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First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02728

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Nuclear Titin interacts with A- and B-type lamins in vitro and in vivo

Michael S. Zastrow^1,*, Denise B. Flaherty^2,, Guy M. Benian² and Katherine L. Wilson^1,

¹ Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 N. Wolfe St, Baltimore, MD 21205, USA
² Department of Pathology, Emory University, Whitehead Biomedical Research Building, Atlanta, GA 30332, USA

View larger version (65K): [in a new window]   Fig. 1. Two-hybrid bait and titin polypeptides identified in the two-hybrid screen. (A) Diagram of the pre-lamin A protein drawn to scale, showing regions encoded by exons 1-12. The exon 8-9 bait polypeptide is underlined (bait). Vertical lines indicate positions of missense mutations that cause EDMD (R527P, R453W), MAD (L530P) or FPLD (R482Q). (B) Schematic drawing of the human titin protein (NP_596869; 34,350 residues) showing the position of the lamin-binding fragment (prey). (C) Amino acid sequence of the lamin-binding region of human titin. The arrow indicates the first residue encoded by the smaller two-hybrid titin isolate. The larger isolate encodes a 61 kDa polypeptide termed titin 1-551. Four predicted Ig-fold domains are underlined. Non-underlined regions correspond to titin-specific regions M-is6 (residues 61-172) and M-is7 (residues 353-462).

View larger version (41K): [in a new window]   Fig. 2. Biochemical confirmation of lamin-titin binding using three independent assays. (A) Titin and lamins interact in Ni2+ pull-down assays. 35S-titin 1-551 was incubated with His-tagged wild-type pre-lamin A tail, and pelleted with Ni2+-NTA agarose beads; 20% of each supernatant and 50% of each pellet were resolved by SDS-PAGE (12% gels) and exposed to X-ray film (autoradiograph is shown). (B) Results of the microtiter-well binding assays, in which either the purified pre-lamin A tail or BSA were immobilized in microtiter wells and incubated with 1 nM 35S-titin 1-551 (see Materials and Methods). The data are representative of five independent experiments, each done in triplicate. (C) Binding to lamin-AffiGel beads. Purified recombinant pre-lamin A tail (wild type or mutants; `prog' indicates pre-lamin A with the 50-residue deletion that causes HGPS) or lamin B1 tail were coupled covalently to AffiGel beads, incubated with 35S-titin 1-551 and pelleted to recover bound probe. Pellets (50% of each sample) were resolved by SDS-PAGE and autoradiographed (35S-titin; upper panel) or immunoblotted with antibodies against the His-tag on each lamin tail (lower panel). (D) Quantification of data from panel C plotted as % of each probe that bound and normalized against titin binding to wild-type pre-lamin A tail. Bars indicate standard deviations from four independent experiments.

View larger version (26K): [in a new window]   Fig. 3. Mapping the lamin-binding regions in titin 1-551. (A) Schematic diagram of titin 1-551, showing locations of predicted Ig-fold domains (gray) and titin-specific domains M-is6 and M-is7. Bars numbered 1-6 represent the titin subfragments tested for binding to the pre-lamin A tail. (B) Autoradiograph showing 35S-labeled titin fragments bound to His-tagged pre-lamin A tail (on Ni2+ beads). Corresponding unbound (supernatant, S) and bound (pellet, P) fractions (20% each) were resolved by SDS-PAGE and exposed to film. (C) Data from panel B are shown as the ratio of bound:unbound probe for 35S-titin fragments 1-6. Bars indicate standard deviations from four independent experiments.

View larger version (58K): [in a new window]   Fig. 4. Localization of M-is6 epitopes in HeLa cells. (A) Immunoblot analysis of lysates from HeLa cells transfected with GFP-is6 and probed with immune rabbit serum 5460 against M-is6 (5460; Im), preimmune serum 5460 (PI), or immune 5460 pretreated with purified M-is6 protein (Inh). (B) Immunoblots of protein lysates of whole HeLa cells (cells) or purified HeLa nuclei (nuclei) separated on 2.5-7.5% SDS-PAGE to resolve high molecular-weight proteins. Strips were probed using serum 5460 as in (A). The 584-kDA molecular mass marker consisted of crosslinked phosphorylase B (Sigma, St Louis, MO). (C) Indirect immunofluorescence localization of endogenous titin M-is6 epitopes in HeLa cells. Cells were triple-stained with immune rabbit serum 5460 against M-is6 (red, Cy3), or serum 5460 preincubated with purified M-is6 antigen (Im + Ag), or pre-immune serum 5460 (PI) and counter-stained using antibodies against lamin A (green; FITC) and DAPI to stain DNA. (D) Indirect immunofluorescence staining of isolated HeLa cell nuclei using the same antibodies as in (A). Arrows indicate three of many puncta in which lamin A and titin potentially colocalized. Bars, 2 µm.

View larger version (74K): [in a new window]   Fig. 5. Phenotype of HeLa cells transiently overexpressing GFP-fused M-is7, PK or M-is6. HeLa cells were examined 24 hours after transfection by fixing and staining with antibodies specific against lamin B (red, Cy3), staining with DAPI for DNA (blue) and visualizing GFP fluorescence (green). Untransfected cells (Un) were included as controls. Cells expressing GFP-is7 had a high frequency of aberrantly-shaped nuclei and nuclear envelope herniations (arrowheads) that contained GFP-is7, but not chromatin or lamin B. (B) HeLa cells overexpressing GFP-is6, which does not bind lamins in vitro, have normal nuclear shape and lamin organization. Bars, 2 µm. (C) Percentage of cells with deformed nuclei; n=250 cells per group. (D) Lysates from HeLa cells overexpressing GFP-is7 were immunoprecipitated with antibodies against GFP, or no antibody as a control; lanes contain supernatant (Sup, 10% of sample) and magnetic-bead-associated pellet (bead, 20% of sample). Fractions were resolved by SDS-PAGE and probed with a mixture of antibodies NC-5 and NC-7 against lamins A/C and B1, respectively.

View larger version (89K): [in a new window]   Fig. 6. (A) Schematic diagram of C. elegans titin protein (Ce-titin) showing the PEVT domain and polypeptide antigens used to raise antibodies EU145, EU102 and EU143 (grey boxes). (B) Adult C. elegans stained by indirect immunofluorescence for endogenous Ce-titin using affinity-purified EU102, EU145 and EU143 antibodies. All three gave typical striated staining in the muscle myofilament lattice. (C) Indirect immunofluorescence staining for endogenous Ce-titin (R, red; Cy3) and Ce-lamin (G, green; FITC) in wild-type embryos stained with affinity-purified EU145 or EU143 antibodies. DNA was stained with DAPI. Ce-lamin was detected with rabbit serum 3932 (Gruenbaum et al., 2002). (D) Indirect immunofluorescence staining for endogenous Ce-titin using rabbit polyclonal EU102 (G, green; FITC) and Ce-lamin rat serum 3933 (R, red; Cy3). DNA was stained with DAPI in wild-type (WT) or lmn-1(RNAi) embryos. Bars, 5 µm.

View larger version (79K): [in a new window]   Fig. 7. Cell-cycle-dependent localization of endogenous Ce-titin in C. elegans embryos stained with antibody EY102 against Ce–titin (green, FITC) and DAPI to visualize DNA (blue). Stages of mitosis were inferred from chromosome morphology: interphase (INT), prophase (PRO), pro metaphase (PROM), metaphase (META), early and late anaphase (ANA), and telophase (TELO). Bar, 5 µm.