Proper contraction of striated muscle requires the assembly of actin filaments with precise spacing, polarity and lengths, however the mechanisms by which the cell accomplishes this remain unclear. In one model, the giant protein nebulin is proposed to function as a "molecular ruler" specifying the final lengths of the actin filaments. This dissertation focuses on determining the mechanisms by which nebulin regulates thin filament assembly. We found that nebulin physically interacts with CapZ, a protein that caps the barbed end of the actin filament within the Z-disc. Reduction of nebulin levels in chick skeletal myocytes via siRNA results in a reduction of assembled CapZ, and a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin-filament barbed ends to the Z-disc via a direct interaction with CapZ. Unexpectedly, the CapZ binding site was mapped to a site on nebulin that was previously predicted to localize outside of the Z-disc. Thus, we also propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres. To determine the mechanism by which nebulin regulates thin filament length and directly test the molecular ruler hypothesis, a unique small nebulin molecule ("mini-nebulin") was constructed. The introduction of mini-nebulin into chick skeletal myocytes, with endogenous nebulin knocked down, does not result in corresponding shorter actin filaments; an observation that is inconsistent with a strict ruler function. Treatment of these cells, however, with the actin depolymerizing agent Latrunculin A produces filaments that match the length of the mini-nebulin molecule, indicating mini-nebulin stabilizes the actin filaments. Furthermore, knockdown of nebulin results in more dynamic populations of the thin filament components actin, tropomyosin and tropomodulin. Strikingly, introduction of mini-nebulin is able to restore the normal stability of the actin filaments. Taken together, these data indicate that nebulin is responsible for proper actin organization within the Z-disc and contributes to actin filament length regulation by stabilizing the filament, preventing actin depolymerization.

Nemaline myopathy is a subtype of congenital myopathy that is clinically characterized by muscle weakness and early hypotonia of variable severity. Pathologically, nemaline myopathy is characterized by the presence of nemaline rods that stain purple in modified Gӧmӧri trichrome dye in patient biopsies under a microscope. Affected individuals experience skeletal muscle weakness and feeding difficulties, but most individuals will also experience respiratory muscle weakness that is disproportional to the weakness in skeletal muscles. Currently, 6 different subtypes of nemaline myopathy have been identified, each caused by mutations in ACTA1, NEB, TPM2, TPM3, TNNT1, KBTBD13, CFL2, KLHL40, KLHL41, or LMOD3, which are genes that encode either thin filament proteins or Kelch-like proteins. Of these genes, mutations in NEB and ACTA1 account for the majority of nemaline myopathy cases. Due to the genetic heterogeneity of nemaline myopathy, it is imperative to discover therapeutic targets and treatments that can universally treat nemaline myopathy patients. Preliminary data from our lab has demonstrated that proteasome complexes are downregulated in nemaline myopathy patients. Further, proteasomal activators improved motor function in neb zebrafish models, demonstrating the potential for proteasome activators to be therapeutics for nemaline myopathy patients. To extend these studies, the effect of proteasome activators, betulinic acid and Rolipram, was evaluated on the motor function in neb zebrafish models. However, in our experimental trials with betulinic acid and Rolipram, no positive effect on motor function in neb zebrafish was observed. In order to confirm our findings for both betulinic acid and Rolipram, additional trials will need to be conducted. / 2019-10-31T00:00:00Z

BACKGROUND:Nemaline myopathy (NM) is a congenital muscle disease associated with weakness and the presence of nemaline bodies (rods) in muscle fibers. Mutations in seven genes have been associated with NM, but the most commonly mutated gene is nebulin (NEB), which is thought to account for roughly 50% of cases.RESULTS:We describe two siblings with severe NM, arthrogryposis and neonatal death caused by two novel NEB mutations: a point mutation in intron 13 and a frameshift mutation in exon 81. Levels of detectable nebulin protein were significantly lower than those in normal control muscle biopsies or those from patients with less severe NM due to deletion of NEB exon 55. Mechanical studies of skinned myofibers revealed marked impairment of force development, with an increase in tension cost.CONCLUSIONS:Our findings demonstrate that the mechanical phenotype of severe NM is the consequence of mutations that severely reduce nebulin protein levels and suggest that the level of nebulin expression may correlate with the severity of disease.

BACKGROUND: Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is characterized by generalized skeletal muscle weakness, often from birth. To date, no therapy exists that enhances the contractile strength of muscles of NM patients. Mutations in NEB, encoding the giant protein nebulin, are the most common cause of NM. The pathophysiology of muscle weakness in NM patients with NEB mutations (NEB-NM) includes a lower calcium-sensitivity of force generation. We propose that the lower calcium-sensitivity of force generation in NEB-NM offers a therapeutic target. Levosimendan is a calcium sensitizer that is approved for use in humans and has been developed to target cardiac muscle fibers. It exerts its effect through binding to slow skeletal/cardiac troponin C. As slow skeletal/cardiac troponin C is also the dominant troponin C isoform in slow-twitch skeletal muscle fibers, we hypothesized that levosimendan improves slow-twitch muscle fiber strength at submaximal levels of activation in patients with NEB-NM. METHODS: To test whether levosimendan affects force production, permeabilized slow-twitch muscle fibers isolated from biopsies of NEB-NM patients and controls were exposed to levosimendan and the force response was measured. RESULTS: No effect of levosimendan on muscle fiber force in NEB-NM and control skeletal muscle fibers was found, both at a submaximal calcium level using incremental levosimendan concentrations, and at incremental calcium concentrations in the presence of levosimendan. In contrast, levosimendan did significantly increase the calcium-sensitivity of force in human single cardiomyocytes. Protein analysis confirmed that the slow skeletal/cardiac troponin C isoform was present in the skeletal muscle fibers tested. CONCLUSIONS: These findings indicate that levosimendan does not improve the contractility in human skeletal muscle fibers, and do not provide rationale for using levosimendan as a therapeutic to restore muscle weakness in NEB-NM patients. We stress the importance of searching for compounds that improve the calcium-sensitivity of force generation of slow-twitch muscle fibers. Such compounds provide an appealing approach to restore muscle force in patients with NEB-NM, and also in patients with other neuromuscular disorders.

In order for cells to migrate, communicate, and facilitate attachment to the surrounding extraceullar matrix, they must form intricate protein complexes called focal adhesions. The number of identified focal adhesion components continues to grow and the field is an area of active study.Lasp-2 is a member of the nebulin family of actin-binding proteins that has been identified as a member of focal adhesion complexes. To gain further insights into the functional role of lasp-2, we identified two additional binding partners of lasp-2, the integral focal adhesion proteins, vinculin and paxillin. Interestingly, the interaction of lasp-2 with its binding partners vinculin and paxillin was significantly reduced in presence of lasp-1, another nebulin family member. The presence of lasp-2 appears to enhance the interaction of vinculin and paxillin with each other, however, as with the interaction of lasp-2 with vinculin or paxillin, this effect is greatly diminished in the presence of excess lasp-1 suggesting the interplay between lasp-2 and lasp-2 could be an adhesion regulatory mechanism. Lasp-2's potential role in metastasis was revealed as overexpression of lasp-2 in SW620 cells, a highly metastatic cancer cell line, increased cell migration, but impeded cell invasion.Lasp-2 transcript and protein is readily detected in neural tissues. Preliminary experiments involving the knockdown of lasp-2- in frog embryos revealed gross morphological abnormalities in the head region as well as the inability to move normally. Neural crest derived melanocytes also failed to migrate normally.Taken together, these data suggest that lasp-2 has an important role in coordinating and regulating the composition and dynamics of focal adhesions.