Journal of Cell Science 113, 759-766 (2000) 759 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0731

COMMENTARY The cytoskeleton of Dictyostelium: a story told by mutants

Angelika A. Noegel1,* and Michael Schleicher2 1Institut für Biochemie I, Medizinische Fakultät, Universität zu Köln, Joseph-Stelzmann-Str. 52, 50931 Köln, Germany 2Adolf-Butenandt-Institut für Zellbiologie, Ludwig-Maximilians-Universität, Schillerstr. 42, 80336 München, Germany *Author for correspondence (e-mail: [email protected])

Published on WWW 14 February 2000

SUMMARY

Actin-binding proteins are effectors of cell signalling and development. Furthermore, the studies have identified coordinators of cellular behaviour. Research on the several cellular and developmental stages that are Dictyostelium actin cytoskeleton has focused both on the particularly sensitive to an unbalanced cytoskeleton. In elucidation of the function of bona fide actin-binding addition, use of GFP fusion proteins is revealing the spatial proteins as well as on proteins involved in signalling to the and temporal dynamics of interactions between actin- cytoskeleton. A major part of this work is concerned with associated proteins and the cytoskeleton. the analysis of Dictyostelium mutants. The results derived from these investigations have added to our understanding Key words: Actin binding protein, Cell signalling, , of the role of the actin cytoskeleton in growth and Morphogenesis, GFP fusion protein

INTRODUCTION antisense strategies, restriction-enzyme-mediated integration (REMI), transposon-tagging-like mutagenesis, and expression The actin cytoskeleton of a cell is required for cell-shape of GFP fusion proteins. The 34-Mb genome is carried on six changes, cell motility and chemotaxis, as well as for chromosomes. The Dictyostelium genome project (see cytokinesis, intracellular transport processes, development and http://www.uni-koeln.de/dictyostelium/) should be completed signal transduction. It is composed of actin and associated by 2002; however, information on more than 90% of the genes proteins. Their interactions are highly dynamic, and constant is already available. reorganization of the actin network is required for it to perform Dictyostelium is therefore a valuable and convenient its functions. experimental system for studies of the role of the actin Dictyostelium has been a model system for the analysis of cytoskeleton in cell motility, chemotaxis, signal transduction, the actin cytoskeleton for some time now, and research in this development and differentiation. Here, we highlight new field has contributed to a general understanding of the structure developments in three areas: (1) cytoskeletal dynamics at leading and function of cytoskeletal proteins (for reviews see Schleicher fronts, where experiments with GFP fusion proteins provided and Noegel, 1992; Noegel and Luna, 1995). Dictyostelium has spectacular data on chemotaxis, cell movement, several features that make it especially well suited to studies of and pinocytosis (Maniak et al., 1995; Parent et al., 1998); (2) the the actin cytoskeleton. It is a unicellular that lives as a roles of actin and actin-binding proteins as effectors of cell natural phagocyte and feeds on yeast and bacteria. When cells signalling; (3) coordination of cellular behaviour by actin and starve, development is initiated, and cells aggregate by actin-binding proteins, where the genome project aided the chemotaxis in response to relayed cAMP signals. Once the identification of new components that reveal additional roles for amoebae have aggregated, they form a the actin cytoskeleton. Moreover, we try to give an update on that in its slug form can migrate towards light and orient in a components of the actin cytoskeleton and putative regulatory thermotactic gradient. During development, cells differentiate components, and their impact on cell biology Ð as taken from into spore and stalk cells and, finally, a fruiting body is mutant analysis (Table 1). Structural analyses of cytoskeletal constructed. In the laboratory this program is completed within proteins have also made significant contributions; these studies, 24 hours. Multicellularity requires specific cell interactions, however, are beyond the scope of this Commentary. extracellular signals and the corresponding receptors, as well as various signal transduction pathways. Dictyostelium is haploid. This facilitates isolation of mutants DYNAMIC EVENTS AT LEADING FRONTS but makes it difficult to study essential genes. A wide spectrum of molecular genetic techniques is available, including gene Components of the signal transduction pathway inactivation by homologous recombination, gene replacement, Chemotaxis in Dictyostelium requires a cAMP receptor and 760 A. A. Noegel and M. Schleicher

Table 1. Mutants in bona fide actin-binding proteins and in components that might be involved in regulation of the actin cytoskeleton Protein Exp. approach Phenotype References α-actinin Knock-out Reduced resistance to shear stress; growth defect under different osmotic conditions; Eichinger et al., 1996; in combination with gelation factor: development arrested at mound stage; small Witke et al., 1992; cell size, motility defect, orientation defect, sensitivity to osmotic shock Rivero et al., 1996a Akt/PKB Knock-out Aberrant chemotaxis, polarisation defect, aggregation-minus at low cell densities Meili et al., 1999 34-kDa bundling protein Knock-out Reduced growth rate at low temperatures, persistence of motility increased Rivero et al., 1996b Calmodulin Antisense Cytokinesis Liu et al., 1992 CAP/ASP56 Knock-out Cell morphology altered, cytokinesis defect, endocytosis defect, reduced motility, Noegel et al., 1999 delay in development Capping protein Antisense Reduced motility; Hug et al., 1995 (cap 32/34) overexpression increased motility, cytoskeletal architecture altered Clathrin heavy chain Knock-out Pinocytosis, cell type differentiation, cytokinesis O’Halloran and Anderson, 1992; Niswonger and O’Halloran, 1997a,b Cofilin Overexpression Stimulation of actin bundling, membrane ruffles, cell movement, reorganises actin Aizawa et al., 1996 into bundles that, in concert with DAip1, contract in a myosin independent way in and 1999 response to hyperosmotic stress Coronin Knock-out Cytokinesis defect, phagocytosis defect, motility defect de Hostos et al., 1993; Maniak et al., 1995 Cortexillin I Knock-out Developmental arrest in Fey and Cox, 1999 Cortexillin I and II Knock-out Cell shape altered, cytokinesis defect Faix et al., 1996 DAip1 Knock-out Impaired growth, endocytosis, phagocytosis and movement, slight cytokinesis defect Konzok et al., 1999 DdLim Overexpression Formation of large lamella, increased ruffling, multinucleated cells Prassler et al., 1998 DdMEK1 Knock-out Chemotaxis defect, aggregation defect, small aggregates formed Ma et al., 1997 DGAP1 Knock-out F-actin level increased; Faix et al., 1998 overexpressor cellular projections suppressed, speed of motility reduced Discoidin Conventional mutant No elongated morphology Alexander et al., 1992 ERK1 Antisense, knock-out Essential for vegetative growth, morphogenesis and cell-type differentiation; Gaskins et al., 1994 overexpression abnormal expression from slug stage on, abnormal morphogenesis in combination and 1996 with PTP2 ko GEF (aimless) Knock-out Chemotaxis defect, aggregation defective Insall et al., 1996 Gelation factor (ABP120) Knock-out Actin cross-linking affected, cytoskeletal structure altered, pseudopod number and Cox et al., 1996; size altered, cell motility, chemotaxis and phagocytosis impaired; phototaxis defect Fisher et al., 1997 G-protein β subunit Knock-out Aggregation minus, chemotaxis defect, phagocytosis defect, no transient actin Lilly et al., 1993; polymeristaion upon stimulation by chemoattractant Peracino et al., 1998 GRP125 Knock-out Phototaxis defect Stocker et al., 1999 Hisactophilin I and II Knock-out Resistance of cells to acidification reduced; Stoeckelhuber et al., overexpression of hisII resistance of cells to acidification increased 1996 IQGAP Knock-out Completion of cytokinesis affected Adachi et al., 1997 Myosin I (A,B, C) Knock-out Multiple knock-outs: fluid phase endocytosis impaired, membrane internalisation Novak et al., 1995; overexpression impaired, rearrangement of cortical actin-rich structures impaired, cortical tension Jung, G. et al., 1996; reduced; overexpressors: cell velocity reduced, delay in aggregation Novak and Titus, 1997; Dai et al., 1999 Myosin II heavy chain Knock-out Cortical motile activities impaired, cytokinesis defect, development arrested at De Lozanne and antisense mound stage Spudich, 1987; Knecht and Loomis, 1987; Fukui et al., 1990 Myosin II heavy chain Knock-out Partial defects in myosin localization; Kolman et al., 1996 kinase overexpression growth in suspension reduced, size increased, multinuclearity, arrest at mound stage Myosin essential light Antisense Increased cell size, multinuclearity, developmental arrest Pollenz et al., 1992 chain (ELC) Myosin regulatory light Knock-out Increased cell size, multinuclearity, developmental arrest Chen et al., 1994 chain (RLC) Myosin VII Knock-out Phagocytosis reduced by 80% Titus, 1999 PAKa Knock-out Cytokinesis in suspension defect, formation of lateral pseudopods not suppressed, Chung and Firtel, 1999 direction of cell movement impaired, myosin II assembly defect PI3 kinases Knock-out Macropinocytosis defect, deficits in certain F-actin-enriched structures such as Buczynski et al., 1997; ruffles, crowns, Zhou et al., 1998 Ponticulin Knock-out Pseudopod stabilisation affected, efficiency of chemotaxis reduced Shutt et al., 1995 Profilin I and II Knock-out and Reduced speed of motility, cell size increase, increase in F-actin content, Haugwitz et al., 1994; antisense cytoskinesis defect, developmental arrest before fruiting body formation Lee et al., 2000 Phosphotyrosine Knock-out Morphologically aberrant fruiting bodies, prolonged tyrosine Howard et al., 1992 phosphatase 1 (PTP1) overexpression phosphorylation of actin; failure to aggregate in overexpressors and 1993 (PTP2) Knock-out Increased slug and fruiting body size; Howard et al., 1994 overexpression slow growth, developmental abnormalities in overexpressors PTP3 Overexpression Arrest in development Gamper et al., 1996 RacC Overexpression Altered morphology, unsusual F-actin rich structures, enhanced phagocytosis Seastone et al., 1998 The actin cytoskeleton of Dictyostelium 761

Table 1. Continued Protein Exp. approach Phenotype References RacE Knock-out Cytokinesis defect Larochelle et al., 1996 and 1997; Gerald et al., 1998 RasG Knock-out Defects in cell movement and regulation of pseudopod extension, lack of polarity, Tuxworth et al., 1997; activated RasG, abnormal actin cytoskeleton and generation of long filopodia, cytokinesis defect Khosla et al., 1996 dominant negative block in aggregation, cytoskeletal changes RasG Rap1 Overexpression of WT, Cell morphology altered, flattened cells, increased F-actin level; Rebstein et al., 1997; constitutively active early events in phagocytosis affected Seastone et al., 1999 and dominant negative SCAR Knock-out Suppresses CAR2-phenotype; knock-out in wild type: small cells, lowered F-actin Bear et al., 1998 content, abnormal morphology, abnormal F-actin distribution during chemotaxis Talin homologue Knock-out Phagocytosis defect, cell-to-substrate interaction impaired, slight defect in Niewöhner et al., TAL A cytoskinesis 1997 Talin homologue Knock-out Multicellular morphogenesis blocked Tsujioka et al., 1999 TAL B

The type of mutation is given and the corresponding phenotype presented (proteins for which mutant analysis has not revealed a noticeable phenotype are not shown). signalling by G proteins. The cell responds to cAMP by filaments and also severs filaments. In species other than polarising and moving up the cAMP gradient. For the cell to Dictyostelium, cofilin is a target of protein kinases and is sense a gradient of chemotactic agents, an asymmetric inactivated by phosphorylation. It is thought to be a key distribution or activation of the components involved is required. regulator of the actin cytoskeleton (Bamburg et al., 1999). In A localised and transient accumulation of several cytoskeletal living Dictyostelium cells a GFP-cofilin fusion protein proteins has been described; however, how this is accomplished accumulates at cell protrusions in chemotaxing cells, and is not known. Studies with GFP fusions containing the cAMP during phagocytosis it accumulates in a broad zone around the receptor CAR1 show that CAR1 is evenly distributed during forming phagosome. The enrichment on pinosomes is much chemotaxis. This might also apply to the G protein β subunits. more distinct, and GFP-cofilin surrounds the pinosome in a In contrast, CRAC, a cytosolic regulator of adenylate cyclase, narrow zone and disappears rapidly once the pinosome is transiently associates with the plasma membrane and might thus completely engulfed. Attempts to isolate mutants lacking activate intracellular signalling events initiated by G proteins at cofilin have been unsuccessful in Dictyostelium (Aizawa et al., the leading edge (Parent et al., 1998). Evidence for an interaction 1997), which is consistent with data from other organisms, between G proteins and CRAC stems from the finding that such as yeast, in which null mutations in the genes that encode receptor-mediated CRAC redistribution requires G protein β cofilin are lethal (Moon et al., 1993). subunits (Lilly and Devreotes, 1995). Aip1 (actin-interacting protein 1), a partner of cofilin, was The events initiated at leading fronts during chemotaxis identified in a two-hybrid screen against yeast actin and parallel those that occur during phagocytosis. Both are subsequently was shown to cause depolymerisation of actin in characterised by polarisation and formation of pseudopods or the presence of cofilin. Aip1 can bind to actin, and this binding a phagocytic cup, which require transient actin polymerisation. is enhanced by cofilin (Rodal et al., 1999). In Dictyostelium, Moreover, analysis of cells that lack G protein β subunits the Aip1 homologue DAip1 redistributes to newly formed indicated that the signal transduction pathways might be pseudopods during chemotaxis. It accumulates at the common to both processes, given that these cells have a phagocytic cup, stays on the phagosome until it is completely chemotaxis defect and exhibit impaired phagocytosis. The engulfed, and is released approximately one minute after underlying defect in phagocytosis and chemotaxis appears to closure of the phagocytic cup. Pinosomes are also surrounded be a failure of actin to polymerise either upon cAMP by DAip1. A GFP-DAip1 fusion disappears quite rapidly after stimulation or upon contact with a particle during complete internalisation. DAip1 mutants exhibit multiple phagocytosis. Spontaneous actin accumulation, however, still cytoskeleton-associated defects. They show reduced growth occurs. This process therefore appears to require different rates, endocytosis and phagocytosis, impaired motility, as well stimuli and/or cytoskeletal organisations. Use of as defective cytokinesis (Konzok et al., 1999). pharmacological agents showed that the G protein does not act DdLim is a multidomain member of the cysteine-rich family directly on the actin cytoskeleton but requires PLC activation of Lim domain proteins that was isolated from a cytoskeletal and Ca2+ mobilisation (Peracino et al., 1998). fraction of Dictyostelium cells. In addition to possessing a single Lim domain, it has a glycine-rich domain and a C- Cytoskeleton-associated proteins terminal coiled-coil domain. GFP-DdLim fusion proteins Cytoskeleton-associated proteins redistribute during localise to the extreme rim of newly formed extensions. During chemotaxis, phagocytosis and fluid-phase uptake, which all pinocytosis, GFP-DdLim stays on the pinosome until the cup involve dynamic rearrangement of the actin cytoskeleton. closes, and then rapidly dissipates. Interestingly, DdLim seems Recent studies have provided data on three such proteins: to interact with Rac proteins: it can be specifically precipitated cofilin, DAip1 and DdLim. from a whole-cell extract with a Rac1-GST fusion protein. This Cofilin is a member of the actin depolymerising interaction is enhanced when Rac1 is charged with GTPγS. It factor/cofilin (AC) family. It causes the disassembly of was proposed that DdLim might act as an adapter protein at 762 A. A. Noegel and M. Schleicher the cytoskeleton-membrane interface, where it is involved in a and colocalises with F actin at the plasma membrane (Gottwald receptor-mediated rac1-signalling pathway that leads to actin et al., 1996). The requirements for this localisation are in contrast polymerisation. Cells that overexpress DdLim have a severe to data published for the yeast protein, in which a proline-rich growth defect and do not reach cell densities observed for wild- SH3-binding region is thought to be responsible for localisation type cells (3-5×106 cells/ml compared with 1.4 ×107 cells/ml (Freeman et al., 1996). for the wild type). In addition, the cells are multinucleated, which indicates that they have a cytokinesis defect, and have ACTIN AND ACTIN-BINDING PROTEINS AS altered morphology (Prassler et al., 1998). EFFECTORS OF CELL SIGNALLING Rac GTPases Initiation of polymerisation RacF1 is one of >10 Rac proteins in Dictyostelium and is most A central theme in cytoskeleton research is the link between closely related to the previously described Rac1A, Rac1B and signal transduction pathways and the actin cytoskeleton. Rac1C proteins. RacF1 has another homologue in Dictyostelium, Significant advances in studies of amoeba and vertebrate cells RacF2, with which it shares 94% identity, and the absence of revealed that the ARP2/3 complex, a complex of several measurable defects in racF1 mutants might be due to the proteins, is involved in signalling to the actin cytoskeleton presence of RacF2 protein. GFP-RacF1 fusion protein is present (reviewed by Machesky and Insall, 1999; Machesky and in the cytosol and localises to the plasma membrane; however, it is absent from newly formed protrusions. This contrasts with the CYTOSKELETAL AND REGULATORY behaviour of the other GFP fusion proteins we have described above. During pinosome PROTEINS INVOLVED IN formation, GFP-RacF1 stays on the pinosome for a comparatively long time until the CAP/ASP56, calmodulin, clathrin heavy pinosome completely internalises. We CYTOKINESIS chain, coronin, cortexillin, DAip1, DdLim, observed a similar time course of association IQGAP, myosin II (heavy chain, ELC, RLC, during phagocytosis (Rivero et al., 1999a). heavy chain kinase), PAKa, profilin, This might indicate that RacF1 signals to Ras (E, G), TAL A components other than those described above. In summary, several actin-binding proteins Ð ENDO- AND PHAGO- alpha-actinin, CAP/ASP56, cofilin, coronin, of which cofilin, DAip1, DdLim and the CYTOSIS previously described coronin (reviewed by de DAip1, DdLim, gelation factor, G protein Hostos, 1999) are just examples (see Fig. 1) Ð beta-subunit, myosin (I, VII), PI3 kinases, are present at sites of localised actin Rac (C, F1), Rap 1, TAL A accumulation during chemotaxis, phagocytosis and pinocytosis. The time courses of their CELL MOVEMENT AND Akt/PKB, alpha actinin + gelation factor, association with these structures appear to CHEMOTAXIS differ, although for definitive conclusions to be 34 kDa bundling protein, CAP/ASP56, drawn, a side-by-side comparison is needed. capping protein cap 32/34, cofilin, coronin, The distinct patterns of association, however, DAip1, DdMEK1, DGAP1, GEF, G protein are consistent with the requirements for and beta-subunit,PAKa, ponticulin, profilin, generation of different actin assemblies during Ras G these processes. PHOTOTAXIS GFP fusion proteins in domain gelation factor analyses GRP125 GFP fusion proteins not only are suitable for studies of the in vivo dynamics of actin-binding proteins but have also been used to analyse the functions of separate domains in vivo. This DEVELOPMENT way Larochelle et al. (1997) have shown that alpha-actinin +gelation factor , cortexillin I, the C-terminal 40 residues of RacE, a regulator DdMEK1, GEF, G protein beta-subunit, of cytokinesis, localises the protein to the myosin II (heavy chain, ELC, RLC, heavy plasma membrane. This domain, although it chain kinase), profilin, PTP (1, 3), Ras G, exhibited the correct localisation, however, TAL B could not rescue the cytokinesis defect with which a lack of RacE is associated. We found that the N-terminal third of the ASP56/CAP homologue of Dictyostelium mediates its Fig. 1. Actin-binding proteins and regulatory proteins as components of the machinery localisation (Noegel et al., 1999). ASP56/CAP involved in cytokinesis, phagocytosis, endocytosis, chemotaxis and cell movement, and is a G-actin-binding protein that relocates to development – as indicated from mutant analysis and immunofluorescene studies. For newly formed pseudopods during chemotaxis references and details please consult Table 1. The actin cytoskeleton of Dictyostelium 763

Cooper, 1999). The ARP2/3 complex binds to pointed ends of severin and provides another direct link between PAK actin filaments and promotes filament elongation at the fast signalling and the cytoskeleton (Eichinger et al., 1998). growing end. Loisel et al. (1999) demonstrated that purified ARP2/3 complex in vitro, together with cofilin and capping protein, induces actin assembly and reconstitutes movement of ACTIN AND ACTIN-BINDING PROTEINS AS Listeria and Shigella, two pathogens that use the actin in living COORDINATORS OF CELLULAR BEHAVIOUR cells to propel themselves. The ARP2/3 complex is localised to leading edges by members of the WASP protein family. Actin-binding proteins in development WASP is a multidomain protein that links upstream signalling The roles of actin-associated proteins in coordination of pathways to cytoskeletal ligands by providing a binding site for cellular behaviour during the developmental processes of Cdc42, a member of the Rho family of small GTP-binding organisms such as Drosophila and Caenorhabditis elegans are proteins (reviewed by Ramesh et al., 1999). Bear et al. (1998) well established: mutations in several proteins cause alterations isolated SCAR, a WASP-related protein from Dictyostelium, in at specific stages of development (Lundquist et al., 1998; a genetic screen for components that suppress the phenotype Straub et al., 1996). The multicellular development of of mutants that lack the cAMP receptor CAR2. These mutants Dictyostelium involves aggregation of approximately 150,000 arrest during development before tip formation. Inactivation of cells, differentiation into mainly two cell types (Kay, 1999), the SCAR in these cells restored tip formation. Cells lacking formation of a photo- and thermotactically-active slug (Fisher, SCAR have altered morphology and are smaller than wild-type 1997) and, finally, formation of the fruiting body. Several cells. Furthermore, the F-actin levels appear to be lower than studies indicate that the multicellular stage of Dictyostelium is in the wild type. Machesky et al. (1999) have recently shown also sensitive to alterations in the regulation of the actin that a human SCAR enhances the ability of the ARP2/3 cytoskeleton. For example, mutations in the myosin II heavy complex to nucleate actin filaments. chain gene of Dictyostelium give rise to a block in multicellular development, and fruiting body formation does not occur (De Small G proteins, kinases and their targets Lozanne and Spudich, 1987). Similar defects are evident in As described above, RacF1 and RacE have roles in different mutants lacking α-actinin and gelation factor/ABP120, myosin actin-associated events. Another Rac, RacC, enhances light chains and profilins (Chen et al., 1994; Haugwitz et al., phagocytosis when overexpressed. RacC-overexpressing cells 1994; Witke et al., 1992; for summary see Fig. 1). exhibit unusual F-actin-rich structures whose formation can be The availability of the slug stage allows investigation of blocked by inhibition of phosphoinositide 3-kinase (PI 3-kinase; motility events during development, and indeed mutants Seastone et al., 1998). PI 3-kinase provides another way of lacking the gelation factor or GRP125, a gelsolin-related signalling to the cytoskeleton. Two PI 3-kinases have been more protein, cannot orient properly in response to light. Normally, closely investigated in Dictyostelium. Inactivation of both genes Dictyostelium slugs move directly towards a light source, generates mutants that cannot rearrange their cytoskeleton for whereas slugs of these mutants move at an angle towards light efficient pinocytosis (Buczynski et al., 1997; Zhou et al., 1998). (Fisher et al., 1997; Stocker et al., 1999). Dictyostelium slugs The mechanism of activation of the Dictyostelium PI 3-kinases can also orient in a temperature gradient, and move to their is thought to occur via G-protein-coupled chemoreceptors (Meili preferred temperature. Gelation factor mutants have a defect in et al., 1999). Similarly, in mammalian cells they are activated in this reaction as well (Fisher et al., 1997). This result is not response to a variety of extracellular stimuli involving receptor unexpected, given that the signal transduction pathways for tyrosine kinases (Downward, 1995). both reactions merge very early. It is, however, not clear Faix and Dittrich (1996) purified DGAP1, a Rac-interacting whether it is defects in the protein’s activity as an actin-binding protein, from actin-myosin complexes. It is related to the GTP- protein that lead to altered motility, and hence affect phototaxis activating protein IQGAP. DGAP1 mutants have cytoskeletal and thermotaxis, or whether a so-far-unknown role in signal defects. In cells overexpressing DGAP1, the formation of transduction pathways is affected. Genetic studies allow us to cellular projections containing F-actin is suppressed, whereas predict that >20 proteins are involved in generation of a DGAP1-null mutants show elevated F-actin levels (Faix et al., phototactic or thermotactic response; among these, there might 1998). Interestingly GAPA (IQGAP), a closely related GAP, is well be more actin-binding proteins (Fisher, 1997). the protein affected in a cytokinesis-deficient mutant (Adachi et al., 1997). Developmentally regulated actin-binding proteins Kinases have recently attracted much interest as direct Several more actin-binding proteins, such as interaptin or the downstream targets of Rho proteins. Cofilin is one of the talin homologue TALB, are now known to show specific substrates of such kinases in non-Dictyostelium cells (Edwards expression patterns during development, and in these cases the et al., 1999; Maekawa et al., 1999), whereas in Dictyostelium mutants exhibit developmental abnormalities (Rivero et al., cofilin the corresponding residues are not present. In 1998; Tsujoka et al., 1999). Dictyostelium a myosin I heavy chain kinase was identified as In Polysphondylium, a close relative of Dictyostelium, lack downstream target. This kinase is activated by Cdc42 and Rac of cortexillin blocks formation of aggregation streams and also and can activate myosin ID heavy chain by phosphorylation of results in formation of abnormal fruiting bodies (Fey and Cox, serine and threonine residues (Lee et al., 1996). This kinase is 1999). Cortexillins have an N-terminal domain closely related a member of the p21-activated kinase (PAK) family that is to the actin-binding site of α-actinin. They dimerise through a involved in modulation of the actin cytoskeleton or gene coiled-coil region, and studies of cortexillin mutants suggest expression (Bagrodia and Cerione, 1999). Another PAK, a 62- that they play a role during cytokinesis (Faix et al., 1996; kDa protein, phosphorylates the actin-fragmenting protein Weber et al., 1999). Cytokinesis has attracted considerable 764 A. A. Noegel and M. Schleicher interest since it was reported that in Dictyostelium it can occur the proposed roles. An important aspect in Dictyostelium research in the absence of myosin II heavy chain and in the absence of is the exploitation of new assays for mutant analysis Ð for the formation of a contractile ring. Other proteins such as the example, osmoshock response (Kuwayama et al., 1996; Zischka cortexillins might take over functions that allow cytokinesis to et al., 1999), life under more natural conditions (Ponte et al., proceed in these instances (reviewed by Eichinger et al., 1999). 1998, 2000) and biophysical assays (Eichinger et al., 1996; Lee et al., 2000). With these assays in combination with GFP fusions FUTURE DIRECTIONS and mutants, we can pinpoint the contributions of particular proteins to the maintenance of a dynamic actin cytoskeleton. Membrane trafficking and sorting events Future cytoskeletal research will also heavily focus on the role of Identification of proteins associated with internal membranes the cytoskeleton during multicellular development, given that will extend the field into membrane trafficking. Examples are highly sophisticated techniques are now available for studies of comitin, an F-actin-binding protein that associates with the behaviour of single fluorescently tagged cells in a membranes owing to its lectin activity (Weiner et al., 1993; multicellular organism (Zimmermann and Siegert, 1998). Jung, E. et al., 1996), and interaptin. Interaptin has an N- A further step of complexity is reached by generation of terminal actin-binding domain of the α-actinin type. multiple knockouts, with which we can address networks, Investigation of this protein showed that it localises to interactions and crosstalk between various actin-binding membranes of the ER. Membrane association is mediated by proteins (Rivero et al., 1996a). The search for links between a C-terminal hydrophobic region (Rivero et al., 1998). An the cytoskeleton and signal transduction is still going on. interesting link to vesicle trafficking has also been established Dictyostelium allows us to use a combination of biochemical, by the isolation of a CD36/Lim-II homologue that can suppress cell biological and genetic approaches that will enable us to the profilin-null phenotype. DdLIMP is present on vesicles and discover how cells coordinate the different regulatory on ring-like structures on the cell surface. A direct interaction mechanisms that modulate actin dynamics. with profilin was not detected; however, DdLIMP and profilin both bind to phosphatidylinositol 4,5-bisphosphate We thank Dr B. Metz for comments and encouragement. Research by the authors is supported by the DFG, Köln Fortune, the EU, and [PtdIns(4,5)P2], which is a major regulator of profilin activity (Karakesisoglou et al., 1999). the Fonds der Chemischen Industrie. Furthermore, investigation of phagocytosis will continue to attract considerable attention. Phagocytic cups have been REFERENCES studied by immunofluorescence microscopy, and several proteins have been localised to the cup at distinct time points Adachi, H., Takahashi, Y., Hasebe, T., Shirouzu, M., Yokoyama, S. and (Noegel and Luna, 1995; Fig. 1). These investigations are now Sutoh, K. (1997). Dictyostelium IQGAP-related protein specifically involved in the completion of cytokinesis. J. Cell Biol. 137, 891-898. being aided by GFP-fusion proteins, as was brilliantly Aizawa, H., Sutoh, K. and Yahara, I. (1996). Overexpression of cofilin demonstrated for coronin (Maniak et al., 1995). stimulates bundling of actin filaments, membrane ruffling, and cell Morrissette et al. 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