Understanding structure and function of the Z-disc in striated muscle
Lead Research Organisation:
University of Leeds
Department Name: Astbury Centre
Abstract
Muscles are essential for movement, and the organisation of proteins within the muscle cells is important for muscles to generate this movement. These proteins are organised with very high precision, into building blocks called muscle sarcomeres. The sarcomeres are arranged end to end along the muscle fibre, and the interaction between the two main contractile proteins, actin and myosin organised into filaments within each sarcomere, causes each sarcomere to shorten by a small amount. These small movements are added together along the length of the fibre, to produce a large movement. It is essential that all the sarcomeres work in the same way, to ensure that the sarcomeres contract uniformly, and this is underpinned by the precision engineering of proteins into each sarcomere, ensuring that every sarcomere is the same. At the ends of each sarcomere are complex structures known as Z-discs. These important structures contain over 40 different proteins that both anchor the contractile proteins in the muscle sarcomere, and detect and respond to forces generated by the sarcomere when muscle contracts. However, these structures are very thin. So far no-one has been able to find out how these proteins are arranged within the Z-disc. Light microscopy cannot see inside the Z-discs with enough detail, and while electron microscopy shows the overall Z-disc organisation, it cannot pinpoint where individual proteins are. Without knowing how these proteins are organised, it is difficult to understand how they interact with each other, and how mutations in these proteins lead to muscle disease. In this new research, we plan to use a novel 'super-resolution' light microscopy microscopy, which is able to determine the positions of proteins much more accurately than normal light microscopy. This approach will allow us to pinpoint the positions of individual proteins and uncover their arrangement in the Z-disc. To help us do this, we will also develop novel types of probes to help us label proteins within the Z-disc more precisely. We will also use new ways to analyse the data to help us understand how the proteins are arranged in this structure. Together, we expect that our new techniques will allow us to see inside the Z-disc and understand its complexity for the first time.
Technical Summary
Cardiac and Skeletal muscles are essential for pumping blood around the body and for movement. Both types of muscle are 'striated', because the proteins within the muscle cells are organised into small (~2 micron) long units, called muscle sarcomeres, which are arranged in series within long structures called myofibrils, that can extend from one end of the muscle cell to the other. A single muscle cell contains many myofibrils. The organisation of proteins into muscle sarcomeres is highly precise and regulated, such that each sarcomere is identical to every other sarcomere, generating similar amounts of force and movement. At the ends of each sarcomere are complex structures known as Z-discs, that anchor the muscle contractile proteins, and also contain many signalling proteins that detect and respond to forces generated by the sarcomere when muscle contracts. However, the Z-disc is a narrow structure (30 -140nm wide), conventional light microscopy cannot resolve the detailed organisation of proteins within it, and Cryo-EM and immunoEM studies cannot identify precisely where proteins are. Thus, we do not know how proteins are organised within the Z-disc, or with respect to each other. This new research will use novel super-resolution single molecule localisation microscopy techniques (3D PALM (photo activated light microscopy) and 3D dSTORM (direct stochastic optical reconstruction microscopy)) to determine the the arrangement of proteins in the Z-disc in cardiac, skeletal and developing muscle with high precision (~5nm in X & Y, ~10nm in Z). Exploiting the development of novel small probes (Affimers; Z~12kDa) that reduce linkage error, and implementing new software and hardware improvements, together with the new pattern analysis techniques we've developed, will help us achieve this goal enabling us to to 'see inside' the Z-disc and understand its complexity for the first time.
Planned Impact
The main beneficiaries of this research will be academics, as well as the wider UK economy and society.
This research is basic in nature, addressing a key biological question - how are proteins in the Z-disc arranged? It will develop new tools to develop this question, including Affimers and novel image pattern analysis techniques. Knowledge of the Z-disc protein arrangement is important to other academics. It also benefits society as the new information it will impact on our understanding of how disease causing mutations in these proteins affect Z-disc structure and function, leading to heart and skeletal muscle disease. It will benefit the economy by providing cutting edge training in new techniques to the staff working on the proposal, and the wider scientific community will benefit from the technical developments proposed. The derivation of novel Affimers are also of strong interest to the wider community, and have the potential to be commercialised. We will use a number of avenues to disseminate the outcomes of our research (small workshops to conferences, and publications in a wide variety of journals and magazines, websites, twitter), to achieve the maximum impact.
This research is basic in nature, addressing a key biological question - how are proteins in the Z-disc arranged? It will develop new tools to develop this question, including Affimers and novel image pattern analysis techniques. Knowledge of the Z-disc protein arrangement is important to other academics. It also benefits society as the new information it will impact on our understanding of how disease causing mutations in these proteins affect Z-disc structure and function, leading to heart and skeletal muscle disease. It will benefit the economy by providing cutting edge training in new techniques to the staff working on the proposal, and the wider scientific community will benefit from the technical developments proposed. The derivation of novel Affimers are also of strong interest to the wider community, and have the potential to be commercialised. We will use a number of avenues to disseminate the outcomes of our research (small workshops to conferences, and publications in a wide variety of journals and magazines, websites, twitter), to achieve the maximum impact.
Organisations
Publications
Prakash K
(2023)
Assessment of 3D MINFLUX data for quantitative structural biology in cells.
in Nature methods
Parker F
(2023)
Affimers targeting proteins in the cardiomyocyte Z-disc: Novel tools that improve imaging of heart tissue.
in Frontiers in cardiovascular medicine
Lin CC
(2022)
Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.
in Molecular cell
Gravett M
(2021)
Moving in the mesoscale: Understanding the mechanics of cytoskeletal molecular motors by combining mesoscale simulations with imaging
in WIREs Computational Molecular Science
Fineberg A
(2024)
Myosin-5 varies its step length to carry cargo straight along the irregular F-actin track.
in Proceedings of the National Academy of Sciences of the United States of America
Curd AP
(2021)
Nanoscale Pattern Extraction from Relative Positions of Sparse 3D Localizations.
in Nano letters
Cordell P
(2022)
Affimers and nanobodies as molecular probes and their applications in imaging.
in Journal of cell science
Chuntharpursat-Bon E
(2023)
PIEZO1 and PECAM1 interact at cell-cell junctions and partner in endothelial force sensing.
in Communications biology
Abad B
(2023)
The 2022 applied physics by pioneering women: a roadmap
in Journal of Physics D: Applied Physics
Description | We have made Affimers (small non-antibody binding proteins) to a range of proteins found in muscle z-discs. We have used one of these Affimers, isolated against alpha-actinin-2 a major Z-disc protein, used this to image this protein using high-resolution light microscopy and then developed new software to uncover how this protein is organised in the Z-disc. We published this work in Nanoletters, and Alistair Curd has used this to analyse further super-resolution data. This technique will be of broad use to others and we are already using it to uncover how other proteins in the muscle Z-discs are organised. We have also show that the Affimers outperform antibodies in imaging proteins in the Z-disc in heart tissue sections, and this is also now published in Frontiers in Cardiovascular Research. The Affimers are thus highly useful tools for understanding heart and skeletal muscle in health and disease. |
Exploitation Route | The software we developed is available for others to use on GitHub, and will be useful to others doing super-resolution imaging. The Affimers will be useful to others to substitute for antibodies. |
Sectors | Digital/Communication/Information Technologies (including Software) |
URL | https://pubs.acs.org/doi/10.1021/acs.nanolett.0c03332 |
Title | Affimers |
Description | Affimers are small non-antibody binding proteins that can be used like antibodies, but are much smaller, better for super-resolution and better for labelling dense cytoskeletal structures. We have isolated Affimers to a wide range of proteins, including many Z-disc proteins, actin, tubulin and others. |
Type Of Material | Technology assay or reagent |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | Replacement of antibodies in research, using Affimers, which are isolated by phage display, and do not require the use of animals. |
Title | PERPL-Python3: for analysis of super-resolution imaging data |
Description | Python code for analysis of image data to find patterns in datasets |
Type Of Technology | Software |
Year Produced | 2020 |
Open Source License? | Yes |
Impact | Paper published in nanoletters, collaborations with others |
URL | https://pubs.acs.org/doi/10.1021/acs.nanolett.0c03332 |
Description | Be Curious |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | There were over 40 stalls allowing everyone to have a go with activities suitable for all ages. We presented a stall to engage people with using microscopes to see 'things' in more detail |
Year(s) Of Engagement Activity | 2019 |
URL | https://biologicalsciences.leeds.ac.uk/biological-sciences/events/event/98/be-curious-2019 |