Golgi localisation of GMAP210 requires two distinct cis-membrane binding mechanisms

<p>Abstract</p> <p>Background</p> <p>The Golgi apparatus in mammals appears as a ribbon made up of interconnected stacks of flattened cisternae that is positioned close to the centrosome in a microtubule-dependent manner. How this organisation is achieved and retained is not well understood. GMAP210 is a long coiled-coil cis-Golgi associated protein that plays a role in maintaining Golgi ribbon integrity and position and contributes to the formation of the primary cilium. An amphipathic alpha-helix able to bind liposomes <it>in vitro </it>has been recently identified at the first 38 amino acids of the protein (amphipathic lipid-packing sensor motif), and an ARF1-binding domain (Grip-related Arf-binding domain) was found at the C-terminus. To which type of membranes these two GMAP210 regions bind <it>in vivo </it>and how this contributes to GMAP210 localisation and function remains to be investigated.</p> <p>Results</p> <p>By using truncated as well as chimeric mutants and videomicroscopy we found that both the N-terminus and the C-terminus of GMAP210 are targeted to the cis-Golgi <it>in vivo</it>. The ALPS motif was identified as the N-terminal binding motif and appeared concentrated in the periphery of Golgi elements and between Golgi stacks. On the contrary, the C-terminal domain appeared uniformly distributed in the cis-cisternae of the Golgi apparatus. Strikingly, the two ends of the protein also behave differently in response to the drug Brefeldin A. The N-terminal domain redistributed to the endoplasmic reticulum (ER) exit sites, as does the full-length protein, whereas the C-terminal domain rapidly dissociated from the Golgi apparatus to the cytosol. Mutants comprising the full-length protein but lacking one of the terminal motifs also associated with the cis-Golgi with distribution patterns similar to those of the corresponding terminal end whereas a mutant consisting in fused N- and C-terminal ends exhibits identical localisation as the endogenous protein.</p> <p>Conclusion</p> <p>We conclude that the Golgi localisation of GMAP210 is the result of the combined action of the two N- and C-terminal domains that recognise different sub-regions of the cis-GA. Based on present and previous data, we propose a model in which GMAP210 would participate in homotypic fusion of cis-cisternae by anchoring the surface of cisternae via its C-terminus and projecting its distal N-terminus to bind the rims or to stabilise tubular structures connecting neighbouring cis-cisternae.</p

Disconnecting the Golgi ribbon from the centrosome prevents directional cell migration and ciliogenesis

AKAP450 is a critical determinant of Golgi ribbon integrity, positioning, and function

Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.This work was supported by Ministerio de Economia y Competitividad, Spain (BFU2012-36717 and CSD2009-00016 to RMR and BFU2011-22916 to JRM) and by Junta de Andalucia (CVI-7256 and CTS-2071), and by a funding GenHomme Network 02490-6088 to Hybrigenics and the Institut Curie. MA and AZ were supported by MEC–FPI Grants.Peer Reviewe

The Centrosomal Protein C-Nap1 Is Required for Cell Cycle–Regulated Centrosome Cohesion

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle–regulated manner. To test this model, we have performed a detailed structure–function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle–regulated structure that mediates centriole–centriole cohesion

Atomic Force Microscopy of height fluctuations of fibroblast cells

We investigated the nanometer scale height fluctuations of 3T3 fibroblast cells with the atomic force microscope (AFM) under physiological conditions. Correlation between these fluctuations and lateral cellular motility can be observed. Fluctuations measured on leading edges appear to be predominantly related to actin polymerization-depolymerization processes. We found fast (5 Hz) pulsatory behavior with 1--2 nm amplitude on a cell with low motility showing emphasized structure of stress fibres. Myosin driven contractions of stress fibres are thought to induce this pulsation.Comment: 6 pages, 5 figures, 1 tabl

Mouse Odf2 localizes to centrosomes and basal bodies in adult tissues and to the photoreceptor primary cilium

Odf2 (outer dense fiber 2) is the major protein of the cytoskeleton of the sperm tail. In somatic cells, it is a component of the centrosome in which it is located in the appendages of the mother centriole. Additionally, as shown previously by forced expression in cultured cells, Odf2 localizes to centrioles, basal bodies, and primary cilia, which are all structurally and functionally interconnected. The importance of Odf2 has become obvious by the absence of primary cilia in Odf2-deficient cells and by the embryonic lethality of the Odf2 gene trap insertional mouse. However, nothing is known about the endogenous localization of Odf2 in the tissues of adult mice. We show here that Odf2 protein localizes to centrosomes, to photoreceptor primary cilia, and to basal bodies of ciliated cells of the respiratory epithelium and of the kidney. Our results thus suggest that Odf2 contributes to assorted ciliopathies

Role of CAP350 in Centriolar Tubule Stability and Centriole Assembly

BACKGROUND: Centrioles are microtubule-based cylindrical structures composed of nine triplet tubules and are required for the formation of the centrosome, flagella and cilia. Despite theirs importance, centriole biogenesis is poorly understood. Centrosome duplication is initiated at the G1/S transition by the sequential recruitment of a set of conserved proteins under the control of the kinase Plk4. Subsequently, the procentriole is assembled by the polymerization of centriolar tubules via an unknown mechanism involving several tubulin paralogs. METHODOLOGY/PRINCIPAL FINDINGS: Here, we developed a cellular assay to study centrosome duplication and procentriole stability based on its sensitivity to the microtubule-depolymerizing drug nocodazole. By using RNA interference experiments, we show that the stability of growing procentrioles is regulated by the microtubule-stabilizing protein CAP350, independently of hSAS-6 and CPAP which initiate procentriole growth. Furthermore, our analysis reveals the critical role of centriolar tubule stability for an efficient procentriole growth. CONCLUSIONS/SIGNIFICANCE: CAP350 belongs to a new class of proteins which associate and stabilize centriolar tubules to control centriole duplication