuclei and majority of organelles surrounded by a thin, organelle-free zone (Fig. 1, c and d; Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200303023/DC1).

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