Ca2+-dependent muscle dysfunction caused by mutation of the Caenorhabditis elegans troponin T-1 gene.

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca2+]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061-1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca2+-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca2+ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca2+ signaling support that CeTnT-1 phenotypes are dependent on Ca2+ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca2+] does not require TnT.

Orientation changes in myosin regulatory light chains following photorelease of ATP in skinned muscle fibers.

The orientation of the light-chain region of myosin heads in muscle fibers was followed by polarized fluorescence from an extrinsic probe during tension transients elicited by photolysis of caged ATP. Regulatory light chain from chicken gizzard myosin was covalently modified with iodoacetamidotetramethylrhodamine and exchanged into skinned fibers from rabbit psoas muscle without significant effect of the tension transients. Fluorescence polarization ratios Q parallel = (parallel I parallel-perpendicular I parallel)/ (parallel I parallel+perpendicular I parallel) and Q perpendicular = perpendicular I perpendicular - parallel I perpendicular)/ (perpendicular I perpendicular + parallel I perpendicular), where mIn denote fluorescence intensities for excitation (pre-subscript) and emission (post-subscript) parallel or perpendicular to the fiber axis, were simultaneously measured at 0.5 ms time resolution. Q perpendicular decreased and Q parallel increased promptly after ATP release in the presence or absence of CA2+, indicating changes in orientation of the light-chain region associated with ATP binding or cross-bridge detachment. Little further change in the Q signals accompanied either active tension development (+Ca2+) or the final relaxation (-Ca2+). The Q and tension transients slowed when liberated ATP concentration was reduced. Assuming that ATP is released at 118 s-1 (20 degrees C), the apparent second-order rate constants were 3-10 x 10(5) M-1 s-1 for Q parallel, 1-5 x 10(5) M-1 s-1 for Q perpendicular, and 0.5-2 x 10(5) M-1 s-1 for the convergence of tension traces starting from different rigor values. Fitting of model orientation distributions to the Q signals indicated that the angular disorder increases after ATP binding. This orientation change is specific to ATP because photo release of ADP caused much smaller changes in the Q signals.

Developmental genetic analysis of troponin T mutations in striated and nonstriated muscle cells of Caenorhabditis elegans.

We have been investigating a set of genes, collectively called mups, that are essential to striated body wall muscle cell positioning in Caenorhabditis elegans. Here we report our detailed characterization of the mup-2 locus, which encodes troponin T (TnT). Mutants for a heat-sensitive allele, called mup-2(e2346ts), and for a putative null, called mup-2(up1), are defective for embryonic body wall muscle cell contraction, sarcomere organization, and cell positioning. Characterizations of the heat-sensitive allele demonstrate that mutants are also defective for regulated muscle contraction in larval and adult body wall muscle, defective for function of the nonstriated oviduct myoepithelial sheath, and defective for epidermal morphogenesis. We cloned the mup-2 locus and its corresponding cDNA. The cDNA encodes a predicted 405-amino acid protein homologous to vertebrate and invertebrate TnT and includes an invertebrate-specific COOH-terminal tail. The mup-2 mutations lie within these cDNA sequences: mup-2(up1) is a termination codon near NH2 terminus (Glu94) and mup-2(e2346ts) is a termination codon in the COOH-terminal invertebrate-specific tail (Trp342). TnT is a muscle contractile protein that, in association with the thin filament proteins tropomyosin, troponin I and troponin C, regulates myosin-actin interaction in response to a rise in intracellular Ca2+. Our findings demonstrate multiple essential functions for TnT and provide a basis to investigate the in vivo functions and protein interactions of TnT in striated and nonstriated muscles.

Transients of fluorescence polarization in skeletal muscle fibers labeled with rhodamine on the regulatory light chain.

Structural changes of the myosin heads were correlated with mechanical events in the cross-bridge cycle by measuring fluorescence polarization signals at high time resolution from rhodamine probes bound to myosin regulatory light chains in skeletal muscle fibers. Motions of the cross-bridges were partially synchronized either by applying quick length changes to the fibers during active contractions or by activating the fibers from rigor by photolysis of caged ATP in the presence of Ca2+. With fibers in rigor, the fluorescence polarization values indicate that the probe dipoles are quite well ordered and are directed away from the muscle fiber axis. After photorelease of ATP from caged ATP, changes in polarization signals are consistent with broadening of the distribution of probe orientations. The signal deflections occur when ATP binds to actomyosin or when the cross-bridges detach, but the orientational distribution changes surprisingly little during active force development. In contrast, when staircases of quick releases are applied to labeled fibers during active contractions, the fluorescence polarization signals suggest a concerted rotation of the probes. The results indicate that the light chain region of myosin tilts during the quick release and/or during the tension recovery phase within the next few ms.

Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle.

Direct measurements were made of the Ca distribution within sarcomeres of glycerinated rabbit psoas muscle fibers in rigor using electron probe x-ray microanalysis. Both analogue raster analysis and digital x-ray imaging were used to quantitate the Ca distribution along thick and thin filaments as a function of the concentration of free Ca2+. Even when corrected for the estimated contribution of Ca bound to thick filaments, the Ca measured in the region of overlap between thick and thin filaments significantly exceeded the Ca in the I-band at subsaturating concentrations of free Ca2+. At saturating levels of free Ca2+, the excess Ca in the overlap region was diminished but still statistically significant. The data thus suggest that the formation of rigor linkages exerts multiple effects on the binding of Ca2+ to thin filaments in the overlap region by increasing the affinity of troponin C for Ca2+ and possibly by unmasking additional Ca2+ binding sites. The data also show that the cooperativity invested in the thin filaments is insufficient to permit the effects of rigor cross-bridge formation on Ca2+ binding to propagate far along the thin filaments into the I-band.

Mechanics and structure of cross-bridges during contractions initiated by photolysis of caged Ca2+.

Cross-bridge structure and mechanics were studied during development of skinned frog muscle fiber contractions initiated by photolysis of DM-nitrophen (a caged Ca2+). Stiffness rises earlier than tension following photo-release of Ca2+. A similar lead of stiffness in electrically stimulated fibers and the early rise of the I11/I10 ratio of equatorial X-ray reflections are thought to signal attachment of cross-bridges into states with lower force than in steady-state contraction. We investigated the structure of the early attachments by electron microscopy of fibers activated by photolysis of DM-nitrophen and then ultra-rapidly frozen and freeze substituted with tannic acid and OsO4. Sections from relaxed fibers show helical tracks of myosin heads on the thick filaments surface. Optical diffraction patterns show strong meridional intensities and layer lines up to the 6th order of 1/43 nm, indicating preservation and resolution of periodic structures smaller than 10 nm. Following photo-release of Ca2+, the 1/43 nm myosin layer line becomes less intense, and higher orders disappear. A approximately 1/36 nm layer line appears early (12-15 ms) and becomes stronger at later times. The 1/14.3 nm meridional spot weakens initially and recovers at a later time, while it broadens laterally. The 1/43 nm meridional spot is present during contraction, but the 2nd order meridional spot (1/21.5 nm) is weak or absent. These results are consistent with time resolved X-ray diffraction data on the periodic structures within the fiber. In sections along the 1,1 plane of activated fibers, the individual cross-bridges have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z-line. Fibers frozen at 13 ms, 33 ms, and 220 ms after photolysis all show surprisingly similar cross-bridges. Thus, a highly variable distribution of cross-bridge shapes and angles is established early in contraction.

Ca(2+)-dependence of structural changes in troponin-C in demembranated fibers of rabbit psoas muscle.

The Ca(2+)-dependence of structural changes in troponin-C (TnC) has been detected by monitoring the fluorescence from TnC labeled at Methionine-25, in the NH2-terminal domain, with danzylaziridine (TnC-DANZ) and then exchanged for endogenous TnC in glycerinated single fibers. The fluorescence-pCa relation obtained from fibers stretched to a sarcomere length greater than 4.0 microns evidenced two transitions: a small one, attributable to the binding of Ca2+ to the high affinity, Ca(2+)-Mg(2+)-binding sites of TnC; and a large one, attributable to the binding of Ca2+ to the low affinity, Ca(2+)-specific binding sites of TnC. In the fluorescence-pCa relation determined with fibers set to a sarcomere length of 2.4 microns, hence obtained in the presence of cycling cross-bridges, the large transition had the same Ca(2+)-dependence as did the development of tension. These results indicate that the NH2-terminal globular domain of TnC is modified by the binding of Ca2+ to sites located in both globular domains and that the structural changes in TnC resulting from the binding of Ca2+ to the low-affinity sites, but not to the high-affinity sites, are directly associated with the triggering of contraction.