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First published online October 12, 2006
doi: 10.1242/10.1242/jcs.03198

Journal of Cell Science 119, 4322-4331 (2006)
Published by The Company of Biologists 2006
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Targeted homozygous deletion of M-band titin in cardiomyocytes prevents sarcomere formation

Hanny Musa1, Stephen Meek2, Mathias Gautel3, Dianna Peddie2, Andrew J. H. Smith2 and Michelle Peckham1,*

1 Institute for Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK
2 Gene Targeting Laboratory, Institute for Stem Cell Research, University of Edinburgh, Edinburgh, UK
3 Kings College London, Muscle Cell Biology, Cardiovascular and Randall Division for Cell and Molecular Biophysics, London, UK


Figure 1
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  		Fig. 1. Generation of ES cells heterozygous and homozygous for a Titin M-band deletion. (A) Generation of an ES cell clone heterozygous for a Titin M-band deletion. A linearised neo/tk Titin targeting vector was integrated by homologous recombination into one WT Titin allele to generate a heterozygous targeted clone, NEOR GANCS 1-F9. (WT – WT cells with two normal chromosomes.) (B) Generation of ES cell clones homozygous for a Titin M-band deletion utilising a puro/tk cassette. A linearised puro/tk targeting vector was integrated by homologous recombination into the remaining WT Titin allele of clone NEOR GANCS 1-F9 to generate homozygous targeted clones NEOR PUROR GANCS 2-C5, 2-B11, 1-H6 and 1-G6 (labelled as 2-C5 etc on the figure). (C) Generation of an ES cell clone homozygous for a Titin M-band deletion utilising Cre recombination. Clone NEOR GANCS 1-F9 was transiently transfected with the pCAGGS-Cre-IRESpuro plasmid to excise the neo/tk cassette by Cre-mediated recombination between the direct-repeat loxP sites flanking the cassette. A resultant clone, NEOS GANCR 2-A10, contained a single loxP site at the deletion position. In a second round of targeting a linearised neo/tk Titin targeting vector was integrated by homologous recombination into the remaining WT Titin allele to generate a homozygous targeted clone, NEOR GANCS 1-D9. Restriction enzyme sites indicated are: AvaI(AV), EcoRI (RI), EcoRV (RV), I-SceI (I), NotI (N) and XbaI (X). Confirmation that the allele had been targeted was performed by Southern blot analysis of EcoRI (see Fig. 2) and EcoRV (data not shown) digested genomic DNA, using probes flanking the 5' and 3' homology arms, respectively. The sizes of the expected restriction fragments are shown in Fig. 2. Exon sequences are shown as red boxes (but with the kinase-encoding domain in one exon highlighted in pale blue), intron sequences as a thick black line, plasmid vector sequences as a thin black line, selection marker sequences as green (PGKneopA), blue (MC1tk) and purple (pApuroPGK) boxes, loxP sites as yellow triangles, and probe sequences as hatched boxes.

 

Figure 2
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  		Fig. 2. Confirmation that the alleles had been targeted to generate heterozygous or homozygous clones as detailed in Fig. 1. (A) Table showing the sizes of the expected EcoRI restriction fragment sizes for each type of clone as detected by the 5' probe. (B) Southern blot analysis of EcoRI digested genomic DNAs from clones heterozygous and homozygous for the deletion using the 5' probe. The result for 1-D9 appears to show a single band, but there are in fact two restriction fragments running very close together (8.5 and 8.4 kb).

 

Figure 3
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  		Fig. 3. Immunostaining of WT and heterozygous targeted cells shows myofibrillogenesis is normal. WT and heterozygous targeted cells (clone 5-A8) were coimmunostained for {alpha}-actinin and skeletal myosin or {alpha}-actinin and M-band titin, and for actin using Alexa-546 phalloidin, or stained for titin (Z1-Z2 antibody) myomesin and myosin, and imaged using a deconvolution microscope. Arrows show the positions of the Z-disks in each of the images. In the top four panels, the Z-disks stain positively for {alpha}-actinin and actin (thus, purple in the merged image). Myosin staining is clearly defined by a doublet between the Z-disks stained for {alpha}-actinin. The M-band region of titin is clearly seen as a stripe at the middle of the sarcomere between the two Z-disks stained for {alpha}-actinin. In the bottom two panels, the Z-disks stain immunopositively for titin (Z1-Z2 antibody), and myomesin is found midway between the Z-disks at the M-band. Occasional weak staining with the Z1-Z2 antibody at the M-band, which we assume is an anomalous cross-reaction, is sometimes observed. Bars, 5 µm.

 

Figure 4
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  		Fig. 4. Immunostaining of WT and heterozygous targeted cells shows normal localisation of obscurin and telethonin, but some mislocalisation of MURF2. WT and heterozygous cells were coimmunostained for obscurin and {alpha}-actinin, Z-disk titin (T12 antibody) and telethonin, or for Z-disk titin (T12 antibody) and MURF2 and imaged using a confocal microscope. The arrows show the alternate localisation of obscurin at the M-band and {alpha}-actinin at the Z-disk in the top pair of panels for WT and heterozygous cells (clone 5-A8). Titin and telethonin are both localised to the Z-disk (arrows in the middle panels) for WT and heterozygous cells (clone 1-F9). There is also a diffuse cytoplasmic pool of telethonin. In the bottom pair of panels, the arrows show the alternate localisation of titin at the Z-disk and MURF2 at the M-band in WT cells, but this alternate localisation is less clear in the heterozygous cells (clone 5-A8). Arrows indicate the M-band localisation, arrowheads show spots and/or streaks in the heterozygous cells. Bars, 5 µm.

 

Figure 5
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  		Fig. 5. Myosin, myomesin, obscurin and titin lack ordered organization in homozygous targeted cells. Confocal images of triple-stained homozygous targeted cells. Cells were co-stained for (A) actin (using Alexa-546 phalloidin), the Z-disk protein {alpha}-actinin, and myosin; (B) actin (using Alexa-546 phalloidin) {alpha}-actinin, and the M-band region of titin; (C) the Z-disk epitope of titin, myomesin and DAPI; or (D) myosin, myomesin and obscurin. Arrows in A and B show stripes of {alpha}-actinin staining that might be immature Z-disks and which have a spacing of approximately 1 µm. Arrows in C show close association of titin spots with myomesin. Arrows in D show occasional colocalisation of myomesin and obscurin. Arrowheads show spots of the rare association of myomesin spots with myosin. Bars, 5 µm.

 

Figure 6
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  		Fig. 6. Mislocalisation of obscurin, telethonin and MURF2 in homozygous targeted cells. Cells were coimmunostained for a range of sarcomeric proteins as indicated. Obscurin is shown for cells co-stained with either {alpha}-actinin or myosin. (A) In the top two panels, arrows indicate puncta that stain positively for obscurin. (B) In the middle two panels, MURF2 is shown for cells co-stained with either the Z-disk epitope of titin (T12) or myosin. Arrows indicate puncta that stain positively for MURF2. (C) In the bottom set of panels, cells are co-stained for Z-disk titin and telethonin, and there is some colocalisation of these two proteins (arrows). Many titin dots do not localise with telethonin, however (arrowheads). In all cases, sarcomeric organisation is lacking, and only sporadic colocalisation with titin is observed even for obscurin and telethonin, both of which contain Z-disk-binding sites. Bars, 5 µm.

 

Figure 7
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  		Fig. 7. MURF2, Nbr1 and p62 fail to assemble with titin in homozygous M-band-deficient cells. WT and homozygous targeted cells stained for the titin Z-disk epitope T12 (red) and for Nbr1 p62, and MURF2 (green) under conditions optimal for any nuclear localisation for MURF2, are shown. Association with sarcomeric structures is completely lost for both proteins, which adopt a punctate and diffuse staining pattern. MURF2 and Nbr1 can sometimes be found weakly in the nuclei of some ES cells (arrows), which are stained by DAPI (blue). Bars, 5 µm.

 

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© The Company of Biologists Ltd 2006