Ohta T, Essner R, Ryu JH, Palazzo RE, Uetake Y, Kuriyama R.

GM55735 NIGMS NIH HHS , R01 GM055735 NIGMS NIH HHS

	Figure 1. Identification of Cep135 in the mammalian centrosome. CHO cells at interphase (A) and mitosis (B) are double immunostained with anti-tubulin (green) and anti-Cep135 (yellow) antibodies. (C) Proteins prepared from isolated mitotic spindles (lanes 1 and 1′) and whole cell lysates (lanes 2 and 2′) were run on 7.5% SDS-PAGE and immunoblotted with polyclonal anti-Cep135 bacterial fusion protein antibodies (lanes 1′ and 2′). An arrowhead indicates the position of the 135-kD band recognized by the anti-Cep135 antibody. The positions of α- and β-tubulin are also indicated by arrows. Bars: (A) 10 μm; (B) 5 μm.
	Figure 2. Localization of Cep135 at the centrosome in nocodazole-treated CHO cells (A, A′, B, and B′), mouse embryonic fibroblasts lacking p53 (C and C′), frog fibroblasts (D), and sea urchin eggs (E). Cells were double stained with anti-Cep135 (A–C) and either anti–β-tubulin (A'), anti–γ-tubulin (B'), or anti-pericentrin (C') antibodies. Cep135 is present in the centrosome in a microtubule-independent manner. Interphase centrosomes and spindle poles in nonmammalian cells also contain the Cep135 antigen (D and E). An arrow indicates mitotic chromosomes aligned at the metaphase plate. Bars: (A–D) 10 μm; (E) 50 μm.
	Figure 3. Immunoelectron microscopy of Cep135 in interphase CHO cells (A) and isolated mitotic spindles (B). 6-nm gold-conjugated Cep135 antibodies were detected around the centrioles (arrowheads) and in the amorphous material (arrows) in the centrosome located next to the nucleus (N). At the pole, in isolated mitotic spindles, Cep135 is present in close association with the centriole, both outside (C) and inside (D and E) the centriole wall. Bars: (A and B) 1 μm; (C–E) 0.2 μm.
	Figure 4. Schematic diagram of the secondary structure of Cep135 (A) and the presence of two types of Cep135 transcripts in CHO cells (B). (A) The protein consists of 1,145-amino acid residues in which extensive α-helical domains (shaded blocks) span almost the continuous length of polypeptide chain. The positions of two leucine zipper consensus motifs and three putative tyrosine phosphorylation motifs are indicated by black boxes and arrows, respectively. Numbers indicate the position of amino acids. (B) 10 cDNA clones isolated by screening of a CHO expression library are classified into two categories. All clones share the overlapping restriction maps and identical Cep135 coding sequence. Indicated are the start and stop codons as well as some restriction sites included in the sequences.
	Figure 5. A map of deletion constructs created for identification of the centrosomal target domain in Cep135. Numbers indicate the positions of amino acids. The results of the immunolocalization of HA-tagged polypeptides at the centrosome and in the cytoplasm are summarized at the right.
	Figure 6. Immunolocalization of truncated Cep135 polypeptides expressed in CHO cells by transient transfection. The same cells are seen by double immunostaining with anti-HA (left side of each pair) and with either anti–γ-tubulin, anti-Cep135, or anti-pericentrin antibodies as indicated in each frame. Although the sequences encoded by Δ1, Δ11, Δ5, and Δ13 are not found in the centrosome, other clones produce proteins recruited to the centrosome. Note the NH2-terminal Δ1 and Δ11 proteins in the cytoplasm are not recognized by the Cep135 antibody raised against the COOH-terminal #0 sequence. Bar, 10 μm.
	Figure 7. Overexpression of Cep135 polypeptides causes the formation of cytoplasmic foci. (A–E) Dots of various size and number were detected in cells overexpresssing different domains of the Cep135 sequence. The foci containing the COOH-terminal domain of Cep135 (#0, #1, and Δ14) show smooth and well-rounded surfaces. Arrows indicate the position of HA-tagged Δ3 and Δ14 that were successfully targeted to the centrosome as determined by γ-tubulin staining. (F–I′) Cytoplasmic dots containing the full-coding sequence of GFP-labeled Cep135 nucleate microtubules in vivo upon release from nocodazole treatment. Cells expressing GFP–Cep135 were treated with nocodazole for 2 h to depolymerize in situ microtubules. 5–15 min after removing nocodazole, cells were fixed, stained with anti-β-tubulin antibodies, and visualized for GFP–Cep135 (F', G', and H'). In addition to Cep135 (F), the dots are associated with pericentrin (G) and γ-tubulin (H). I and I′ show the control cell expressing a GFP leader sequence only. Bars, 10 μm.
	Figure 8. Induction of fibrous aggregates in the centrosome (A and B) and the cytoplasm (C and D). (A) A cell expressing GFP-tagged Δ3 includes a centrosome that appears as a large electron-dense cloud (oval area within inset at lower magnification) containing characteristic whorl-like particles (A, arrowheads). (B) Similar whorl-like particles of various shapes and sizes (arrowheads; B' and B” at higher magnification) and 6-nm fibers (small arrows) are seen in the centriole (large arrow)-containing centrosome. (C and D) The cytoplasmic dots are composed of electron-dense, curved aggregates. Similar to the whorls seen in A and B, the filamentous aggregates are composed of dense, curved parallel lines arranged with a periodicity of 6 nm. Bars: (A and B) 0.5 μm; (B' and B”) 0.1 μm; (C and D) 0.5 μm.
	Figure 9. Abnormal mitotic spindles induced in cells overexpressing HA-tagged Δ3 polypeptides of Cep135. The same cells are seen by fluorescence microscopy after double immunostaining with anti-tubulin and anti-HA antibodies as indicated in each figure. A′–C″ and F1–F2′ represent the images at different focal planes. D″ shows a double image of phase-contrast and fluorescence microscopy. Exogenous Δ3 Cep135 causes the formation of extra dots to which spindle microtubules are frequently attached (C, E, and F). Some dots are also labeled by anti–β-tubulin staining (D). Quantitative analysis of normal and abnormal spindles is summarized in the table. Note that the nocodazole treatment necessary to synchronize M phase cells resulted in the production of more abnormal cells (17–23%) than nontreated controls, which generally include less than 2–5% abnormal cells (Matuliene et al., 1999). Bar, 10 μm.
	Figure 10. Suppression of Cep135 expression by RNAi. (A) Immunoblotting analysis: equal amounts of proteins prepared from CHO cells at 27 (lane 2), 48 (lane 3), and 83 h (lanes 1 and 4) after mock (lane 1), single (RNAi-I; lanes 2 and 3), and double (RNAi-II; lane 4) siRNA transfections were run on a 7.5% protein gel. Arrow indicates the position of Cep135 bands. (B) Immunofluorescence staining: mock- (a) and siRNA-treated (b–d) CHO cells were fixed at 72 (b and c) and 96 h (a and d) after transfection, and immunostained with Cep135; single (RNAi-I; b), double (RNAi-II; c), and triple (RNAi-III; d) RNAi. To correctly represent the amount of endogenous Cep135 in all cells, both mock- and siRNA-transfected cells were fixed and immunostained in an identical manner, and all images were captured and printed under identical conditions. Nontransfected cells in RNAi-I formed a colony as indicated by arrowheads (b), and arrows in RNAi-III indicate the position of weakly stained centrosomes (d). Bar, 50 μm. (C) Frequency histogram of Cep135 immunofluorescence intensity at the centrosome in mock- (Mock) and siRNA-transfected cells (RNAi-I, -II, and -III). Cells were classified into four categories: 100–75, 75–50, 50–25, and 25–0% of the Cep135 fluorescence intensity at the centrosome relative to the mean value of the centrosomal Cep135 fluorescence mock cells.
	Figure 11. RNAi causes abnormal organization of interphase and mitotic microtubules in CHO cells. The same cells were stained with anti-Cep135 (A–F), anti–α-tubulin (A'–F') antibodies, and DAPI (A”–F”). D” shows a double image of phase-contrast and fluorescence microscopy. siRNA-transfected cells tended to lose the microtubule focal point during interphase (B'). Abnormal spindles in monopolar (C–C″ and E–E″) and multipolar (C–C″ and D–D″) orientation were frequently seen in mitotic cells. (F–F”) Cep135 was still weakly detected at each pole (arrows), but central spindle microtubules were severely disorganized. Tables summarize normal and abnormal microtubule patterns scored in interphase (RNAi-II and RNAi-III) and mitotic (RNAi-I + RNAi-II) cells. Bars: (A–B″) 50 μm; (C–F″) 10 μm.

Andersen, Molecular characteristics of the centrosome. 1999, Pubmed

Andersen, Molecular characteristics of the centrosome. 1999, Pubmed
Atkinson, Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation. 1991, Pubmed
Bornens, Is the centriole bound to the nuclear membrane? 1977, Pubmed
Buendia, A centrosomal antigen localized on intermediate filaments and mitotic spindle poles. 1990, Pubmed , Xenbase
Dictenberg, Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome. 1998, Pubmed , Xenbase
Doxsey, Re-evaluating centrosome function. 2001, Pubmed
Elbashir, Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. 2001, Pubmed
Francis, The spindle pole body of Saccharomyces cerevisiae: architecture and assembly of the core components. 2000, Pubmed
Fry, C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2. 1998, Pubmed
Fukasawa, Abnormal centrosome amplification in the absence of p53. 1996, Pubmed
Heitlinger, Expression of chicken lamin B2 in Escherichia coli: characterization of its structure, assembly, and molecular interactions. 1991, Pubmed
Hobohm, A sequence property approach to searching protein databases. 1995, Pubmed
Ishikawa, Prediction of the coding sequences of unidentified human genes. X. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro. 1998, Pubmed
Kellogg, Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo. 1989, Pubmed
Kilmartin, A spacer protein in the Saccharomyces cerevisiae spindle poly body whose transcript is cell cycle-regulated. 1993, Pubmed
Kimble, Functional components of microtubule-organizing centers. 1992, Pubmed
Kuriyama, 225-Kilodalton phosphoprotein associated with mitotic centrosomes in sea urchin eggs. 1989, Pubmed
Kuriyama, Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells. 1995, Pubmed
Kuriyama, Methods for identification of centrosome-associated proteins. 2001, Pubmed
Kuriyama, Centriole cycle in Chinese hamster ovary cells as determined by whole-mount electron microscopy. 1981, Pubmed
Kuriyama, Obtaining antibodies to spindle components. 1999, Pubmed
Lange, A molecular marker for centriole maturation in the mammalian cell cycle. 1995, Pubmed
Lupas, Predicting coiled coils from protein sequences. 1991, Pubmed
Matuliene, Function of a minus-end-directed kinesin-like motor protein in mammalian cells. 1999, Pubmed
Mazia, The chromosome cycle and the centrosome cycle in the mitotic cycle. 1987, Pubmed
Mogensen, Microtubule minus-end anchorage at centrosomal and non-centrosomal sites: the role of ninein. 2000, Pubmed
Nakagawa, Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: its identification from isolated centrosomes. 2001, Pubmed
Nakamura, When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin. 1998, Pubmed
Oegema, The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains. 1995, Pubmed
Paoletti, Most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles. 1996, Pubmed , Xenbase
Paul, Identification of a 102 kDa protein (cytocentrin) immunologically related to keratin 19, which is a cytoplasmically derived component of the mitotic spindle pole. 1993, Pubmed
Ryu, Filamentous polymers induced by overexpression of a novel centrosomal protein, Cep135. 2000, Pubmed
Saredi, NuMA assembles into an extensive filamentous structure when expressed in the cell cytoplasm. 1996, Pubmed
Schatten, Centrosome detection in sea urchin eggs with a monoclonal antibody against Drosophila intermediate filament proteins: characterization of stages of the division cycle of centrosomes. 1987, Pubmed
Schmidt, AKAP350, a multiply spliced protein kinase A-anchoring protein associated with centrosomes. 1999, Pubmed
Schnackenberg, The disassembly and reassembly of functional centrosomes in vitro. 1998, Pubmed
Sellitto, Heterogeneity of microtubule organizing center components as revealed by monoclonal antibodies to mammalian centrosomes and to nucleus-associated bodies from dictyostelium. 1992, Pubmed
Shu, Gamma-tubulin can both nucleate microtubule assembly and self-assemble into novel tubular structures in mammalian cells. 1995, Pubmed
Stearns, The cell center at 100. 1997, Pubmed
Stewart, Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure. 1989, Pubmed
Sym, Zip1-induced changes in synaptonemal complex structure and polycomplex assembly. 1995, Pubmed
Tassin, Identification of an Spc110p-related protein in vertebrates. 1997, Pubmed , Xenbase
Vassilev, Identification of intrinsic dimer and overexpressed monomeric forms of gamma-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas gamma-tubulin sequence. 1995, Pubmed
Vogel, Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin. 1997, Pubmed
Wiese, Gamma-tubulin complexes and their interaction with microtubule-organizing centers. 1999, Pubmed
Wigge, Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. 1998, Pubmed
Xie, Coiled-coil packing in spermine-induced tropomyosin crystals. A comparative study of three forms. 1994, Pubmed
Zhou, Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. 1998, Pubmed