Differentiation of X chromosomes in early female mouse embryos

The stalk segment of the heterotrich ciliate, Stentor coeruleus, appears nearly isotropic in the contracted state and develops a characteristic birefringence during extension. The birefringence occurs in stripes and is associated with the myonemes, one of the two longitudinally running subpellicular fiber systems. Electron microscopical investigations reveal changes in the ultrastructure of the myonemes from the extended to the contracted state. The relaxed myonemes consist mainly of 3 nm filaments running in the longitudinal direction, while the contracted myonemes show 10 nm tubular-like filaments, more randomly oriented. It is suggested that during elongation the randomly oriented tubular filaments undergo a conformational change to more regularly arranged thin filaments, thus causing development of birefringence.     

THE CONTRACTILE BASIS OF AMOEBOID MOVEMENT : I. The Chemical Control of Motility in Isolated Cytoplasm

Cytoplasm has been isolated from single amoeba (Chaos carolinensis) in physiological solutions similar to rigor, contraction, and relaxation solutions designed to control the contractile state of vertebrate striated muscle. Contractions of the isolated cytoplasm are elicited by free calcium ion concentrations above ca. 7.0 x 10^-7 M. Amoeba cytoplasmic contractility has been cycled repeatedly through stabilized (rigor), contracted, and relaxed states by manipulating the exogenous free calcium and ATP concentrations. The transition from stabilized state to relaxed state was characterized by a loss of viscoelasticity which was monitored as changes in the capacity of the cytoplasm to exhibit strain birefringence when stretched. When the stabilized cytoplasm was stretched, birefringent fibrils were observed. Thin sections of those fibrils showed thick (150–250 Å) and thin (70 Å) filaments aligned parallel to the long axis of fibrils visible with the light microscope. Negatively stained cytoplasm treated with relaxation solution showed dissociated thick and thin filaments morphologically identical with myosin aggregates and purified actin, respectively, from vertebrate striated muscle. In the presence of threshold buffered free calcium, ATP, and magnesium ions, controlled localized contractions caused membrane-less pseudopodia to extend into the solution from the cytoplasmic mass. These experiments shed new light on the contractile basis of cytoplasmic streaming and pseudopod extension, the chemical control of contractility in the amoeba cytoplasm, the site of application of the motive force for amoeboid movement, and the nature of the rheological transformations associated with the circulation of cytoplasm in intact amoeba.     

GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA

The RhoA GTPase plays a vital role in assembly of contractile actin-myosin filaments (stress fibers) and of associated focal adhesion complexes of adherent monolayer cells in culture. GEF-H1 is a microtubule-associated guanine nucleotide exchange factor that activates RhoA upon release from microtubules. The overexpression of GEF-H1 deficient in microtubule binding or treatment of HeLa cells with nocodazole to induce microtubule depolymerization results in Rho-dependent actin stress fiber formation and contractile cell morphology. However, whether GEF-H1 is required and sufficient to mediate nocodazole-induced contractility remains unclear. We establish here that siRNA-mediated depletion of GEF-H1 in HeLa cells prevents nocodazole-induced cell contraction. Furthermore, the nocodazole-induced activation of RhoA and Rho-associated kinase (ROCK) that mediates phosphorylation of myosin regulatory light chain (MLC) is impaired in GEF-H1–depleted cells. Conversely, RhoA activation and contractility are rescued by reintroduction of siRNA-resistant GEF-H1. Our studies reveal a critical role for a GEF-H1/RhoA/ROCK/MLC signaling pathway in mediating nocodazole-induced cell contractility.     

Effect of External Calcium and of Temperature on Contraction in Snake Muscle Fibers

The effect of external calcium and of temperature on the contractile responses has been studied in voltage clamped snake twitch muscle fibers. Increasing [Ca⁺⁺]_o from 0.2 to 7.0 mM raised contractile threshold by 15–20 mV, the latter coinciding with the appearance of delayed rectification. The duration of contracture, the rates of rise and decay of tension depended on the level of depolarization and [Ca⁺⁺]_o. The minimum duration of repolarization necessary to restore the contractile response was much shorter in high [Ca⁺⁺]_o. When the bathing solution was cooled to 10 from 20°C the time-course of contracture was markedly prolonged and the outward current was reduced without significant change in maximum tension. The threshold for contraction tended to be somewhat lower at the lower temperature. The contractile repriming was much slower at low temperature. However, reduction in temperature slowed the rate of recovery much less at low [Ca⁺⁺]_o than at normal [Ca⁺⁺]_o.     

Interplay of Troponin- and Myosin-Based Pathways of Calcium Activation in Skeletal and Cardiac Muscle: The Use of W7 as an Inhibitor of Thin Filament Activation

To investigate the interplay between the thin and thick filaments during calcium activation in striated muscle, we employed n-(6-aminohexyl) 5-chloro-1-napthalenesulfonamide (W7) as an inhibitor of troponin C and compared its effects with that of the myosin-specific inhibitor, 2,3-butanedione 2-monoxime (BDM). In both skeletal and cardiac fibers, W7 reversibly inhibited ATPase and tension over the full range of calcium activation between pCa 8.0 and 4.5, resulting in reduced calcium sensitivity and cooperativity of ATPase and tension activations. At maximal activation in skeletal fibers, the W7 concentrations for half-maximal inhibition (K_I) were 70–80 μM for ATPase and 20–30 μM for tension, nearly >200-fold lower than BDM (20 mM and 5–8 mM, respectively). When W7 (50 μM) and BDM (20 mM) were combined in skeletal fibers, the ATPase and tension-pCa curves exhibited lower apparent cooperativity and maxima and higher calcium sensitivity than expected from two independent activation pathways, suggesting that the interplay between the thin and thick filaments varies with the level of activation. Significantly, the inhibition of W7 increased the ATPase/tension ratio during activation in both muscle types. W7 holds much promise as a potent and reversible inhibitor of thin filament-mediated calcium activation of skeletal and cardiac muscle contraction.     

THE SYNTHESIS OF MICROTUBULE AND OTHER PROTEINS OF THE ORAL APPARATUS IN TETRAHYMENA PYRIFORMIS

Several proteins, including microtubule proteins, have been isolated from the oral apparatus of the ciliate Tetrahymena. The synthesis of these proteins has been studied in relation to formation of this organelle system by the cell. Electron microscopy has shown that the isolated oral apparatus consists primarily of basal bodies, pellicular membranes, and a system of subpellicular microtubules and filaments. Cilia were removed during the isolation; therefore none of the proteins studied was from these structures. Evidence was obtained from the study of total oral apparatus protein which indicates that at least some of the proteins involved in formation of this organelle system may be synthesized and stored in the cytoplasm for use over long periods. This pattern of regulation was found for three individual proteins isolated from the oral apparatus fraction after extraction with a phenol-acetic acid solvent. A different pattern of regulation was found for microtubule proteins isolated from the oral apparatus of Tetrahymena. The data suggest that microtubule proteins, at least in logarithmically growing cells, are not stored in a cytoplasmic pool but are synthesized in the same cell cycle in which they are assembled into oral structures.     

FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo–electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule.     

The presence of cyclic AMP, and its effects on oral regeneration and in situ ciliary regeneration in Stentor coeruleus.

We have found cyclic AMP in the large, heterotrichous ciliate Stentor coeruleus in amounts per milligram protein similar to those found in another ciliate, Tetrahymena pyriformis. The possible function of cyclic AMP in Stentor was first examined by determining its effects on oral regeneration, the process by which Stentor can replace a missing oral apparatus in eight to ten hours. Once begun (by brief exposure to a 15% sucrose solution, causing shedding of the oral apparatus) regeneration follows eight specific morphological stages visible with the dissecting microscope. Continuous exposure of regenerating cells to either N⁶, 2′-0-dibutyryl adenosine cyclic 3′:5′-monophosphate (DBC) or theophylline begun at the onset of oral regeneration (stage 0) caused delays in the completion of regeneration. The delays induced by DBC occurred in the early stages prior to stage 5. Regenerating cells exposed to DBC or theophylline at various stages of development were delayed, even at stages 5 and 6. Both DBC and theophylline reversibly bleached the cortical pigment of the cells. Guanosine 3′:5′-cyclic monophosphate (cyclic GMP), AMP, GMP, and sodium butyrate neither delayed oral regeneration nor bleached the cortical pigment. Excess extracellular calcium ions alone had no effect on oral regeneration, but 10 mM calcium and DBC caused more delay than DBC alone. Thus, the delay of oral regeneration in Stentor caused by cyclic AMP may involve calcium ions. To determine if cyclic AMP can retard in situ ciliary regeneration by Stentor, as it does in Tetrahymena, a new technique, more accurate than past methods, was developed to monitor ciliary regrowth. Using this procedure we found that both DBC and theophylline significantly delayed the in situ ciliary regeneration by Stentor.     

The fine structure of euchromatin and centromeric heterochromatin in Tenebrio molitor chromosomes

The fine structure of constitutive heterochromatin and euchromatin was compared in electron microscope whole-mount preparations of Tenebrio molitor (Insecta, Coleoptera) spermatocyte nuclei. Tenebrio molitor pachytene chromosomes display extended segments of centromeric heterochromatin and thus are especially suitable for this purpose. When nuclei were incubated in solutions containing different concentrations of NaCl or of MgCl₂, two levels of chromatin fine structures were observed in the euchromatic segments: nucleosome fibers (0.1 mM–20 mM NaCl) and supranucleosomal fibers with 28 nm in diameter (40 mM–100 mM NaCl, 0.2 mM–1.0 mM MgCl₂). The fine structure in the heterochromatic segments was the same as that in the euchromatic segments in all NaCl concentrations and in MgCl₂ concentrations up to 0.4 mM. In higher MgCl₂ concentrations the heterochromatin remained more compact than the euchromatin and consisted of 37-nm-thick fibers in 0.6 mM MgCl₂ and of 65-nm-thick fibers in 1.0 mM MgCl₂. After the 37-nm and the 65-nm fibers had been dispersed in Mg²⁺-free solutions they could be recondensed by incubation in 0.6 mM and 1.0 mM MgCl₂, respectively. It is concluded that a Mg²⁺-sensitive component of the heterochromatin is responsible for the folding of the nucleosome chain to heterochromatin-specific supranucleosomal structures.     

Motility processes in Acantharia (protozoa) III. Calcium regulation of the contraction - relaxation cycles of in vivo myonemes

The myonemes in the marine pelagic protozoa Acantharia are contractile organelles involved in buoyancy regulation. It was previously shown that they can perform three kinds of movement: rapid contraction, slow undulation and slow relaxation. They consist of a periodically striated bundle of 2–4 nm nonactin filaments that are twisted in pairs and shortened by a coiling mechanism. After permeabilization or demembranation, contraction and relaxation can still be performed by varying Ca²⁺ concentration and ATP is not needed. In the present paper, we have studied the role of Ca²⁺ and inhibitors of energy production in intact cells. Our data suggest that; (i) the in vivo rapid contraction subsequent to mechanical or electrical stimulation is triggered by Ca²⁺ influx across the cell membrane; (ii) the slow contraction that takes place during the undulating movement depends on Ca²⁺ release provided by internal calcium stores; (iii) the rapid contraction as well as the progressive shortening that occurs during the slow undulating movement are caused by Ca²⁺ binding to the myoneme filaments; (iv) ATP is not directly involved in the saturation by Ca²⁺ of Ca²⁺ sensitive sites located along the myoneme microstrands; (v) regulation of the movements of Ca²⁺ within the cytoplasm depends mainly upon the alternative pathway of ATP production; (vi) calmodulin is presumably involved in this regulation. A tentative cytophysiologic interpretation of the mechanism of contractility is proposed.     

Calcium Efflux from Barnacle Muscle Fibers : Dependence on External Cations

Calcium-45 was injected into single giant barnacle muscle fibers, and the rate of efflux was measured under a variety of conditions. The rate constant (k) for ⁴⁵Ca efflux into standard seawater averaged 17 x 10^–4 min^–1 which corresponds to an efflux of about 1–2 pmol/cm²·s. Removal of external Ca (Ca_o) reduced the efflux by 50%. In most fibers about 40% of the ⁴⁵Ca efflux into Ca-free seawater was dependent on external Na (Na_o); treatment with 3.5 mM caffeine increased the magnitude of the Na_o-dependent efflux. In a few fibers removal of Na_o, in the absence of Ca_o, either had no effect or increased k; caffeine (2–3.5 mM) unmasked an Na_o-dependent efflux in these fibers. The Na_o-dependent Ca efflux had a Q₁₀ of about 3.7. The data are consistent with the idea that a large fraction of the Ca efflux may be carrier-mediated, and may involve both Ca-Ca and Na-Ca counterflow. The relation between the Na_o-dependent Ca efflux and the external Na concentration is sigmoid, and suggests that two, or more likely three, external Na⁺ ions may activate the efflux of one Ca⁺². With a three-for-one Na-Ca exchange, the Na electrochemical gradient may be able to supply sufficient energy to maintain the Ca gradient in these fibers. Other, more complex models are not excluded, however, and may be required to explain some puzzling features of the Ca efflux such as the variable Na_o-dependence.     

Organization of cytoskeleton during the tentacle contraction and cytostome movement in the dinoflagellate Noctiluca scintillans McCartney

Summary Electron microscopy of Noctiluca scintillans reveals that the cytoskeleton of the tentacle involved in the motor action of the prey capture consists of three characteristic elements: a deformable peripheral fibrillo-granular ectosarc, abundant underlying microtubules organized in several rows on the convex side, and helicoid filaments about 8 nm in diameter organized into striated myonemes. Microtubules of the external row are crossed-linked with each other by fibrous elements 5 nm in diameter and 10–15 nm long, their links with the second row result in a Y-shaped binding. Bonds of the other rows are linked to each other irregularly between those of the same row. Striated myonemes are regularly inserted between the rows of microtubules on the ectosarc and between its pleats, joining together in a knot of disarrayed filaments with multidirectional orientation in the central axis of the tentacle. Striation of myonemes is based on an alternation of thick striae (TS) 40 nm wide with a periodicity of about 200 nm, and of some intermediary fine striae (FS) 10 nm wide. The events during tentacle contraction are: (1) Rotation of the tentacle, bringing the convex side to the inner side of it. Here, large numbers of microtubules have been visualized by optical immunocytochemistry after labelling with Paramecium antitubulin antiserum. (2) Increase of pleat amplitude (200–300 nm to 600 nm) in concomitance with a decrease of its period (500–700 nm to 250 nm). (3) Apparent modification of the microtubule orientation. (4) Transformation of some TS in several FS without modification of the striation periodicity.Near the cytostome, the cytoskeleton consists of a number quantity of microtubules underlying a non-pleated ectosarc and long tracts of contractile myonemes formed by 6-nm helicoid filaments linking the internal side of the cytostome of the supporting rod. Semirelaxed myonemes show an alternation of fine striae (FS) 35 nm wide between two clear areas (CA) with a periodicity of about 300 nm, plus an incipient dark area (DA) lying between them; together they are transformed into a thick stria (TS) during maximal contraction; the striation periodicity thus decreases by one half. These two systems are compared with one another and with other motile systems.     

A new fiber-forming protein from Tetrahymena pyriformis

A new fiber-forming protein was isolated from the acetone powder of Tetrahymena pyriformis by co-precipitating with skeletal muscle myosin while trials were made to find actin or actin-like protein in Tetrahymena. It has a molecular weight of 38000 D and forms a tetramer (140000 D, 9 S) in physiological conditions. Its isoelectric point (pH 6.7), amino acid composition and antigenic determinant(s) differ significantly from those of non-muscle actin and skeletal muscle actin. It does not undergo G-F conversion while actin does, and does not activate Mg²⁺-ATPase of skeletal muscle myosin. The protein localizes in the oral apparatus and division furrow as revealed by fluorescent antibody method. The protein can be assembled into 14-nm filaments in a reassembly buffer. The in vitro filaments appear to correspond to some filaments included in the oral apparatus and the contractile ring. The fiber-forming protein from Tetrahymena may play important roles in cell motility including cell division.     

The Kinase Regulator Mob1 Acts as a Patterning Protein for Stentor Morphogenesis

Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1—with conserved functions in cell division and morphogenesis from plants to humans—as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system.     

Microtubule-Dependent Modulation of Adhesion Complex Composition

The microtubule network regulates the turnover of integrin-containing adhesion complexes to stimulate cell migration. Disruption of the microtubule network results in an enlargement of adhesion complex size due to increased RhoA-stimulated actomyosin contractility, and inhibition of adhesion complex turnover; however, the microtubule-dependent changes in adhesion complex composition have not been studied in a global, unbiased manner. Here we used label-free quantitative mass spectrometry-based proteomics to determine adhesion complex changes that occur upon microtubule disruption with nocodazole. Nocodazole-treated cells displayed an increased abundance of the majority of known adhesion complex components, but no change in the levels of the fibronectin-binding α₅β₁ integrin. Immunofluorescence analyses confirmed these findings, but revealed a change in localisation of adhesion complex components. Specifically, in untreated cells, α₅-integrin co-localised with vinculin at peripherally located focal adhesions and with tensin at centrally located fibrillar adhesions. In nocodazole-treated cells, however, α₅-integrin was found in both peripherally located and centrally located adhesion complexes that contained both vinculin and tensin, suggesting a switch in the maturation state of adhesion complexes to favour focal adhesions. Moreover, the switch to focal adhesions was confirmed to be force-dependent as inhibition of cell contractility with the Rho-associated protein kinase inhibitor, Y-27632, prevented the nocodazole-induced conversion. These results highlight a complex interplay between the microtubule cytoskeleton, adhesion complex maturation state and intracellular contractile force, and provide a resource for future adhesion signaling studies. The proteomics data have been deposited in the ProteomeXchange with identifier PXD001183.     

A tale of the ciliate tail: investigation into the adaptive significance of this sub-cellular structure

Ciliates can form an important link between the microbial loop and higher trophic levels primarily through consumption by copepods. This high predation pressure has resulted in a number of ciliate species developing rapid escape swimming behaviour. Several species of these escaping ciliates also possess a long contractile tail for which the functionality remains unresolved. We use high-speed video, specialized optics and novel fluid visualization tools to evaluate the role of this contractile appendage in two free-swimming ciliates, Pseudotontonia sp. and Tontonia sp., and compare the performance to escape swimming behaviour of a non-tailed species, Strobilidium sp. Here, we show that ‘tailed’ species respond to hydrodynamic disturbances with extremely short response latencies (less than or equal to 0.89 ms) by rapidly contracting the tail which carries the cell body 2–4 cell diameters within a few milliseconds. This provides an advantage over non-tailed species during the critical first 10–30 ms of an escape. Two small, short-lived vortex rings are created during contraction of the tail. The flow imposed by the ciliate jumping can be described as two well-separated impulsive Stokeslets and the overall flow attenuates spatially as r⁻³. The high initial velocities and spatio-temporal arrangement of vortices created by tail contractions appear to provide a means for rapid escape as well as hydrodynamic ‘camouflage’ against fast striking, mechanoreceptive predators such as copepods.     

Ca2+ -binding proteins and contractility of the infraciliary lattice in Paramecium

The infraciliary lattice (ICL) is the innermost cortical cytoskeletal network of Paramecium. Its meshes which run around the proximal end of basal bodies form a continuous contractile network beneath the cell surface. We had previously shown that the network, which could be recovered in a contracted form and selectively solubilized by EGTA from an ICL-enriched cell fraction, was principally composed of 23–24 kDa polypeptides cross-reacting with antibodies raised against the 22 kDa Ca²⁺ -binding proteins of the ecto-endoplasmic boundary (EEB), a contractile cytoskeletal network of another ciliate Isotricha prostoma. We show here 1) that the ICL also comprises a 220 kDa polypeptide; 2) that the 23–24 kDa polypeptides are resolved in 2D gels into 11 spots of acidic pI, 7 of which are both Ca²⁺ -binding and cross-reacting with the anti EEB polypeptides; 3) that the network displays a high Ca²⁺ -affinity as the treshold for solubilization/co-precipitation of both high and low MW polypeptides is around 10⁻⁸ M free Ca²⁺ ; 4) that in vivo contraction of the network occurs upon physiological increase of internal calcium concentration. The likely phylogenetic relationships of the 23–24 kDa ICL polypeptides with the calmodulin related family of Ca²⁺ -modulated polypeptides and the functions of the ICL in cell contractility and Ca²⁺ homeostasis are discussed.     

A quantitative analysis of contractility in active cytoskeletal protein networks

Cells actively produce contractile forces for a variety of processes including cytokinesis and motility. Contractility is known to rely on myosin II motors which convert chemical energy from ATP hydrolysis into forces on actin filaments. However, the basic physical principles of cell contractility remain poorly understood. We reconstitute contractility in a simplified model system of purified F-actin, muscle myosin II motors, and α-actinin cross-linkers. We show that contractility occurs above a threshold motor concentration and within a window of cross-linker concentrations. We also quantify the pore size of the bundled networks and find contractility to occur at a critical distance between the bundles. We propose a simple mechanism of contraction based on myosin filaments pulling neighboring bundles together into an aggregated structure. Observations of this reconstituted system in both bulk and low-dimensional geometries show that the contracting gels pull on and deform their surface with a contractile force of ∼1 μN, or ∼100 pN per F-actin bundle. Cytoplasmic extracts contracting in identical environments show a similar behavior and dependence on myosin as the reconstituted system. Our results suggest that cellular contractility can be sensitively regulated by tuning the (local) activity of molecular motors and the cross-linker density and binding affinity.     

Regulation of Cardiac Muscle Contractility

The heart's physiological performance, unlike that of skeletal muscle, is regulated primarily by variations in the contractile force developed by the individual myocardial fibers. In an attempt to identify the basis for the characteristic properties of myocardial contraction, the individual cardiac contractile proteins and their behavior in contractile models in vitro have been examined. The low shortening velocity of heart muscle appears to reflect the weak ATPase activity of cardiac myosin, but this enzymatic activity probably does not determine active state intensity. Quantification of the effects of Ca⁺⁺ upon cardiac actomyosin supports the view that myocardial contractility can be modified by changes in the amount of calcium released during excitation-contraction coupling. Exchange of intracellular K⁺ with Na⁺ derived from the extracellular space also could enhance myocardial contractility directly, as highly purified cardiac actomyosin is stimulated when K⁺ is replaced by an equimolar amount of Na⁺. On the other hand, cardiac glycosides and catecholamines, agents which greatly increase the contractility of the intact heart, were found to be without significant actions upon highly purified reconstituted cardiac actomyosin.     

Instability in the Central Region of Tropomyosin Modulates the Function of Its Overlapping Ends

The causal link between disparate tropomyosin (Tm) functions and the structural instability in Tm is unknown. To test the hypothesis that the structural instability in the central region of Tm modulates the function of the overlapping ends of contiguous Tm dimers, we used transgenic mice (Tm_DM) that expressed a mutant α-Tm in the heart; S229E and H276N substitutions induce structural instability in the central region and the overlapping ends of Tm, respectively. In addition, two mouse cardiac troponin T mutants (TnT_1–44Δ and TnT_45–74Δ) that have a divergent effect on the overlapping ends of Tm were employed. The S229E-induced instability in the central region of Tm_DM altered the overlapping ends of Tm_DM, thereby it negated the attenuating effect of H276N on Ca²⁺-activated maximal tension. The rate of cross-bridge detachment (g) decreased in Tm_DM+TnT_WT and Tm_H276N+TnT_WT fibers but increased in Tm_DM+TnT_45–74Δ fibers; however, TnT_45–74Δ did not alter g, demonstrating that S229E in Tm_DM had divergent effects on g. The S229E substitution in Tm_DM ablated the H276N-induced desensitization of myofilament Ca²⁺ sensitivity in Tm_DM+TnT_1–44Δ fibers. To our knowledge, novel findings from this study show that the structural instability in the central region of Tm modifies cardiac contractile function via its effect on the overlapping ends of contiguous Tm.