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1.  Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle 
Molecular Biology of the Cell  2014;25(6):852-865.
Calpain-mediated proteolysis of the thin filament pointed-end–capping protein tropomodulin results in actin subunit association onto pointed ends and increased thin filament lengths in two different murine models of Duchenne muscular dystrophy. This mechanism affects different skeletal muscles in a use- and disease severity–dependent manner.
Duchenne muscular dystrophy (DMD) induces sarcolemmal mechanical instability and rupture, hyperactivity of intracellular calpains, and proteolytic breakdown of muscle structural proteins. Here we identify the two sarcomeric tropomodulin (Tmod) isoforms, Tmod1 and Tmod4, as novel proteolytic targets of m-calpain, with Tmod1 exhibiting ∼10-fold greater sensitivity to calpain-mediated cleavage than Tmod4 in situ. In mdx mice, increased m-calpain levels in dystrophic soleus muscle are associated with loss of Tmod1 from the thin filament pointed ends, resulting in ∼11% increase in thin filament lengths. In mdx/mTR mice, a more severe model of DMD, Tmod1 disappears from the thin filament pointed ends in both tibialis anterior (TA) and soleus muscles, whereas Tmod4 additionally disappears from soleus muscle, resulting in thin filament length increases of ∼10 and ∼12% in TA and soleus muscles, respectively. In both mdx and mdx/mTR mice, both TA and soleus muscles exhibit normal localization of α-actinin, the nebulin M1M2M3 domain, Tmod3, and cytoplasmic γ-actin, indicating that m-calpain does not cause wholesale proteolysis of other sarcomeric and actin cytoskeletal proteins in dystrophic skeletal muscle. These results implicate Tmod proteolysis and resultant thin filament length misspecification as novel mechanisms that may contribute to DMD pathology, affecting muscles in a use- and disease severity–dependent manner.
doi:10.1091/mbc.E13-10-0608
PMCID: PMC3952854  PMID: 24430868
2.  Congenital myopathy-causing tropomyosin mutations induce thin filament dysfunction via distinct physiological mechanisms 
Human Molecular Genetics  2012;21(20):4473-4485.
In humans, congenital myopathy-linked tropomyosin mutations lead to skeletal muscle dysfunction, but the cellular and molecular mechanisms underlying such dysfunction remain obscure. Recent studies have suggested a unifying mechanism by which tropomyosin mutations partially inhibit thin filament activation and prevent proper formation and cycling of myosin cross-bridges, inducing force deficits at the fiber and whole-muscle levels. Here, we aimed to verify this mechanism using single membrane-permeabilized fibers from patients with three tropomyosin mutations (TPM2-null, TPM3-R167H and TPM2-E181K) and measuring a broad range of parameters. Interestingly, we identified two divergent, mutation-specific pathophysiological mechanisms. (i) The TPM2-null and TPM3-R167H mutations both decreased cooperative thin filament activation in combination with reductions in the myosin cross-bridge number and force production. The TPM3-R167H mutation also induced a concomitant reduction in thin filament length. (ii) In contrast, the TPM2-E181K mutation increased thin filament activation, cross-bridge binding and force generation. In the former mechanism, modulating thin filament activation by administering troponin activators (CK-1909178 and EMD 57033) to single membrane-permeabilized fibers carrying tropomyosin mutations rescued the thin filament activation defect associated with the pathophysiology. Therefore, administration of troponin activators may constitute a promising therapeutic approach in the future.
doi:10.1093/hmg/dds289
PMCID: PMC3459469  PMID: 22798622
3.  Thin filament architecture in skeletal muscle: a two-segment model 
PREFACE
Correct specification of myofilament lengths is essential for efficient skeletal muscle contraction. Thin filament lengths are best specified by a novel ‘two-segment’ model, wherein the thin filament consists of two concatenated segments, of either constant or variable length, rather than by the classical ‘nebulin ruler’ model, wherein the thin filament is a uniform structure whose length is dictated by nebulin. The two-segment model implicates position-specific ‘differential microregulation’ of actin dynamics as a general principle underlying actin filament lengths and stability.
doi:10.1038/nrm3510
PMCID: PMC3711620  PMID: 23299957
actin; tropomodulin; tropomyosin; nebulin; proximal segment; distal segment
4.  Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types 
Cytoskeleton (Hoboken, N.J.)  2012;69(6):337-370.
Tropomodulins are a family of four proteins (Tmods 1–4) that cap the pointed ends of actin filaments in actin cytoskeletal structures in a developmentally regulated and tissue-specific manner. Unique among capping proteins, Tmods also bind tropomyosins (TMs), which greatly enhance the actin filament pointed-end capping activity of Tmods. Tmods are defined by a tropomyosin (TM)-regulated/Pointed-End Actin Capping (TM-Cap) domain in their unstructured N-terminal portion, followed by a compact, folded Leucine-Rich Repeat/Pointed-End Actin Capping (LRR-Cap) domain. By inhibiting actin monomer association and dissociation from pointed ends, Tmods regulate regulate actin dynamics and turnover, stabilizing actin filament lengths and cytoskeletal architecture. In this review, we summarize the genes, structural features, molecular and biochemical properties, actin regulatory mechanisms, expression patterns, and cell and tissue functions of Tmods. By understanding Tmods’ functions in the context of their molecular structure, actin regulation, binding partners, and related variants (leiomodins 1–3), we can draw broad conclusions that can explain the diverse morphological and functional phenotypes that arise from Tmod perturbation experiments in vitro and in vivo. Tmod-based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology.
doi:10.1002/cm.21031
PMCID: PMC3444156  PMID: 22488942
5.  Tmod1 and CP49 Synergize to Control the Fiber Cell Geometry, Transparency, and Mechanical Stiffness of the Mouse Lens 
PLoS ONE  2012;7(11):e48734.
The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α2β2-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.
doi:10.1371/journal.pone.0048734
PMCID: PMC3492431  PMID: 23144950
6.  The sarcoplasmic reticulum 
Bioarchitecture  2011;1(4):175-179.
Skeletal muscle exhibits strikingly regular intracellular sorting of actin and tropomodulin (Tmod) isoforms, which are essential for efficient muscle contraction. A recent study from our laboratory demonstrates that the skeletal muscle sarcoplasmic reticulum (SR) is associated with cytoplasmic γ-actin (γcyto-actin) filaments, which are predominantly capped by Tmod3. When Tmod3 is experimentally induced to vacate its SR compartment, the cytoskeletal organization of SR-associated γcyto-actin is perturbed, leading to SR swelling, depressed SR Ca2+ release and myofibril misalignment. Based on these findings, Tmod3-capped γcyto-actin filaments mechanically stabilize SR structure and regulate SR function via a novel lateral linkage. Furthermore, by placing these findings in the context of studies in nonmuscle cells, we conclude that Tmodcapped actin filaments are emerging as critical regulators of membrane stability and physiology in a broad assortment of cell types.
doi:10.4161/bioa.1.4.17533
PMCID: PMC3210515  PMID: 22069510
cell membrane; cytoskeletal connections; muscle contraction; sarcomere; sarcoplasmic reticulum; thin filament
7.  Cytoplasmic γ-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers 
The Journal of Cell Biology  2011;194(1):105-120.
Tropomodulins, cytoplasmic γ-actin, and small ankyrin 1.5 mechanically stabilize the sarcoplasmic reticulum and maintain myofibril alignment in skeletal muscle fibers.
The sarcoplasmic reticulum (SR) serves as the Ca2+ reservoir for muscle contraction. Tropomodulins (Tmods) cap filamentous actin (F-actin) pointed ends, bind tropomyosins (Tms), and regulate F-actin organization. In this paper, we use a genetic targeting approach to examine the effect of Tmod1 deletion on the organization of cytoplasmic γ-actin (γcyto-actin) in the SR of skeletal muscle. In wild-type muscle fibers, γcyto-actin and Tmod3 defined an SR microdomain that was distinct from another Z line–flanking SR microdomain containing Tmod1 and Tmod4. The γcyto-actin/Tmod3 microdomain contained an M line complex composed of small ankyrin 1.5 (sAnk1.5), γcyto-actin, Tmod3, Tm4, and Tm5NM1. Tmod1 deletion caused Tmod3 to leave its SR compartment, leading to mislocalization and destabilization of the Tmod3–γcyto-actin–sAnk1.5 complex. This was accompanied by SR morphological defects, impaired Ca2+ release, and an age-dependent increase in sarcomere misalignment. Thus, Tmod3 regulates SR-associated γcyto-actin architecture, mechanically stabilizes the SR via a novel cytoskeletal linkage to sAnk1.5, and maintains the alignment of adjacent myofibrils.
doi:10.1083/jcb.201011128
PMCID: PMC3135406  PMID: 21727195
8.  Tropomodulin Capping of Actin Filaments in Striated Muscle Development and Physiology 
Efficient striated muscle contraction requires precise assembly and regulation of diverse actin filament systems, most notably the sarcomeric thin filaments of the contractile apparatus. By capping the pointed ends of actin filaments, tropomodulins (Tmods) regulate actin filament assembly, lengths, and stability. Here, we explore the current understanding of the expression patterns, localizations, and functions of Tmods in both cardiac and skeletal muscle. We first describe the mechanisms by which Tmods regulate myofibril assembly and thin filament lengths, as well as the roles of closely related Tmod family variants, the leiomodins (Lmods), in these processes. We also discuss emerging functions for Tmods in the sarcoplasmic reticulum. This paper provides abundant evidence that Tmods are key structural regulators of striated muscle cytoarchitecture and physiology.
doi:10.1155/2011/103069
PMCID: PMC3196151  PMID: 22013379
9.  Tropomodulin isoforms regulate thin filament pointed-end capping and skeletal muscle physiology 
The Journal of Cell Biology  2010;189(1):95-109.
In skeletal muscle fibers, tropomodulin 1 (Tmod1) can be compensated for, structurally but not functionally, by Tmod3 and -4.
During myofibril assembly, thin filament lengths are precisely specified to optimize skeletal muscle function. Tropomodulins (Tmods) are capping proteins that specify thin filament lengths by controlling actin dynamics at pointed ends. In this study, we use a genetic targeting approach to explore the effects of deleting Tmod1 from skeletal muscle. Myofibril assembly, skeletal muscle structure, and thin filament lengths are normal in the absence of Tmod1. Tmod4 localizes to thin filament pointed ends in Tmod1-null embryonic muscle, whereas both Tmod3 and -4 localize to pointed ends in Tmod1-null adult muscle. Substitution by Tmod3 and -4 occurs despite their weaker interactions with striated muscle tropomyosins. However, the absence of Tmod1 results in depressed isometric stress production during muscle contraction, systemic locomotor deficits, and a shift to a faster fiber type distribution. Thus, Tmod3 and -4 compensate for the absence of Tmod1 structurally but not functionally. We conclude that Tmod1 is a novel regulator of skeletal muscle physiology.
doi:10.1083/jcb.201001125
PMCID: PMC2854367  PMID: 20368620
10.  Syncoilin is required for generating maximum isometric stress in skeletal muscle but dispensable for muscle cytoarchitecture 
Syncoilin is a striated muscle-specific intermediate filament-like protein, which is part of the dystrophin-associated protein complex (DPC) at the sarcolemma and provides a link between the extracellular matrix and the cytoskeleton through its interaction with α-dystrobrevin and desmin. Its upregulation in various neuromuscular diseases suggests that syncoilin may play a role in human myopathies. To study the functional role of syncoilin in cardiac and skeletal muscle in vivo, we generated syncoilin-deficient (syncoilin−/−) mice. Our detailed analysis of these mice up to 2 yr of age revealed that syncoilin is entirely dispensable for cardiac and skeletal muscle development and maintenance of cellular structure but is required for efficient lateral force transmission during skeletal muscle contraction. Notably, syncoilin−/− skeletal muscle generates less maximal isometric stress than wild-type (WT) muscle but is as equally susceptible to eccentric contraction-induced injury as WT muscle. This suggests that syncoilin may play a supportive role for desmin in the efficient coupling of mechanical stress between the myofibril and fiber exterior. It is possible that the reduction in isometric stress production may predispose the syncoilin skeletal muscle to a dystrophic condition.
doi:10.1152/ajpcell.00049.2008
PMCID: PMC2749034  PMID: 18367591
intermediate filament; sarcomere; mutant mouse

Results 1-10 (10)