Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of ∼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.

pull-down experiments with the same extracts. Few proteins (Ͻ10) were eluted from the GST column, and these San Francisco, California 94107 2 Section for Developmental Biology were excluded from analysis. Twenty "EB1-specific" proteins were identified over Department of Cell and Molecular Biology Lund University the course of five independent pull-down experiments. However, of these, only six candidates were identified BMC B13 22184 Lund in all five trials: CLIP190 (Mr [relative molecular weight] ϭ 190 kDa; the Drosophila ortholog of vertebrate CLIP-Sweden 170, which localizes to the plus ends of microtubules), Orbit/MAST (Mr ϭ 165 kDa; a microtubule plus-endassociated protein that interacts with CLIP-170), non-Summary muscle myosin II heavy chain (Mr ϭ 230 kDa), the minusend-directed kinesin, Ncd (Mr ϭ 75 kDa), Shortstop Members of the Rho/Rac/Cdc42 superfamily of (Mr ϭ 590 kDa; a member of the spectraplakin family of GTPases [1,2] and their upstream activators, guanine actin/microtubule cross-linking proteins), and DRhonucleotide exchange factors (GEFs) [3], have emerged GEF2 (Mr ϭ 280 kDa; a member of the Dbl family of as key regulators of actin and microtubule dynamics. Rho GEFs). In this paper, we focused on DRhoGEF2In their GTP bound form, these proteins interact with for further study; analyses of the other candidate EB1 downstream effector molecules that alter actin and binding factors will be described elsewhere. microtubule behavior. During Drosophila embryogen-The association of DRhoGEF2 with EB1 in vitro raised esis, a G␣ subunit (Concertina) and a Rho-type guathe possibility that this protein may localize to the tips of nine nucleotide exchange factor (DRhoGEF2) have microtubules. To test this idea, we generated polyclonal been implicated in the dramatic epithelial-cell shape antibodies against the C-terminal 720 amino acid resichanges that occur during gastrulation [4-6] and mordues of DRhoGEF2. These antibodies recognized a phogenesis [7]. Using Drosophila S2 cells as a model ‫082ف‬ kDa polypeptide on immunoblots of S2 cell exsystem, we show that DRhoGEF2 induces contractile tracts; this polypeptide was eliminated after DRhoGEF2 cell shape changes by stimulating myosin II via the RNAi treatment, indicating that the antibodies were re-Rho1 pathway. Unexpectedly, we found that DRhoacting with the correct polypeptide (Figure S1B). GEF2 travels to the cell cortex on the tips of growingBy immunofluorescence, anti-DRhoGEF2 antibodies microtubules by interaction with the microtubule plusrecognized punctate structures distributed throughout end tracking protein EB1. The upstream activator Conthe cell (Figure 1A). Superimposed upon this punctate certina, in its GTP but not GDP bound form, dissociates pattern, however, were short ‫1ف(‬ m) linear tracks that DRhoGEF2 from microtubule tips and also causes celcolocalized with the tips of microtubules. Moreover, imlular contraction. We propose that DRhoGEF2 uses munofluorescent staining of DRhoGEF2 in S2 cells exmicrotubule dynamics to search for cortical subdop...

Three genome-wide RNA interference screens were performed in Drosophila S2 cells to dissect the contribution of host processes to Listeria monocytogenes entry, vacuolar escape, and intracellular growth. Among the 116 genes identified, several host pathways previously unrecognized as playing a role in listerial pathogenesis were identified: knockdowns affecting vacuolar trafficking to and from the multivesicular body bypassed the requirement for the essential pore-forming toxin listeriolysin O in mediating escape from phagocytic vacuoles and knockdowns affecting either subunit of serine palmitoyltransferase, a key enzyme in ceramide and sphingolipid biosynthesis, enhanced the toxicity of listeriolysin O expressed in the host cell cytosol, leading to lack of appropriate toxin activity compartmentalization and host cell death. Genomewide RNA interference screens using Drosophila S2 cells proved to be a powerful approach to dissect host-pathogen interactions.Listeria monocytogenes ͉ listeriolysin O ͉ multivesicular bodies ͉ serine palmitoyltransferase I nfectious diseases caused by intracellular pathogens are responsible for an enormous amount of worldwide morbidity and mortality. These pathogens exploit the basic processes of host cells to establish their intracellular niche (1). Listeria monocytogenes, a facultative intracellular Gram-positive bacterial pathogen, thrives in the cytosol of host cells. The intracellular life cycle of L. monocytogenes has been well defined (2) and can be summarized as follows. Bacteria enter cells by either phagocytosis or bacteria-mediated internalization. Subsequent to internalization, the bacteria produce a cholesterol-dependent pore-forming cytolysin, termed listeriolysin O (LLO), and two phospholipases C (PLCs) that mediate rupture of the resulting phagosome, thereby allowing bacteria access to the rich milieu of the host cytosol. Once in the cytosol, bacteria grow rapidly and exploit a host system of actin-based motility to move intracellularly and spread from cell to cell. Mutants lacking LLO cannot escape from the phagosome, whereas those lacking PLCs are partially defective in escape. In some mammalian epithelial cells, however, a requirement for LLO can be bypassed, and in that case, PLCs are required for vacuolar escape (3, 4). Nevertheless, LLO is absolutely required for pathogenicity and is essential in the vast majority of cells analyzed. However, LLO is a doubleedged sword that can kill the host cell if expressed inappropriately. Mutations affecting its acidic pH optimum or in a PESTlike sequence result in inappropriate LLO expression in the cytosol, leading to plasma membrane damage, premature cell death, and severe attenuation in experimental listeriosis (5-7).Although much has been learned about the cellular microbiology of L. monocytogenes infection, the characterization of host processes contributing to pathogenesis has been hampered by the lack of tools for whole-genome genetic manipulations of the host. Many unanswered questions remain, such as how do bacteria e...