Component Of Filaments Research Articles

Immunoaffinity purification and characterization of specific antibodies against recombinant tau protein from intravenous immunoglobulin product

There is evidence that humoral immunity is involved in some processes associated with neurodegenerative diseases. Antibodies against many molecules occurring predominantly in brain tissue are detected also in cerebrospinal fluid as well as in plasma and serum. One of these molecules is tau protein, the integral component of paired helical filaments (PHF) which were identified as the major part of neurofibrillary pathology in Alzheimer's disease (AD). It is also known that specific antibodies against tau protein are contained in intravenous immunoglobulin (IVIG) products.

Nestin involvement in tissue injury and cancer--a potential tumor marker?

In eukaryotic cells, the cytoskeleton contains three major filamentous components: actin microfilaments, microtubules and intermediate filaments. Nestin represents one of the class VI intermediate filament proteins. Clinical and molecular analyses have revealed substantial information regarding the presence of Nestin in cells with progenitor or stem cell properties. During tissue injury Nestin is expressed in cells with progenitor cell-like properties. These cells may serve as a tissue reserve and, as such, may contribute to tissue repair. Based on currently available data, Nestin also appears to be implicated in two oncogenic processes. First, Nestin has been found to be expressed in cancer stem-like cells and poorly differentiated cancer cells and, as such, Nestin is thought to contribute to the aggressive behavior of these cells. Second, Nestin has been found to be involved in tumor angiogenesis through an interaction of cancer cells and blood vessel endothelial cells and, as such, Nestin is thought to facilitate tumor growth. We conclude that Nestin may serve as a promising tumor marker and as a potential therapeutic target amenable to tumor suppression and angiogenesis inhibition.

Soluble components of the flagellar export apparatus, FliI, FliJ, and FliH, do not deliver flagellin, the major filament protein, from the cytosol to the export gate

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook–filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear.We have used continuous ATPase activity measurements and quartz crystal microbalance (QCM) studies to characterize interactions between the soluble export components and flagellin or the FliC:FliS substrate–chaperone complex. As controls, interactions between soluble export component pairs were characterized providing Kd values. FliC or FliC:FliS did not influence the ATPase activity of FliI alone or in complex with FliH and/or FliJ suggesting lack of interaction in solution. Immobilized FliI, FliH, or FliJ did not interact with FliC or FliC:FliS detected by QCM. The lack of interaction in the fluid phase between FliC or FliC:FliS and the soluble export components, in particular with the ATPase FliI, suggests that cells use different mechanisms for the export of late minor substrates, and the major substrate, FliC. It seems that the abundantly produced flagellin does not require the assistance of the soluble export components to efficiently reach the export gate.

Understanding Nanocalcification: A Role Suggested for Crystal Ghosts

The present survey deals with the initial stage of the calcification process in bone and other hard tissues, with special reference to the organic-inorganic relationship and the transformation that the early inorganic particles undergo as the process moves towards completion. Electron microscope studies clearly exclude the possibility that these particles might be crystalline structures, as often believed, by showing that they are, instead, organic-inorganic hybrids, each comprising a filamentous organic component (the crystal ghost) made up of acidic proteins. The hypothesis is suggested that the crystal ghosts bind and stabilize amorphous calcium phosphate and that their subsequent degradation allows the calcium phosphate, once released, to acquire a hydroxyapatite, crystal-like organization. A conclusive view of the mechanism of biological calcification cannot yet be proposed; even so, however, the role of crystal ghosts as a template of the structures usually called “crystallites” is a concept that has gathered increasing support and can no longer be disregarded.

Abstract 121: Combinatorial Tailored Polymers Enhanced Maturation of Human iPSC-CMs

There is a tremendous interest in human cardiomyocytes generated from patient-derived induced pluripotent stem cells (iPSC-CMs) for the study and possible treatment of human heart diseases. Despite their vast potential, a significant impediment to a broader application of iPSC-CMs to study human myocyte biology is the structural and functional immaturity of iPSC-CMs. Growing evidence indicates that synthetic polymers utilized as extracellular substrates can exert significant effects on in vitro tissue generation, although the underlying mechanisms remain largely unknown. Based on the profound impact of the extracellular matrix of developing embryos on in vivo organogenesis, we hypothesize that engineered polymer substrates will likewise influence in vitro maturation of iPSC-CMs. A subset of combinatorial polymers was synthesized by polymerizing poly(ε-caprolacton) (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL), abbreviated as x%PEG-y%PCL-z%cPCL (x, y, and z: molar %). We investigated effects of the polymer composition on maturation of iPSC-CMs with respect to the beating behavior, mitochondrial function and molecular profiles after 30 days in culture on polymer scaffolds. Results showed the 4%PEG-96%PCL scaffold promoted the most active beating in iPSC-CMs at 30 days and further, that the mitochondrial function, as assessed by tetramethyl rhodamine methylester (TMRM) was significantly increased in the iPSC-CMs cultured on 4%PEG-96%PCL over other polymers. Molecular profiling analysis indicates 4%PEG-96%PCL scaffolds enhanced the expression of MYL2 (a commonly accepted marker of mature ventricular myocytes) as well as of components of the intermediate filaments linking the plasma membrane to the myofilament. In summary, although the polymers we used here exhibit similar physicochemical properties, they have divergent effects on iPSC-CM differentiation. Thus, specific chemical compositions of synthetic substrates can exert profound influence on in vitro maturation of hiPSC-CMs. Our work exploring the effects of synthetic biomaterials on human stem cell differentiation could pave the way for a successful translation of ongoing advances in tissue engineering to new treatments for human heart diseases.

Microboring in a Freshwater Fluvial Unionid Bivalve Substrate

Samples of the unionid bivalve Elliptio complanata were collected from the channel of the freshwater Saint John River, from Fredericton, New Brunswick, Canada. Scanning electron microscopy imaging of prepared shell samples revealed an assemblage of microborings. No borings are noted on the periostracum or prismatic shell layers. Boring structures are instead confined to the underlying nacreous aragonitic shell material, together with its associated organic conchiolin layers. Three main styles of boring are encountered, encompassing both predominantly surficial structures and penetrative tubular borings. Surficial structures are represented by a polygonal network on an exposed conchiolin shell layer. The penetrative borings take two forms, one being simple unbranched tubes, steeply aligned (perpendicular to the shell surface) and traversing the full thickness of the nacreous shell layer. The other penetrative boring style, again occurring within the nacreous layer, comprises a complex irregular network of randomly oriented rarely branching tubular borings. Borings generally display diameters of micron scale. Biofilm and extracellular polymeric substances, with bacterial, diatomaceous and filamentous components are also observed, often displaying a close association with both the borings and the conchiolin layers within the shell. The formation of the borings may be attributed to cyanobacteria, cyanophyte or fungal progenitors.

A Novel High Throughput High Content Analysis Assay for Intermediate Filament Perturbing Drugs

Keratins form the intermediate filament (IF) component of the epithelial cytoskeleton. Depolymerisation of these filaments causes the cell to collapse and become more plastic. We have previously shown that short chain fatty acids may trigger depolymerisation of keratins through altered protein acetylation. Currently, there is no single functional assay for screening of the cytoskeleton. The aim of this study is to develop a high-throughput assay to quantify IF depolymerisation and to apply as a screen for IF-perturbing nutrients and drugs. Three treatments were used in a proof-of-principle study: the anti-fungal drugs griseofulvin and cordycepin (the former a c-mitotic drug known to suppress microtubule growth, the latter inducing abnormal mitosis by suppressing microtubule dynamics) and sodium butyrate, a histone deacetylase inhibitor which disrupts IF formation in cancer cells via post-translational modification of keratins. Methods were optimised for cell fixation using methanol or formalin, permeabilising agents for Keratin 8 (krt8) antibody (triton-x100, digitonin and saponin) and blocking of non-specific binding prior to cell staining using BSA. High Content Analysis (HCA) was employed to quantify cell staining intensities by comparing co-occurrence of adjacent pixel intensities. Immunocytochemistry was used to identify Krt 8 intermediate filaments. Indicators of depolymerisation include Krt 8 fluorescence intensity, filamentousness or texture intensity, fibre spot count and fibre spot total area. All treatments resulted in significant decreases for texture intensity. Assessment of this assay using a Z prime calculation recorded gace a statistic of 0.95, indicating suitability for high-throughput applications. In conclusion, a HCA assay for intermediate filament integrity has been demonstrated, establishing proof of principle with griseofulvin, and cross-validating with two further treatments assayable using this method. Normal 0 false false false ES X-NONE X-NONE

Rotaxane-based molecular muscles.

CONSPECTUS: More than two decades of investigating the chemistry of bistable mechanically interlocked molecules (MIMs), such as rotaxanes and catenanes, has led to the advent of numerous molecular switches that express controlled translational or circumrotational movement on the nanoscale. Directed motion at this scale is an essential feature of many biomolecular assemblies known as molecular machines, which carry out essential life-sustaining functions of the cell. It follows that the use of bistable MIMs as artificial molecular machines (AMMs) has been long anticipated. This objective is rarely achieved, however, because of challenges associated with coupling the directed motions of mechanical switches with other systems on which they can perform work. A natural source of inspiration for designing AMMs is muscle tissue, since it is a material that relies on the hierarchical organization of molecular machines (myosin) and filaments (actin) to produce the force and motion that underpin locomotion, circulation, digestion, and many other essential life processes in humans and other animals. Muscle is characterized at both microscopic and macroscopic length scales by its ability to generate forces that vary the distance between two points at the expense of chemical energy. Artificial muscles that mimic this ability are highly sought for applications involving the transduction of mechanical energy. Rotaxane-based molecular switches are excellent candidates for artificial muscles because their architectures intrinsically possess movable filamentous molecular components. In this Account, we describe (i) the different types of rotaxane "molecular muscle" architectures that express contractile and extensile motion, (ii) the molecular recognition motifs and corresponding stimuli that have been used to actuate them, and (iii) the progress made on integrating and scaling up these motions for potential applications. We identify three types of rotaxane muscles, namely, "daisy chain", "press", and "cage" rotaxanes, and discuss their mechanical actuation driven by ions, pH, light, solvents, and redox stimuli. Different applications of these rotaxane-based molecular muscles are possible at various length scales. On a molecular level, they have been harnessed to create adjustable receptors and to control electronic communication between chemical species. On the mesoscale, they have been incorporated into artificial muscle materials that amplify their concerted motions and forces, making future applications at macroscopic length scales look feasible. We emphasize how rotaxanes constitute a remarkably versatile platform for directing force and motion, owing to the wide range of stimuli that can be used to actuate them and their diverse modes of mechanical switching as dictated by the stereochemistry of their mechanical bonds, that is, their mechanostereochemistry. We hope that this Account will serve as an exposition that sets the stage for new applications and materials that exploit the capabilities of rotaxanes to transduce mechanical energy and help in paving the path going forward to genuine AMMs.

CH–π interaction in VQIVYK sequence elucidated by NMR spectroscopy is essential for PHF formation of tau

One of the histopathological features of Alzheimer's disease (AD) is higher order neurofibrillary tangles formed by abnormally aggregated tau protein. Investigation of the mechanism of tau aggregation is important for the clarifying the cause of AD and the development of therapeutic drugs. The microtubule-binding domain, which consists of repeats of similar amino acids (R1-R4) is thought to form the core component of paired helical filament (PHF). The hexapeptide(306) VQIVYK(311) of R3 has been shown to take a key role of promoting tau aggregation and assumed that its CH-π interaction between the side chains of Ile308 and Tyr310 would contribute in stabilizing the filament. In this work, we investigated a short isoform of tau (4RTau), R3, VQIVYK peptide and their mutants by thioflavin S (ThS) fluorescence, and NMR measurements, and proved for the first time that this CH-π interaction stabilizes the filament at the atomic level. In addition, by molecular modeling, we revealed that this interaction further supports an extended amphipathic structure for molecular self-association during the process of PHF formation of tau protein. The present work indicates new approach that inhibits the CH-π interaction for developing a therapeutic agent for AD.

Post-translational modifications of tubulin

Microtubules are the largest filamentous components of the eukaryotic cytoskeleton. In spite of their extraordinary level of structural conservation, microtubules fulfill a vast range of different functions in cells. How this functional diversity is achieved remains an open question; however, recent advances point towards post-translational modifications (PTMs) of tubulin as a potent mechanism to generate microtubule identities. As many microtubule functions have direct implications for development and homeostasis of organisms, understanding the molecular functions of tubulin PTMs could provide a more differentiated view on the role of microtubules in both normal and pathological aspects of organism development.

Site-specific phosphorylation of Tau protein is associated with deacetylation of microtubules in mouse spermatogenic cells during meiosis.

Tau is one of the microtubule-associated proteins and a major component of paired helical filaments, a hallmark of Alzheimer's disease. Its expression has also been indicated in the testis. However, its function and modification in the testis have not been established. Here, we analyzed the dynamics of phosphorylation patterns during spermatogenesis. The expression of Tau protein and its phosphorylation were shown in the mouse testis. Immunohistochemistry revealed that the phosphorylation was strongly detected during meiosis. Correspondingly, the expression of acetylated tubulin was inversely weakened during meiosis. These results suggest that phosphorylation of Tau protein contributes to spermatogenesis, especially in meiosis.

Exon 12 of slow skeletal troponin t affects calcium sensitivity of force development and interactions with other thin filament components (1102.30)

Little information exists concerning the functional roles of the human slow skeletal troponin T isoforms (HSSTnT isoforms) in slow skeletal muscle. Three HSSTnT isoforms have been found in human slow skeletal muscle: HSSTnT1 (+ exons 5 and 12), HSSTnT2 (+5, ‐12), and HSSTnT3 (‐5, ‐12). Another potential isoform HSSTnT‐Hyp (‐5, +12) was recently found at the mRNA level. The objective of this study was to determine the physiological role of these SSTnT isoforms in slow skeletal muscle. To investigate these SSTnT isoforms several methods including skinned fiber mechanics, peptide spot blot, and mammalian two‐hybrid assays were utilized. Skinned rabbit slow soleus muscle fibers were displaced with HSSTnT1, 2, 3 or Hyp and reconstituted with the human slow skeletal troponin I (HSSTnI)/human cardiac troponin C (HCTnC) complex. The calcium sensitivity increased between SSTnT isoforms: isoform 1 (pCa50 = 5.74) < Hyp isoform (pCa50 = 5.80) < isoform 2 (pCa50 = 5.81) < isoform 3 (pCa50 = 5.84). In a reconstituted skeletal muscle system, the HSSTnT 1‐3 isoforms had similar actomyosin ATPase activation or inhibition in the presence or absence of calcium. Peptide spot blot analysis revealed novel interactions between HSSTnT peptides and HCTnC, RSTm and HCTnI. Mammalian two‐hybrid studies showed that the alternatively spliced regions affect the interaction of HSSTnT with other thin filament components. These results suggest that the functional differences that occur between HSSTnT isoforms may be partially due to alternative splicing of exon 12. Overall, HSSTnT isoforms were found to have distinct functional properties in slow skeletal muscle regulation and are likely to be relevant for the understanding of skeletal muscle diseases.Grant Funding Source: UC Davis research funds and NIH

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca(2+) binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca(2+) conditions, cMyBP-C displaces tropomyosin toward its high Ca(2+) position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca(2+), cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca(2+) but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca(2+) position on actin, and govern contractile speed by an independent molecular mechanism.

Cardiac myosin-binding protein-C is a critical mediator of diastolic function

Diastolic dysfunction prominently contributes to heart failure with preserved ejection fraction (HFpEF). Owing partly to inadequate understanding, HFpEF does not have any effective treatments. Cardiac myosin-binding protein-C (cMyBP-C), a component of the thick filament of heart muscle that can modulate cross-bridge attachment/detachment cycling process by its phosphorylation status, appears to be involved in the diastolic dysfunction associated with HFpEF. In patients, cMyBP-C mutations are associated with diastolic dysfunction even in the absence of hypertrophy. cMyBP-C deletion mouse models recapitulate diastolic dysfunction despite in vitro evidence of uninhibited cross-bridge cycling. Reduced phosphorylation of cMyBP-C is also associated with diastolic dysfunction in patients. Mouse models of reduced cMyBP-C phosphorylation exhibit diastolic dysfunction while cMyBP-C phosphorylation mimetic mouse models show enhanced diastolic function. Thus, cMyBP-C phosphorylation mediates diastolic function. Experimental results of both cMyBP-C deletion and reduced cMyBP-C phosphorylation causing diastolic dysfunction suggest that cMyBP-C phosphorylation level modulates cross-bridge detachment rate in relation to ongoing attachment rate to mediate relaxation. Consequently, alteration in cMyBP-C regulation of cross-bridge detachment is a key mechanism that causes diastolic dysfunction. Regardless of the exact molecular mechanism, ample clinical and experimental data show that cMyBP-C is a critical mediator of diastolic function. Furthermore, targeting cMyBP-C phosphorylation holds potential as a future treatment for diastolic dysfunction.

In Situ Structural Changes in Thick and Thin Filaments of Cardiac Muscle Induced by Fragments of Myosin Binding Protein C (MyBP-C)

MyBP-C is a component of the thick filaments of striated muscle, and mutations in the cardiac MyBP-C gene are the second most common cause of hypertrophic cardiomyopathy. The C-terminus of MyBP-C is bound to the thick filament, but the N-terminus is believed to interact with both the thin filaments and the S2 domain of myosin in the thick filaments. These interactions are phosphorylation-dependent, and are thought to regulate cardiac contractility. In this study, polarized fluorescence was used to monitor structural changes of the cardiac myosin regulatory light chain (cRLC) in the thick filaments and cardiac troponin C (cTnC) in the thin filaments on activation of skinned cardiac muscle either by calcium or by exogenous N-terminal fragments of cardiac MyBP-C. The structural changes in both proteins produced by activation with 50 μM C1-C2 fragment of cMyBPC at sarcomere length 2.1 μm, 20°C, pCa 9 were similar to those produced by calcium activation (pCa 4.5). However, only the cTnC probes showed significant structural changes when contraction was inhibited by blebbistatin, suggesting that the activating effect of C1-C2 is mediated by binding to the thin filament.Supported by the British Heart Foundation

Cytokeratin 14 and cytokeratin 18 expressions in reduced enamel epithelium and dentigerous cyst: Possible role in oncofetal transformation and histogenesis- of follicular type of adenomatoid odontogenic tumor.

Introduction:Odontogenic cysts and tumors arise from the structures and remnants associated with tooth development. Cysts and tumors derived from the odontogenic tissues constitute an unusually diverse group of lesions. This diversity reflects the complex development of the dental structures, since all these lesions originate through some alteration from the normal pattern of odontogenesis. Cytokeratin (CK) 14 is the typical intermediary filament of odontogenic epithelium, CK 18 is the major components of the intermediate filaments of simple or single layered epithelial tissue; it is not expressed in stratified squamous epithelium. The present study was undertaken to understand the expression pattern of these cytokeratins in dentigerous cyst, dental follicular tissue, adenomatoid odontogenic tumor (AOT) and unicystic ameloblastoma.Materials and Methods:The present study consists of 60 specimens consisting of 20 samples of Dentigerous cyst, 20 samples of Reduced enamel epithelium/dental follicles, 10 samples of Follicular type of AOT, 10 samples of unicystic ameloblastoma. The sections of these specimens were stained for CK 14 and CK 18. The number of cells positive for CK 14 and CK 18 was counted per 100 cells. The cells were counted in four randomly selected high-power fields and the mean was calculated. Scoring of cytokeratin 14 expressions was done using Remmele score.Results:The highest expression of cytokeratin 14 was noted in AOT, least was seen in dental follicle/Reduced enamel epithelium (REE). CK18 was negative in all the cases included in the present study.Conclusion:In the present study, the expression of CK14 was noted in AOT, Dentigerous cyst (DC), Unicystic Ameloblastoma (UCA) and Dental follicle/REE. The expressions between these lesions were compared. These expression pattern may provide an insight to the histogenesis of AOT.

Schistosome Muscles Contain Striated Muscle-Like Myosin Filaments in a Smooth Muscle-Like Architecture

Schistosomes are parasitic worms infecting 200 million humans worldwide. Schistosomiasis is treated with the drug praziquantel, which affects the parasite muscle, possibly by binding to myosin light chains. To understand the molecular functioning of Schistosome muscles, we have studied their cellular and molecular makeup. EM sections of adult worms reveal exclusively smooth muscle throughout the body wall (thick filaments not in register, dense bodies instead of Z-lines). We carried out 3D-reconstruction of negatively stained, relaxed Schistosome thick filaments. Surprisingly, the reconstruction was indistinguishable from those obtained previously for arthropod striated muscles, showing a 4-fold helical arrangement of myosin interacting-head motifs. Published data show that only one myosin heavy chain (MHC) gene is expressed in Schistosoma mansoni. By proteomic-Mass Spectrometry (MS) analysis of myosin from the adult parasite stage we showed that this was similar to a striated, not smooth, MHC. Analysis of published protein sequences supports this finding. A percentage-of-identity distance tree, and alignment of multiple diverse MHC sequences, showed two main branches: one for vertebrate and invertebrate striated muscles, and a smooth and nonmuscle branch. The Schistosome MHC lies in the striated muscle group, completely separate from the smooth/nonmuscle group. The in vitro motility assay showed movement of Schistosome thin filaments over unregulated tarantula striated myosin filaments independent of calcium and similar to rabbit F-actin motility, suggesting the absence of thin filament regulation. In agreement with this result, proteomic-MS analysis of Schistosome filament homogenates demonstrated the expression of the thin filament components tropomyosin and actin, but no signal for troponin was detected. We conclude that Schistosome muscles are hybrids, containing striated muscle-like thick filaments (with striated muscle MHC) and smooth muscle-like thin filaments (no troponin), arranged in a smooth muscle-like architecture.

Mg2+ Dependent Modulation of Striated Muscle Myosin ATPase by Thin Filament Components

While it's been reported that non-muscle myosin such as myosin V can be regulated by Mg2+, striated muscle myosin modulation by Mg2+-concentration dependency, especially in relation to thin filament components, has not been well studied up to now. It has been suggested that ADP release (product release) from myosin V is affected by Mg2+ concentration. Therefore, Mg2+-dependent modulation of striated muscle myosin ATPase was explored in the presence of reconstituted muscle thin filament components, i.e. actin, tropomyosin and troponin. ATPase activity assays and Nuclear Magnetic Resonance (NMR) spectroscopy were employed to understand the mechanisms of ATP hydrolysis by striated muscle myosin and the role of Mg2+ in the enzymatic activity. An NMR technique called Water Ligand-Observed Gradient Spectroscopy (WaterLOGSY) was utilized to probe if the interaction of myosin with ADP (one of the products resulted from ATP hydrolysis) is different depending on Mg2+ concentration as well as the presence of muscle thin filament components. Our WaterLOGSY data demonstrated that this technique is a promising method to monitor ADP binding to myosin head S1 even in the presence of actin filaments and that there was a change in the appearance of peaks between with and without tropomyosin at a low Mg2+ concentration. This set of results indicates that the way by which myosin binds ADP is different depending on the states of thin filaments. Moreover, actomyosin S1 ATPase rate decreased as Mg2+ concentration increased. When tropomyosin was reconstituted in the thin filaments, the ATPase rate decreased more rapidly compared with actomyosin alone. Thus we conclude that Mg2+ plays an important role in striated muscle myosin ATPase and that regulatory components such as tropomyosin modulate Mg2+ dependency of myosin ATPase in the striated muscle.

The nebulin SH3 domain is dispensable for normal skeletal muscle structure but is required for effective active load bearing in mouse

Nemaline myopathy (NM) is a congenital myopathy with an estimated incidence of 150,000 live births. It is caused by mutations in thin filament components, including nebulin, which accounts for about 50% of the cases. The identification of NM cases with nonsense mutations resulting in loss of the extreme C-terminal SH3 domain of nebulin suggests an important role of the nebulin SH3 domain, which is further supported by the recent demonstration of its role in IGF-1-induced sarcomeric actin filament formation through targeting of N-WASP to the Z-line. To provide further insights into the functional significance of the nebulin SH3 domain in the Z-disk and to understand the mechanisms by which truncations of nebulin lead to NM, we took two approaches: (1) an affinity-based proteomic screening to identify novel interaction partners of the nebulin SH3 domain; and (2) generation and characterization of a novel knockin mouse model with a premature stop codon in the nebulin gene, eliminating its C-terminal SH3 domain (NebΔSH3 mouse). Surprisingly, detailed analyses of NebΔSH3 mice revealed no structural or histological skeletal muscle abnormalities and no changes in gene expression or localization of interaction partners of the nebulin SH3 domain, including myopalladin, palladin, zyxin and N-WASP. Also, no significant effect on peak isometric stress production, passive tensile stress or Young's modulus was found. However, NebΔSH3 muscle displayed a slightly altered force-frequency relationship and was significantly more susceptible to eccentric contraction-induced injury, suggesting that the nebulin SH3 domain protects against eccentric contraction-induced injury and possibly plays a role in fine-tuning the excitation-contraction coupling mechanism.

Human cardiac troponin complex. Structure and functions

Troponin complex is a component of skeletal and cardiac muscle thin filaments. It consists of three subunits - troponin I, T, and C, and it plays a crucial role in muscle activity, connecting changes in intracellular Ca2+ concentration with generation of contraction. In spite of more than 40 years of studies, many aspects of troponin functioning are still not completely understood, and several models describing the mechanism of muscle contraction exist. Being a key factor in the regulation of cardiac muscle contraction, troponin complex is utilized in medicine as a target for some cardiotonic drugs used in the treatment of heart failure. A number of mutations in troponin subunits are associated with development of different types of cardiomyopathy. Moreover, for the last 25 years cardiac isoforms of troponin I and T have been widely used for immunochemical diagnostics of pathologies associated with cardiomyocyte death (myocardial infarction, myocardial trauma, and others). This review summarizes the existing evidence on the structure and function of troponin complex subunits, their role in the regulation of cardiac muscle contraction, and their clinical applications.