The role of DIF-1 signaling in Dictyostelium development

We have constructed a mutant blocked in the biosynthesis of DIF-1, a chlorinated signal molecule proposed to induce differentiation of both major prestalk cell types formed during Dictyostelium development. Surprisingly, the mutant still forms slugs retaining one prestalk cell type, the pstA cells, and can form mature stalk cells. However, the other major prestalk cell type, the pstO cells, is missing. Normal pstO cell differentiation and their patterning in the slug are restored by development on a uniform concentration of DIF-1. We conclude that pstO and pstA cells are in fact induced by separate signals and that DIF-1 is the pstO inducer. Positional information, in the form of DIF-1 gradients, is evidently not required for pstO cell induction.     

A novel disintegrin domain protein affects early cell type specification and pattern formation in Dictyostelium

The cellular slime mold, Dictyostelium discoideum is a non-metazoan organism, yet we now demonstrate that a disintegrin domain-containing protein, the product of the ampA gene, plays a role in cell type specification. Disintegrin domain-containing proteins are involved in Notch signaling in Drosophila and C. elegans via an ectodomain shedding mechanism that depends on a metalloprotease domain. The Dictyostelium protein lacks a metalloprotease domain. Nonetheless, analysis of cell type specific reporter gene expression during development of the ampA null strain identifies patterning defects that define two distinct roles for the AmpA protein in specifying cell fate. In the absence of a functional ampA gene, cells prematurely specify as prespore cells. Prestalk cell differentiation and migration are delayed. Both of these defects can be rescued by the inclusion of 10% wild-type cells in the developing null mutant aggregates, indicating that the defect is non-cell autonomous. The ampA gene is also demonstrated to be necessary in a cell-autonomous manner for the correct localization of anterior-like cells to the upper cup of the fruiting body. When derived from ampA null cells, the anterior-like cells are unable to localize to positions in the interior of the developing mounds. Wild-type cells can rescue defects in morphogenesis by substituting for null cells when they differentiate as anterior-like cells, but they cannot rescue the ability of ampA null cells to fill this role. Thus, in spite of its simpler structure, the Dictyostelium ampA protein carries out the same diversity of functions that have been observed for the ADAM and ADAMTS families in metazoans.     

Cell type-specific filamin complex regulation by a novel class of HECT ubiquitin ligase is required for normal cell motility and patterning

Differential cell motility, which plays a key role in many developmental processes, is perhaps most evident in examples of pattern formation in which the different cell types arise intermingled before sorting out into discrete tissues. This is thought to require heterogeneities in responsiveness to differentiation-inducing signals that result in the activation of cell type-specific genes and 'salt and pepper' patterning. How differential gene expression results in cell sorting is poorly defined. Here we describe a novel gene (hfnA) that provides the first mechanistic link between cell signalling, differential gene expression and cell type-specific sorting in Dictyostelium. HfnA defines a novel group of evolutionarily conserved HECT ubiquitin ligases with an N-terminal filamin domain (HFNs). HfnA expression is induced by the stalk differentiation-inducing factor DIF-1 and is restricted to a subset of prestalk cells (pstO). hfnA(-) pstO cells differentiate but their sorting out is delayed. Genetic interactions suggest that this is due to misregulation of filamin complex activity. Overexpression of filamin complex members phenocopies the hfnA(-) pstO cell sorting defect, whereas disruption of filamin complex function in a wild-type background results in pstO cells sorting more strongly. Filamin disruption in an hfnA(-) background rescues pstO cell localisation. hfnA(-) cells exhibit altered slug phototaxis phenotypes consistent with filamin complex hyperactivity. We propose that HfnA regulates filamin complex activity and cell type-specific motility through the breakdown of filamin complexes. These findings provide a novel mechanism for filamin regulation and demonstrate that filamin is a crucial mechanistic link between responses to differentiation signals and cell movement in patterning based on 'salt and pepper' differentiation and sorting out.     

Chemotaxis of Dictyostelium discoideum: Collective Oscillation of Cellular Contacts

Chemotactic responses of Dictyostelium discoideum cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single Dictyostelium discoideum cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of Dictyostelium discoideum cells.     

A novel Akt/PKB-related kinase is essential for morphogenesis in Dictyostelium

BACKGROUND: Dictyostelium Akt/PKB is homologous to mammalian Akt/PKB and is required for cell polarity and proper chemotaxis during early development. The kinase activity of Akt/PKB kinase is activated in response to chemoattractants in neutrophils and in Dictyostelium by the chemoattractant cAMP functioning via a pathway involving a heterotrimeric G protein and PI3-kinase. Dictyostelium contains several kinases structurally related to Akt/PKB, one of which, PKBR-1, is investigated here for its role in cell polarity, movement and cellular morphogenesis during development. RESULTS: PKBR-1 has a kinase and a carboxy-terminal domain related to those of Akt/PKB, but no PH domain. Instead, it has an amino-terminal myristoylation site, which is required for its constitutive membrane localization. Like Akt/PKB, PKBR-1 is activated by cAMP through a G-protein-dependent pathway, but does not require PI3-kinase, probably because of the constitutive membrane localization of PKBR-1. This is supported by experiments demonstrating the requirement for membrane association for activation and in vivo function of PKBR-1. PKBR-1 protein is found in all cells throughout early development but is then restricted to the apical cells in developing aggregates, which are thought to control morphogenesis. PKBR-1 null cells arrest development at the mound stage and are defective in morphogenesis and multicellular development. These phenotypes are complemented by Akt/PKB, suggesting functional overlap between PKBR-1 and Akt/PKB. Akt/PKB PKBR-1 double knockout cells exhibit growth defects and show stronger chemotaxis and cell-polarity defects than Akt/PKB null cells. CONCLUSIONS: Our results expand the previously known functions of Akt/PKB family members in cell movement and morphogenesis during Dictyostelium multicellular development. The results suggest that Akt/PKB and PKBR-1 have overlapping effectors and biological function: Akt/PKB functions predominantly during aggregation to control cell polarity and chemotaxis, whereas PKBR-1 is required for morphogenesis during multicellular development.     

Topographical and physicochemical modification of material surface to enable patterning of living cells.

Precise control of the architecture of multiple cells in culture and in vivo via precise engineering of the material surface properties is described as cell patterning. Substrate patterning by control of the surface physicochemical and topographic features enables selective localization and phenotypic and genotypic control of living cells. In culture, control over spatial and temporal dynamics of cells and heterotypic interactions draws inspiration from in vivo embryogenesis and haptotaxis. Patterned arrays of single or multiple cell types in culture serve as model systems for exploration of cell-cell and cell-matrix interactions. More recently, the patterned arrays and assemblies of tissues have found practical applications in the fields of Biosensors and cell-based assays for Drug Discovery. Although the field of cell patterning has its origins early in this century, an improved understanding of cell-substrate interactions and the use of microfabrication techniques borrowed from the microelectronics industry have enabled significant recent progress. This review presents the important early discoveries and emphasizes results of recent state-of-the-art cell patterning methods. The review concludes by illustrating the growing impact of cell patterning in the areas of bioelectronic devices and cell-based assays for drug discovery.     

Topographical and Physicochemical Modification of Material Surface to Enable Patterning of Living Cells

ABSTRACT:?

Precise control of the architecture of multiple cells in culture and in vivo via precise engineering of the material surface properties is described as cell patterning. Substrate patterning by control of the surface physicochemical and topographic features enables selective localization and phenotypic and genotypic control of living cells. In culture, control over spatial and temporal dynamics of cells and heterotypic interactions draws inspiration from in vivo embryogenesis and haptotaxis. Patterned arrays of single or multiple cell types in culture serve as model systems for exploration of cell-cell and cell-matrix interactions. More recently, the patterned arrays and assemblies of tissues have found practical applications in the fields of Biosensors and cell-based assays for Drug Discovery. Although the field of cell patterning has its origins early in this century, an improved understanding of cell-substrate interactions and the use of microfabrication techniques borrowed from the microelectronics industry have enabled significant recent progress. This review presents the important early discoveries and emphasizes results of recent state-of-the-art cell patterning methods. The review concludes by illustrating the growing impact of cell patterning in the areas of bioelectronic devices and cell-based assays for drug discovery.     

Phg2, a kinase involved in adhesion and focal site modeling in Dictyostelium

The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility.     

Loss of FrmB results in increased size of developmental structures during the multicellular development of <Emphasis Type="Italic">Dictyostelium</Emphasis> cells

FERM domain-containing proteins are involved in diverse biological and pathological processes, including cell-substrate adhesion, cell-cell adhesion, multicellular development, and cancer metastasis. In this study, we determined the functions of FrmB, a FERM domain-containing protein, in the cell morphology, cell adhesion, and multicellular development of Dictyostelium cells. Our results show that FrmB appears to play an important role in regulating the size of developmental structures. frmB null cells showed prolonged aggregation during development, resulting in increased size of developmental structures, such as mounds and fruiting bodies, compared to those of wild-type cells, whereas FrmB overexpressing cells exhibited decreased size of developmental structures. These results suggest that FrmB may be necessary for limiting the sizes of developmental structures. Loss of FrmB also resulted in decreased cell-substrate adhesion and slightly increased cell area, suggesting that FrmB had important roles in the regulation of cell adhesion and cell morphology. These studies would contribute to our understanding of the intertwined and overlapped functions of FERM domain-containing proteins.     

Rac regulation of chemotaxis and morphogenesis in Dictyostelium

Chemotaxis requires localized F-actin polymerization at the site of the plasma membrane closest to the chemoattractant source, a process controlled by Rac/Cdc42 GTPases. We identify Dictyostelium RacB as an essential mediator of this process. RacB is activated upon chemoattractant stimulation, exhibiting biphasic kinetics paralleling F-actin polymerization. racB null cells have strong chemotaxis and morphogenesis defects and a severely reduced chemoattractant-mediated F-actin polymerization and PAKc activation. RacB activation is partly controlled by the PI3K pathway. pi3k1/2 null cells and wild-type cells treated with LY294002 exhibit a significantly reduced second peak of RacB activation, which is linked to pseudopod extension, whereas a PTEN hypomorph exhibits elevated RacB activation. We identify a RacGEF, RacGEF1, which has specificity for RacB in vitro. racgef1 null cells exhibit reduced RacB activation and cells expressing mutant RacGEF1 proteins display chemotaxis and morphogenesis defects. RacGEF1 localizes to sites of F-actin polymerization. Inhibition of this localization reduces RacB activation, suggesting a feedback loop from RacB via F-actin polymerization to RacGEF1. Our findings provide a critical linkage between chemoattractant stimulation, F-actin polymerization, and chemotaxis in Dictyostelium.     

A temperature-sensitive adenylyl cyclase mutant of Dictyostelium

Dictyostelium development starts with the chemotactic aggregation of up to 10(6) amoebae in response to propagating cAMP waves. cAMP is produced by the aggregation stage adenylyl cyclase (ACA) and cells lacking ACA (aca null) cannot aggregate. Temperature-sensitive mutants of ACA were selected from a population of aca null cells transformed with a library of ACA genes, a major segment of which had been amplified by error-prone PCR. One mutant (tsaca2) that can complement the aggregation null phenotype of aca null cells at 22 degrees C but not at 28 degrees C was characterized in detail. The basal catalytic activity of the enzyme in this mutant was rapidly and reversibly inactivated at 28 degrees C. Using this mutant strain we show that cell movement in aggregates and mounds is organized by propagating waves of cAMP. Synergy experiments between wild-type and tsaca2 cells, shifted to the restrictive temperature at various stages of development, showed that ACA plays an important role in the control of cell sorting and tip formation.     

The CNS midline cells coordinate proper cell cycle progression and identity determination of the Drosophila ventral neuroectoderm

The CNS midline cells, specified by the single-minded (sim) gene, are required for the proper patterning of the ventral CNS and epidermis, which are derived from the Drosophila ventral neuroectoderm. Defects in the sim mutant are characterized by the loss of the gene expression, which is required for the proper formation of the ventral neurons and epidermis, and by a decrease in the spacing of longitudinal and commissural axon tracks. Molecular and cellular mechanisms for these defects were analyzed to elucidate the precise role of the CNS midline cells in proper patterning of the ventral neuroectoderm during embryonic neurogenesis. These analyses showed that the ventral neuroectoderm in the sim mutant fails to carry out its proper formation and characteristic cell division cycle. This resulted in the loss of the dividing neuroectodermal cells that are located ventral to the CNS midline. The CNS midline cells are also required for the cell cycle-independent expression of the neural and epidermal markers. This indicates that the CNS midline cells are essential for the establishment and maintenance of the ventral epidermal and neuronal cell lineage by cell-cell interaction. On the other hand, the CNS midline cells do not cause extensive cell death in the ventral neuroectoderm. This study indicates that the CNS midline cells play important roles in the coordination of the proper cell cycle progression and the correct identity determination of the adjacent ventral neuroectoderm along the dorsoventral axis.     

A SET/MYND chromatin re-modelling protein regulates Dictyostelium prespore patterning

SmdA is a Dictyostelium orthologue of the SET/MYND chromatin re-modelling proteins. In developing structures derived from a null mutant for smdA (a smdA- strain), prestalk patterning is normal, but using a prespore lacZ reporter fusion, there is ectopic accumulation of beta-galactosidase in the prestalk region. As wild type slugs migrate, there is continual forward movement and re-differentiation of prespore cells into prestalk cells. Thus, a potential explanation for the ectopic reporter localization in smdA null prestalk cells is an increased rate of re-differentiation and anterior movement of prespore cells. In support of this notion, analysis of an unstable lacZ reporter, driven by the prespore promoter, reveals a normal staining pattern in the smdA- strain. We suggest that one or more genes regulated by SmdA acts to repress prespore re-specification.     

Two Dictyostelium tyrosine kinase–like kinases function in parallel,stress-induced STAT activation pathways

When Dictyostelium cells are hyperosmotically stressed, STATc is activated by tyrosine phosphorylation. Unusually, activation is regulated by serine phosphorylation and consequent inhibition of a tyrosine phosphatase: PTP3. The identity of the cognate tyrosine kinase is unknown, and we show that two tyrosine kinase–like (TKL) enzymes, Pyk2 and Pyk3, share this function; thus, for stress-induced STATc activation, single null mutants are only marginally impaired, but the double mutant is nonactivatable. When cells are stressed, Pyk2 and Pyk3 undergo increased autocatalytic tyrosine phosphorylation. The site(s) that are generated bind the SH2 domain of STATc, and then STATc becomes the target of further kinase action. The signaling pathways that activate Pyk2 and Pyk3 are only partially overlapping, and there may be a structural basis for this difference because Pyk3 contains both a TKL domain and a pseudokinase domain. The latter functions, like the JH2 domain of metazoan JAKs, as a negative regulator of the kinase domain. The fact that two differently regulated kinases catalyze the same phosphorylation event may facilitate specific targeting because under stress, Pyk3 and Pyk2 accumulate in different parts of the cell; Pyk3 moves from the cytosol to the cortex, whereas Pyk2 accumulates in cytosolic granules that colocalize with PTP3.     

A novel Dictyostelium gene encoding multiple repeats of adhesion inhibitor-like domains has effects on cell-cell and cell-substrate adhesion

The Dictyostelium protein AmpA (adhesion modulation protein A) is encoded by the gene originally identified by the D11 cDNA clone. AmpA contains repeated domains homologous to a variety of proteins that influence cell adhesion. The protein accumulates during development, reaching a maximal level at the finger stage. Much of the AmpA protein is found extracellularly during development, and in culminants, AmpA is found in association with anterior-like cells. Characterization of an ampA- strain generated by gene replacement reveals a significant increase in cell-cell clumping when cells are starved in nonnutrient buffer suspensions. Developing ampA- cells are also more adhesive to the underlying substrate and are delayed in developmental progression, with the severity of the delay increasing as cells are grown in the presence of bacteria or on tissue culture dishes rather than in suspension culture. Reintroduction of the ampA gene rescues the developmental defects of ampA- cells; however, expression of additional copies of the gene in wild-type cells results in more severe developmental delays and decreased clumping in suspension culture. We propose that the AmpA protein functions as an anti-adhesive to limit cell-cell and cell-substrate adhesion during development and thus facilitates cell migration during morphogenesis.     

Activation of beta1 integrins induces cell-cell adhesion

Integrins are highly regulated receptors that can function in both cell-substrate and cell-cell adhesion. We have found that the activating anti-beta1 mAb, 12G10, can specifically and rapidly induce both cell-substrate and cell-cell adhesion of HT-1080 human fibrosarcoma and other cell types. Binding of mAb 12G10 induced clustering of cell-surface integrins, and the preferential localization of beta1 integrins expressing the 12G10 epitope at cell-cell adhesion sites. Fab fragments of mAb 12G10 induced HT-1080 cell-cell adhesion as effectively as did intact antibodies, suggesting that integrin clustering was not due to direct antibody crosslinking. Latrunculin B, an inhibitor of F-actin polymerization, inhibited cell-cell adhesion but not the clustering of integrins. Results from a novel, two-color cell-cell adhesion assay suggested that nonactivated cells can bind to activated cells and that integrin activation-induced HT-1080 cell-cell adhesion minimally requires the interaction of activated alpha2beta1 with nonactivated alpha3beta1. These findings suggest that HT-1080 cell-cell adhesion induced by integrin activation require a signaling process involving integrin clustering and the subsequent organization of the cytoskeleton. Integrin activation could therefore play a key role in cell-cell adhesion.     

The SUN family of Saccharomyces cerevisiae: the double knock-out of UTH1 and SIM1 promotes defects in nucleus migration and increased drug sensitivity

UTH1 and SIM1 are two of four 'SUN' genes (SIM1, UTH1, NCA3 and SUN4/SCW3) whose products are involved in different cellular processes such as DNA replication, lifespan, mitochondrial biogenesis or cell septation. UTH1 or SIM1 inactivation did not affect cell growth, shape or nuclear migration, whereas the double null mutant presented phenotypes of numerous binucleate cells and benomyl sensitivity, suggesting that microtubule function could be altered; the uth1Deltasim1Delta strain also presented defects which could be related to the Ras/cAMP pathway: pet phenotype, heat shock sensitivity, inability to store glycogen, sensitivity to starvation and failure of spores to germinate. These observations suggested that Uth1p could be involved as a connection step between pathways controlling growth and those controlling division.