We have developed a novel system for the study of actin cytoskeletal dynamics that is amenable to in vivo imaging and targeted inhibition of proteins via RNAi. From a set of 90 proteins implicated in actin dynamics, we have found 13 proteins that contribute to normal lamellae formation and seven proteins that are involved in cytokinesis. However, we cannot rule out the involvement of other proteins from our tested list. As with all RNAi-mediated “knockdown” screens, negative results are not definitively conclusive without demonstrating that the actual target protein is depleted. Moreover, it also remains possible that a small amount of residual protein that remains after RNAi treatment is sufficient for cellular function. In addition, it is possible that some actin dynamics phenotypes may only become apparent by time-lapse microscopy. For example, perturbation of mammalian enabled/VASP did not cause gross changes in cellular morphology (Bear et al., 2002
), as we have found for S2 cells, but drastically altered lamellae behavior and membrane ruffling. Time-lapse observation is very time consuming for the relatively large number of genes investigated in this study, however, we plan more detailed examination of actin dynamics by live cell microscopy for a smaller number of RNAi experiments in the future.
In this work, we found that a relatively small number of the proteins tested is essential for lamella protrusion. These proteins include: (a) an actin-nucleating factor (Arp2/3) and one of its activators (SCAR), (b) a factor that caps barbed ends of newly formed actin filaments (capping protein), (c) proteins involved in severing and depolymerizing actin filaments to allow turnover (cofilin and Aip1), and (d) factors that sequester actin monomers and promote nucleotide exchange (profilin and CAP). This set of proteins and observed RNAi phenotypes are consistent with current models for the cycle of actin dynamics in lamellae (Pollard et al., 2000
; Pollard and Borisy, 2003
). Moreover, these proteins are similar to the minimal set needed to reconstitute actin-based propulsion of Listeria
in vitro (Loisel et al., 1999
Although our results largely agree with the protein requirements for reconstitution of actin-based motility in vitro as described above, some observations reveal that actin dynamics in the cell are more complex. For example, two actin monomer–binding proteins (profilin and CAP) are required for normal lamella formation, whereas profilin alone is sufficient to facilitate movement in vitro, although the role of CAP has not yet been tested (Loisel et al., 1999
). Our result indicates that these two proteins play distinct and nonredundant roles in cells, although loss of either yields a similar phenotype consisting of uniformly distributed actin filaments throughout the cytoplasm, as opposed to being confined to the leading edge, and a failure to spread. CAP was shown to bind actin monomers and inhibit polymerization in vitro (Gieselmann and Mann, 1992
; Freeman et al., 1995
) but has been less well studied biochemically than profilin. A careful side-by-side comparison of the effects of CAP and profilin on actin dynamics and nucleotide exchange may provide insight into why the cell needs both proteins to recycle actin for polymerization at the leading edge. In addition, we find that cells require the depolymerizing protein cofilin as well as the cofilin-interacting protein Aip1 to form lamella, whereas cofilin alone suffices in vitro (Loisel et al., 1999
). Previous biochemical studies have suggested that Aip1 acts synergistically with cofilin to promote actin filament disassembly (Okada et al., 1999
; Rodal et al., 1999
), and this synergy may be essential for cofilin function in vivo. We have also observed a role for slingshot, a cofilin phosphatase, for normal lamellae morphology, further underscoring the role for actin disassembly for S2 cell spreading. A third unanticipated result was the partial defect in lamella organization in cytoplasmic myosin II RNAi cells. Cytoplasmic myosin II is generally believed to be important for the retraction of the trailing end of migrating cells (Eddy et al., 2000
) and in generating the fan-shaped appearance of the lamella in migrating keratocytes (Pollard and Borisy, 2003
). Our phenotype shows that myosin II plays a role in the organization of actin filaments in the lamella of nonmigrating cell types as well, although it is not essential for cell spreading.
Our studies also have provided new insight into the activation of SCAR, which is summarized in the model shown in . 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.
Figure 5. 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 (more ...)
Our work also has uncovered an additional role of the kette–Sra-1–Abi complex in protecting SCAR from degradation. Unlike the WASP protein, which is autoinhibited (Pollard and Borisy, 2003
), SCAR is constitutively active (Machesky et al., 1999
). Therefore, long-lived, uncomplexed SCAR may be detrimental, as it would cause uncontrolled actin polymerization. Thus, the degradation of free SCAR would ensure a proper stoichiometry of SCAR to its inhibitory complex. It is also possible that the kinetics of SCAR degradation may be regulated under some circumstances to modulate actin cytoskeleton dynamics. Further studies are underway to explore this potential avenue of SCAR regulation and understand the mechanism of SCAR degradation.
Given the relative ease and effectiveness of RNAi-mediated gene inhibition, we foresee that S2 and other Drosophila tissue culture cells can be used to explore other aspects of the actin cytoskeleton, such as filopodia formation. If proper cues are provided to these cells, cell migration and cell polarity may be amenable to investigation as well. Moreover, although we restricted our studies to known actin-binding proteins, genome-wide screens can be performed to identify novel genes associated with cytoskeletal regulation.