Cardiac muscles are made up of repeating units of muscle sarcomeres, arranged end to end, along the length of each cell, in myofibrils. Each sarcomere is about 2 m long and bounded by a Z-disc at each end. In the Z-disc, actin filaments are arranged in an anti-parallel organization and are cross-linked by -actinin such that there is about one -actinin every 19-20 nm moving from one side of the Z-disc to the other. In addition, the Z-disc contains at least 30 different proteins, which contribute to the overall architecture, stability and organization of the muscle sarcomere and to signalling. Thus, the Z-disc is a key organizing and signalling centre for muscle structure and function. The narrow width of the Z-disc, about 100 nm, means that conventional light microscopy does not have enough resolution to localise proteins within it. To overcome this limitation, we have developed the use of 3-D Photoactivated Localisation Microscopy (PALM 1,2) to image two specific proteins within the Z-disc;-actinin 2 and Lasp-2 (LIM and SH3 containing protein 2). -actinin 2 forms an anti-parallel dimer that cross-links adjacent actin filaments from neighbouring sarcomeres. Lasp-2 binds to -actinin 2 3. Recombinant DNA techniques were used to generate adenoviral expression constructs for -actinin and LASP fused to mEos2, and purified adenovirus was used to express these tagged proteins in either cultured embryonic mouse or isolated adult rat cardiomyocytes. Cells were then fixed using 4% Paraformaldehyde for 10 minutes and imaged. 100 nm gold fiducials, added prior to imaging, were used to generate a measured point spread function 2 for localization, and for correcting drift during image acquisition. A weak cylindrical lens was inserted into the light path between the specimen and the camera to obtain 3D information from a single 2D plane. The resulting images show individual molecules of mEos2--actinin within the Z-disc to a localization precision of 20nm (X,Y) and 50nm in Z, with an estimated width for the Z-disc of 100 nm. The molecules are not evenly distributed along the length of the Z-disc (Figure. 1) but show clusters of -actinin with a higher density separated by regions of lower density. Areas of lower density could represent regions between myofibrils. We also successfully obtained images of single molecules of mEos2-LASP within the Z-disc to a similar localization precision (20nm in XY, and 50nm in Z). The density of these molecules appears to be lower than that for mEos2--actinin, and also shows variation along the length of the Z-disc. These results demonstrate that PALM can be used to localise specific proteins within the Z-disc, and that two different Z-disc proteins have an apparently different distribution or density within this narrow structure. This approach therefore has great potential for investigating the organisation of component proteins within the Z-disc. Neutrophils have been isolated from the bone marrow of mice expressing a GFP-tagged myosin IIA. We are imaging myosin with a custom built structure illumination microscopy (SIM) microscope designed by Hari Shroff (NIBIB) and hope to follow the polymerization and depolymerization of single myosin filaments in this chemotactic cell.

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National Heart, Lung, and Blood Institute
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