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NUANCE, a giant protein connecting the nucleus and actin cytoskeleton

Institute for Biochemistry, Medical Faculty, University of Cologne, 50931 Cologne, Germany

View larger version (23K): [in a new window]   Fig. 1. Cloning of the human NUANCE gene. (A) The 21.8 kb cDNA was assembled from 13 overlapping clones obtained by RT- and RACE-PCR using mRNA from BL-60 cells as the template. (B) The exon-intron organization of the human NUANCE gene. The NUACE gene contains 114 protein-coding exons. Introns, represented by lines, are drawn to scale; exons are shown as vertical bars. Exon 1a, found in the EST BI026470, exon 8a, found in the short isoform NUANCE-ABD-S, exon 103a, found in the KIAA1011 protein (accession number AB023228), and the longer form of exon 104, which is found in DKFZp434H2235 mRNA (accession number AL117404), are represented by empty bars. The positions of Start and Stop codons are indicated by arrows. The annotated sequences of DKFZp434G173 (accession number NM_015180) and DKFZp434H2235 are underlined by solid lines. The estrogen receptor 2 gene (ESR2) located downstream is boxed. Alternative splicing at the 5' end (C) and at the 3' end (D) of the NUANCE gene. An additional exon 1a that encodes an alternative transcriptional start was found in EST BI026470. The boxes represent exons drawn to scale. Gray shading indicate coding regions; white boxes indicate untranslated regions. Aternatively spliced coding exons are shown in black. The locations of the ATG start codon and the TGA stop codon are indicated. The size of each exon and intron is given in Table 1.

View larger version (77K): [in a new window]   Fig. 9. Localization of GFP-fused chimeric proteins in transfected COS7 cells. Protein domains are presented as in Fig. 3. All GFP fusion protein comprising ABDs (A-F) were colocalized with actin stress fibers (A,C,D,F, big arrows) and actin meshwork throughout the cell as well as with the cortical actin in lamellae (C,F, arrowheads). The GFP-ABD-S was enriched at the tips of stress fibers (B,arrow) and diminished at the plasmalemma (B), whereas the GFP-ABDsr1-2 (C) associated stronger with the submembrane actin. Nascent focal complexes did not attract GFP-ABD-S (small arrowheads). The amino acids AYKN at the C-terminus of the GFP-ABD-S protein were encoded by the additional exon 8a found by PCR analysis. GFP-ABDsr1-2 protein (C,F) harbors two spectrin repeats in addition to the ABD. GFP-sr15-21, corresponding to the middle part of NUANCE from spectrin repeat 15 to 20, seems to target intracellular membranes and vesicular structures colocalizing with ß-COP-positive structures (G,J, arrow) but not to the NE. By contrast, the GFP-Cterm1 protein harboring the TMD together with the two preceding spectrin repeats is recruited to the NE and adjacent ER (H). Note the lack of nuclear rim staining in the transfected cell (K, arrowhead). The GFP-Cterm2tm fusion protein is associated with vesicles but not with the NE (I,L, arrowheads). Cells transfected with the GFP-Cterm2tm construct were fixed with methanol (see details in the text). The transfected COS7 cells were double-labeled with TRITC-phalloidin (D), vinculin (E) and NUANCE (F,K,L) and ß-COP (J) mAbs. Bar, 20 µm.

View larger version (137K): [in a new window]   Fig. 2. Deduced polypeptide sequence of human NUANCE. Amino acid residues are numbered on the left. The actin binding domain of NUANCE is highlighted in green; 22 detected dystrophin-like spectrin repeats are in cyan, the transmembrane region is in yellow, Klarsicht-like domain is in black and underlined. Predicted nuclear localization signals are shown in blue and are underlined, leucine zippers are in red and underlined.

View larger version (70K): [in a new window]   Fig. 3. Structural features of human NUANCE. (A) The ABD is represented by an empty box; 22 spectrin repeats with considerable homology to dystrophin are shown as filled ovals; and the TMD is indicated by a black bar. The positions of nuclear localization signals and leucine zippers are indicated. Coiled-coil regions were detected by the MultiCoil program (Wolf et al., 1997) with a window size of 21. Blue and red lines mark the location of predicted dimeric or trimeric coiled coils, respectively. (B) Alignment of the ABDs of NUANCE, enaptin, calmin (BAB59010), ß-spectrin (AAA60580) and MACF (AAD32244). NUANCE, enaptin and calmin harbor long stretches between both CH domains unlike conventional ABDs of ß-spectrin and MACF. (C) A phylogenetic tree of the ABDs of NUANCE and other proteins of the -actinin superfamily on the basis of calculations from ClustalW alignment of these domains. The accession numbers are: human filamin (AF184126), human dystrophin (P11532), Dictyostelium cortexillin (L49527), human ß-spectrin (M96803), Drosophila kakapo (AJ011924), Dictyostelium interaptin (AF057019), chicken fimbrin (A37097), mouse -actinin (P12814), human utrophin (P46939), mouse plectin (AF188012), mouse MACF (AF150755), mouse dystonin (AF252549) and human calmin (BAB59010). (D) Klarsicht-like domain of NUANCE. The C-termini of NUANCE, human Syne-1 (KIAA0796 protein, BAA34516), mouse Syne-1 (AAG24392), human lymphocyte membrane associated protein (LMAP, AAC02992), an uncharacterised C. elegans protein similar to myosin-like proteins (AAF40010) and D. melanogaster Klarsicht protein (AAD43129) are aligned with ClustalW version 4.2. The amino acids similar in more than 40% of the sequences are shaded. Three parts of the Klarsicht-like domain are marked. (E) Alignment of 22 selected spectrin repeats of human NUANCE. The multiple alignment was made using the CLUSTAL W program (EMBL). The bars indicate positions of three helices according to the structure-based alignment of Winder and colleagues (Winder et al., 1995).

View larger version (60K): [in a new window]   Fig. 4. Expression analysis of NUANCE. (A) A northern dot-blot of a variety of human tissues and cell lines, probed with a fragment corresponding to the ABD of NUANCE. These results are summarized in Table 2. Lane 12 represents controls: A12, yeast total RNA; B12, yeast tRNA; C12, E. coli rRNA; D12, E. coli DNA; E12, poly(A); F12 human C0t_1 DNA; G12, human DNA 100 ng; H12, human DNA 500 ng. (B) Immunoblots of the COS7 cells homogenate. (C) Cells fractionated into nuclei, cytosol and cytoplasmic membranes. Each fraction was separated on 3-15% gradient SDS-PAGE and immunoblotted with anti-NUANCE, anti-lamin B and anti-annexin A7 mAbs as indicated. The positions of molecular mass marker proteins are shown on the left.

View larger version (84K): [in a new window]   Fig. 5. Nuclear localization of NUANCE in COS7 cells (C,G) immunolabeled with the anti-NUANCE mAb K20-478. NUANCE remains associated with NE during prophase (A,B, arrow) and with chromosomes during prometaphase (A,B, large arrowhead). At later stages NUANCE is diffusely distributed throughout the cytoplasm (A, B, small arrowhead). NUANCE is also detected in bridges, which were occasionally seen between the nuclei of two cells (C,D, arrow). DNA was visualized with DAPI (B,D). Bar, 20 µm.

View larger version (135K): [in a new window]   Fig. 6. Localization of NUANCE compared with that of nuclear pores. Cells were double labelled with anti-NUANCE (A,D,G,J) and anti-nucleoporin358 (Nup385) (B,E,K) antibodies. A-C, confocal sections of the apical part of a COS7 cell nucleus. The optical surfaces of nuclei are shown with enlargements of the regions indicated. NUANCE- (arrowheads, A-C) and Nup358-positive spots (arrows, A-C) are only partially colocalized. (D-L) The equatorial optical sections from the nuclei of COS7 cells, treated with 0.5% Triton X-100 (D-I) and 4 µM digitonin (J-L). Visualization of NUANCE at the NE in digitonin-treated cells (J-L, arrowheads) suggests its localization on the cytoplasmic face of the ONM. Note the polar distribution of cytoplasmic NUANCE (D, J, asterisk) and Nup385. In Triton X-100-treated cells, the intranuclear labeling was enriched in the nucleoli (D-I, arrows), which was verified by anti-NO38 staining (H). Bar, 5 µm.

View larger version (101K): [in a new window]   Fig. 7. Effect of LatA treatment on the subcellular distribution of endogenous NUANCE. COS7 cells were incubated in the absence (A-C) and presence (D-I) of 1 µM LatA for 30 minutes (D-F) and 60 minutes (G-I). (J-L) COS7 cells were stimulated to migrate by wounding a confluent monolayer of cells. 6 hours after wounding, the cells were processed for immunofluorescence microscopy. Cells were stained with an anti-NUANCE mAb followed by an Alexa-488-conjugated secondary antibody (A,D,G,J) and TRITC-phalloidin (B,E,H,K) to visualize actin filaments. (C-L) overlays. Bars, 20 µM.

View larger version (35K): [in a new window]   Fig. 8. Binding of 6xHis-ABD to actin in vitro. (A) Cosedimentation of 10 µM 6xHis-ABD with 2.5 µM F-actin by ultracentrifugation. (B) Bundling of 10 µM actin in the presence of 6xHis-ABD analyzed by low-speed centrifugation. S, supernatant; P, pellet. Positions of ABD and actin are indicated on the right. (C) The effect on polymerization kinetics of 8 µM actin in the absence or presence of various amounts of 6xHis-ABD observed by the change in pyrenyl-actin fluorescence.

View larger version (93K): [in a new window]   Fig. 10. The C-terminal domain of NUANCE targets the NE. (A-C) Confocal optical section through the apical part of the COS7 nucleus showing that the overexpressed GFP-Cterm1 protein was arranged in patches displacing nuclear pores (arrows), detected by anti-Nup358 antibody (B). (D-F) The GFP-Cterm2 remained associated with the NE in prophase (D, arrow) when the nuclear pores begin to disassemble as indicated by diffuse anti-Nup358 staining (E). DNA was visualized with DAPI (F,I,L). (J-L) Two examples of the cellular localization of the GFP-NUA460-6643 overexpressed in COS7 cells stained with TRITC-phalloidin (H,K). The GFP-NUA460-6643 possesses the N-terminal ABD and the C-terminal Klarsicht-like domain but lacks most of the central rod domain. The GFP-NUA460-6643 is arranged in fibers (G-I, small arrowheads) or perinuclear aggregates (J-L, arrows) partially colocalizing with actin. Dynamic cortical cytoskeleton (G-I, arrow) and some of the stress fibers (G-I, large arrowhead) do not recruit GFP-NUA460-6643. (I,L) show overlays of the GFP, TRITC and DAPI channels. Bars (A-C) 5 µM; (D-F) 10 µM; (J-L) 20 µM.

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