[show abstract][hide abstract] ABSTRACT: Rhizaria are a major branch of eukaryote evolution with an extensive microfossil record, but only scarce molecular data are available. The rhizarian species Reticulomyxa filosa, belonging to the Foraminifera, is free-living in freshwater environments. In culture, it thrives only as a plasmodium with thousands of haploid nuclei in one cell. The R. filosa genome is the first foraminiferal genome to be deciphered.
The genome is extremely repetitive, and the large amounts of identical sequences hint at frequent amplifications and homologous recombination events. Presumably, these mechanisms are employed to provide more gene copies for higher transcriptional activity and to build up a reservoir of gene diversification in certain gene families, such as the kinesin family. The gene repertoire indicates that it is able to switch to a single-celled, flagellated sexual state never observed in culture. Comparison to another rhizarian, the chlorarachniophyte alga Bigelowiella natans, reveals that proteins involved in signaling were likely drivers in establishing the Rhizaria lineage. Compared to some other protists, horizontal gene transfer is limited, but we found evidence of bacterial-to-eukaryote and eukaryote-to-eukaryote transfer events.
The R. filosa genome exhibits a unique architecture with extensive repeat homogenization and gene amplification, which highlights its potential for diverse life-cycle stages. The ability of R. filosa to rapidly transport matter from the pseudopodia to the cell body may be supported by the high diversification of actin and kinesin gene family members.
Current biology: CB 12/2013; · 10.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dictyostelium harbors several paralogous Sec7 genes that encode members of three subfamilies of the Sec7 superfamily of guanine nucleotide exchange factors. One of them is the cytohesin family represented by three members in D. discoideum, SecG, Sec7 and a further protein distinguished by several transmembrane domains. Cytohesins are characterized by a Sec7-PH tandem domain and have roles in cell adhesion and migration.
We study here Sec7. In vitro its PH domain bound preferentially to phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). When following the distribution of GFP-Sec7 in vivo we observed the protein in the cytosol and at the plasma membrane. Strikingly, when cells formed pseudopods, macropinosomes or phagosomes, GFP-Sec7 was conspicuously absent from areas of the plasma membrane which were involved in these processes. Mutant cells lacking Sec7 exhibited an impaired phagocytosis and showed significantly reduced speed and less persistence during migration. Cellular properties associated with mammalian cytohesins like cell-cell and cell-substratum adhesion were not altered. Proteins with roles in membrane trafficking and signal transduction have been identified as putative interaction partners consistent with the data obtained from mutant analysis.
Sec7 is a cytosolic component and is associated with the plasma membrane in a pattern distinctly different from the accumulation of PI(3,4,5)P3. Mutant analysis reveals that loss of the protein affects cellular processes that involve membrane flow and the actin cytoskeleton.
Cell Communication and Signaling 08/2013; 11(1):54. · 5.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: Diaphanous-related formins (DRFs) act as downstream effectors of Rho family GTPases and drive the formation and elongation of linear actin filaments in various cellular processes. Here we analyzed the DRF dDia1 from Dictyostelium cells. The biochemical characterization of recombinant dDia1-FH1FH2 by bulk polymerization assays and single filament TIRF microscopy revealed that dDia1 is a rather weak nucleator. Addition of any of the three Dictyostelium profilin isoforms, however, markedly accelerated formin-mediated actin filament barbed end elongation in TIRF assays. Interestingly, filament elongation was significantly faster in presence of DdPFN I (profilin I) when compared to the other two isoforms, suggesting selectivity of dDia1 for DdPFN I. Additionally, we frequently observed dissociation of the formin from growing barbed ends. These findings are consistent with dilution-induced depolymerization assays in presence of dDia1-FH1FH2 showing that dDia1 is a weak capper in comparison with heterodimeric capping protein. To study the physiological role of this formin, we created cell lines lacking dDia1 or overexpressing GFP-tagged dDia1. Of note, constitutively active dDia1 accumulated homogenously in the entire pseudopod suggesting that it controls microfilament architecture to regulate cell migration. Comparison of wild type and dDia1-null cells in random cell migration and chemotaxis toward a cAMP gradient revealed no major differences. By contrast, phototaxis of dDia1-deficient cells during the multicellular stage was markedly impaired. While wild type slugs moved with high directionality toward the light source, the trails of dDia1-null slugs displayed a characteristic V-shaped profile and deviated in angles between 50° and 60° from the path of the incident light. Possibly in conjunction with this defect, dDia1-null cells also formed substantially smaller fruiting bodies. These findings demonstrate dDia1 to be critically involved in collective cell migration during terminal differentiation.
European journal of cell biology 01/2013; · 3.31 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cyclase-associated proteins are highly conserved proteins that have a role in the regulation of actin dynamics. Higher eukaryotes have two isoforms, CAP1 and CAP2. To study the in vivo function of CAP2, we generated mice in which the CAP2 gene was inactivated by a gene-trap approach. Mutant mice showed a decrease in body weight and had a decreased survival rate. Further, they developed a severe cardiac defect marked by dilated cardiomyopathy (DCM) associated with drastic reduction in basal heart rate and prolongations in atrial and ventricular conduction times. Moreover, CAP2-deficient myofibrils exhibited reduced cooperativity of calcium-regulated force development. At the microscopic level, we observed disarrayed sarcomeres with development of fibrosis. We analyzed CAP2's role in actin assembly and found that it sequesters G-actin and efficiently fragments filaments. This activity resides completely in its WASP homology domain. Thus CAP2 is an essential component of the myocardial sarcomere and is essential for physiological functioning of the cardiac system, and a deficiency leads to DCM and various cardiac defects.
Cellular and Molecular Life Sciences CMLS 09/2012; · 5.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chemotaxis depends on a network of parallel pathways that coordinate cytoskeletal events to bias cell movement along a chemoattractant gradient. Using a forward genetic screen in Dictyostelium discoideum, we identified the Ste20 kinase KrsB, a homolog of tumor suppressors Hippo and MST1/2, as a negative regulator of cell spreading and substrate attachment. The excessive adhesion of krsB(-) cells reduced directional movement and prolonged the streaming phase of multicellular aggregation. These phenotypes depended on an intact kinase domain and phosphorylation of a conserved threonine (T176) within the activation loop. Chemoattractants triggered a rapid, transient autophosphorylation of T176 in a heterotrimeric G protein-dependent and PI3K- and TorC2-independent manner. The active phosphorylated form of KrsB acts to decrease adhesion to the substrate. Taken together these studies suggest that cycling between active and inactive forms of KrsB may provide the dynamic regulation of cell adhesion needed for proper cell migration and chemotaxis. KrsB interacts genetically with another D. discoideum Hippo/MST homolog, KrsA, but the two genes are not functionally redundant. These studies show that Hippo/MST proteins, like the tumor suppressor PTEN and oncogenes Ras and PI3K, play a key role in cell morphological events in addition to their role in regulating cell growth.
Proceedings of the National Academy of Sciences 07/2012; 109(34):13632-7. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cell adhesion, an integral part of D. discoideum 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.
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 aca- mutant. We show that ectopic expression of GFP-CAP improves cell polarization, streaming and aggregation in aca- 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 aca- 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.
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.
[show abstract][hide abstract] ABSTRACT: The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs--even a single WH2 repeat--sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.
Proceedings of the National Academy of Sciences 11/2011; 108(49):19575-80. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dictyostelium discoideum (DD), an extensively studied model organism for cell and developmental biology, belongs to the most derived group 4 of social amoebas, a clade of altruistic multicellular organisms. To understand genome evolution over long time periods and the genetic basis of social evolution, we sequenced the genomes of Dictyostelium fasciculatum (DF) and Polysphondylium pallidum (PP), which represent the early diverging groups 1 and 2, respectively. In contrast to DD, PP and DF have conventional telomere organization and strongly reduced numbers of transposable elements. The number of protein-coding genes is similar between species, but only half of them comprise an identifiable set of orthologous genes. In general, genes involved in primary metabolism, cytoskeletal functions and signal transduction are conserved, while genes involved in secondary metabolism, export, and signal perception underwent large differential gene family expansions. This most likely signifies involvement of the conserved set in core cell and developmental mechanisms, and of the diverged set in niche- and species-specific adaptations for defense and food, mate, and kin selection. Phylogenetic dating using a concatenated data set and extensive loss of synteny indicate that DF, PP, and DD split from their last common ancestor at least 0.6 billion years ago.
Genome Research 07/2011; 21(11):1882-91. · 14.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dictyostelium discoideum cells are professional phagocytes that provide an easily accessible system to gain insights into the mechanisms and the regulatory machinery controlling phagocytosis. Here, we describe a novel function for nuclear Dbf2-related (NDR) family kinases in phagocytosis of D. discoideum. Deletion of one of the four NDR kinases of D. discoideum, NdrA, resulted in impaired cell growth caused by reduced phagocytosis rates. Detailed analysis of NdrA-null cells revealed that the formation of phagocytic cups was delayed. Microscopic investigations showed that NdrA localizes to centrosomes, and NdrA was also identified in isolated centrosome preparations. The localization of NdrA is regulated during the cell cycle. In prophase, NdrA disappears from the centrosome and forms a cloud-like structure around the spindle, which is totally absent during later stages until completion of mitosis. Our results suggest that a signal which originates from the NdrA kinase at the centrosome affects the efficiency of phagocytosis. We assume that in NdrA-null cells the defects in phagocytosis may be caused by an impairment of vesicle trafficking, which is involved in providing new membrane at the sites of particle uptake.
[show abstract][hide abstract] ABSTRACT: Dictyostelium discoideum harbors a short (CRN12) and a long coronin (CRN7) composed of one and two beta-propellers, respectively. They are primarily
present in the cell cortex and cells lacking CRN12 (corA
−) or CRN7 (corB
−) have defects in actin driven processes. We compared the characteristics of a mutant cell line (corA
−) lacking CRN12 and CRN7 with the single mutants focusing on cytokinesis, phagocytosis, chemotaxis and development. Cytokinesis,
uptake of small particles, and developmental defects were not enhanced in the corA
− strain as compared to the single mutants, whereas motility and phagocytosis of yeast particles were more severely impaired.
It appears that although both proteins affect the same processes they do not act in a redundant manner. Rather, they often
act antagonistically, which is in accordance with their proposed roles in the actin cytoskeleton where CRN12 acts in actin
disassembly whereas CRN7 stabilizes actin filaments and protects them from disassembly.
KeywordsActin cytoskeleton–Coronin–Cell motility–Phagocytosis–Development
Cellular and Molecular Life Sciences CMLS 01/2011; 68(2):303-313. · 5.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: Diaphanous-related formins (DRFs) are large multi-domain proteins that nucleate and assemble linear actin filaments. Binding of active Rho family proteins to the GTPase-binding domain (GBD) triggers localization at the membrane and the activation of most formins if not all. In recent years GTPase regulation of formins has been extensively studied, but other molecular mechanisms that determine subcellular distribution or regulate formin activity have remained poorly understood. Here, we provide evidence that the activity and localization of mouse formin mDia1 can be regulated through interactions with phospholipids. The phospholipid-binding sites of mDia1 are clusters of positively charged residues in the N-terminal basic domain (BD) and at the C-terminal region. Upon binding to the lipid bilayer the N-terminal region of mDia1 induces strong clustering of phosphatidylinositol-4,5-bisphosphate (PIP(2)) and subsequently inserts into the membrane bilayer thus anchoring mDia1 to the reconstituted plasma membrane. In addition, an interaction of phospholipids with the C-terminal region of mDia1 causes a drastic reduction of its actin filament assembly activity. Our data suggest that the N-terminal phospholipid-binding sites help to anchor formins at the plasma membrane, and the interaction with phospholipids in the C-terminus functions as a switch for transient inactivation.
European journal of cell biology 10/2010; 89(10):723-32. · 3.31 Impact Factor
[show abstract][hide abstract] ABSTRACT: Three classes of proteins are known to nucleate new filaments: the Arp2/3 complex, formins, and the third group of proteins that contain ca. 25 amino acid long actin-binding Wiskott-Aldrich syndrome protein homology 2 domains, called the WH2 repeats. Crystal structures of the complexes between the actin-binding WH2 repeats of the Spire protein and actin were determined for the Spire single WH2 domain D, the double (SpirCD), triple (SpirBCD), quadruple (SpirABCD) domains, and an artificial Spire WH2 construct comprising three identical D repeats (SpirDDD). SpirCD represents the minimal functional core of Spire that can nucleate actin filaments. Packing in the crystals of the actin complexes with SpirCD, SpirBCD, SpirABCD, and SpirDDD shows the presence of two types of assemblies, "side-to-side" and "straight-longitudinal," which can serve as actin filament nuclei. The principal feature of these structures is their loose, open conformations, in which the sides of actins that normally constitute the inner interface core of a filament are flipped inside out. These Spire structures are distant from those seen in the filamentous nuclei of Arp2/3, formins, and in the F-actin filament.
Proceedings of the National Academy of Sciences 06/2010; 107(26):11757-62. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dictyostelium discoideum Coronin7 (DdCRN7) together with human Coronin7 (CRN7) and Pod-1 of Drosophila melanogaster and Caenorhabditis elegans belong to the coronin family of WD-repeat domain-containing proteins. Coronin7 proteins are characterized by two WD-repeat domains that presumably fold into two beta-propeller structures. DdCRN7 shares highest homology with human CRN7, a protein with roles in membrane trafficking. DdCRN7 is present in the cytosol and accumulates in cell surface projections during movement and phago- and pinocytosis. Cells lacking CRN7 have altered chemotaxis and phagocytosis. Furthermore, loss of CRN7 affects the infection process by the pathogen Legionella pneumophila and allows a more efficient internalization of bacteria. To provide a mechanism for CNR7 action, we studied actin-related aspects. We could show that CRN7 binds directly to F-actin and protects actin filaments from depolymerization. CRN7 also associated with F-actin in vivo. It was present in the Triton X-100-insoluble cytoskeleton, colocalized with F-actin, and its distribution was sensitive to drugs affecting the actin cytoskeleton. We propose that the CRN7 role in chemotaxis and phagocytosis is through its effect on the actin cytoskeleton.
Journal of Biological Chemistry 03/2010; 285(12):9249-61. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin.
PLoS ONE 01/2010; 5(11):e15440. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration.
Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG(-) cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced.
The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote.
PLoS ONE 01/2010; 5(2):e9378. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Dictyostelium centrosome is a nucleus associated body consisting of a box-shaped core surrounded by the corona, an amorphous matrix functionally equivalent to the pericentriolar material of animal centrosomes which is responsible for the nucleation and anchoring of microtubules. Here we describe CP250 a component of the corona, an acidic coiled coil protein that is present at the centrosome throughout interphase while disappearing during prophase and reappearing at the end of late telophase. Amino acids 756-1148 of the 2110 amino acids are sufficient for centrosomal targeting and cell cycle-dependent centrosome association. Mutant cells lacking CP250 are smaller in size, growth on bacteria is delayed, chemotaxis is altered, and development is affected, which, in general, are defects observed in cytoskeletal mutants. Furthermore, loss of CP250 affected the nuclear envelope and led to reduced amounts and altered distribution of Sun-1, a conserved nuclear envelope protein that connects the centrosome to chromatin.
Molecular biology of the cell 09/2009; 20(20):4348-61. · 5.98 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days' knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin.
[show abstract][hide abstract] ABSTRACT: Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes.
PLoS ONE 02/2008; 3(7):e2654. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The genome of the social amoeba Dictyostelium discoideum encodes approximately 285 kinases, which represents approximately 2.6% of the total genome and suggests a signaling complexity similar to that of yeasts and humans. The behavior of D. discoideum as an amoeba and during development relies heavily on fast rearrangements of the actin cytoskeleton. Here, we describe the knockout phenotype of the svkA gene encoding severin kinase, a homolog of the human MST3, MST4 and YSK1 kinases. SvkA-knockout cells show drastic defects in cytokinesis, development and directed slug movement. The defect in cytokinesis is most prominent, leading to multinucleated cells sometimes with >30 nuclei. The defect arises from the frequent inability of svkA-knockout cells to maintain symmetry during formation of the cleavage furrow and to sever the last cytosolic connection. We demonstrate that GFP-SvkA is enriched at the centrosome and localizes to the midzone during the final stage of cell division. This distribution is mediated by the C-terminal half of the kinase, whereas a rescue of the phenotypic changes requires the active N-terminal kinase domain as well. The data suggest that SvkA is part of a regulatory pathway from the centrosome to the midzone, thus regulating the completion of cell division.
[show abstract][hide abstract] ABSTRACT: Cyclase-associated proteins (CAPs) are evolutionarily conserved proteins with roles in regulating the actin cytoskeleton and in signal transduction. Mammals have two CAP genes encoding the related CAP1 and CAP2. We studied the distribution and subcellular localization of CAP1 and CAP2 using specific antibodies. CAP1 shows a broad tissue distribution, whereas CAP2 is significantly expressed only in brain, heart and skeletal muscle, and skin. CAP2 is found in the nucleus in undifferentiated myoblasts and at the M-line of differentiated myotubes. In PAM212, a mouse keratinocyte cell line, CAP2 is enriched in the nucleus, and sparse in the cytosol. By contrast, CAP1 localizes to the cytoplasm in PAM212 cells. In human skin, CAP2 is present in all living layers of the epidermis localizing to the nuclei and the cell periphery. In in vitro studies, a C-terminal fragment of CAP2 interacts with actin, indicating that CAP2 has the capacity to bind to actin.
Cellular and Molecular Life Sciences CMLS 11/2007; 64(19-20):2702-15. · 5.62 Impact Factor