Cell adhesion: ushering in a new understanding of myosin VII.

The myosin VII motor protein has recently been found to have a role in cell adhesion. This new function is conserved from amoebae to man and provides an explanation for deafness in Usher syndrome patients.

Fluid-phase uptake and transit in axenic Dictyostelium cells.

The main route for fluid-phase uptake in Dictyostelium is macropinocytosis, a process powered by the actin cytoskeleton. Nutrients within the endocytosed fluid are digested and resorbed, disposal of remnants follows by exocytosis. Along the endocytic pathway, membrane fusion and fission events take place at multiple steps. The regulator and effector molecules involved in uptake and transit are largely conserved between higher and lower eukaryotes. This feature, together with its accessibility by molecular genetics, recommend Dictyostelium as a valuable model system for mammalian cells.

DAip1, a Dictyostelium homologue of the yeast actin-interacting protein 1, is involved in endocytosis, cytokinesis, and motility.

The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes. We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia. In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions. Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.

Patterns of cellular activities based on protein sorting in cell motility, endocytosis and cytokinesis.

In order to move persistently, a cell has to harmonize its protrusion and retraction with attachment and detachment from the substrate. Time-series analyses based on fluctuations in these activities are being used in combination with advanced imaging techniques to unravel the network of protein-protein interactions that tune the activities in a motile cell and co-ordinate them in space and time. Fusions with the green fluorescent protein have helped to visualize the recruitment of cytoskeletal proteins from a soluble pool and their transient assembly into supramolecular structures. Using a series of mutants deficient in specific cytoskeletal proteins has revealed common themes and interrelationships between cell motility, endocytosis and cytokinesis. For instance, a phagocytic cup competes with leading-edge formation, and recruits the same actin-associated proteins. Cytokinesis is based on the fine tuning of activities in the microtubule system and the actin network in the cell cortex. Cells dividing on a substrate apply tension to the surface on which they adhere, as determined by the silicone rubber technique. Actin-associated proteins are sorted during cytokinesis either to the extensions formed at the poles of a dividing cell or to the cleavage furrow. A major effort will be required to elucidate the mechanisms that dictate the pattern of local activities and drive the translocation of proteins in cell motility, endocytosis and cytokinesis.

Ethane-freezing/methanol-fixation of cell monolayers: a procedure for improved preservation of structure and antigenicity for light and electron microscopies.

In order to dissect at the ultrastructural level the morphology of highly dynamic processes such as cell motility, membrane trafficking events, and organelle movements, it is necessary to fix/stop time-dependent events in the millisecond range. Ideally, immunoelectron microscopical labeling experiments require the availability of high-affinity antibodies and accessibility to all compartments of the cell. The biggest challenge is to define an optimum between significant preservation of the antigenicity in the fixed material without compromising the intactness of fine structures. Here, we present a procedure which offers an opportunity to unify preparation of cell monolayers for immunocytochemistry in fluorescence and electron microscopy. This novel strategy combines a rapid ethane-freezing technique with a low temperature methanol-fixation treatment (EFMF) and completely avoids chemical fixatives. It preserves the position and delicate shape of cells and organelles and leads to improved accessibility of the intracellular antigens and to high antigenicity preservation. We illustrate the establishment of this procedure using Dictyostelium discoideum, a powerful model organism to study molecular mechanisms of membrane trafficking and cytoskeleton.

Targeted gene disruption reveals a role for vacuolin B in the late endocytic pathway and exocytosis.

Cells of Dictyostelium discoideum take up fluid by macropinocytosis. The contents of macropinosomes are acidified and digested by lysosomal enzymes. Thereafter, an endocytic marker progresses in an F-actin dependent mechanism from the acidic lysosomal phase to a neutral post-lysosomal phase. From the post-lysosomal compartment indigestible remnants are released by exocytosis. This compartment is characterised by two isoforms of vacuolin, A and B, which are encoded by different genes. Fusions of the vacuolin isoforms to the green fluorescent protein associate with the cytoplasmic side of post-lysosomal vacuoles in vivo. Vacuolin isoforms also localise to patches at the plasma membrane. Since vacuolins have no homologies to known proteins and do not contain domains of obvious function, we investigated their role by knocking out the genes separately. Although the sequences of vacuolins A and B are about 80% identical, only deletion of the vacuolin B gene results in a defect in the endocytic pathway; the vacuolin A knock-out appeared to be phenotypically normal. In vacuolin B- mutants endocytosis is normal, but the progression of fluid-phase marker from acidic to neutral pH is impaired. Furthermore, in the mutants post-lysosomal vacuoles are dramatically increased in size and accumulate endocytic marker, suggesting a role for vacuolin B in targeting the vacuole for exocytosis.

Talin-null cells of Dictyostelium are strongly defective in adhesion to particle and substrate surfaces and slightly impaired in cytokinesis.

Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones. Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous cross talk between the cell surface and underlying cytoskeleton. The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed. Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast with the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin dependent. The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface. With their high proportion of binucleated cells, the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells.

Coronin and vacuolin identify consecutive stages of a late, actin-coated endocytic compartment in Dictyostelium.

Cells of the unicellular eukaryote Dictyostelium discoideum take up all their nutrients by endocytosis. Both particle- and fluid-containing vacuoles are transiently surrounded by a cytoskeletal coat [1] [2]. When this coat has dissociated, acidification and digestion of the vesicle contents occur, followed by exocytosis of the indigestible remnants after 60-90 minutes. At least nine compartments are needed for mathematical modelling of endocytic transit [3], suggesting that markers associate for only a few minutes with a specific endocytic compartment. Among the proteins that have been identified as components of endocytic vesicles are actin, subunits of the V-H+ ATPase and small GTP-binding proteins of the Rab family [4] [5] [6] [7]. Using a monoclonal antibody produced against Dictyostelium endocytic vesicles, we have isolated a cDNA corresponding to a novel protein that we have named vacuolin. In order to determine the precise step along the endocytic pathway that involves vacuolin, we generated a fusion protein of the green fluorescent protein (GFP) and vacuolin. GFP-vacuolin-decorated vesicles were identified as a post-lysosomal compartment that acquires endocytic markers shortly before exocytosis. At earlier stages, this post-lysosomal compartment was identified by the binding of a tagged cytoskeletal protein, coronin-GFP. Vacuoles were coated with filamentous actin along the entire post-lysosomal pathway, and the integrity of the actin coat was required for exocytosis.

Fluid-phase uptake by macropinocytosis in Dictyostelium.

To study fluid-phase endocytosis in living cells and its relationship to changes in the cell cortex, we have used a green fluorescent protein (GFP)-tagged version of coronin, an actin-associated protein that localises to dynamic regions of the Dictyostelium cell cortex. In the confocal microscope, internalisation of fluorescently labelled dextran as a fluid-phase marker can be recorded simultaneously with the recruitment of the coronin-GFP fusion-protein from the cytoplasm of the phagocyte. At crown-shaped surface protrusions, extracellular medium is taken up into vesicles with an average diameter of 1.6 microns, which is significantly larger than the 0.1 microns diameter of clathrin-coated pinosomes. The observed frequency of macropinosome formation can account for a large portion, if not all, of the fluid-phase uptake. The redistribution of coronin-GFP strongly resembles cytoskeletal rearrangements during phagocytosis. Scanning-electron micrographs indicate that crown-shaped cell-surface extensions can undergo shape changes, without a particle bound, that are similar to shape changes that occur during phagocytosis. In quantitative assays, the uptake of particles and fluid are about equally dependent on F-actin and coronin.

Dictyostelium discoideum protein disulfide isomerase, an endoplasmic reticulum resident enzyme lacking a KDEL-type retrieval signal.

The primary activity of protein disulfide isomerase (PDI), a multifunctional resident of the endoplasmic reticulum (ER), is the isomerization of disulfide bridges during protein folding. We isolated a cDNA encoding Dictyostelium discoideum PDI (Dd-PDI). Phylogenetic analyses and basic biochemical properties indicate that it belongs to a subfamily called P5, many members of which differ from the classical PDIs in many respects. They lack an intervening inactive thioredoxin module, a C-terminal acidic domain involved in Ca2+ binding and a KDEL-type retrieval signal. Despite the absence of this motif, the ER is the steady-state location of Dd-PDI, suggesting the existence of an alternative retention mechanism for P5-related enzymes.

Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag.

Coronin is a protein involved in cell locomotion and cytokinesis of Dictyostelium discoideum. Here we show that coronin is strongly enriched in phagocytic cups formed in response to particle attachment. A fusion of coronin with green fluorescent protein (GFP) accumulates in the cups within less than 1 min upon attachment of a particle and is gradually released from the phagosome within 1 min after engulfment is completed. Phagocytic cup formation competes with leading edge formation and can be interrupted at any stage. When the cup regresses, coronin dissociates from the site of accumulation. TRITC-labeled yeast cells have been used to assay phagocytosis quantitatively in wild-type and coronin-null cells. In the mutant, the rate of uptake is reduced to about one third, which shows that coronin contributes to the efficiency of phagocytosis to about the same extent as it improves the speed of cell locomotion.

Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored using a green fluorescent protein-coronin fusion protein.

BACKGROUND: The highly motile cells of Dictyostelium discoideum rapidly remodel their actin filament system when they change their direction of locomotion either spontaneously or in response to chemoattractant. Coronin is a cytoplasmic actin-associated protein that accumulates at the coritcal sites of moving cells and contributes to the dynamics of the actin system. It is a member of the WD-repeat family of proteins and is known to interact with actin-myosin complexes. In coronin null mutants, cell locomotion is slowed down and cytokinesis is impaired. RESULTS: We have visualized the redistribution of coronin by fluorescence imaging of motile cells that have been transfected with an expression plasmid containing the coding sequence of coronin fused to the sequence encoding the green fluorescent protein (GFP). This coronin-GFP fusion protein (GFP). This coronin-GFP fusion protein transiently accumulates in the front regions of growth-phase cells, reflecting the changing positions of leading edges and the competition between them. During the aggregation stage, local accumulation of coronin-GFP is biased by chemotactic orientation of the cells in gradients of cAMP. The impairment of cell motility in coronin null mutants shows that coronin has an important function at the front region of the cells. The mutant cells are distinguished by the formation of extended particle-free zones at their front regions, from where pseudopods often break out as blebs. Cytochalasin A reduces the size of these zones, indicating that actin filaments prevent entry of the particles. CONCLUSIONS: These data demonstrate that coronin is reversibly recruited from the cytoplasm and is incorporated into the actin network of a nascent leading edge, where it participates in the reorganization of the cytoskeleton. Monitoring the dynamics of protein assembly using GFP fusion proteins and fluorescence microscopy promises to be a generally applicable method for studying the dynamics of cytoskeletal proteins in moving and dividing cells.

Developmental regulation of antisense-mediated gene silencing in Dictyostelium.

In Dictyostelium, the expression of antisense transcripts has been successfully used to reduce or eliminate gene expression. In most cases this occurs on the level of RNA stability resulting in a loss of both sense and antisense transcript accumulation. We here show that the antisense effect is regulated during the developmental cycle, i.e., in certain developmental stages and under certain developmental conditions, complementary RNAs appear not to interact with each other, resulting in a failure to abolish expression of the gene of interest. We find that this is not only the case with artificially introduced antisense constructs but also with the endogenous, antisense-regulated PSV-A gene. Our data demonstrate that antisense-mediated gene silencing is conferred by a biochemical machinery that is subject to regulation in vivo. The results provide a basis to better understand this machinery and to dissect the components. They may also explain the failure of some antisense experiments in Dictyostelium and possibly in other organisms.

3' processing in Dictyostelium: unusual sequence requirements and interaction with a downstream promoter.

We have determined the sequence requirements for 3'-processing in Dictyostelium and find that a single AATAAA site, embedded in an A/T-rich environment is sufficient. A synthetic oligonucleotide containing the additional GT/T-rich element, which is necessary for 3'-processing in higher eukaryotes, is not used in Dictyostelium. On the basis of reports suggesting termination signals (upstream terminators) in Dictyostelium promoters, we investigated possible interactions between processing signals and regulatory elements. Our data suggest that upstream termination enhances transcription from a downstream promoter.

Evidence for a feedback regulated back-up promoter which controls permanent expression of a Dictyostelium gene.

We have previously described the elements of two overlapping promoters regulating differential expression of the Dictyostelium P8A7 gene. One promoter was constitutively active but stimulated during development (S-promoter), while the other (L-promoter) was found to be induced by a variety of stimuli. This effect was mediated by the induction (i-) element. By a series of antisense transformation experiments we here provide evidence that this element is the target of a feedback control circuit. In wild type cells, feedback regulation activates the L-promoter in unfavourable environmental situations when transcription from the S-promoter is insufficient to generate adequate amounts of protein. A model for the function of the feedback loop under natural and experimental conditions is presented.

Two separable promoters control different aspects of expression of a Dictyostelium gene.

A single copy Dictyostelium gene was dissected and elements responsible for its complex pattern of regulation were defined by transcript analysis of gene fusions. Two overlapping promoters responsible for the transcription of an 'L' and an 'S' mRNA could be defined. Further dissection of the P8A7 L promoter resulted in the identification of a sequence necessary for stress induction and an element required for vegetative expression. The P8A7 S promoter could be reduced to 449 bp which were sufficient for expression in developing cells. The sequence element required for this transcriptional activity was shown to reside in a 51 bp fragment. Our results show that differential expression of the P8A7 gene is mediated by two independently functioning promoters which, however, share some regulatory elements. A third nuclear RNA species 'P' was due to the stress-sensitivity of the 3' processing signal.

A colony-blot technique for the detection of specific transcripts in eukaryotes.

We present a simple and rapid technique for the detection of specific transcripts in eukaryotic cells. The method allows the screening of large numbers of clones for the expression of a gene of interest, similar to the colony blotting techniques described for prokaryotes. We have used this method to monitor developmentally regulated transcription of endogenous genes and the expression of foreign genes in transformed Dictyostelium discoideum cells. The same procedure can be applied to detect specific transcripts in yeast and should thus provide a useful molecular tool for most biological systems.