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First published online 5 December 2006
doi: 10.1242/jcs.03325

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Specific and conserved sequences in D. melanogaster and C. elegans lamins and histone H2A mediate the attachment of lamins to chromosomes

Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904 Israel

View larger version (44K): [in this window] [in a new window]   Fig. 1. Expression of wild-type and mutant lamin Dm0 constructs. (A) Schematic view of the lamin monomer, which is composed of a short N-terminal head, -helical rod and carboxyl terminal tail domains. The major region required for binding of lamin Dm0 to chromosomes is enlarged (below) and compared with those of other species. ClustalW (http://www.ebi.ac.uk/clustalw) was used to align the sequences. Conserved residues in that region are highlighted in black (homology) or in gray (similarity). (B) The QuickChange site-directed mutagenesis method was used to produce different mutant constructs of the lamin Dm0 tail domain. These include deletions () and substitutions with alanine or aspartate residues. (C) Wild-type and mutant proteins were expressed, purified to near homogeneity and subjected to 12% SDS-PAGE analysis. Proteins were stained with Coomassie Brilliant Blue.

View larger version (58K): [in this window] [in a new window]   Fig. 2. The binding of lamin Dm0 to chromosomes requires both the KRKR (NLS) and the RAT sequences. (A,B) The lamin Dm0 tail domains (residues 425-622), wild-type and containing the indicated mutations, were used to bind mitotic CHO chromosomes in the presence of 10% BSA. The control did not include the lamin Dm0 protein. Chromosomes were co-stained with DAPI (left) and with 611A3A6 monoclonal anti-lamin Dm0 antibody as primary antibody and Cy3-conjugated anti-mouse antibodies as secondary antibodies (right). Quantification of these experiments is shown in Table 1.

View larger version (44K): [in this window] [in a new window]   Fig. 3. The Ce-lamin tail domain binds to mitotic chromosomes and the NLS motif is important for this binding. Binding of full-length Ce-lamin, Ce-lamin tail domain and tail domain mutated in its NLS (mNLS) to mitotic CHO chromosomes was performed in the presence of 10% BSA. The control did not include the Ce-lamin protein. Chromosomes were co-stained with DAPI (left) and with affinity purified polyclonal Ce-lamin antibody as primary antibody and Cy3-conjugated anti-rabbit antibodies as secondary antibodies (right). Quantification of the fluorescent signal (far right) was performed using the Science Laboratory 99 Image Gauge software and is given as the average fluorescence signal of binding to mitotic chromosomes in arbitrary units/sq. pixel. Data was taken from at least three independent experiments. SD, standard deviation.

View larger version (42K): [in this window] [in a new window]   Fig. 4. The tail domains of lamin Dm0 and Ce-lamin bind directly to histone H2A in vitro and the NLS motif is important for this binding. Blot overlay analysis of binding interactions between histone H2A and the lamin Dm0 tail domain; wild-type and NLS and the Ce-lamin tail domain; wild-type and mNLS. Equal amounts of protein (5 µg) were resolved using SDS-PAGE and then transferred to a nitrocellulose membrane. Relative protein content was confirmed by Ponceau S staining of the membrane before incubation with the overlay solution (Ponceau, upper panel). Immunostaining was performed with anti-histone H2A antibody after incubation with overlay buffer containing 12 µg /ml histone H2A (ECL, lower panel). BSA was used as a negative control (not shown). Purified proteins and their corresponding overlay signals are indicated above the lanes.

View larger version (53K): [in this window] [in a new window]   Fig. 5. Blot overlay assay showing that lamin Dm0 binding to histone H2A requires both the N- and C-tail domains of histone H2A. (A) Schematic view of the different histone H2A-derived polypeptides used in the blot overlay and chromosome binding experiments. (B,D,E) Equal amounts of protein (6 µg) were resolved using SDS-PAGE and transferred to a nitrocellulose membrane. Relative protein content was confirmed by staining the membrane with Ponceau S before incubation with the overlay solution (Ponceau, top panels). Immunostaining was performed with 611A3A6 monoclonal lamin Dm0 antibody after incubation with overlay buffer containing 12 µg/ml lamin Dm0 tail domain protein (ECL, bottom panels). Purified proteins and their corresponding overlay signals are indicated above the lanes. (C) Increasing amounts of the synthetic H2A N-tail peptide (N-tail) were used in the blot overlay experiments. BSA was used as a negative control (shown only in C).

View larger version (16K): [in this window] [in a new window]   Fig. 6. Relative binding intensity of histone H2A-derived polypeptides to the lamin Dm0 tail domain. The relative average intensity was calculated from three to six independent blot overlay experiments and was compared to that of the full-length histone H2A. The blot overlays reaction products were visualized with an ECL western blotting detection system and a luminoimage analyzer (LAS-1000 plus). Densitometric quantification was performed using the Science Laboratory 99 Image Gauge software. Bars indicate the standard deviation.

View larger version (46K): [in this window] [in a new window]   Fig. 7. Chromosome-binding assay showing that lamin Dm0 binding to histone H2A requires both the N- and C-tail domains of histone H2A. The lamin Dm0 tail domain (residues 425-622) was used to bind mitotic CHO chromosomes in the presence of 10% BSA with or without 100-fold molar excess of competitor histone H2A-derived proteins. Chromosomes were co-stained with DAPI (left) and with 611A3A6 monoclonal anti lamin Dm0 antibody as primary antibody and Cy3-conjugated anti-mouse antibodies as secondary antibodies (right). The control did not include the lamin Dm0 protein. Quantification of the fluorescent signal was performed using the Science Laboratory 99 Image Gauge software and is given as the average fluorescence signal of binding to mitotic chromosomes in arbitrary units/sq. pixel. Data was taken from at least three independent experiments. SD, standard deviation.