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Motor proteins of microtubules, kinesin and dynein superfamily proteins play an important role in the intracellular transport. Inside a neuron they are involved in the transport of organelles, proteins and mRNAs along the axons and dendrites to the nerve terminals and back to the cell bodies. Disturbance of axonal transport may affect neurotransmitter release and short-term presynaptic plasticity. Disturbance of dendritic transport, in particular the recycling of synaptic receptors, affects postsynaptic plasticity. The review attempts to trace the connections between the motor proteins of microtubules and mechanisms of synaptic plasticity from the perspective of their involvement in the intracellular transport of proteins and organelles, which play role in the mechanisms of synaptic plasticity.

Seasonal changes of the isoform composition of myosin heavy chains in skeletal muscles (m. triceps, m. longissimus dorsi, m. soleus, m. gastrocnemius, m. vastus lateralis) of hibernating ground squirrels Spermophilus undulatus were studied. Functional properties of myosin (the actin-activated ATPase activity and its Ca(2+)-sensitivity in vitro) were also examined. It was observed that the content of slow myosin heavy chain I isoform increased and the content of fast IIx/d isoform decreased in muscles of torpid ground squirrels and animals which are active in autumn and winter. In muscles of these animals the content of N2A-titin isorfom decreased although the relative content of NT-titin isoform, observed in striated muscles of mammals in our previous experimental works, increased. Actin-activated ATPase activity and Ca(2+)-sensitivity of myosin isolated from skeletal muscles of torpid and interbout ground squirrels were found to reduce. The changes observed are discussed in the context of adaptation of skeletal muscles of ground squirrels to hibernation conditions.

The interference fine structure of the M3 reflection on the low-angle x-ray diffraction patterns of muscle fibres is used for the measurements of axial movements of myosin heads with a precision of 0.1-0.2 nm. We have measured changes in the M3 interference profile during tension rise induced by a 5 to 30 degrees C temperature jump in thin bundles of contracting fibers from rabbit skeletal muscle. Interpreting the data with a point diffractor model gives an estimate for the axial movement of the myosin heads during force rise of less than 0.6 nm. Modifications of the point diffractor model are discussed. We show that our experimental data can be explained by a model where myosin heads bind actin in a number of structurally different states.

The review is devoted to analysis of sequential modifications of our views on structural organization and functional significance of actin-myosin system in vital activity of a cell. Originally it was considered as a contractile apparatus of muscle cells only. Then actin, myosin and their accompanying proteins have been found in the cytoplasm of all non-muscle cells and shown to take part and be responsible for different types of cell motility. Later on, it has been found that actin is a highly polymorphic protein, which is able to form different structures and to play a role in a broad spectrum of cellular functions up to signal transduction and chromatin reorganization. And finally, the latest data demonstrate that actin and myosin directly participate in gene transcription and messenger RNA splicing. Thus, the data accumulated to date allow us to consider these proteins as the universal structural-regulatory system which provides a way to unite various poorly combined biochemical processes on the same molecular basis.

The review is concerned with the optical trap method, a modern experimental tool based on the recently discovered ability of light to trap and hold micron and submicron particles in the focused beam. The physical principle of operation of the optical trap and the possibilities of using the tool in studies of the molecular nature of biological motility have been considered. Some studies devoted to the physical characteristics and functioning of single molecules of motor proteins have been analyzed that have been performed with the optical trap method, which allows one to record nanometer displacements and piconewton forces.

In a series of experiments on regulated contractile systems (i.e., in vitro mobile systems with reconstructed thin filaments), the velocities of the movement of a thin filament on the surface covered by either rabbit skeletal or rat cardiac myosin at various concentrations of calcium ions in solution (in the pCa range from 4 to 8) were assessed. The corresponding "pCa-velocity" relationships were plotted, which proved to be of the sigmoid form. It was found that, at a saturating calcium concentration (pCa 4), the velocity of regulated thin filaments was 65% higher than for unregulated ones in the case of skeletal myosin and 87% higher than for unregulated thin filaments in the case of cardiac myosin. It was also found that the Hill coefficient was 1.95 and 2.5 for skeletal and cardiac myosins, respectively. The difference in the Hill coefficients for skeletal and cardiac myosins is discussed in terms of the difference in contribution of cooperativity mechanisms of contractile and regulatory proteins in the regulation of contraction in these types of muscles.

The structure of the strongly bound complex of the globular myosin head and F-actin is a key for understanding some important details of the mechanism of the actin-myosin motor. Current knowledge about the structure is based on the docking of known atomic structures of actin and myosin heads into low-resolution EM electron density maps. To refine the structure, we suggested a new approach based on energy minimization using the ICM-Pro software. The minimization includes rigid-body movement of protein backbone and side chain optimization on the protein interface. Our best model structure is similar to that obtained from EM. It also provides the highest calculated interaction energy and agrees with a number of mutagenesis experiments. Using the structure, we suggest molecular explanations for actin activation of product release from myosin and actin-induced myosin dissociation.

The effect of N-ethylmaleimide on the polymerization of myorod, a protein of molluscan smooth muscles, which is colocalized with myosin on the surface of paramyosin core of thick filaments and is a product of the alternative splicing of the gene of heavy myosin chains, was studied. It was shown that myorod modified by N-ethylmaleimide completely loses the polymerization ability but acquires the ability to aggregate in the presence of Mg2+. At the same time, treatment of molluscan myosin with N-ethylmaleimide did not affect its polymerization. It was supposed that the effect of N-ethylmaleimide on myorod polymerization is related to the modification of the myorod SH domain containing Cys722.

To elucidate the role of titin in the onset and development of dilated cardiomyopathy, the structure and functional properties of this protein from pathological myocardium (human left ventricle) were studied. By the use of SDS gel electrophoresis, a decrease in molecular weight of titin in dilated cardiomyopathy compared with norm (pig left ventricle) was revealed. The decrease correlated with the stage of the disease. A decrease in the length of molecules of pathological forms of titin was also found by electron microscopy, which confirms the results of electrophoresis tests. It was shown that, unlike titin from healthy muscle, pathological forms of titin do not activate but inhibit the main functional properties of control myosin: the actin-activated ATPase activity and its Ca2+ sensitivity. The direction of the changes in structure and functional properties of titin allows one to conclude about its contribution to the development of the pathology studied.

Two-dimensional maps of birefringence in sarcomers of a single fiber of rabbit m.psoas were obtained by an automated interference microscope developed at our laboratory. The changes in birefringence of muscle fibers reflect the movement of myosin cross-bridges. The orientation of cross-bridges was modified by varying the pH (pH 7.0, 6.0, 8.0) and ionic strength (mu = 0.115, 0.085, 0.235) of the bathing rigor solution. The maximum value of total birefringence in the rigor state was observed at neutral pH. Total birefringence markedly decreased (by 40%) as pH was changed from 7.0 to both 8.0 and 6.0. No significant changes in light phase shifts were found at a 1.5 reduction of ionic strength in the rigor solution. The calculated birefringence values were 45% higher in rigor solutions of a high (mu = 0.235) ionic strength. The results observed are discussed in terms of changes in the orientation of cross-bridges due to the movement of the alpha-helical subfragment-2 away from the filament shaft (pH 8) or coming closer to it (mu = 0.235). The available data do not allow one to explain the results obtained at pH 6.0.

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.

This review describes the main ultrastructural features, molecular organization and regulation of smooth muscle contractile machinery. Possible molecular mechanisms of smooth muscle sensitization to Ca2+, Ca(2+)-independent contraction and latch state are discussed.

The investigations of the molecular bases of muscle contraction posed the following question: in which part of the myosin cross-bridge, the head or the fibrillar part, the conformational changes occur that lead to the generation of force. The paper deals with experimental and theoretical data that are in some way related to the solution of this question. Based on the analysis of recent data, an alternative pattern of ATP hydrolysis-coupled structural alternations in the myosin bridge during the mechanochemical act is proposed.

Recent data about the structural and functional features of protein motors (myosin, dynein, kinesin) are presented. The mechanism by which the chemical energy of ATP is transduced into mechanical work is discussed.

A simple kinetic scheme of the mechano-chemical cycle of the cross-bridge is proposed. It was found on the experiments with using of caged ATP and the experiments with the limited number of the myosin molecules in motility assay in vitro. The model reproduces the results of the experiments in motility assay in vitro and allows to investigate the actin-myosin complexes at an external load condition.

The process of sarcomere membranes approaching under the action of actin and myosin filament stretching is studied theoretically. In elaboration of previous studies it is supposed that this stretching is due to the formation of hydrogen bonds excited by the energy of ATP splitting. A possibility for the hydrogen bond breakage at different stages of contraction is considered to be the greater the more are the polymers stretched, and the larger statistical scatter in the moments of H-bonds formation at different filaments if it is supposed that sarcomere contraction occurs much slower than stretching of each separate filament. As a result, one obtains a relationship between the rate of contraction and the external force which is close to the Hill's law, and the external force efficiency of a muscle contraction and heat extraction value are related to parameters of polymers and their properties.

In one of author's previous work the stepping cross-bridge model has been proposed. It was shown that the structure of actin filaments and their arrangement in muscle sarcomere ensure the whole necessary condition for the stepping cross-bridges movement in contracting muscle. In this work the requirements put on the thick filament structure by the stepping cross-bridge model are determined. The consideration leads to the conclusion on the rearrangement of myosin cross-bridges on the thick filament surface caused by the activation of the muscle.

A mathematical model of an actin filament sliding along myosin-coated surface in the in vitro motility assay is considered. The frontal end of the filament is supposed to be affected by a random Brownian force. The relation between the surface density of the myosin heads and average distance between the neighbour heads propelling the filament is calculated and compared with those predicted by earlier simplified models.

A new in vitro approach to study biological motility called movement or motility assay has been developed in last ten years. The method enabling direct observation of actin filament movement through the light microscope permits rather simple measurements of sliding velocity, force and stiffness on particular molecules. The aim of the review is to show the main features and achievements in this area.

A model of myosin complex with two active sites is considered. Each of the sites is supposed to pass through four states during a cycle of ATP hydrolysis. The model describes the distribution of the complex between states and the ATP hydrolysis rate as dependent on ATP concentration and reaction constants. Some of the constants were determined experimentally [6], but these values reproduce experimental curves [5] only with the assumption of cooperativity. If the first site is bind to actin, the rate of binding for the second one is shown to increase tenfold. If one of the sites is bind to actin by a "pulling" bridge, and the second site is in "rigor" state, then the ATP binding for the second site is about ten times faster. The transition into the rigor state proceeds much faster if both sites form pulling bridges. The rate of binding to actin is the same for one and for two sites.

In Harrington's model, local helix-coil transition in coiled-coil portion of myosin is considered to be an origin of force generation in muscle. However, the detailed analysis of this model leads to the conclusion about the different functional role of the helix-coil transition in contracting muscle.

Statistics of the hydrogen bond formation and break in a bundle of actin and myosin filaments realizing the attractive force in the sarcomere of a muscle is studied. Purely mechanical problem of the attractive-force formation and motion of myosin heads and action globules under their action is supplemented by accounting for the irreversible processes: 1. Thermal de-excitation of the latter in the chain of hydrogen bond during the elementary act of the ATP energy use resulting in fixing the extended actin filament. 2. Break of the hydrogen bonds, realizing this fixing, due to thermal fluctuations for the time tau. The average life-time turns out to be the order of time necessary for the movement of z-membrane sarcomere for the value of action filament extension delta 1, which is necessary for the process of muscle contraction to be continued.

It has been shown that breaks in Arrhenius plots take place only when the two-stage mechanism of actomyosin ATPase is realized. Removal of two-stage behavior of ATPase leads to the strengthening of Arrhenius plots. It can be achieved by decomposition of two-stage kinetic curves into two one-stage (higher and lower temperature) constituent curves, or by "switching off" ATPase reaction over one of the two one-stage mechanisms by changing KC1 or ATP concentrations. In both cases Arrhenius plots are linear and Ea values, as well as maximal ATPase activity for higher temperature mechanism are twice as much as those for the lower temperature mechanism. On the contrary, non-linear Arrhenius plots show the lowering of Ea with an increase of temperature. We conclude that the breaks in Arrhenius plots can be due to the shift of equilibrium between two one-stage mechanisms of actomyosin ATPase, whose simultaneous course leads to the two-stage kinetics of this reaction.

Study of myosin and actomyosin preparations photocleavage conditioned by polyvanadates confirmed the data on V1 and V2 centre cleavage independence of bivalent cations. Actin does not change sufficiently the reaction in V1 centre and considerably slows down the reaction in V2 centre. These actin properties do not depend on bivalent cation (Mg2+), nor on preliminary incubation with vanadate. It was also discovered that preincubation with vanadate in EDTA medium results in myosin molecule cleavage with producing light (M 18 kD) fragments in both cases: with myosin and actomyosin preparations. Besides vanadate-dependent photocleavage of myosin peptide bonds, there were discovered photocrosslinkings of polypeptide chains in myosin and actomyosin preparations also depending on the presence of vanadate. In actomyosin preparations they probably lead to crosslinking of heavy minor proteins to heavy myosin chains.

A theory of muscle contraction developed by the authors in their earlier papers based on the idea of hydrogen-bond attractive force increase under excitation is modified for the model of myosin head swivel. The hydrogen bonds are supposed to be located at a hinge or near actin-myosin contact. The energy of ATP decay is transferred to the hydrogen bonds whose operation results in myosin head swivel. Equation of actin-chain globules vibrations is considered together with the equation for myosin head swivel by an angle psi approximately pi/4. Solution of the set of equations gives the time dependence of a globule displacement and of the force acting on the transverse z-membrane of the muscle fibre. Here the force and displacement in one act of ATP decay are obtained to be of the correct order of magnitude, F approximately 13 kg per 1 cm2 of muscle section and delta 1-10(-6) cm.

Morphometric and histochemical methods were used to examine the soleus, gastrocnemius (medial portion), quadriceps femoris (central portion) and biceps brachii muscles of Wistar SPF rats two days after the 13-day flight on Cosmos-1887. It was found that significant atrophy developed only in the soleus muscle. The space flight did not change the percentage content of slow (type I) and fast (type II) fibers in fast twitch muscles. During two days at 1 g the slow soleus muscle developed substantial circulation disorders, which led to interstitial edema and necrotic changes. The gastrocnemius muscle showed small foci containing necrotic myofibers. Two days after recovery no glycogen aggregates were seen in myofibers, which were previously observed in other rats examined 4--8 hours after flight. An initial stage of muscle readaptation to 1 g occurred, when NAD.H2-dehydrogenase activity was decreased.