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

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

Cytoskeletal interactions at the nuclear envelope mediated by Nesprins

Nesprin-1 is a giant tail-anchored nuclear envelope protein composed of an N-terminal F-actin binding domain, a long linker region formed by multiple spectrin repeats and a C-terminal transmembrane domain. Based on this structure, it connects the nucleus to the actin cytoskeleton. Earlier reports had shown that Nesprin-1 binds to nuclear envelope proteins emerin and lamin through C-terminal spectrin repeats. These repeats can also self-associate. We focus on the N-terminal Nesprin-1 sequences and show that they interact with Nesprin-3, a further member of the Nesprin family, which connects the nucleus to the intermediate filament network. We show that upon ectopic expression of Nesprin-3 in COS7 cells, which are nearly devoid of Nesprin-3 in vitro, vimentin filaments are recruited to the nucleus and provide evidence for an F-actin interaction of Nesprin-3 in vitro. We propose that Nesprins through interactions amongst themselves and amongst the various Nesprins form a network around the nucleus and connect the nucleus to several cytoskeletal networks of the cell

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into earlyeukaryoteevolution.Wedescribeextensiveuseofhistidinekinase-basedtwo-componentsystemsandtyrosinekinasesignaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases inAcanthamoeba and Physarum as representatives of two distantly related subdivisions ofAmoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfe

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

KASH-domain proteins and the cytoskeletal landscapes of the nuclear envelope

Over the last few years, several novel proteins have been identified that facilitate the physical integration of the nucleus with the cytoplasmic compartment. The majority belong to the evolutionarily conserved KASH [klarsicht/ANC-1 (anchorage 1)/SYNE (synaptic nuclear envelope protein) homology]-domain family, which function primarily as exclusive outer nuclear membrane scaffolds that associate with the cytoskeleton, the centrosome and the motor protein apparatus. In the present paper, we propose a novel model, which may explain why these proteins also determine nuclear architecture. Moreover, we discuss further nuclear membrane-tethering devices, which indicate collectively the presence of specific molecular mechanisms that organize the cytoplasmic-nuclear membrane interface in mammalian cells

Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin content, cytokinesis, and development

To study in vivo functions of the ubiquitous actin-binding protein profilin, we generated by antisense and gene disruption techniques Dictyostelium mutants that lack one or both of the profilin isoforms. Whereas the single mutants showed an essentially unchanged phenotype, the behavior of the double mutant was drastically altered. Motility was significantly reduced, single cells were up to 10 times larger than wild-type cells and showed a broad rim of filamentous actin below the plasma membrane, the filamentous actin concentration was increased by about 60%-70%, and development was blocked prior to fruiting body formation. Furthermore, double mutants could not be grown in shaking culture under normal conditions, reflecting an impaired cytokinesis. The aberrant phenotype could be rescued by reintroducing a functional profilin I or profilin II gene. The data in this study suggest that profilin functions in Dictyostelium amoebae primarily as an actin-sequestering protein

Novel phosphatidylinositol phosphate kinases with a G-protein coupled receptor signature are shared by Dictyostelium and Phytophthora

G-protein coupled receptors (GPCR) and phosphatidylinositol phosphate kinases (PIPK) are important key switches in signal transduction pathways. A novel class of proteins was identified in the genomes of two unrelated organisms that harbor both a GPCR and a PIPK domain. Dictyostelium discoideum contains one GPCR¿PIPK, which is crucial in cell-density sensing, and the genomes of Phytophthora sojae and Phytophthora ramorum each encode twelve GPCR¿PIPKs. Intriguingly, these are currently the only species that have these two domains combined in one protein. Here, the structural and regulatory characteristics of GPCR¿PIPKs are presented and discussed. It is hypothesized that, upon activation, GPCR¿PIPKs are able to trigger heterotrimeric G-protein signaling and phosphoinositide second-messenger synthesis

Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin

In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner