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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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Related in: MedlinePlus

Model for the signaling pathway leading to SCAR activation during S2 cell lamella formation. The con A–coated coverslip activates both Rac proteins and Nck by initially cross-linking an unidentified cell surface receptor(s). The Rac proteins and Nck signal through parallel pathways to cause dissociation of trans-inhibited SCAR bound by a complex of kette, Sra-1, and Abi. After dissociation, SCAR is able to promote actin nucleation by Arp2/3 at the cell membrane. SCAR may then be inactivated either by reassociation with its inhibitory complex or by degradation.
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fig5: Model for the signaling pathway leading to SCAR activation during S2 cell lamella formation. The con A–coated coverslip activates both Rac proteins and Nck by initially cross-linking an unidentified cell surface receptor(s). The Rac proteins and Nck signal through parallel pathways to cause dissociation of trans-inhibited SCAR bound by a complex of kette, Sra-1, and Abi. After dissociation, SCAR is able to promote actin nucleation by Arp2/3 at the cell membrane. SCAR may then be inactivated either by reassociation with its inhibitory complex or by degradation.

Mentions: Our studies also have provided new insight into the activation of SCAR, which is summarized in the model shown in Fig. 5. Plasma membrane receptors on S2 cells (currently unknown) are activated, perhaps by cross-linking upon contact with con A–treated coverslips. Subsequently, two parallel pathways transduce this stimulus. One is mediated by small GTPases belonging to the Rac family. Our results show that three Rac GTPases (Rac1, Rac2, and Mtl1) participate in the transduction pathway, confirming the functional redundancy of these proteins reported in many fly tissues (Hakeda-Suzuki et al., 2002). A second transduction pathway is mediated by the SH2-SH3 adaptor protein Nck, which has been shown to activate SCAR in vitro (Eden et al., 2002). Our results confirm that this Nck-mediated activation of SCAR is important in vivo as well. The Rac and SCAR pathways probably converge in activating actin polymerization by dissociating SCAR from its trans-inhibited kette–Sra-1–Abi-bound complex and allowing it to bind to Arp2/3 (Eden et al., 2002). Moreover, the finding that simultaneous inhibition of Rac-like proteins and Nck does not completely mimic SCAR RNAi treatment raises the possibility that additional SCAR activators exist.


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

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

Model for the signaling pathway leading to SCAR activation during S2 cell lamella formation. The con A–coated coverslip activates both Rac proteins and Nck by initially cross-linking an unidentified cell surface receptor(s). The Rac proteins and Nck signal through parallel pathways to cause dissociation of trans-inhibited SCAR bound by a complex of kette, Sra-1, and Abi. After dissociation, SCAR is able to promote actin nucleation by Arp2/3 at the cell membrane. SCAR may then be inactivated either by reassociation with its inhibitory complex or by degradation.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Model for the signaling pathway leading to SCAR activation during S2 cell lamella formation. The con A–coated coverslip activates both Rac proteins and Nck by initially cross-linking an unidentified cell surface receptor(s). The Rac proteins and Nck signal through parallel pathways to cause dissociation of trans-inhibited SCAR bound by a complex of kette, Sra-1, and Abi. After dissociation, SCAR is able to promote actin nucleation by Arp2/3 at the cell membrane. SCAR may then be inactivated either by reassociation with its inhibitory complex or by degradation.
Mentions: Our studies also have provided new insight into the activation of SCAR, which is summarized in the model shown in Fig. 5. Plasma membrane receptors on S2 cells (currently unknown) are activated, perhaps by cross-linking upon contact with con A–treated coverslips. Subsequently, two parallel pathways transduce this stimulus. One is mediated by small GTPases belonging to the Rac family. Our results show that three Rac GTPases (Rac1, Rac2, and Mtl1) participate in the transduction pathway, confirming the functional redundancy of these proteins reported in many fly tissues (Hakeda-Suzuki et al., 2002). A second transduction pathway is mediated by the SH2-SH3 adaptor protein Nck, which has been shown to activate SCAR in vitro (Eden et al., 2002). Our results confirm that this Nck-mediated activation of SCAR is important in vivo as well. The Rac and SCAR pathways probably converge in activating actin polymerization by dissociating SCAR from its trans-inhibited kette–Sra-1–Abi-bound complex and allowing it to bind to Arp2/3 (Eden et al., 2002). Moreover, the finding that simultaneous inhibition of Rac-like proteins and Nck does not completely mimic SCAR RNAi treatment raises the possibility that additional SCAR activators exist.

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