The mechanism regulating the dissociation of the centrosomal protein C-Nap1 from mitotic spindle poles

The centrosomal protein C-Nap1 is thought to play an important role in centrosome cohesion during interphase of the cell cycle. At the onset of mitosis, when centrosomes separate for bipolar spindle formation, C-Nap1 dissociates from centrosomes. Here we report the results of experiments aimed at determining whether the dissociation of C-Nap1 from mitotic centrosomes is triggered by proteolysis or phosphorylation. Specifically, we analyzed both the cell cycle regulation of endogenous C-Nap1 and the fate of exogenously expressed full-length C-Nap1. Western blot analyses suggested a reduction in the endogenous C-Nap1 level during M phase, but studies using proteasome inhibitors and destruction assays performed in Xenopus extracts argue against ubiquitin-dependent degradation of C-Nap1. Instead, our data indicate that the mitotic C-Nap1 signal is reduced as a consequence of M-phase-specific phosphorylation. Overexpression of full-length C-Nap1 in human U2OS cells caused the formation of large structures that embedded the centrosome and impaired its microtubule nucleation activity. Remarkably, however, these centrosome-associated structures did not interfere with cell division. Instead, centrosomes were found to separate from these structures at the onset of mitosis, indicating that a localized and cell-cycle-regulated activity can dissociate C-Nap1 from centrosomes. A prime candidate for this activity is the centrosomal protein kinase Nek2, as the formation of large C-Nap1 structures was substantially reduced upon co-expression of active Nek2. We conclude that the dissociation of C-Nap1 from mitotic centrosomes is regulated by localized phosphorylation rather than generalized proteolysis

Purification and characterization of a tartrate-sensitive acid phosphatase of Trypanosoma brucei

AbstractIn search for invariant surface proteins in Trypanosoma brucei bloodstream forms, acid phosphatase was investigated. Earlier work had shown that part of the cellular phosphatase activity is associated with the flagellar pocket of the parasite. It is demonstrated that T. brucei contains at least two membrane-bound enzymes, one is sensitive to the inhibitor L-(+)-tartrate while the other is resistant. The tartrate-sensitive phosphatase was purified to homogeneity by monoclonal antibody affinity chromatography and shown to be a glycoprotein of low abundance (13,000 molecules/ cell). It has an apparent molecular weight of 70,000 Da. The usefulness of acid phosphatase as a marker for characterizing the membrane lining the flagellar pocket is discussed

H+-Independent Glutamine Transport in Plant Root Tips

BACKGROUND: Glutamine is one of the primary amino acids in nitrogen assimilation and often the most abundant amino acid in plant roots. To monitor this important metabolite, a novel genetically encoded fluorescent FRET-reporter was constructed and expressed in Arabidopsis thaliana. As a candidate for the glutamine fluxes, the root tip localized, putative amino acid transporter CAT8 was analyzed and heterologously expressed in yeast and oocytes. PRINCIPAL FINDINGS: Rapid and reversible in vivo fluorescence changes were observed in reporter-expressing root tips upon exposure and removal of glutamine. FRET changes were detected at acid and neutral pH and in the presence of a protonophore, suggesting that part of the glutamine fluxes were independent of the pH. The putative amino acid transporter CAT8 transported glutamine, had a half maximal activity at approximately 100 microM and the transport was independent of external pH. CAT8 localized not only to the plasma membrane, but additionally to the tonoplast, when tagged with GFP. Ultrastructural analysis confirmed this dual localization and additionally identified CAT8 in membranes of autophagosomes. Loss-of function of CAT8 did not affect growth in various conditions, but over-expressor plants had increased sensitivity to a structural substrate analog, the glutamine synthetase inhibitor L-methionine sulfoximine. CONCLUSIONS: The combined data suggest that proton-independent glutamine facilitators exist in root tips

Rootletin forms centriole-associated filaments and functions in centrosome cohesion

After duplication of the centriole pair during S phase, the centrosome functions as a single microtubule-organizing center until the onset of mitosis, when the duplicated centrosomes separate for bipolar spindle formation. The mechanisms regulating centrosome cohesion and separation during the cell cycle are not well understood. In this study, we analyze the protein rootletin as a candidate centrosome linker component. As shown by immunoelectron microscopy, endogenous rootletin forms striking fibers emanating from the proximal ends of centrioles. Moreover, rootletin interacts with C-Nap1, a protein previously implicated in centrosome cohesion. Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis. Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA–mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion. The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers

Direct Visualization and Silver Enhancement of Ultra-Small Antibody-Bound Gold Particles on Immunolabeled Ultrathin Resin Sections

Ultra-small gold colloids bound to immunolabeled ultrathin resin sections were visualized using transmission, scanning, and scanning transmission electron microscopy (TEM, SEM, STEM). The best marker contrast is obtained in a field emission STEM (200 kV) equipped with a high-angle annular dark-field (HAADF) detector. HAADF STEM renders possible the simultaneous visualization of ultra-small gold and ultrastructural details in unstained resin sections, and an overall presentation of a labeled E. coli cell. For routine work, an enhancement step is a prerequisite for easy detection of bound marker molecules. Five different silver enhancing solutions were tested for their suitability for ultra-small gold intensification. Enhancers lacking the protective colloid gum arabic exhibit lower quality with regard to efficiency and homogeneity of enhancement. This problem can be overcome by adding gum arabic. Silver enhancement generally results in heterogeneously sized particles. This is most probably due to the heterogeneous original gold colloid probe. In general, an estimation of enhancement efficiency is associated with difficulties depending on experimental conditions and the electron microscopic imaging modes used. Only a low number of the ultra-small gold particles seems to remain unenhanced or poorly enhanced when treated with high-quality enhancers. On-section labeling of ultrathin resin sections with silver-enhanced ultra-small gold markers also offers the possibility of high-resolution immunolabeling experiments at the light microscopic level

Topologically Restricted Appearance in the Developing Chick Retinotectal System of Bravo, a Neural Surface Protein: Experimental Modulation by Environmental Cues

A novel neural surface protein, Bravo, shows a pattern of topological restriction in the embryonic chick retinotectal system. Bravo is present on the developing optic fibers in the retina; however, retinal axons in the tectum do not display Bravo. The appearance of Bravo in vitro is modulated by environmental cues. Axons growing out from retinal explants on retinal basal lamina, their natural substrate, express Bravo, whereas such axons growing on collagen do not. Retinal explants provide a valuable system to characterize the mechanism of Bravo restriction, as well as the cellular signals controlling it. Bravo was identified with monoclonal antibodies from a collection generated against exposed molecules isolated by using a selective cell surface biotinylation procedure. The NH2-terminal sequence of Bravo shows similarity with L1, a neural surface molecule which is a member of the immunoglobulin superfamily. This possible relationship to L1, together with its restricted appearance, suggests an involvement of Bravo in axonal growth and guidance

Molecular Effects of Biogenic Zinc Nanoparticles on the Growth and Development of Brassica napus L. Revealed by Proteomics and Transcriptomics

Plants are indispensable on earth and their improvement in terms of food security is a need of time. The current study has been designed to investigate how biogenic zinc nanoparticles (Zn NPs) can improve the growth and development of Brassica napus L. In this study, Zn NPs were synthesized utilizing Mentha arvensis aqueous extracts, and their morphological and optical properties were assessed using UV-Visible spectrophotometry, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD). The synthesized Zn NPs were irregular in shape, indicating aggregation in pattern, with an average particle size of 30 nm, while XRD analysis revealed the crystalline structure of nanoparticles. The growth and development of B. napus varieties (Faisal canola and Shiralee) were assessed after foliar treatments with different concentrations of biogenic Zn NPs. In B. napus varieties, exposure to 15 mg/L Zn NPs dramatically increased chlorophyll, carotenoid content, and biomass accumulation. Similarly, proteomic analyses, on the other hand, revealed that proteins associated with photosynthesis, transport, glycolysis, and stress response in both Brassica varieties were substantially altered. Such exposure to Zn NPs, differential expression of genes associated with photosynthesis, ribosome structural constituents, and oxidative stress response were considerably upregulated in B. napus var. (Faisal and Shiralee canola). The results of this study revealed that foliar applications of biogenic Zn NPs influence the transcriptome and protein profiling positively, therefore stimulating plant growth and development.Peer Reviewe

Cep164, a novel centriole appendage protein required for primary cilium formation

Primary cilia (PC) function as microtubule-based sensory antennae projecting from the surface of many eukaryotic cells. They play important roles in mechano- and chemosensory perception and their dysfunction is implicated in developmental disorders and severe diseases. The basal body that functions in PC assembly is derived from the mature centriole, a component of the centrosome. Through a small interfering RNA screen we found several centrosomal proteins (Ceps) to be involved in PC formation. One newly identified protein, Cep164, was indispensable for PC formation and hence characterized in detail. By immunogold electron microscopy, Cep164 could be localized to the distal appendages of mature centrioles. In contrast to ninein and Cep170, two components of subdistal appendages, Cep164 persisted at centrioles throughout mitosis. Moreover, the localizations of Cep164 and ninein/Cep170 were mutually independent during interphase. These data implicate distal appendages in PC formation and identify Cep164 as an excellent marker for these structures

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

Function of the anion transporter AtCLC-d in the trans-Golgi network

Anion transporting proteins of the CLC type are involved in anion homeostasis in a variety of organisms. CLCs from Arabidopsis have been shown to participate in nitrate accumulation and storage. In this study, the physiological role of the functional chloride transporter AtCLC-d from Arabidopsis was investigated. AtCLC-d is weakly expressed in various tissues, including the root. When transiently expressed as a GFP fusion in protoplasts, it co-localized with the VHA-a1 subunit of the proton-transporting V-type ATPase in the trans-Golgi network (TGN). Stable expression in plants showed that it co-localized with the endocytic tracer dye FM4-64 in a brefeldin A-sensitive compartment. Immunogold electron microscopy confirmed the localization of AtCLC-d to the TGN. Disruption of the AtCLC-d gene by a T-DNA insertion did not affect the nitrate and chloride contents. The overall morphology of these clcd-1 plants was similar to that of the wild-type, but root growth on synthetic medium was impaired. Moreover, the sensitivity of hypocotyl elongation to treatment with concanamycin A, a blocker of the V-ATPase, was stronger in the clcd-1 mutant. These phenotypes could be complemented by overexpression of AtCLC-d in the mutant background. The results suggest that the luminal pH in the trans-Golgi network is adjusted by AtCLC-d-mediated transport of a counter anion such as Cl− or NO3−