Using interferometric photoactivated localisation microscopy (iPALM) and small engineered binders, researchers located three Z‑disc proteins in pig cardiac myofibrils with sub‑10 nm precision and analysed their spatial patterns with PERPL to reveal distinct, regular organisations.
iPALM with Adhiron labelling produced <10 nm 3D localisations of ZASP, α‑actinin‑2 and titin Z1Z2 in cardiac Z‑discs.
Raw localisation data and the analysis software are publicly available for reuse and further modelling.
Z‑discs are the narrow (~100 nm) boundaries that link sarcomeres in striated muscle. They contain dozens of structural and signalling proteins that influence contractile mechanics and cardiac signalling. Conventional light microscopy cannot resolve details within the Z‑disc due to its sub‑diffraction width. This study applies super‑resolution 3D imaging and pattern analysis to map where specific Z‑disc proteins sit relative to each other.
The authors labelled three Z‑disc components in isolated pig cardiac myofibrils: ZASP (a multi‑functional Z‑disc protein), α‑actinin‑2 (the actin cross‑linker that anchors filaments), and the Z1Z2 epitope of titin (titin's N‑terminal region that helps regulate sarcomere length). Labels were Adhirons — small binding proteins conjugated to fluorescent probes — chosen for their compact size to reduce localisation offsets.
The pattern analysis identified repeating organisational motifs for each protein across the Z‑disc: ZASP and α‑actinin‑2 exhibited similar repeating arrangements, indicating these two proteins occupy comparable positions within the Z‑disc lattice. By contrast, localisations of the titin Z1Z2 epitope showed a distinct but still regular organisation, implying titin's N‑terminus is positioned differently relative to the other two proteins.
These spatial relationships clarify how specific structural and regulatory proteins are positioned in the Z‑disc at near‑molecular resolution. Knowing that ZASP and α‑actinin‑2 share a repeating geometry while titin's Z1Z2 is arranged differently refines models of Z‑disc architecture and can inform hypotheses about how mechanical load or signalling inputs are routed through the Z‑disc.
The paper provides the iPALM localisation data via a DOI and makes the PERPL‑based analysis code available on GitHub (release v0.0.3). Procedural modelling capabilities are available in later PERPL releases. These resources allow independent reanalysis, integration with other datasets, or use as input for mechanical or structural modelling.
Takeaway for researchers and modelers If you need detailed spatial constraints for Z‑disc proteins, this dataset supplies sub‑10 nm 3D positions for three core components in cardiac myofibrils. Use the provided PERPL workflows to test alternative structural hypotheses or to build constrained computational models of Z‑disc mechanics and signalling.
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