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First published online 16 November 2004
doi: 10.1242/jcs.01529

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LAP2 and BAF transiently localize to telomeres and specific regions on chromatin during nuclear assembly

¹ Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, 1030 Vienna, Austria
² Gene Expression and Cell Biology/Biophysics Programmes, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
³ CREST of JST Kansai Advanced Research Center, Kobe 651-2492, Japan
⁴ Department of Chemistry, Faculty of Sciences, Niigata University, Niigata 950-2181, Japan

View larger version (109K): [in a new window]   Fig. 1. LAP2 dynamics during nuclear assembly. (A-D) HeLa cells stably expressing YFP-LAP2 were transiently transfected with constructs encoding CFP-histone 2B and analyzed by live cell imaging. Confocal images of cells in interphase and metaphase (A) or at various stages during anaphase to G1 phase progression (B) or anaphase (C) are shown. Arrowheads indicate LAP2 at the tips of chromatin extensions; arrows indicate outer core regions. See also Movie 1 in supplementary material. (D) Isosurface reconstruction of z-stacks of images. Non-chromosome-associated clusters were excluded from the isosurface reconstruction. Numbers in images indicate elapsed time from anaphase onset (B) or from first image (D). Bars, 10 µm.

View larger version (79K): [in a new window]   Fig. 2. LAP2 associates with telomeres in stable structures. (A) HeLa cells were fixed and processed for immunofluorescence using antibodies to LAP2 (green) and TRF2 (red). Arrows indicate areas shown at higher magnifications in inserts. (B) Mitotic HeLa cells were incubated for 10 minutes (a-c) or 20 minutes (d,e) in hypotonic buffer, followed by preparation of chromosome spreads. Samples were processed for immunofluorescence microscopy using monoclonal (a) or polyclonal (b-d) antibodies to LAP2 or monoclonal antibody to LAP2ß (e) and the Hoechst DNA stain. Confocal images are shown. (C) FRAP analyses. The boxed areas were bleached to background levels (0 s) and recovery monitored after indicated time points. Fluorescence recovery after 8 seconds as a percentage of the prebleach intensity was corrected for intensity changes in unbleached zones and plotted. Bars, 10 µm.

View larger version (82K): [in a new window]   Fig. 3. LAP2 dynamics in relation to lamins. (A) HeLa cells stably expressing YFP-LAP2 were transiently transfected with constructs encoding either CFP-lamin C or CFP-lamin B1 and analyzed by live cell imaging. Confocal images of cells in interphase and various stages during anaphase to G1 phase progression are shown. Time points show elapsed time relative to first image. Arrowheads denote LAP2 in the inner core region; arrows, lamin structures. (B) Immunofluorescence microscopy of fixed GFP-LAP2-expressing cells using antibodies to lamins A/C. Bars, 10 µm.

View larger version (75K): [in a new window]   Fig. 4. LAP2 dynamics in relation to membrane proteins. HeLa cells stably expressing YFP-LAP2 were transiently transfected with CFP-LAP2ß or CFP-LBR or CFP-emerin constructs and analyzed by live cell imaging. Confocal images were taken of cells in interphase and various stages during telophase to G1 phase progression. Arrowheads denote LAP2 structures in the inner core region. Bars, 10 µm. Movies 2 and 3 in supplementary material show dynamics of LAP2-LAP2ß and LAP2-LBR, respectively.

View larger version (85K): [in a new window]   Fig. 5. LAP2 and BAF colocalize at core regions. (a-e) HeLa cells at various stages of mitosis were analyzed by immunofluorescence microscopy using antibodies to LAP2 (a-d) and LAP2ß (e) (green), BAF (red) and Hoechst DNA stain (blue). Arrowheads show first BAF accumulation at sites of LAP2 association. Confocal images are shown with merged images in right hand panels. Bar, 10 µm.

View larger version (28K): [in a new window]   Fig. 6. LAP2 interacts with BAF. (A) In vitro translated BAF (input) was mixed with buffer (-), recombinant full length LAP2 (1-693) or LAP2 C-terminal fragment (410-693) and complexes were precipitated with immobilized monoclonal antibodies to LAP2. Panels show immunoblots using antibodies to BAF (upper) and to LAP2 (lower). (B) LAP2 was immunoprecipitated from metaphase cell lysates. Controls were performed in the absence of antibodies. Supernatant (S) and pellet (P) fractions were analyzed by immunoblotting using antiserum to BAF and to LAP2. IgG, immunoglobulin heavy chain.

View larger version (47K): [in a new window]   Fig. 7. Model depicting the major association sites (arrows) of lamina proteins on chromatin during assembly. Red and green boxes represent the two major protein groups preferentially associating at telomeres/core regions and peripheral chromatin regions, respectively. Numbers denote the temporal sequence of detectable chromatin association. For details, see text.

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