In vitro motility of native thin filaments from Drosophila indirect flight muscles reveals that the held-up 2 TnI mutation affects calcium activation.

A procedure for the isolation of regulated native thin filaments from the indirect flight muscles (IFM) of Drosophila melanogaster is described. These are the first striated invertebrate thin filaments to show Ca-regulated in vitro motility. Regulated native thin filaments from wild type and a troponin I mutant, held-up-2, were compared by in vitro motility assays that showed that the mutant troponin I caused activation of motility at pCa values higher than wild type. The held-up2 mutation, in the sole troponin I gene (wupA) in the Drosophila genome, is known to cause hypercontraction of the IFM and other muscles in vivo leading to their eventual destruction. The mutation causes substitution of alanine by valine at a homologous and completely conserved troponin I residue (A25) in the vertebrate skeletal muscle TnI isoform. The effects of the held-up 2 mutation on calcium activation of thin filament in vitro motility are discussed with respect to its effects on hypercontraction and dysfunction. Previous electron microscopy and 3-dimensional reconstruction studies showed that the tropomyosin of held-up 2 thin filaments occupies positions associated with the so-called 'closed' state, but independently of calcium concentration. This is discussed with respect to calcium dependent regulation of held-up-2 thin filaments in in vitro motility.

Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin.

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.

C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states.

Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments' interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506-793) and CaDH2 (residues 683-767) on the structure of actin-tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin-tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle 'ghost' fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)-phalloidin or fluorescein-5'-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Phi(A), Phi(E), Theta(1/2) and Nu were calculated. Phi(A) and Phi(E) are angles between the fiber axis and the absorption and emission dipoles, respectively; Theta(1/2) is the angle between the F-actin filament axis and the fiber axis; Nu is the relative number of randomly oriented fluorophores. Actin-tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the 'on' conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca(2+)) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca(2+)) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between 'off' (caldesmon and CaDH1) and 'on' (S-1 and CaDH2) states in a manner analogous to troponin.

[Caldesmon inhibits formation of strongly bound myosin cross-bridges and activates an ability of weakly bound cross-bridges to transform actin monomers to the off-conformation]. / Kal'desmon podavliaet formirovanie sil'nosviazannykh poperechnykh miozinovykh mostikov i aktiviruet sposobnost' slabosviazannykh mostikov transformirovat' sub"edinitsy aktina v vykliuchennuiu konformatsiiu.

The effect of caldesmon and its actin-binding C-terminal 35 kDa fragment on conformational alterations of actin in a muscle fiber at relaxation, rigor and at simulation of strong and weak binding of myosin heads to actin was studied by polarizational fluorimetry technique. The strong and weak binding forms were mimicked during binding of F-actin of ghost muscle fibers to myosin subfragment-1 modified with NEM (NEM-S1) or pPDM (pPDM-S1), respectively. As a test for alterations in actin conformation, changes in orientation and mobility of a fluorescent probe, TRITC-phalloidin, bound specifically to F-actin were used. The results obtained have shown that during transition of the muscle fiber from the relaxed state into the rigor and during binding of actin filaments to NEM-S1, changes of polarization parameters take place, which are characteristic of formation between actin and myosin of the strong binding and of transformation of actin subunits from the "turned-off" (inactive) to the "turned-on" (active) conformation. Binding of pPDM-S1 to actin and relaxation of the muscle fiber are accompanied, on the contrary, by the changes of orientation and of the fluorescent probe mobility, which are typical of formation of the weak ("non-force-producing") form of actin-myosin binding and of transformation of actin subunits from the active conformation into the inactive one. Caldesmon and its C-terminal fragment markedly inhibit formation of the strong binding at rigor and activate transition of actin monomers to the switched off conformation at relaxation of muscle fiber. In parallel experiments, these regulatory proteins have been shown to inhibit an active force developed at the transition of a muscle fiber from relaxation to rigor. Besides, caldesmon and its fragment decrease the rate of actin filament sliding over myosin in an in vitro motility assay. Caldesmon is suggested to regulate the smooth muscle contraction in an allosterical manner. The alterations in actin conformation inhibit formation of strong binding of myosin cross bridges to actin and activate the ability of weakly bound cross bridges to switch actin monomers from the "on" to the "off" conformation.

[Effect of the C-terminal actin-binding sites of caldesmon on the interaction of actin with myosin]. / Vliianie C-kontsevykh aktinsviazyvaiushchikh saitov kal'desmona na vzaimodeistvie aktina s miozinom.

TRITC-phalloidin or FITC-labeled F-actin of ghost muscle fibers was bound to tropomyosin and C-terminal recombinant fragments of caldesmon CaDH1 (residues 506-793) or CaDH2 (residues 683-767). After that the fibers were decorated with myosin subfragment 1. In the absence of caldesmon fragments, subfragment 1 interaction with F-actin caused changes in parameters of polarized fluorescence, that were typical of "strong" binding of myosin heads to F-actin and of the "switched on" conformational state of actin. CaDH1 inhibited, whereas CaDH2 activated the effect of subfragment 1. It is suggested that C-terminal part of caldesmon may modulate the transition of F-actin subunits from the "switched on" to the "switched off" state.