Understanding structure and function of the Z-disc in striated muscle

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.

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.

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.