Summary This proposal focuses on the structure and function of nebulin, an unusually large sarcomeric protein that is expressed in skeletal muscle. The giant size of nebulin has made it challenging to elucidate its functions but its importance is supported by the many nebulin mutations that cause nemaline myopathy (NEM2), the most common non-dystrophic congenital myopathy. The protein structure of nebulin consists of a large number of simple repeats that are actin-binding, most of which are organized into super-repeats (SRs). Approaches to treat NEM2 are sorely lacking and gaining an in-depth understanding of the many roles of nebulin in muscle structure and function is essential. We will comprehensively study nebulin, building on major advances that we and others have made in recent years. To help achieve our goals we utilize mouse models, some of which mimic severe and others milder NEM2, as powerful tools for our basic science and translational studies. We will investigate nebulin's functions from the single-molecule to the intact muscle levels, using multidisciplinary approaches that involve transcriptomics, proteomics, super-resolution imaging, low-angle X-ray diffraction, and biomechanics. Aim 1 focuses on thin filament length regulation. Our recent work supports that in slow muscle, nebulin collaborates with leiomodin-2 (Lmod2), with nebulin regulating the length of a proximal thin filament segment and Lmod2 regulating the length of a distal segment that is nebulin-free. Here we will critically test this dual length regulation model and study whether it has translational potential, by determining whether upregulating Lmod2 is an effective treatment for severe nebulin-based nemaline myopathy. Aim 2 studies the functional significance of weak actin-binding of centrally-located nebulin SRs that bind actin more weakly than those near the ends of the molecule. This is likely functionally important, considering that a mutation that increases the binding affinity of a central SR causes a skeletal muscle myopathy in patients world-wide. We will study mouse models in which centrally located weak-binding SRs have been converted into strong-binding SRs using mechanical assays and X-ray diffraction on intact muscle. Aim 3 studies the C-terminus of nebulin, its layout in the Z-disk, and the functions of nebulin's differentially expressed Z-repeats. Many NEM2 patients have truncating mutations that result in the loss of most of the C-terminus (located within the Z disk), yet few studies have investigated this region of the molecule. Nebulin's C-terminus contains Z-disk repeats that are alternatively spliced (expressed at high levels in muscles with wide Z-disks). We will establish the layout of nebulin in the Z-disk, the location of the Z-repeats, identify protein binding partners, and study the effects of deleting differentially expressed nebulin Z-repeats on the Z-disk structure and function. Capitalizing on our >15-year track record of innovative nebulin research, and utilizing our team of experienced scientists and talented trainees, this proposal sets ambitious goals that are expected to accelerate the understanding of the biology of nebulin, its role in disease, and to identify possible novel therapeutic avenues.

Narrative This proposal focuses on nebulin, a skeletal muscle protein that causes the debilitating nemaline myopathy disease, NEM2. Using innovative research directions and novel tools we will investigate the nebulin/Lmod2 dual regulation model of thin filament length regulation, the significance of weak actin-binding super-repeats in nebulin, and the role of the C-terminus of nebulin in the Z-disk structure and function. We will also investigate novel avenues for restoring force generation in NEM2.