Regulation of Dictyostelium morphogenesis by RapGAP3

Rap1 is a key regulator of cell adhesion and cell motility in Dictyostelium. Here, we identify a Rap1-specific GAP protein (RapGAP3) and provide evidence that Rap1 signaling regulates cell-cell adhesion and cell migration within the multicellular organism. RapGAP3 mediates the deactivation of Rap1 at the late mound stage of development and plays an important role in regulating cell sorting during apical tip formation, when the anterior-posterior axis of the organism is formed, by controlling cell-cell adhesion and cell migration. The loss of RapGAP3 results in a severely altered morphogenesis of the multicellular organism at the late mound stage. Direct measurement of cell motility within the mound shows that rapGAP3⁻ cells have a reduced speed of movement and, compared to wild-type cells, have a reduced motility towards the apex. rapGAP3⁻ cells exhibit some increased EDTA/EGTA sensitive cell-cell adhesion at the late mound stage. RapGAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation, which is dependent on F-actin polymerization. We suggest that the altered morphogenesis and the cell-sorting defect of rapGAP3⁻ cells may result in reduced directional movement of the mutant cells to the apex of the mound.     

Dynamic localization of the actin-bundling protein cortexillin I during cell migration

The circadian clock in plants regulates many important physiological and biological processes, including leaf movement. We have used an imaging system to genetically screen Arabidopsis seedlings for altered leaf movement with the aim of identifying a circadian clock gene. A total of 285 genes were selected from publicly available microarrays that showed an expression pattern similar to those of the Arabidopsis core oscillator genes. We subsequently isolated 42 homozygous recessive mutants and analyzed their leaf movements. We also analyzed leaf movements of activation tagging mutants that showed altered flowering time. We found that agl6-1D plants, in which AGAMOUS-LIKE 6 (AGL6) was activated by the 35S enhancer, showed a shortened period of leaf movement as well as a high level of ZEITLUPE (ZTL) expression, reduced amplitude of LATE ELONGATED HYPOCOTYL (LHY) expression, and arrhythmic TIMING OF CAB EXPRESSION1 (TOC1)/CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression. A shortened period of leaf movement was also seen in 35S-AGL6-myc plants, although 35S-amiRAGL6 plants, transgenic plants overexpressing an artificial miRNA (amiR) targeting AGL6, showed unaltered leaf movement. The amplitude of CHLOROPHYLL A/B BINDING PROTEIN 2 (CAB2) expression, a circadian output gene, was also reduced in agl6-1D plants. Taken together, these results suggest that AGL6 plays a potential role in the regulation of the circadian clock by regulating ZTL mRNA level in Arabidopsis.     

RapGAP9 regulation of the morphogenesis and development in Dictyostelium

Recent reports have demonstrated that the importance of Rap1-specific GTPase-activating proteins (GAPs) in the spatial and temporal regulation of Rap1 activity during cell migration and development in Dictyostelium. Here, we identified another putative Rap1 GAP-domain containing protein, showing high sequence homologies with those of human Rap1GAP and Dictyotelium RapGAP3, by bioinformatic search. Loss of RapGAP9 resulted in some defects in morphogenesis and development in Dicytostelium. rapGAP9 null cells were more flattened and spread, and highly multinucleated. Compared to wild-type cells, cells lacking RapGAP9 exhibited increased levels of F-actin and more filopodia. These results suggest that RapGAP9 is involved in the regulation of cytoskeleton reorganization and cytokinesis. rapGAP9 null cells showed a small increase of cell–substratum attachment and slightly lower moving speed and directionality compared to wild-type cells. In addition, the loss of RapGAP9 resulted in an altered morphology of fruiting body with a shorter length of stalk and spore. Identification and characterization of RapGAP9 in this study will provide further insights into the molecular mechanism by which Rap1 regulates cytoskeleton reorganization and morphogenesis in Dictyostelium.     

The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains.

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the alpha and beta subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH(2)-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end-directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.     

Regulation of actin cytoskeleton by Rap1 binding to RacGEF1

Rap1 is rapidly and transiently activated in response to chemoattractant stimulation and helps establish cell polarity by locally modulating cytoskeletons. Here, we investigated the mechanisms by which Rap1 controls actin cytoskeletal reorganization in Dictyostelium and found that Rap1 interacts with RacGEF1 in vitro and stimulates F-actin polymerization at the sites where Rap1 is activated upon chemoattractant stimulation. Live cell imaging using GFP-coronin, a reporter for F-actin, demonstrates that cells expressing constitutively active Rap1 (Rap1CA) exhibit a high level of F-actin uniformly distributed at the cortex including the posterior and lateral sides of the chemotaxing cell. Examination of the localization of a PH-domain containing PIP3 reporter, PhdA-GFP, and the activation of Akt/Pkb and other Ras proteins in Rap1CA cells reveals that activated Rap1 has no effect on the production of PIP3 or the activation of Akt/Pkb and Ras proteins in response to chemoattractant stimulation. Rac family proteins are crucial regulators in actin cytoskeletal reorganization. In vitro binding assay using truncated RacGEF1 proteins shows that Rap1 interacts with the DH domain of RacGEF1. Taken together, these results suggest that Rap1-mediated F-actin polymerization probably occurs through the Rac signaling pathway by directly binding to RacGEF1.     

Interferon α Inhibits a Src-mediated Pathway Necessary for Shigella-induced Cytoskeletal Rearrangements in Epithelial Cells

Shigella flexneri, the causative agent of bacillary dysentery, has the ability to enter nonphagocytic cells. The interferon (IFN) family of cytokines was found to inhibit Shigella invasion of cultured epithelial cells. We show here that IFN-α inhibits a Src-dependent signaling cascade triggered by Shigella that leads to the reorganization of the host cell cytoskeleton. Immunofluorescence studies showed that IFN-α inhibits Shigella-induced actin polymerization required for bacterial entry into cells. Phosphorylation of cortactin, a Src-substrate specifically tyrosyl-phosphorylated during Shigella entry, was inhibited by IFN-α. Overexpression of a dominant interfering form of pp60c-src led to inhibition of Shigella-induced cytoskeletal rearrangements and decreased cortactin phosphorylation indicating a role for Src in Shigella entry. Also, Shigella uptake in cells that expressed constitutively active Src was unaffected by IFN-α treatment. We conclude that Src kinase activity is necessary for Shigella invasion of epithelial cells and that IFN-α inhibits this Src-dependent signaling pathway.     

The “8-kD” Cytoplasmic Dynein Light Chain Is Required for Nuclear Migration and for Dynein Heavy Chain Localization in Aspergillus nidulans

The heavy chain of cytoplasmic dynein is required for nuclear migration in Aspergillus nidulans and other fungi. Here we report on a new gene required for nuclear migration, nudG, which encodes a homologue of the “8-kD” cytoplasmic dynein light chain (CDLC). We demonstrate that the temperature sensitive nudG8 mutation inhibits nuclear migration and growth at restrictive temperature. This mutation also inhibits asexual and sexual sporulation, decreases the intracellular concentration of the nudG CDLC protein and causes the cytoplasmic dynein heavy chain to be absent from the mycelial tip, where it is normally located in wild-type mycelia. Coimmunoprecipitation experiments with antibodies against the cytoplasmic dynein heavy chain (CDHC) and the nudG CDLC demonstrated that some fraction of the cytoplasmic dynein light chain is in a protein complex with the CDHC. Sucrose gradient sedimentation analysis, however, showed that not all of the NUDG protein is complexed with the heavy chain. A double mutant carrying a cytoplasmic dynein heavy chain deletion plus a temperature-sensitive nudG mutation grew no more slowly at restrictive temperature than a strain with only the CDHC deletion. This result demonstrates that the effect of the nudG mutation on nuclear migration and growth is mediated through an interaction with the CDHC rather than with some other molecule (e.g., myosin-V) with which the 8-kD CDLC might theoretically interact.     

SCAR,a WASP-related Protein,Isolated as a Suppressor of Receptor Defects in Late Dictyostelium Development

G protein–coupled receptors trigger the reorganization of the actin cytoskeleton in many cell types, but the steps in this signal transduction cascade are poorly understood. During Dictyostelium development, extracellular cAMP functions as a chemoattractant and morphogenetic signal that is transduced via a family of G protein–coupled receptors, the cARs. In a strain where the cAR2 receptor gene is disrupted by homologous recombination, the developmental program arrests before tip formation. In a genetic screen for suppressors of this phenotype, a gene encoding a protein related to the Wiskott-Aldrich Syndrome protein was discovered. Loss of this protein, which we call SCAR (suppressor of cAR), restores tip formation and most later development to cAR2⁻ strains, and causes a multiple-tip phenotype in a cAR2⁺ strain as well as leading to the production of extremely small cells in suspension culture. SCAR⁻cells have reduced levels of F-actin staining during vegetative growth, and abnormal cell morphology and actin distribution during chemotaxis. Uncharacterized homologues of SCAR have also been identified in humans, mouse, Caenorhabditis elegans, and Drosophila. These data suggest that SCAR may be a conserved negative regulator of G protein-coupled signaling, and that it plays an important role in regulating the actin cytoskeleton.     

kakapo, a Gene Required for Adhesion Between and Within Cell Layers in Drosophila, Encodes a Large Cytoskeletal Linker Protein Related to Plectin and Dystrophin

Mutations in kakapo were recovered in genetic screens designed to isolate genes required for integrin-mediated adhesion in Drosophila. We cloned the gene and found that it encodes a large protein (>5,000 amino acids) that is highly similar to plectin and BPAG1 over the first 1,000–amino acid region, and contains within this region an α-actinin type actin-binding domain. A central region containing dystrophin-like repeats is followed by a carboxy domain that is distinct from plectin and dystrophin, having neither the intermediate filament-binding domain of plectin nor the dystroglycan/syntrophin-binding domain of dystrophin. Instead, Kakapo has a carboxy terminus similar to the growth arrest–specific protein Gas2. Kakapo is strongly expressed late during embryogenesis at the most prominent site of position-specific integrin adhesion, the muscle attachment sites. It is concentrated at apical and basal surfaces of epidermal muscle attachment cells, at the termini of the prominent microtubule bundles, and is required in these cells for strong attachment to muscles. Kakapo is also expressed more widely at a lower level where it is essential for epidermal cell layer stability. These results suggest that the Kakapo protein forms essential links among integrins, actin, and microtubules.     

A myosin I is involved in membrane recycling from early endosomes

Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin-myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA(-)/B(-), and myoB(-) cells) and wild-type cells treated with the myosin inhibitor butanedione monoxime accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryoelectron microscopy method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA(-)/B(-) cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment.     

In Vitro Repression of Brca1-associated RING Domain Gene,Bard1, Induces Phenotypic Changes in Mammary Epithelial Cells

BRCA1-associated RING domain (BARD1) was identified as a protein interacting with the breast cancer gene product BRCA1. The identification of tumorigenic missense mutations within BRCA1 that impair the formation of BARD1–BRCA1 complexes, and of BARD1 mutations in breast carcinomas, sustain the view that BARD1 is involved in BRCA1-mediated tumor suppression. We have cloned the murine Bard1 gene and determined that its expression in different tissues correlates with the expression profile of Brca1. To investigate the function of Bard1, we have reduced Bard1 gene expression in TAC-2 cells, a murine mammary epithelial cell line that retains morphogenetic properties characteristic of normal breast epithelium. Partial repression of Bard1, achieved by the transfection of TAC-2 cells with plasmids constitutively expressing ribozymes or antisense RNAs, resulted in marked phenotypic changes, consisting of altered cell shape, increased cell size, high frequency of multinucleated cells, and aberrant cell cycle progression. Furthermore, Bard1-repressed cell clones overcame contact inhibition of cell proliferation when grown in monolayer cultures and lost the capacity to form luminal structures in three-dimensional collagen gels. These results demonstrate that Bard1 repression induces complex changes in mammary epithelial cell properties which are suggestive of a premalignant phenotype.     

Dorsoventral Differences in Cell-Cell Interactions Modulate the Motile Behaviour of Cells from the Xenopus Gastrula

When groups of cells from the inner marginal zone (mesendoderm) of the early Xenopus gastrula are placed on a fibronectin-coated substratum, the explants of the dorsal region spread into monolayers whereas those from the ventral region, though they adhere to the substratum, do not show this spreading reaction. This different behaviour is not reflected in the in vitro behaviour of the respective cells kept in isolation. No difference between dorsal and ventral cells was observed, when they were tested for lamellipodia-driven spreading, movement over the substratum or properties of integrin- and cadherin-mediated adhesion. However, cell contacts between individual dorsal cells are significantly less stable than those between ventral cells. The higher flexibility of the cell-cell contacts seems to determine the spreading behaviour of the dorsal explants, which includes lamellipodia-driven outward movement of the peripheral cells, rearrangements of the cells, building up a horizontal tension within the aggregate and intercalation of cells from above into the bottom layer. Ventral explants lack these properties. Staining for F-actin revealed a decisive difference of the supracellular organisation of the cytoskeleton that underlies the morphology of the different types of explants. Evidence for a higher flexibility of cell-cell contacts in the dorsal mesendoderm was also obtained in SEM studies on gastrulating embryos. Dorsal mesendodermal cells show stronger protrusive activity as compared to ventral mesendodermal cells. The meaning of these observations for the mechanisms of morphogenetic movements during gastrulation is central to the discussion.     

Localization of cytoskeletal proteins in Pneumocystis carinii by immuno-electron microscopy

Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms.     

The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions

Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16⁻ and ATG9⁻/16⁻ cells and compare them to the previously reported ATG9⁻ mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9⁻ and ATG16⁻ cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9⁻/16⁻ double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9⁻/16⁻ cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9⁻/16⁻ cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes.     

MicroFilament Analyzer identifies actin network organizations in epidermal cells of Arabidopsis thaliana roots

The plant cytoskeleton plays a crucial role in the cells’ growth and development during different developmental stages and it undergoes many rearrangements. In order to describe the arrangements of the F-actin cytoskeleton in root epidermal cells of Arabidopsis thaliana, the recently developed software MicroFilament Analyzer (MFA) was exploited. This software enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way. Using confocal microscopy and transgenic GFP-FABD2-GFP plants the actin cytoskeleton was visualized in the root epidermis. MFA analysis revealed that during the early stages of cell development F-actin is organized in a mainly random pattern. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. In the evolution from young to old cells, an approximately even distribution of transverse, oblique or combined orientations is always present besides the switch from random to a longitudinal oriented actin cytoskeleton.