The XMAP215-family protein DdCP224 is required for cortical interactions of microtubules

BACKGROUND: Interactions of peripheral microtubule tips with the cell cortex are of crucial importance for nuclear migration, spindle orientation, centrosome positioning and directional cell movement. Microtubule plus end binding proteins are thought to mediate interactions of microtubule tips with cortical actin and membrane proteins in a dynein-dependent manner. XMAP215-family proteins are main regulators of microtubule plus end dynamics but so far they have not been implicated in the interactions of microtubule tips with the cell cortex. RESULTS: Here we show that overexpression of an N-terminal fragment of DdCP224, the Dictyostelium XMAP215 homologue, caused a collapse of the radial microtubule cytoskeleton, whereby microtubules lost contact with the cell cortex and were dragged behind like a comet tail of an unusually motile centrosome. This phenotype was indistinguishable from mutants overexpressing fragments of the dynein heavy chain or intermediate chain. Moreover, it was accompanied by dispersal of the Golgi apparatus and reduced cortical localization of the dynein heavy chain indicating a disrupted dynein/dynactin interaction. The interference of DdCP224 with cortical dynein function is strongly supported by the observations that DdCP224 and its N-terminal fragment colocalize with dynein and coimmunoprecipitate with dynein and dynactin. CONCLUSIONS: Our data show that XMAP215-like proteins are required for the interaction of microtubule plus ends with the cell cortex in interphase cells and strongly suggest that this function is mediated by dynein

Anionic polymer brushes for biomimetic calcium phosphate mineralization : a surface with application potential in biomaterials

This article describes the synthesis of anionic polymer brushes and their mineralization with calcium phosphate. The brushes are based on poly(3-sulfopropyl methacrylate potassium salt) providing a highly charged polymer brush surface. Homogeneous brushes with reproducible thicknesses are obtained via surface-initiated atom transfer radical polymerization. Mineralization with doubly concentrated simulated body fluid yields polymer/inorganic hybrid films containing AB-Type carbonated hydroxyapatite (CHAP), a material resembling the inorganic component of bone. Moreover, growth experiments using Dictyostelium discoideum amoebae demonstrate that the mineral-free and the mineral-containing polymer brushes have a good biocompatibility suggesting their use as biocompatible surfaces in implantology or related fields

Dynamics of a novel centromeric histone variant CenH3 reveals the evolutionary ancestral timing of centromere biogenesis

The centromeric histone H3 variant (CenH3) serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. The Dictyostelium H3-like variant H3v1 was identified as the CenH3 ortholog. Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins and a histone fold domain at its C-terminus. Within the histone fold, α-helix 2 (α2) and an extended loop 1 (L1) have been shown to be required for targeting CenH3 to centromeres. Compared to other known and putative CenH3 histones, Dictyostelium CenH3 has a shorter L1, suggesting that the extension is not an obligatory feature. Through ChIP analysis and fluorescence microscopy of live and fixed cells, we provide here the first survey of centromere structure in amoebozoa. The six telocentric centromeres were found to mostly consist of all the DIRS-1 elements and to associate with H3K9me3. During interphase, the centromeres remain attached to the centrosome forming a single CenH3-containing cluster. Loading of Dictyostelium CenH3 onto centromeres occurs at the G2/prophase transition, in contrast to the anaphase/telophase loading of CenH3 observed in metazoans. This suggests that loading during G2/prophase is the ancestral eukaryotic mechanism and that anaphase/telophase loading of CenH3 has evolved more recently after the amoebozoa diverged from the animal linage

Evolution of the nucleus

Under a Creative Commons license.The nucleus represents a major evolutionary transition. As a consequence of separating translation from transcription many new functions arose, which likely contributed to the remarkable success of eukaryotic cells. Here we will consider what has recently emerged on the evolutionary histories of several key aspects of nuclear biology; the nuclear pore complex, the lamina, centrosomes and evidence for prokaryotic origins of relevant players.Work in our laboratories was supported by the following agencies, and which is gratefully acknowledged; MRC and Wellcome Trust (MR/K008749/1 and 090007/Z/09/Z respectively, to MCF), C2A Junta de Andalucia to DPD and DFG GR1642/4-1 to RG.Open Access funded by Wellcome Trust.Peer Reviewe

Comparative Biology of Centrosomal Structures in Eukaryotes

The centrosome is not only the largest and most sophisticated protein complex within a eukaryotic cell, in the light of evolution, it is also one of its most ancient organelles. This special issue of “Cells„ features representatives of three main, structurally divergent centrosome types, i.e., centriole-containing centrosomes, yeast spindle pole bodies (SPBs), and amoebozoan nucleus-associated bodies (NABs). Here, I discuss their evolution and their key-functions in microtubule organization, mitosis, and cytokinesis. Furthermore, I provide a brief history of centrosome research and highlight recently emerged topics, such as the role of centrioles in ciliogenesis, the relationship of centrosomes and centriolar satellites, the integration of centrosomal structures into the nuclear envelope and the involvement of centrosomal components in non-centrosomal microtubule organization

CDK5RAP2 Is an Essential Scaffolding Protein of the Corona of the Dictyostelium Centrosome

Dictyostelium centrosomes consist of a nucleus-associated cylindrical, three-layered core structure surrounded by a corona consisting of microtubule-nucleation complexes embedded in a scaffold of large coiled-coil proteins. One of them is the conserved CDK5RAP2 protein. Here we focus on the role of Dictyostelium CDK5RAP2 for maintenance of centrosome integrity, its interaction partners and its dynamic behavior during interphase and mitosis. GFP-CDK5RAP2 is present at the centrosome during the entire cell cycle except from a short period during prophase, correlating with the normal dissociation of the corona at this stage. RNAi depletion of CDK5RAP2 results in complete disorganization of centrosomes and microtubules suggesting that CDK5RAP2 is required for organization of the corona and its association to the core structure. This is in line with the observation that overexpressed GFP-CDK5RAP2 elicited supernumerary cytosolic MTOCs. The phenotype of CDK5RAP2 depletion was very reminiscent of that observed upon depletion of CP148, another scaffolding protein of the corona. BioID interaction assays revealed an interaction of CDK5RAP2 not only with the corona markers CP148, γ-tubulin, and CP248, but also with the core components Cep192, CP75, and CP91. Furthermore, protein localization studies in both depletion strains revealed that CP148 and CDK5RAP2 cooperate in corona organization

The Dictyostelium Centrosome

The centrosome of Dictyostelium amoebae contains no centrioles and consists of a cylindrical layered core structure surrounded by a corona harboring microtubule-nucleating γ-tubulin complexes. It is the major centrosomal model beyond animals and yeasts. Proteomics, protein interaction studies by BioID and superresolution microscopy methods led to considerable progress in our understanding of the composition, structure and function of this centrosome type. We discuss all currently known components of the Dictyostelium centrosome in comparison to other centrosomes of animals and yeasts

In Vivo Assembly of a Dictyostelium Lamin Mutant Induced by Light, Mechanical Stress, and pH

We expressed Dictyostelium lamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-ΔNLSΔCLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of the Dictyostelium lamin, they are likely relevant also for wild-type lamin