SummaryThis proposal focuses on the structure and function of nebulin an unusually large sarcomeric protein that isexpressed in skeletal muscle. The giant size of nebulin has made it challenging to elucidate its functions but itsimportance is supported by the many nebulin mutations that cause nemaline myopathy (NEM2) the mostcommon non-dystrophic congenital myopathy. The protein structure of nebulin consists of a large number ofsimple repeats that are actin-binding most of which are organized into super-repeats (SRs). Approaches totreat NEM2 are sorely lacking and gaining an in-depth understanding of the many roles of nebulin in musclestructure and function is essential. We will comprehensively study nebulin building on major advances that weand others have made in recent years. To help achieve our goals we utilize mouse models some of whichmimic severe and others milder NEM2 as powerful tools for our basic science and translational studies. Wewill investigate nebulin's functions from the single-molecule to the intact muscle levels using multidisciplinaryapproaches that involve transcriptomics proteomics super-resolution imaging low-angle X-ray diffraction andbiomechanics. Aim 1 focuses on thin filament length regulation. Our recent work supports that in slow musclenebulin collaborates with leiomodin-2 (Lmod2) with nebulin regulating the length of a proximal thin filamentsegment and Lmod2 regulating the length of a distal segment that is nebulin-free. Here we will critically testthis dual length regulation model and study whether it has translational potential by determining whetherupregulating Lmod2 is an effective treatment for severe nebulin-based nemaline myopathy. Aim 2 studies thefunctional significance of weak actin-binding of centrally-located nebulin SRs that bind actin more weakly thanthose near the ends of the molecule. This is likely functionally important considering that a mutation thatincreases the binding affinity of a central SR causes a skeletal muscle myopathy in patients world-wide. Wewill study mouse models in which centrally located weak-binding SRs have been converted into strong-bindingSRs using mechanical assays and X-ray diffraction on intact muscle. Aim 3 studies the C-terminus of nebulinits layout in the Z-disk and the functions of nebulin's differentially expressed Z-repeats. Many NEM2 patientshave truncating mutations that result in the loss of most of the C-terminus (located within the Z disk) yet fewstudies have investigated this region of the molecule. Nebulin's C-terminus contains Z-disk repeats that arealternatively spliced (expressed at high levels in muscles with wide Z-disks). We will establish the layout ofnebulin in the Z-disk the location of the Z-repeats identify protein binding partners and study the effects ofdeleting 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 andtalented trainees this proposal sets ambitious goals that are expected to accelerate the understanding of thebiology of nebulin its role in disease and to identify possible novel therapeutic avenues.