3D Palm Shows Distinct Distributions of Z-Disc Proteins with the Z-Discs in Cardiomyocytes

Abstract

Z-discs are important structural and signalling structures that form the boundaries of each muscle sarcomere in striated muscle. within the Z-disc, electron microscopy indicates that actin filaments, arranged in an anti-parallel organization, are cross-linked by α-actinin dimers arranged at ∼20 nm intervals. At least 30 different proteins reside in the Z-disc, and their organisation is not well understood. Conventional light microscopy cannot resolve the localisation of proteins within the Z-disc, as it is too narrow ∼100 nm wide.We have developed the use of 3-D Photoactivated Localisation Microscopy (PALM) to image two specific proteins within the Z-disc; α-actinin2 and Lasp-2 (LIM and SH3 containing protein 2), which binds to α-actinin. α-actinin and LASP fused to mEos2 were expressed in cultured embryonic mouse or isolated adult rat cardiomyocytes. Fixed cells were imaged using PALM, in which a weak cylindrical lens in the light path between the specimen and the camera was used to obtain 3D information from a single 2D plane.The resulting images show individual molecules of mEos2-α-actinin2 and mEos2-LASP within the Z-disc, using light microscopy, for the first time. The localization precision was 20nm (X,Y) and 50nm in Z. The density of mEos2-α-actinin2 molecules was higher than that for mEos2-LASP, and the densities of both molecules was non-uniform throughout the Z-disc structures. A quantitative analysis of these molecules provides new insight into the organisation of these molecules within the Z-disc structure. These results demonstrate that PALM can be used to localise specific proteins within the narrow Z-disc and thus it has great potential for investigating the organisation of component proteins within this structure.