Using iPALM to determine protein organisation in cardiac muscle Z-discs

Fig 1. iPALM imaging of Z-disc proteins. A: Example iPALM data for ɑ-actinin-2, Z1Z2 and ZASP in multiple Z-discs along a myofibril. The z axis is into the plane. B,C: Localisations for individual Z discs from A (blue arrows) in xy (B) and yz (C) planes. Yellow arrows: localisations removed from further analysis by Z-disc segmentation and denoising. Plots show maximum projection in the orthogonal plane to the view.

Fig 2. Experimental and best model Relative Position Distributions (RPDs). Total model RPD contains terms for the background from randomly distributed localisations, repeated localisation of the same dye molecule and the characteristic distances (d1, d2, d3) between localisations (Tables 2,3, S3-S5). Ratios between modelled distances are shown (e.g., 2d1, = 2 x d1). 95% confidence intervals shown on total model RPD, only noticeable in A (*).

Fig 3. Model for organisation of ɑ-actinin-2, ZASP and titin Z1Z2 domains in the Z-disc (cardiac myofibrils). A: Arrangement of actin, ɑ-actinin-2, ZASP and titin Z1Z2 in the axial view. The Z-disc in cardiac muscle contains 4-6 ɑ-actinin-2 repeats [5], and here 6 repeats are shown. The ɑ-actinin-2 molecules can also be organised into doublets of the homodimer, spaced 6 nm apart ([13] not illustrated here for simplicity). B: Arrangement of actin, ɑ‑actinin‑2, ZASP and titin Z1Z2 in the transverse view (basketweave lattice). The lattice constants for titin Z1Z2 and parallel actin filaments are stable across the Z-disc, while the sets of anti-parallel actin filaments may move relative to one another. The rotation of the square lattice between different locations on the Z-disc is also shown [5].