The SCAR/WAVE complex is necessary for proper regulation of traction stresses during amoeboid motility

Cell migration requires a tightly regulated, spatiotemporal coordination of underlying biochemical pathways. Crucial to cell migration is SCAR/WAVE-mediated dendritic F-actin polymerization at the cell's leading edge. Our goal is to understand the role the SCAR/WAVE complex plays in the mechanics of amoeboid migration. To this aim, we measured and compared the traction stresses exerted by Dictyostelium cells lacking the SCAR/WAVE complex proteins PIR121 (pirA(-)) and SCAR (scrA(-)) with those of wild-type cells while they were migrating on flat, elastic substrates. We found that, compared to wild type, both mutant strains exert traction stresses of different strengths that correlate with their F-actin levels. In agreement with previous studies, we found that wild-type cells migrate by repeating a motility cycle in which the cell length and strain energy exerted by the cells on their substrate vary periodically. Our analysis also revealed that scrA(-) cells display an altered motility cycle with a longer period and a lower migration velocity, whereas pirA(-) cells migrate in a random manner without implementing a periodic cycle. We present detailed characterization of the traction-stress phenotypes of the various cell lines, providing new insights into the role of F-actin polymerization in regulating cell-substratum interactions and stresses required for motility.     

The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis

The WAVE/SCAR complex promotes actin nucleation through the Arp2/3 complex, in response to Rac signaling. We show that loss of WVE-1/GEX-1, the only C. elegans WAVE/SCAR homolog, by genetic mutation or by RNAi, has the same phenotype as loss of GEX-2/Sra1/p140/PIR121, GEX-3/NAP1/HEM2/KETTE, or ABI-1/ABI, the three other components of the C. elegans WAVE/SCAR complex. We find that the entire WAVE/SCAR complex promotes actin-dependent events at different times and in different tissues during development. During C. elegans embryogenesis loss of CED-10/Rac1, WAVE/SCAR complex components, or Arp2/3 blocks epidermal cell migrations despite correct epidermal cell differentiation. 4D movies show that this failure occurs due to decreased membrane dynamics in specific epidermal cells. Unlike myoblasts in Drosophila, epidermal cell fusions in C. elegans can occur in the absence of WAVE/SCAR or Arp2/3. Instead we find that subcellular enrichment of F-actin in epithelial tissues requires the Rac-WAVE/SCAR-Arp2/3 pathway. Intriguingly, we find that at the same stage of development both F-actin and WAVE/SCAR proteins are enriched apically in one epithelial tissue and basolaterally in another. We propose that temporally and spatially regulated actin nucleation by the Rac-WAVE/SCAR-Arp2/3 pathway is required for epithelial cell organization and movements during morphogenesis.     

Regulation through phosphorylation/dephosphorylation cascade systems

The cyclic interconversion of enzymes between phosphorylated and unphosphorylated forms comprises a major mechanism of cellular regulation. A theoretical analysis of reversible covalent modification systems (Stadtman, E.R., and Chock, P.B. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2761-2765) revealed that they are endowed with extraordinary regulatory capacities; they may exhibit smooth, flexible responses to changes in single and multiple metabolite levels, signal amplification, and apparent positive cooperativity. To test qualitatively and quantitatively the theories and equations involved in this analysis, a model in vitro phosphorylation/dephosphorylation cyclic cascade was developed in which the converter enzymes catalyzing the covalent modifications were cAMP-dependent protein kinase (EC 2.7.1.37; type II) and phosphoprotein phosphatase (EC 3.1.3.16; Mr = 38,000), both purified to near homogeneity from bovine heart. The kinetic constants for both enzymes were fully characterized using the nanopeptide Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu as the interconvertible substrate, cAMP as an activator for the kinase, and Pi as an inhibitor for the phosphatase. In the presence of a nearly constant concentration of ATP, a steady-state level of phosphorylation of the peptide was attained which was determined by the relative concentrations of the kinase, phosphatase, and effectors. As predicted by the cyclic cascade model, this monocyclic cascade exhibited both signal amplification and an increase in sensitivity to variations in multiple effector concentrations. In addition, the data show that the steady-state level of phosphorylation obtained in the presence of an activator of the kinase (e.g. cAMP) and an inhibitor of the phosphatase (e.g. Pi) is a function of the product of the relative effector concentrations. Finally, the results reveal that when the concentration of enzyme-substrate complex is not negligible, cyclic cascades are potentially more sensitive to variations in effector concentrations and can achieve even greater signal amplification than predicted previously.     

Myosin-II reorganization during mitosis is controlled temporally by its dephosphorylation and spatially by Mid1 in fission yeast

Cytokinesis in many eukaryotes requires an actomyosin contractile ring. Here, we show that in fission yeast the myosin-II heavy chain Myo2 initially accumulates at the division site via its COOH-terminal 134 amino acids independently of F-actin. The COOH-terminal region can access to the division site at early G2, whereas intact Myo2 does so at early mitosis. Ser1444 in the Myo2 COOH-terminal region is a phosphorylation site that is dephosphorylated during early mitosis. Myo2 S1444A prematurely accumulates at the future division site and promotes formation of an F-actin ring even during interphase. The accumulation of Myo2 requires the anillin homologue Mid1 that functions in proper ring placement. Myo2 interacts with Mid1 in cell lysates, and this interaction is inhibited by an S1444D mutation in Myo2. Our results suggest that dephosphorylation of Myo2 liberates the COOH-terminal region from an intramolecular inhibition. Subsequently, dephosphorylated Myo2 is anchored by Mid1 at the medial cortex and promotes the ring assembly in cooperation with F-actin.     

DISTORTED3/SCAR2 is a putative arabidopsis WAVE complex subunit that activates the Arp2/3 complex and is required for epidermal morphogenesis

In a plant cell, a subset of actin filaments function as a scaffold that positions the endomembrane system and acts as a substrate on which organelle motility occurs. Other actin filament arrays appear to be more dynamic and reorganize in response to growth signals and external cues. The distorted group of trichome morphology mutants provides powerful genetic tools to study the control of actin filament nucleation in the context of morphogenesis. In this article, we report that DISTORTED3 (DIS3) encodes a plant-specific SCAR/WAVE homolog. Null alleles of DIS3, like those of other Arabidopsis thaliana WAVE and Actin-Related Protein (ARP) 2/3 subunit genes, cause trichome distortion, defects in cell-cell adhesion, and reduced hypocotyl growth in etiolated seedlings. DIS3 efficiently activates the actin filament nucleation and branching activity of vertebrate Arp2/3 and functions within a WAVE-ARP2/3 pathway in vivo. DIS3 may assemble into a WAVE complex via a physical interaction with a highly diverged Arabidopsis Abi-1-like bridging protein. These results demonstrate the utility of the Arabidopsis trichome system to understand how the WAVE and ARP2/3 complexes translate signaling inputs into a coordinated morphogenetic response.     

Localization of caldesmon and its dephosphorylation during cell division

Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle caldesmon to dissociate from microfilaments during prometaphase. (Yamashiro, S., Y. Yamakita, R. Ishikawa, and F. Matsumura. 1990. Nature (Lond.). 344:675-678; Yamashiro, S., Y. Yamakita, H. Hosoya, and F. Matsumura. 1991. Nature (Lond.) 349:169-172). To explore the functions of caldesmon phosphorylation during cytokinesis, we have examined the relationship between the phosphorylation level, actin- binding, and in vivo localization of caldesmon in cultured cells after their release of metaphase arrest. Immunofluorescence studies have revealed that caldesmon is localized diffusely throughout cytoplasm in metaphase. During early stages of cytokinesis, caldesmon is still diffusely present and not concentrated in contractile rings, in contrast to the accumulation of actin in cleavage furrows during cytokinesis. In later stages of cytokinesis, most caldesmon is observed to be yet diffusely localized although some concentration of caldesmon is observed in cortexes as well as in cleavage furrows. When daughter cells begin to spread, caldesmon shows complete colocalization with F- actin-containing structures. These observations are consistent with changes in the levels of microfilament-associated caldesmon during synchronized cell division. Caldesmon is missing from microfilaments in prometaphase cells arrested by nocodazole treatment, as shown previously (Yamashiro, S., Y. Yamakita, R. Iskikawa, and F. Matsumura. 1990. Nature (Lond.). 344:675-678). The level of microfilament- associated caldesmon stays low (12% of that of interphase cells) when some cells start cytokinesis at 40 min after the release of metaphase arrest. When 60% of cells finish cytokinesis at 60 min, the level of microfilament-associated caldesmon is recovered to 50% of that of interphase cells. The level of microfilament-associated caldesmon is then gradually increased to 80% when cells show spreading at 120 min. Dephosphorylation appears to occur during cytokinesis. It starts when cells begin to show cytokinesis at 40 min and completes when most cells finish cytokinesis at 60 min. These results suggest that caldesmon is not associated with microfilaments of cleavage furrows at least in initial stages of cytokinesis and that dephosphorylation of caldesmon appears to couple with its reassociation with microfilaments. Because caldesmon is known to inhibit actomyosin ATPase and/or regulate actin assembly, its continued dissociation from microfilaments may be required for the assembly and/or activation of contractile rings.     

SCAR/WAVE complex recruitment to a supracellular actomyosin cable by myosin activators and a junctional Arf-GEF during Drosophila dorsal closure

Expansive Arp2/3 actin networks and contractile actomyosin networks can be spatially and temporally segregated within the cell, but the networks also interact closely at various sites, including adherens junctions. However, molecular mechanisms coordinating these interactions remain unclear. We found that the SCAR/WAVE complex, an Arp2/3 activator, is enriched at adherens junctions of the leading edge actomyosin cable during Drosophila dorsal closure. Myosin activators were both necessary and sufficient for SCAR/WAVE accumulation at leading edge junctions. The same myosin activators were previously shown to recruit the cytohesin Arf-GEF Steppke to these sites, and mammalian studies have linked Arf small G protein signaling to SCAR/WAVE activation. During dorsal closure, we find that Steppke is required for SCAR/WAVE enrichment at the actomyosin-linked junctions. Arp2/3 also localizes to adherens junctions of the leading edge cable. We propose that junctional actomyosin activity acts through Steppke to recruit SCAR/WAVE and Arp2/3 for regulation of the leading edge supracellular actomyosin cable during dorsal closure.     

The zebrafish down syndrome cell adhesion molecule is involved in cell movement during embryogenesis

The Down syndrome cell adhesion molecule (Dscam) is a protein overexpressed in the brains of Down syndrome patients and implicated in mental retardation. Dscam is involved in axon guidance and branching in Drosophila, but cellular roles in vertebrates have yet to be elucidated. To understand its role in vertebrate development, we cloned the zebrafish homolog of Dscam and showed that it shares high amino acid identity and structure with the mammalian homologs. Zebrafish dscam is highly expressed in developing neurons, similar to what has been described in Drosophila and mouse. When dscam expression is diminished by morpholino injection, embryos display few neurons and their axons do not enter stereotyped pathways. Zebrafish dscam is also present at early embryonic stages including blastulation and gastrulation. Its loss results in early morphogenetic defects. dscam knockdown results in impaired cell movement during epiboly as well as in subsequent stages. We propose that migrating cells utilize dscam to remodel the developing embryo.     

Topoisomerase II is regulated by translationally controlled tumor protein for cell survival during organ growth in Drosophila

Regulation of cell survival is critical for organ development. Translationally controlled tumor protein (TCTP) is a conserved protein family implicated in the control of cell survival during normal development and tumorigenesis. Previously, we have identified a human Topoisomerase II (TOP2) as a TCTP partner, but its role in vivo has been unknown. To determine the significance of this interaction, we examined their roles in developing Drosophila organs. Top2 RNAi in the wing disc leads to tissue reduction and caspase activation, indicating the essential role of Top2 for cell survival. Top2 RNAi in the eye disc also causes loss of eye and head tissues. Tctp RNAi enhances the phenotypes of Top2 RNAi. The depletion of Tctp reduces Top2 levels in the wing disc and vice versa. Wing size is reduced by Top2 overexpression, implying that proper regulation of Top2 level is important for normal organ development. The wing phenotype of Tctp RNAi is partially suppressed by Top2 overexpression. This study suggests that mutual regulation of Tctp and Top2 protein levels is critical for cell survival during organ development.Subject terms: Cell growth, Organogenesis     

Water movement through Quercus rubra I. Leaf water potential and conductance during polycyclic growth

Two experiments examined simultaneous changes in leaf area (A_L), root length (L_r), stomatal conductance (g_s), leaf water potential (Ψ_L), transpiration and hydraulic plant conductance per unit leaf area (G) during the first three shoot cycles of northern red oak (Quercus rubra L.) grown under favourable and controlled conditions. Each shoot cycle consisted of bud swell, stem elongation, leaf expansion and rest; roots grew almost continuously. The g_s of all leaves decreased substantially while leaves of the newest flush were expanding and increased modestly when seedling leaf area remained constant. Overall, g_s decreased. The Ψ_L of mature leaves decreased during leaf expansion and increased by an equivalent amount during intervening periods. Possible explanations for the paired changes in g_s and Ψ_L are considered. Changes in G closely paralleled those of canopy g_s. These parallel changes during polycyclic seedling growth should act to keep seedling Ψ_L relatively constant as plant size increases and thereby help prevent Ψ_L from dropping to levels that would cause runaway embolism.     

SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodeling at the site of myoblast fusion

Myoblast fusion is crucial for formation and repair of skeletal muscle. Here we show that active remodeling of the actin cytoskeleton is essential for fusion in Drosophila. Using live imaging, we have identified a dynamic F-actin accumulation (actin focus) at the site of fusion. Dissolution of the actin focus directly precedes a fusion event. Whereas several known fusion components regulate these actin foci, others target additional behaviors required for fusion. Mutations in kette/Nap1, an actin polymerization regulator, lead to enlarged foci that do not dissolve, consistent with the observed block in fusion. Kette is required to positively regulate SCAR/WAVE, which in turn activates the Arp2/3 complex. Mutants in SCAR and Arp2/3 have a fusion block and foci phenotype, suggesting that Kette-SCAR-Arp2/3 participate in an actin polymerization event required for focus dissolution. Our data identify a new paradigm for understanding the mechanisms underlying fusion in myoblasts and other tissues.     

UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph Polarize F-Actin during Embryonic Morphogenesis by Regulating the WAVE/SCAR Actin Nucleation Complex

Many cells in a developing embryo, including neurons and their axons and growth cones, must integrate multiple guidance cues to undergo directed growth and migration. The UNC-6/netrin, SLT-1/slit, and VAB-2/Ephrin guidance cues, and their receptors, UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph, are known to be major regulators of cellular growth and migration. One important area of research is identifying the molecules that interpret this guidance information downstream of the guidance receptors to reorganize the actin cytoskeleton. However, how guidance cues regulate the actin cytoskeleton is not well understood. We report here that UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph differentially regulate the abundance and subcellular localization of the WAVE/SCAR actin nucleation complex and its activator, Rac1/CED-10, in the Caenorhabditis elegans embryonic epidermis. Loss of any of these three pathways results in embryos that fail embryonic morphogenesis. Similar defects in epidermal enclosure have been observed when CED-10/Rac1 or the WAVE/SCAR actin nucleation complex are missing during embryonic development in C. elegans. Genetic and molecular experiments demonstrate that in fact, these three axonal guidance proteins differentially regulate the levels and membrane enrichment of the WAVE/SCAR complex and its activator, Rac1/CED-10, in the epidermis. Live imaging of filamentous actin (F-actin) in embryos developing in the absence of individual guidance receptors shows that high levels of F-actin are not essential for polarized cell migrations, but that properly polarized distribution of F-actin is essential. These results suggest that proper membrane recruitment and activation of CED-10/Rac1 and of WAVE/SCAR by signals at the plasma membrane result in polarized F-actin that permits directed movements and suggest how multiple guidance cues can result in distinct changes in actin nucleation during morphogenesis.     

Phosphorylation of NDRG1 is temporally and spatially controlled during the cell cycle

The tumour metastasis suppressor, N-myc Downstream Regulated Gene (NDRG) 1, is a by the protein kinases SGK1 and GSK3β, but the relevance of its phosphorylation remains unclear. Analysis of HCT116 cells, either proficient or deficient for p53 revealed NDRG1 protein expression and phosphorylation by SGK1 was increased basally in p53-deficient cells. Treatment with the cell cycle inhibitors, aphidicolin or nocodazole also revealed increased NDRG1 phosphorylation in p53-deficient cells. Finally, phosphorylated NDRG1 was found to co-localise with γ-tubulin on centromeres and also to the cleavage furrow during cytokinesis. Taken together, this work demonstrates that NDRG1 phosphorylation, by the protein kinase SGK1, is temporally and spatially controlled during the cell cycle, suggesting a role for NDRG1 in successful mitosis.     

Fat2 acts through the WAVE regulatory complex to drive collective cell migration during tissue rotation

Directional cell movements during morphogenesis require the coordinated interplay between membrane receptors and the actin cytoskeleton. The WAVE regulatory complex (WRC) is a conserved actin regulator. Here, we found that the atypical cadherin Fat2 recruits the WRC to basal membranes of tricellular contacts where a new type of planar-polarized whip-like actin protrusion is formed. Loss of either Fat2 function or its interaction with the WRC disrupts tricellular protrusions and results in the formation of nonpolarized filopodia. We provide further evidence for a molecular network in which the receptor tyrosine phosphatase Dlar interacts with the WRC to couple the extracellular matrix, the membrane, and the actin cytoskeleton during egg elongation. Our data uncover a mechanism by which polarity information can be transduced from a membrane receptor to a key actin regulator to control collective follicle cell migration during egg elongation. 4D-live imaging of rotating MCF10A mammary acini further suggests an evolutionary conserved mechanism driving rotational motions in epithelial morphogenesis.     

Regulation of sperm flagellar movement by protein phosphorylation and dephosphorylation

Flagellar motility of Triton models of sea urchin spermatozoa was reactivated by cyclic AMP-dependent protein kinase and a protein factor, termed motility activator, both of which were prepared from the detergent-extract of sea urchin spermatozoa. It was shown that phosphorylation of the motility activator by the protein kinase is necessary for the reactivation of flagellar motility [Ishiguro et al, J. Cell Biol. 92:777-782, 1982; Murofushi et al, in "Biological Functions of Microtubules and Related Structures," Academic Press, 1982]. Reactivating factor was also detected in a KCl-extract of the axoneme fraction devoid of the detergent-extractable materials. The activity of this factor was also cyclic AMP- and protein kinase-dependent. Furthermore, when freshly prepared Triton models were treated with phosphoprotein phosphatase prepared from bovine cardiac muscle, the flagellar motility was drastically suppressed. This inhibition of the motility was partially recovered by the addition of cyclic AMP and protein kinase to the phosphatase-treated models.     

PHLPP-mediated dephosphorylation of S6K1 inhibits protein translation and cell growth

PHLPP is a family of Ser/Thr protein phosphatases that contains PHLPP1 and PHLPP2 isoforms. We have shown previously that PHLPP functions as a tumor suppressor by negatively regulating Akt signaling in cancer cells. Here we report the identification of ribosomal protein S6 kinase 1 (S6K1) as a novel substrate of PHLPP. Overexpression of both PHLPP isoforms resulted in a decrease in S6K1 phosphorylation in cells, and this PHLPP-mediated dephosphorylation of S6K1 was independent of its ability to dephosphorylate Akt. Conversely, S6K1 phosphorylation was increased in cells depleted of PHLPP expression. Furthermore, we showed that the insulin receptor substrate 1 (IRS-1) expression and insulin-induced Akt phosphorylation were significantly decreased as the result of activation of the S6K-dependent negative feedback loop in PHLPP knockdown cells. Functionally, the phosphorylation of ribosomal protein S6 (rpS6) and the amount of phosphorylated rpS6 bound to the translation initiation complex were increased in PHLPP-knockdown cells. This correlated with increased cell size, protein content, and rate of cap-dependent translation. Taken together, our results demonstrate that loss of PHLPP expression activates the S6K-dependent negative feedback loop and that PHLPP is a novel player involved in regulating protein translation initiation and cell size via direct dephosphorylation of S6K1.