The role of the cortical cytoskeleton

We generated Dictyostelium double mutants lacking the two F-actin crosslinking proteins alpha-actinin and gelation factor by inactivating the corresponding genes via homologous recombination. Here we investigated the consequences of these deficiencies both at the single cell level and at the multicellular stage. We found that loss of both proteins severely affected growth of the mutant cells in shaking suspension, and led to a reduction of cell size from 12 microns in wild-type cells to 9 microns in mutant cells. Moreover the cells did not exhibit the typical polarized morphology of aggregating Dictyostelium cells but had a more rounded cell shape, and also exhibited an increased sensitivity towards osmotic shock and a reduced rate of phagocytosis. Development was heavily impaired and never resulted in the formation of fruiting bodies. Expression of developmentally regulated genes and the final developmental stages that were reached varied, however, with the substrata on which the cells were deposited. On phosphate buffered agar plates the cells were able to form tight aggregates and mounds and to express prespore and prestalk cell specific genes. Under these conditions the cells could perform chemotactic signalling and cell behavior was normal at the onset of multicellular development as revealed by time-lapse video microscopy. Double mutant cells were motile but speed was reduced by approximately 30% as compared to wild type. These changes were reversed by expressing the gelation factor in the mutant cells. We conclude that the actin assemblies that are formed and/or stabilized by both F-actin crosslinking proteins have a protective function during osmotic stress and are essential for proper cell shape and motility

GxcDD, a putative RacGEF, is involved in Dictyostelium development

<p>Abstract</p> <p>Background</p> <p>Rho subfamily GTPases are implicated in a large number of actin-related processes. They shuttle from an inactive GDP-bound form to an active GTP-bound form. This reaction is catalysed by Guanine nucleotide exchange factor (GEFs). GTPase activating proteins (GAPs) help the GTPase return to the inactive GDP-bound form. The social amoeba <it>Dictyostelium discoideum </it>lacks a Rho or Cdc42 ortholog but has several Rac related GTPases. Compared to our understanding of the downstream effects of Racs our understanding of upstream mechanisms that activate Rac GTPases is relatively poor.</p> <p>Results</p> <p>We report on GxcDD (<b>G</b>uanine e<b>x</b>change factor for Ra<b>c </b>GTPases), a <it>Dictyostelium </it>RacGEF. GxcDD is a 180-kDa multidomain protein containing a type 3 CH domain, two IQ motifs, three PH domains, a RhoGEF domain and an ArfGAP domain. Inactivation of the gene results in defective streaming during development under different conditions and a delay in developmental timing. The characterization of single domains revealed that the CH domain of GxcDD functions as a membrane association domain, the RhoGEF domain can physically interact with a subset of Rac GTPases, and the ArfGAP-PH tandem accumulates in cortical regions of the cell and on phagosomes. Our results also suggest that a conformational change may be required for activation of GxcDD, which would be important for its downstream signaling.</p> <p>Conclusion</p> <p>The data indicate that GxcDD is involved in proper streaming and development. We propose that GxcDD is not only a component of the Rac signaling pathway in <it>Dictyostelium</it>, but is also involved in integrating different signals. We provide evidence for a Calponin Homology domain acting as a membrane association domain. GxcDD can bind to several Rac GTPases, but its function as a nucleotide exchange factor needs to be studied further.</p

cDNA and derived amino acid sequence of the hypusine containing protein from Dictyostelium discoideum

AbstractThe eukaryotic translation initiation factor eIF-4D is the only protein known to contain the unusual amino acid hypusine, a posttranslationally modified lysine. For the production of monoclonal antibodies the hypusine-containing protein (HP) was isolated from Dictyostelium discoideum. Using these monoclonal antibodies, a full-length cDNA clone was isolated from a λgt11 library. The D. discoideum HP consists of 169 amino acids and has a molecular mass of 18.3 kDa. It is encoded by a single gene. Tryptic and cyanogen bromide peptides were prepared from the purified protein and sequenced. The hypusine residue is located at amino acid position 65 of the HP. The corresponding mRNA of approx. 0.6 kb is present throughout the life cycle of D. discoideum

The 100 kDa F-actin capping protein of Dictyostelium amoebae is a villin prototype (‘protovillin’)

AbstractThe 100 kDa actin-binding protein from Dictyostelium amoebae is an F-actin capping protein that displays neither severing nor crosslinking nor nucleating activities [Hofmann et al. (1992) Cell Motil. Cytoskel. 23,133-144]. Cloning and sequencing of the gene revealed that the protein is highly homologous to vertebrate villin, a unique component of brush border microvilli and contains six domains fused to a villin-like headpiece domain via a threonine/proline rich neck region. The functional differences and similarities between the 100 kDa protein and villin are reflected in the amino acid sequences. We draw from the data the following conclusions, (i) The presence of a six domain protein in Dictyostelium suggests that in contrast to the current view gene duplications must have happened before Dictyostelium branched off during evolution, (ii) The villin-like molecule in Dictyostelium appears to be a premature villin (‘protovillin’) which is able to cap actin filaments but still lacks the other villin-type actin-binding activities. This renders capping of actin filaments as the evolutionarily oldest function of an F-actin binding protein

A set of genes conserved in sequence and expression traces back the establishment of multicellularity in social amoebae

Background: The developmental cycle of Dictyostelid amoebae represents an early form of multicellularity with cell type differentiation. Mutant studies in the model Dictyostelium discoideum revealed that its developmental program integrates the actions of genes involved in signal transduction, adhesion, motility, autophagy and cell wall and matrix biosynthesis. However, due to functional redundancy and fail safe options not required in the laboratory, this single organism approach cannot capture all essential genes. To understand how multicellular organisms evolved, it is essential to recognize both the conserved core features of their developmental programs and the gene modifications that instigated phenotypic innovation. For complex organisms, such as animals, this is not within easy reach, but it is feasible for less complex forms, such as the Dictyostelid social amoebas. Results: We compared global profiles of gene expression during the development of four social amoebae species that represent 600 mya of Dictyostelia evolution, and identified orthologous conserved genes with similar developmental up-regulation of expression using three different methods. For validation, we disrupted five genes of this core set and examined the phenotypic consequences. Conclusion: At least 71 of the developmentally regulated genes that were identified with all methods were likely to be already present in the last ancestor of all Dictyostelia. The lack of phenotypic changes in null mutants indicates that even highly conserved genes either participate in functionally redundant pathways or are necessary for developmental progression under adverse, non-standard laboratory conditions. Both mechanisms provide robustness to the developmental program, but impose a limit on the information that can be obtained from deleting single genes

‘Proclaiming it to Greeks and Natives, along the rows of the chequer-board': readers and viewers of Greek, Latin and Demotic Acrostich inscriptions

Hellenistic and Roman acrostich inscriptions are usually full of verbal and visual clues, which point the reader in the direction of the ‘hidden message’ contained in the vertical lines of the text. The authors of such inscriptions want their audiences to appreciate the skill that has gone into their composition. There are several complementary ways in which the presence of an acrostich might be signalled to the reader or viewer and their attention directed towards it. These include direct verbal statements, or more subtle allusions, within the text of the inscription. But, even without having read its text, the viewer of an inscription containing a ‘hidden message’ is often immediately aware that some kind of wordplay is at work. Acrostichs, palindromes and various kinds of word square are all graphically striking, or their appearance may be enhanced to make them more so. Regular spacing, the repetition of the acrostich in a separate column and the use of painted or incised grids are all ways in which the layout of the text on the stone can invite the viewer to play a word game. In some cases, as I will argue in this paper, acrostich makers envisaged—even intended—the participants in this game to include the illiterate as well as the literate

Ectopic expression of cyclase associated protein CAP restores the streaming and aggregation defects of adenylyl cyclase a deficient Dictyostelium discoideum cells

<p>Abstract</p> <p>Background</p> <p>Cell adhesion, an integral part of <it>D. discoideum </it>development, is important for morphogenesis and regulated gene expression in the multicellular context and is required to trigger cell-differentiation. G-protein linked adenylyl cyclase pathways are crucially involved and a mutant lacking the aggregation specific adenylyl cyclase ACA does not undergo multicellular development.</p> <p>Results</p> <p>Here, we have investigated the role of cyclase-associated protein (CAP), an important regulator of cell polarity and F-actin/G-actin ratio in the <it>aca^-</it>mutant. We show that ectopic expression of GFP-CAP improves cell polarization, streaming and aggregation in <it>aca^-</it>cells, but it fails to completely restore development. Our studies indicate a requirement of CAP in the ACA dependent signal transduction for progression of the development of unicellular amoebae into multicellular structures. The reduced expression of the cell adhesion molecule DdCAD1 together with csA is responsible for the defects in <it>aca^-</it>cells to initiate multicellular development. Early development was restored by the expression of GFP-CAP that enhanced the DdCAD1 transcript levels and to a lesser extent the csA mRNA levels.</p> <p>Conclusions</p> <p>Collectively, our data shows a novel role of CAP in regulating cell adhesion mechanisms during development that might be envisioned to unravel the functions of mammalian CAP during animal embryogenesis.</p

The Dictyostelium discoideum RACK1 orthologue has roles in growth and development

YesBackground: The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes. Results: We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gβ function in D. discoideum as developmental and other defects were not rescued in Gβ null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development. Conclusion: DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins.This work was supported by the DFG and SFB670. TYR acknowledges support from the Professorinnen Program of the University of Cologne

Linking Ras to myosin function: RasGEF Q, a Dictyostelium exchange factor for RasB, affects myosin II functions

Ras guanine nucleotide exchange factor (GEF) Q, a nucleotide exchange factor from Dictyostelium discoideum, is a 143-kD protein containing RasGEF domains and a DEP domain. We show that RasGEF Q can bind to F-actin, has the potential to form complexes with myosin heavy chain kinase (MHCK) A that contain active RasB, and is the predominant exchange factor for RasB. Overexpression of the RasGEF Q GEF domain activates RasB, causes enhanced recruitment of MHCK A to the cortex, and leads to cytokinesis defects in suspension, phenocopying cells expressing constitutively active RasB, and myosin-null mutants. RasGEF Q− mutants have defects in cell sorting and slug migration during later stages of development, in addition to cell polarity defects. Furthermore, RasGEF Q− mutants have increased levels of unphosphorylated myosin II, resulting in myosin II overassembly. Collectively, our results suggest that starvation signals through RasGEF Q to activate RasB, which then regulates processes requiring myosin II