肌动蛋白的聚合动力学研究进展

肌动蛋白的聚合和解聚动力学过程与其功能的行使有密不可分的关系:肌动蛋白如要在细胞内行使其功能就一定涉及到其聚合动力学过程.肌动蛋白的聚合过程可分为4个步骤:肌动蛋白单体的活化;肌动蛋白单体聚合成核;肌动蛋白纤维生长的过程;聚合达到动态平衡,肌动蛋白纤维不再生长.一些影响肌动蛋白聚合过程的因素,比如,核酸和肌动蛋白相关蛋白也在文中做了讨论.其目的在于更深入地了解生物大分子如何组装成更复杂的体系以及这些体系在细胞中怎么行使功能.     

Formation of reverse rigor chevrons by myosin heads

The uniform angle and conformation of myosin subfragment 1 (S1) bound to actin filaments (F-actin) attest to the precise alignment and stereospecificity of the binding of these two contractile proteins. Because actin filaments are polar, myosin heads must swing or rotate about the head-tail junction in order to bind. Electron microscopy of isolated thick filaments and of myosin molecules suggests that the molecules are flexible, but myosin fragments and crossbridges have been reported not to interact with inappropriately oriented actin filaments. Here we describe myofibrillar defects engendered by a site-directed mutation within the flight-muscle-specific actin gene of the fruitfly Drosophila. The mutation apparently retards sarcomere assembly: peripheral thick and thin filaments are misregistered and not incorporated into the Z-line. Therefore, a myosin filament encounters thin filaments with the 'wrong' polarity. We show that myosin heads tethered in a single thick filament can bind with opposite rigor crossbridge angles to flanking thin filaments, which are apparently of opposite polarities. Preservation of identical actomyosin interfaces requires that sets of heads originating from opposite sides of the thick filament swivel 180 degrees relative to each other, implying that myosin crossbridges are as flexible as isolated molecules.     

Force measurements by micromanipulation of a single actin filament by glass needles

Single actin filaments (approximately 7 nm in diameter) labelled with fluorescent phalloidin can be clearly seen by video-fluorescence microscopy. This technique has been used to observe motions of single filaments in solution and in several in vitro movement assays. In a further development of the technique, we report here a method to catch and manipulate a single actin filament (F-actin) by glass microneedles under conditions in which external force on the filament can be applied and measured. Using this method, we directly measured the tensile strength of a filament (the force necessary to break the bond between two actin monomers) and the force required for a filament to be moved by myosin or its proteolytic fragment bound to a glass surface in the presence of ATP. The first result shows that the tensile strength of the F-actin-phalloidin complex is comparable with the average force exerted on a single thin filament in muscle fibres during isometric contraction. This force is increased only slightly by tropomyosin. The second measurement shows that the myosin head (subfragment-1) can produce the same ATP-dependent force as intact myosin. The magnitude of this force is comparable with that produced by each head of myosin in muscle during isometric contraction.     

Structure of the actin-myosin interface

The topography of the rigor complex between F-actin and myosin heads (S1) has been investigated by carbodiimide zero-length cross-linking. The results demonstrate for the first time that the 95,000-molecular weight (95K) heavy chain of the myosin head enters into van der Waals contact with two neighbouring actin monomers; one is bound to the 50K domain and the other to the 20K domain of the myosin chain. The covalent F-actin-S1 complex can be isolated; it shows a vastly elevated Mg2+-ATPase. Each pair of actin subunits in the thin filament seems to act as a functional unit for specific binding of a myosin head and stimulation of its Mg2+-ATPase activity.     

Tying a molecular knot with optical tweezers.

Filamentous structures are abundant in cells. Relatively rigid filaments, such as microtubules and actin, serve as intracellular scaffolds that support movement and force, and their mechanical properties are crucial to their function in the cell. Some aspects of the behaviour of DNA, meanwhile, depend critically on its flexibility-for example, DNA-binding proteins can induce sharp bends in the helix. The mechanical characterization of such filaments has generally been conducted without controlling the filament shape, by the observation of thermal motions or of the response to external forces or flows. Controlled buckling of a microtubule has been reported, but the analysis of the buckled shape was complicated. Here we report the continuous control of the radius of curvature of a molecular strand by tying a knot in it, using optical tweezers to manipulate the strand's ends. We find that actin filaments break at the knot when the knot diameter falls below 0.4 microm. The pulling force at breakage is around 1 pN, two orders of magnitude smaller than the tensile stress of a straight filament. The flexural rigidity of the filament remained unchanged down to this diameter. We have also knotted a single DNA molecule, opening up the possibility of studying curvature-dependent interactions with associated proteins. We find that the knotted DNA is stronger than actin.     

Novel form of growth cone motility involving site-directed actin filament assembly.

Regulation of cytoskeletal structure and motility by extracellular signals is essential for all directed forms of cell movement and underlies the developmental process of axonal guidance in neuronal growth cones. Interaction with polycationic microbeads can trigger morphogenic changes in neurons and muscle cells normally associated with formation of pre- and postsynaptic specializations. Furthermore, when various types of microscopic particles are applied to the lamellar surface of a neuronal growth cone or motile cell they often exhibit retrograde movement at rates of 1-6 microns min-1 (refs 3-6). There is strong evidence that this form of particle movement results from translocation of membrane proteins associated with cortical F-actin networks, not from bulk retrograde lipid flow and may be a mechanism behind processes such as cell locomotion, growth cone migration and capping of cell-surface antigens. Here we report a new form of motility stimulated by polycationic bead interactions with the growth-cone membrane surface. Bead binding rapidly induces intracellular actin filament assembly, coincident with a production of force sufficient to drive bead movements. These extracellular bead movements resemble intracellular movements of bacterial parasites known to redirect host cell F-actin assembly for propulsion. Our results suggest that site-directed actin filament assembly may be a widespread cellular mechanism for generating force at membrane-cytoskeletal interfaces.     

Drosophila Spire is an actin nucleation factor

The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factors--the Arp2/3 complex and the formin family of proteins--that nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.     

Prokaryotic origin of the actin cytoskeleton.

It was thought until recently that bacteria lack the actin or tubulin filament networks that organize eukaryotic cytoplasm. However, we show here that the bacterial MreB protein assembles into filaments with a subunit repeat similar to that of F-actin-the physiological polymer of eukaryotic actin. By elucidating the MreB crystal structure we demonstrate that MreB and actin are very similar in three dimensions. Moreover, the crystals contain protofilaments, allowing visualization of actin-like strands at atomic resolution. The structure of the MreB protofilament is in remarkably good agreement with the model for F-actin, showing that the proteins assemble in identical orientations. The actin-like properties of MreB explain the finding that MreB forms large fibrous spirals under the cell membrane of rod-shaped cells, where they are involved in cell-shape determination. Thus, prokaryotes are now known to possess homologues both of tubulin, namely FtsZ, and of actin.     

Molecular structure of F-actin and location of surface binding sites

Comparisons of three-dimensional maps of vertebrate muscle thin filaments obtained by cryo-electron microscopy and image analysis, reveal the molecular structure of F-actin, the location of the C terminus of the monomer and the positions of the binding sites of tropomyosin, the myosin head and the N-terminal portion of the myosin A1 light chain on the filament. These data provide strong constraints for evaluating models built from the atomic structure of the monomer and the subsequent identification of molecular contacts.     

Cytochalasin D does not produce net depolymerization of actin filaments in HEp-2 cells

The altered morphology, disappearance or 'disruption' of actin filaments (microfilaments) in cells treated with cytochalasin has sometimes been attributed to depolymerization of filamentous actin (F-actin) to its globular subunit (G-actin), but attempts to confirm that mechanism have been inconclusive. Treatment of purified actin filaments with cytochalasin B (CB) decreased their viscosity, consistent with depolymerization, which was not, however, revealed by electron microscopy, although the filaments appeared abnormal. CB also increased the ATP-ase activity of F-actin, suggesting that it had been destabilized, while actin filaments in the acrosomal process were not depolymerized. CB or cytochalasin D (CD) can dissolve actin gels (reviewed in ref. 7, see also refs 8 and 9) without depolymerizing their filaments. The 'disrupted' actin structures in CD-treated cells bound heavy meromysin, indicating that at least some of the cellular actin was filamentous. Using a rapid assay for G- and F-actin in cell extracts, based on the inhibition of DNase I, we have found that neither short-nor long-term exposure of HEp-2 cells to CD produce net depolymerization of actin filaments.     

Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain

The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. Here we report the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine-actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus. Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation. Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain. Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function.     

Direct observation of motion of single F-actin filaments in the presence of myosin

Actin is found in almost all kinds of non-muscle cells where it is thought to have an important role in cell motility. A proper understanding of that role will only be possible when reliable in vitro systems are available for investigating the interaction of cellular actin and myosin. A start has been made on several systems, most recently by Sheetz and Spudich who demonstrated unidirectional movement of HMM-coated beads along F-actin cables on arrays of chloroplasts exposed by dissection of a Nitella cell. As an alternative approach, we report here the direct observation by fluorescence microscopy of the movements of single F-actin filaments interacting with soluble myosin fragments energized by Mg2+-ATP.     

Expression ofChlamydomonas actin-gfp fusion gene in tobacco suspension cell and polymerization of the actin-gfp proteinin vitro

The fusion gene of actin (cDNA ofChlamydomonas reinhardtii) and green fluorescence protein (gfp) had been constructed into two expression vectors which could be expressed inE. coli and tobacco suspension cells BY2. The correct expression was observed inE. coli and BY2 with a fluorescence microscopy. The fusion protein, which took part in the membrane skeleton, was mainly located peripherally along the membrane, specially the fusion protein was distributed around nucleus and cell plate, while the fusion protein also forms F-actin in the cell. The fusion protein was purified from Bl21plus by ammonium sulfate fractionation, ion exchange chromatography and hydrophobic interaction chromatography. The purified production could polymerize into F-actin when the actin polymerizing buffer was added. It was demonstrated that the characteristics and function of actin inChlamydomonas was similar with those of animals and higher plants.     

Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle

Muscle contraction results from a sliding movement of actin filaments induced by myosin crossbridges on hydrolysis of ATP, and many non-muscle cells are thought to move using a similar mechanism. The molecular mechanism of muscle contraction, however, is not completely understood. One of the major problems is the mechanochemical coupling at high velocity under near-zero load. Here, we report measurements of the sliding distance of an actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle in an unloaded condition. We used single sarcomeres from which the Z-lines, structures which anchor the thin filaments in the sarcomere, had been completely removed by calcium-activated neutral protease (CANP) and trypsin, and measured both the sliding velocity of single actin filaments along myosin filaments and the ATPase activity during sliding. Our results show that the average sliding distance of the actin filament is less than or equal to 600 A during one ATP cycle, much longer than the length of power stroke of myosin crossbridges deduced from mechanical studies of muscle, which is of the order of 80 A (for example, ref. 15).     

Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins

Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.     

Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases

Rab5 is a small GTPase involved in the control of intracellular trafficking, both at the level of receptor endocytosis and endosomal dynamics. The finding that Rab5 can be activated by receptor tyrosine kinases (RTK) raised the question of whether it also participates in effector pathways emanating from these receptors. Here we show that Rab5 is indispensable for a form of RTK-induced actin remodelling, called circular ruffling. Three independent signals, originating from Rab5, phosphatidylinositol-3-OH kinase and Rac, respectively, are simultaneously required for the induction of circular ruffles. Rab5 signals to the actin cytoskeleton through RN-tre, a previously identified Rab5-specific GTPase-activating protein (GAP). Here we demonstrate that RN-tre has the dual function of Rab5-GAP and Rab5 effector. We also show that RN-tre is critical for macropinocytosis, a process previously connected to the formation of circular ruffles. Finally, RN-tre interacts with both F-actin and actinin-4, an F-actin bundling protein. We propose that RN-tre establishes a three-pronged connection with Rab5, F-actin and actinin-4. This may aid crosslinking of actin fibres into actin networks at the plasma membrane. Thus, we have shown that Rab5 is a signalling GTPase and have elucidated the major molecular elements of its downstream pathway.     

Structure of the acrosomal bundle

In the unactivated Limulus sperm, a 60- micro m-long bundle of actin filaments crosslinked by the protein scruin is bent and twisted into a coil around the base of the nucleus. At fertilization, the bundle uncoils and fully extends in five seconds to support a finger of membrane known as the acrosomal process. This biological spring is powered by stored elastic energy and does not require the action of motor proteins or actin polymerization. In a 9.5-A electron cryomicroscopic structure of the extended bundle, we show that twist, tilt and rotation of actin-scruin subunits deviate widely from a 'standard' F-actin filament. This variability in structural organization allows filaments to pack into a highly ordered and rigid bundle in the extended state and suggests a mechanism for storing and releasing energy between coiled and extended states without disassembly.     

Rapid regeneration of the actin-myosin power stroke in contracting muscle.

At the molecular level, muscle contraction is the result of cyclic interaction between myosin crossbridges, which extend from the thick filament, and the thin filament, which consists mainly of actin. The energy for work done by a single crossbridge during a cycle of attachment, generation of force, shortening and detachment is believed to be coupled to the hydrolysis of one molecule of ATP. The distance the actin filament slides relative to the myosin filament in one crossbridge cycle has been estimated as 12 nm by step-length perturbation studies on single fibres from frog muscle. The 'mechanical' power stroke of the attached crossbridge can therefore be defined as 12-nm shortening with a force profile like that shown by the quick recovery of force following a length perturbation. According to this definition, power strokes cannot be repeated faster than the overall ATPase rate. Here, however, we show that the power stroke can be regenerated much faster than expected from the ATPase rate. This contradiction can be resolved if, in the shortening muscle, the free energy of ATP hydrolysis is used in several actin-myosin interactions consisting of elementary power strokes each of 5-10 nm.     

Intensification of the 5.9-nm actin layer line in contracting muscle

According to the cross-bridge model of muscle contraction, an interaction of myosin heads with interdigitating actin filaments produces tension. Although X-ray equatorial diffraction patterns of active (contracting) muscle show that the heads are in the vicinity of the actin filaments, structural proof of actual attachment of heads to actin during contraction has been elusive. We show here that during contraction of frog skeletal muscle, the 5.9-nm layer line arising from the genetic helix of actin is intensified by as much as 56% of the change which occurs when muscle enters rigor, using a two-dimensional X-ray detector. This provides strong structural evidence that myosin heads do in fact attach during contraction.