Using iPALM to determine protein organisation in cardiac muscle Z-discs
4 affiliations
- 1School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom
- 2Engineering and Physical Sciences Faculty Services, Faculty of Engineering and Physical Sciences, University of Leeds, Leeds, United Kingdom
- 3Astbury Centre for Structural and Molecular Biology University of Leeds, Leeds, United Kingdom
- 4Advanced Imaging Center, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States of America
Abstract
Sarcomeres, the basic repeating unit of striated muscle, are joined together by crosslinked actin filaments found at the boundaries of muscle sarcomeres, termed Z-discs. Z-discs play a key role in cardiac signalling and disease, however, the arrangement and function of many of the proteins present in the Z-disc remain to be understood. Here, we determined the organisation of 3 key proteins, ZASP, ɑ-actinin-2 and the Z1Z2 epitope of titin, located within the Z-disc. We fluorescently labelled these proteins in cardiac myofibrils using Adhirons specific to each protein and used interferometric photoactivated localization microscopy (iPALM) to obtain the 3D position of these proteins to a high precision (<10nm in x,y,z). We then used PERPL (Pattern Extraction from Relative Positions of Localisations: available at https://github.com/oubino/z_disk/releases/tag/v0.0.3) to analyse patterns in the relative positions of the proteins and reveal their underlying organisation. This analysis revealed that ZASP and ɑ-actinin-2 have a similar repeating organisation, but that the organisation of Z1Z2 is different. Author summary: Z-discs connect myosin- and actin-containing filaments from adjacent sarcomeres in striated (cardiac and skeletal) muscle. They contain over 50 different interacting proteins that have structural and/or signalling roles. Understanding their organisation in Z-discs is important for understanding their role. However, it is challenging to image Z-discs as they are narrower (~100 nm wide) than the resolution limit of conventional light microscopes (~250 nm). To overcome this, we used super-resolution imaging (iPALM: interferomic photoactivation and localisation microscopy), which has a resolution better than 10 nm in all three dimensions (x,y and z) to image specific proteins the z-discs of myofibrils from pig cardiac myocytes. We used small (< 4 nm in size) fluorescently-labelled binding proteins (Adhirons) to label ɑ‑actinin-2, a protein that cross-links actin-containing filaments from adjacent sarcomeres; the N-terminal region (Z1Z2) of titin, a large protein that regulates the length of myosin‑containing filaments; or ZASP (Z-band Alternative Spliced PDZ motif), which might help to regulate Z-disc width. We performed a tailored downstream analysis of the iPALM single molecule localisation data to uncover the detailed organisation of each protein. This revealed that ZASP and ɑ-actinin-2 have a similar repeating organisation, while titin Z1Z2 has a different, yet regular organisation throughout the Z-disc.
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BibTeX
@article{umney2026using,
title = {{Using iPALM to determine protein organisation in cardiac muscle Z-discs}},
author = {Oliver Umney and Alistair P. Curd and Heather L. Martin and Tarek Lewis and Anna Ah-San Tang and Thembaninkosi Gaule and Iain W. Manfield and Harikrushnan Balasubramanian and Satya Khuon and Jesse Aaron and Michelle Peckham},
journal = {PLOS Computational Biology},
year = {2026},
month = sep,
doi = {10.1371/journal.pcbi.1014309},
url = {https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1014309},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Using iPALM to determine protein organisation in cardiac muscle Z-discs
AU - Oliver Umney
AU - Alistair P. Curd
AU - Heather L. Martin
AU - Tarek Lewis
AU - Anna Ah-San Tang
AU - Thembaninkosi Gaule
AU - Iain W. Manfield
AU - Harikrushnan Balasubramanian
AU - Satya Khuon
AU - Jesse Aaron
AU - Michelle Peckham
PY - 2026
DA - 2026/09/28
JO - PLOS Computational Biology
DO - 10.1371/journal.pcbi.1014309
UR - https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1014309
AB - Sarcomeres, the basic repeating unit of striated muscle, are joined together by crosslinked actin filaments found at the boundaries of muscle sarcomeres, termed Z-discs. Z-discs play a key role in cardiac signalling and disease, however, the arrangement and function of many of the proteins present in the Z-disc remain to be understood. Here, we determined the organisation of 3 key proteins, ZASP, ɑ-actinin-2 and the Z1Z2 epitope of titin, located within the Z-disc. We fluorescently labelled these proteins in cardiac myofibrils using Adhirons specific to each protein and used interferometric photoactivated localization microscopy (iPALM) to obtain the 3D position of these proteins to a high precision (<10nm in x,y,z). We then used PERPL (Pattern Extraction from Relative Positions of Localisations: available at https://github.com/oubino/z_disk/releases/tag/v0.0.3) to analyse patterns in the relative positions of the proteins and reveal their underlying organisation. This analysis revealed that ZASP and ɑ-actinin-2 have a similar repeating organisation, but that the organisation of Z1Z2 is different. Author summary: Z-discs connect myosin- and actin-containing filaments from adjacent sarcomeres in striated (cardiac and skeletal) muscle. They contain over 50 different interacting proteins that have structural and/or signalling roles. Understanding their organisation in Z-discs is important for understanding their role. However, it is challenging to image Z-discs as they are narrower (~100 nm wide) than the resolution limit of conventional light microscopes (~250 nm). To overcome this, we used super-resolution imaging (iPALM: interferomic photoactivation and localisation microscopy), which has a resolution better than 10 nm in all three dimensions (x,y and z) to image specific proteins the z-discs of myofibrils from pig cardiac myocytes. We used small (< 4 nm in size) fluorescently-labelled binding proteins (Adhirons) to label ɑ‑actinin-2, a protein that cross-links actin-containing filaments from adjacent sarcomeres; the N-terminal region (Z1Z2) of titin, a large protein that regulates the length of myosin‑containing filaments; or ZASP (Z-band Alternative Spliced PDZ motif), which might help to regulate Z-disc width. We performed a tailored downstream analysis of the iPALM single molecule localisation data to uncover the detailed organisation of each protein. This revealed that ZASP and ɑ-actinin-2 have a similar repeating organisation, while titin Z1Z2 has a different, yet regular organisation throughout the Z-disc.
SN - 1553-7358
ER -