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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01079

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A novel isoform of sarcolemmal membrane-associated protein (SLMAP) is a component of the microtubule organizing centre

Department of Cellular and Molecular Medicine, 451 Smyth Road, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada

View larger version (26K): [in a new window]   Fig. 2. SLMAP3M1 is characterized by a unique N-terminal extension. (A) Amino acid composition of the N-terminal extension identified in SLMAP3. M1 (red) and M2 (blue) represent the two initiating methionines used to generate SLMAPs (Wigle et al., 1997). The consensus fork head associated (FHA) domain is demarcated by italics in bold print. Peptide sequence used to generate anti-SLMAP(N) rabbit antiserum is underlined (RLSRGSEESPPCEI). (B) Immunoblot analysis of SLMAP expression in NIH 3T3 cells. NIH 3T3 cells were lysed in RIPA buffer; 15 µg of total protein was separated by 10% SDS-PAGE then electrotransferred to PVDF membranes. Immunoblotting with anti-SLMAP(C) antibody (lane 1) or anti-SLMAP(N) antisera (lane 2) detected a single band at 91 kDa. Prior incubation of the anti-SLMAP(N) with SLMAP peptide abolished the immunoreactivity (lane 3). Positions of molecular mass standards are indicated (BENCHMARK Prestained Protein ladder, Gibco BRL).

View larger version (11K): [in a new window]   Fig. 1. Genomic organization of the SLMAP gene. (A) The SLMAP gene is composed of 24 exons and introns spanning over 122 kb of genomic DNA. Exon-exon (exons I-XXIV) boundaries are depicted. Exons XI (red), XII (black), XIII (blue), XVII (orange) and XXIII (green) encode alternative exons. Transmembrane domains are encoded by exon XXIII and exon XXIV. Exon XX and exon XXI contain the leucine zipper motifs (*). ATG1 (M1, red arrow) is encoded in exon I, whereas ATG2 (M2; green arrow) is present in exon II. (B) Amino acid composition of alternative exons XI, XII and XIII. Consensus casein kinase 2 phosphorylation sites are underlined; protein kinase C phosphorylation sites are indicated in blue; N-glycosylation site is in italics; and the N-myristolyation site is shown in red.

View larger version (66K): [in a new window]   Fig. 3. Subcellular localization of endogenous SLMAP in NIH 3T3 cells. (A) Immunofluorescent labeling of SLMAP proteins using anti-SLMAP(C) antiserum revealed staining of reticular formations, the cytosol and perinuclear sites in paraformaldehyde fixed NIH 3T3 cells (a). Reduced cytosolic SLMAP staining and labeling of distinct foci at the perinuclear region was observed in detergent-extracted, paraformaldehyde-fixed cells (b). No immunoreactivity was observed with prior incubation of SLMAP(C) antisera with purified SLMAP protein (e) or in cells incubated with the pre-immune rabbit serum (f). Perinuclear foci staining was also evident in detergent extracted COS-7 cells stained with anti-SLMAP(N) (c). This staining pattern was not observed in COS-7 cells stained with the pre-immune serum (d). (B) SLMAP colocalizes with -tubulin at centrosomes throughout the cell cycle. Paraformaldehyde-fixed NIH 3T3 cells were co-stained with anti-SLMAP (a-e), anti--tubulin (f-j) and DAP1 (k-o). Representative cells in interphase (a,f,k), prophase (b,g,l), metaphase (c,h,m), anaphase (d,i,n) and telophase (e,j,o) are shown. Bars, 20 µm.

View larger version (60K): [in a new window]   Fig. 4. SLMAP is a component of centrosomes. (A) SLMAP localization at centrosomes is not affected by nocodazole or taxol treatment. NIH 3T3 cells were untreated (a,d,g,j) or treated with either taxol (5 µM, 4 hours; b,e,h,k) to stabilize microtubules or nocodazole (6 µg/ml, 4 hours; c,f,i,l) to depolymerize microtubules. Cells were prepared for immunofluorescence microscopy and co-stained with anti-SLMAP (a,b,c), anti--tubulin (d,e,f) and DAP1 (g,h,i). To show that the drugs affected cytoplasmic microtubules, cells were stained with anti--tubulin monoclonal antibodies (j,k,l). Bar, 25 µm. (B) Centrosomes were isolated from exponentially growing NIH 3T3 cells by fractionation on a sucrose gradient as described previously (Moudjou and Bornens, 1998). Equivalent amount (15 µg) of each protein fraction was analyzed by SDS-PAGE followed by western blotting with anti-SLMAP or anti--tubulin. Lane 1 (lysed extract), lane 2 (crude centrosomes) and lanes 3-6 represent fractions obtained from sucrose density gradients.

View larger version (32K): [in a new window]   Fig. 5. SLMAP3M1 sequences target GFP to centrosomes. (A) GFP-SLMAPM1 fusion protein used for transient transfection studies. Depicted are the GFP tag, the FHA domain, PEST motif, leucine zippers and the transmembrane (TM) domain. (B) Localization of GFP-SLMAP fusion proteins transiently expressed in NIH 3T3 cells was visualized by live microscopy. GFP fluorescence was detected at centrosomes in mitotic cells expressing GFP-SLMAP3M1 (a, two closely spaced dots). No green fluorescence was observed at centrosomes in control mitotic cells transfected with GFP-pcDNA alone (c). Co-incidence of the -tubulin signal (d) and GFP fluorescence (b) occurred in paraformaldehyde fixed mitotic cells expressing GFP-SLMAP3M1. Bar, 25 µm.

View larger version (42K): [in a new window]   Fig. 6. N-terminal sequences of SLMAP are required for targeting GFP to centrosomes. (A) Transient expression of GFP-tagged SLMAP mutants showed that green fluorescence was observed at centrosomes in live cells expressing GFP-SLMAP3M1LZ (a) and GFP-SLMAP3M1C (b) but not in mitotic cells expressing GFP-SLMAP3M2C (c). Corresponding phase contrast images are shown for SLMAP3M1LZ (d), GFP-SLAP3M1C (e) and GFP-SLMAP3M2C (f). Bar, 25 µm. (B) N-terminal SLMAP3 constructs encoding amino acids 1-163 (a), 1-183 (b) or 1-206 (c) were expressed in fibroblast cells. GFP fluorescence was detected throughout the cell, including the nucleus in (a) or within detergent-insoluble aggregates (b, c). Bar, 10 µm. (C) Transient expression of the full-length GFP-SLMAP fusion protein, GFP-SLMAP3M1 (a), was detected at perinuclear sites in interphase cells, indicative of membrane localization. Diffuse cytosolic localization was observed for both C-terminal mutants, GFP-SLMAP3M1C (b) and SLMAP3M2C (c). Bar, 50 µm. (D) At 36 hours post-transfection, cells expressing GFP-SLMAP3M1 were rounded (b), whereas those cells expressing either GFP-pcDNA3 (a), GFP-SLMAP3M1C (c) or GFP-SLMAP3M2C appeared normal. Bar, 10 µm.

View larger version (22K): [in a new window]   Fig. 7. SLMAP overexpression affects cell proliferation. (A) Overexpression of GFP-tagged SLMAPs inhibits BrdU uptake in NIH 3T3 cells. NIH 3T3 cells were transfected with either GFPpcDNA3 (green bar), GFP-SLMAP3M1 (red bar), GFP-SLMAP3M2 (yellow bar) or GFP-SLMAP3M2C (blue bar). Thirty-six hours post-transfection, cells were labeled with BrdU (100 mmol/l) for 2, 8, 12 or 24 hours. Ethanol fixed cells were co-stained with anti-mouse BrdU antibody and anti-rabbit GFP antibody. Transfected cells were identified by their green fluorescence and the number of cells with co-existing nuclear red fluorescence (BrdU positive cells) was determined. The mean results of at least three independent experiments for each labeling period are shown. For each transfectant and condition, a minimum of 200 cells were scored. Error bars represent standard deviations. (B) FACS profiles of GFP-pcDNA3 (i) and GFP-SLMAP3M2 (ii) transfected NIH 3T3 cells. At 48 hours post-transfection, cells were incubated with propidium iodide to stain DNA (Pestov et al., 1999).

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