A gelsolin-like Ca2+-dependent actin-binding domain in villin

Villin is an actin-binding protein of relative molecular mass (Mr) 95,000 found in the core bundle of microfilaments in brush border microvilli from intestine. In physiological calcium concentrations (less than 1 microM), villin crosslinks actin filaments into bundles. However, in free calcium concentrations (greater than 1 microM), villin severs actin filaments into short pieces. To understand how villin can sever and bundle actin filaments, we are studying the molecular basis of villin-actin binding interactions by identifying important actin-binding domains in villin. Here, we report the purification and preliminary characterization of a 44,000-Mr fragment of villin which contains a calcium-dependent actin-severing activity. In addition, the partial amino-acid sequence from the amino terminus of this fragment reveals homology with a 16-residue region near the amino terminus of gelsolin, an actin-severing protein found in many cells and sera. The sequence homology suggests a common structural basis for the calcium-regulated actin-severing properties shared by villin and gelsolin.     

Effect of ATP on actin filament stiffness

Actin is an adenine nucleotide-binding protein and an ATPase. The bound adenine nucleotide stabilizes the protein against denaturation and the ATPase activity, although not required for actin polymerization, affects the kinetics of this assembly Here we provide evidence for another effect of adenine nucleotides. We find that actin filaments made from ATP-containing monomers, the ATPase activity of which hydrolyses ATP to ADP following polymerization, are stiff rods, whereas filaments prepared from ADP-monomers are flexible. ATP exchanges with ADP in such filaments and stiffens them. Because both kinds of actin filaments contain mainly ADP, we suggest the alignment of actin monomers in filaments that have bound and hydrolysed ATP traps them conformationally and stores elastic energy. This energy would be available for release by actin-binding proteins that transduce force or sever actin filaments. These data support earlier proposals that actin is not merely a passive cable, but has an active mechanochemical role in cell function.     

Actin dynamics in the contractile ring during cytokinesis in fission yeast

Cytokinesis in many eukaryotes requires a contractile ring of actin and myosin that cleaves the cell in two. Little is known about how actin filaments and other components assemble into this ring structure and generate force. Here we show that the contractile ring in the fission yeast Schizosaccharomyces pombe is an active site of actin assembly. This actin polymerization activity requires Arp3, the formin Cdc12, profilin and WASP, but not myosin II or IQGAP proteins. Both newly polymerized actin filaments and pre-existing actin cables can contribute to the initial assembly of the ring. Once formed, the ring remains a dynamic structure in which actin and other ring components continuously assemble and disassemble from the ring every minute. The rate of actin polymerization can influence the rate of cleavage. Thus, actin polymerization driven by the Arp2/3 complex and formins is a central process in cytokinesis. Our studies show that cytokinesis is a more dynamic process than previously thought and provide a perspective on the mechanism of cell division.     

Spatial control of actin organization at adherens junctions by a synaptotagmin-like protein Btsz

Epithelial tissues maintain a robust architecture during development. This fundamental property relies on intercellular adhesion through the formation of adherens junctions containing E-cadherin molecules. Localization of E-cadherin is stabilized through a pathway involving the recruitment of actin filaments by E-cadherin. Here we identify an additional pathway that organizes actin filaments in the apical junctional region (AJR) where adherens junctions form in embryonic epithelia. This pathway is controlled by Bitesize (Btsz), a synaptotagmin-like protein that is recruited in the AJR independently of E-cadherin and is required for epithelial stability in Drosophila embryos. On loss of btsz, E-cadherin is recruited normally to the AJR, but is not stabilized properly and actin filaments fail to form a stable continuous network. In the absence of E-cadherin, actin filaments are stable for a longer time than they are in btsz mutants. We identify two polarized cues that localize Btsz: phosphatidylinositol (4,5)-bisphosphate, to which Btsz binds; and Par-3. We show that Btsz binds to the Ezrin-Radixin-Moesin protein Moesin, an F-actin-binding protein that is localized apically and is recruited in the AJR in a btsz-dependent manner. Expression of a dominant-negative form of Ezrin that does not bind F-actin phenocopies the loss of btsz. Thus, our data indicate that, through their interaction, Btsz and Moesin may mediate the proper organization of actin in a local domain, which in turn stabilizes E-cadherin. These results provide a mechanism for the spatial order of actin organization underlying junction stabilization in primary embryonic epithelia.     

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.     

Cross-bridge interaction with oppositely polarized actin filaments in double-overlap zones of insect flight muscle

An unresolved problem in understanding muscular contraction is why the internal resistance to sarcomere shortening increases progressively during contraction. We have addressed this problem here by investigating the movement of detached acting filaments in the sarcomeres of insect flight muscle. The final position of the detached actin filaments shows that they were able to slide freely into regions where they have the wrong polarity to interact actively with myosin (double-overlap zones) but where they prevent the exertion of force by cross-bridges between myosin and the correctly polarized acting filaments. These observations indicate that the isometric tension at all sarcomere lengths is directly proportional to the number of cross-bridges in the region of single-overlap of correctly polarized actin and myosin filaments. The decrease in tension as sarcomeres shorten is thus the result of the decrease in the number of effective cross-bridges as actin filaments slide into regions where they are of the wrong polarity to form cross-bridges, and where they inhibit the existing cross-bridges.     

Polymerization of fluorescent analogue of plant actinin vitro andin vivo

Maize pollen actin has been labeled with Oregon Green 488 iodoacetamide. A yield of 3 mg fluorescent actin analogue has been obtained from 10 mg of maize pollen actin, which is 99% in purity and the dye/protein ratio is 72%. In the presence of Mg²⁺ and K⁺, the fluorescent actin analogue polymerized into filamentsin vitro. Green fluorescent filaments were observed when the fluorescent actin was introduced into living plant cells by microinjection, indicating that the fluorescent actin analogue functions similarly to the native actin.     

Bidirectional movement of actin filaments along tracks of myosin heads

It is well established that muscle contraction results from the relative sliding of actin and myosin filaments. Both filaments have definite polarities and well-ordered structures. Thick filaments, however, are not vital for supporting movement in vitro. Previously we have demonstrated that actin filaments can move continuously on myosin fragments (subfragment-1 or heavy meromyosin (HMM] that are bound to a nitrocellulose surface. Here we report that actin filaments can move in opposite directions on tracks of myosin heads formed when actin filaments decorated with HMM are placed on a nitrocellulose surface. The actin filaments always move forward, frequently changing the direction of the movement, but never move backward reversing the polarity of the movement. The direction of movement is therefore determined by the polarity of the actin filament. These results indicate that myosin heads have considerable flexibility.     

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.     

Reversible stress softening of actin networks

The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.     

In vitro formation of a complex between cytoskeletal proteins of the human erythrocyte.

The formation of a high-molecular weight complex between spectrin and F-actin depends on the presence of a third cytoskeletal constituent, protein 4.1. Electron microscopy shows that in this ternary complex the actin filaments are linked by bridges, which have the appearance of spectrin. The spectrin must be in the tetrameric state for such bridges to form: the dimer is evidently univalent, for it binds but forms no cross-links. G-actin also fails to form extended complexes. It is inferred that in the native cytoskeleton the spectrin is tetrameric and associated with 4.1 and probably oligomers of actin.     

Dependence of the mechanical properties of actin/alpha-actinin gels on deformation rate

The cortical cytoplasm, including the cleavage furrow, is largely composed of a network of actin filaments that is rigid even as it is extensively deformed during cytokinesis. Here we address the question of how actin-filament networks such as those in the cortex can be simultaneously rigid (solid-like) and fluid-like. Conventional explanations are that actin filaments rearrange by some combination of depolymerization and repolymerization; fragmentation and annealing of filaments; and inactivation and reestablishment of crosslinks between filaments. We describe the mechanical properties of a model system consisting of actin filaments and Acanthamoeba alpha-actinin, one of several actin crosslinking proteins found in amoeba and other cells. The results suggest another molecular mechanism that may account for the paradoxical mechanical properties of the cortex. When deformed rapidly, these mixtures are 40 times more rigid than actin filaments without alpha-actinin, but when deformed slowly these mixtures were indistinguishable from filaments alone. These time-dependent mechanical properties can be explained by multiple, rapidly rearranging alpha-actinin crosslinks between the actin filaments, a mechanism proposed by Frey-Wyssling to account for the behaviour of cytoplasm long before the discovery of cytoplasmic actin or alpha-actinin. If other actin-filament crosslinking proteins behave like Acanthamoeba alpha-actinin, this mechanism may explain how the cortex recoils elastically from small rapid insults but deforms extensively when minute forces are applied over long periods of time.     

Redistribution of intermediate filaments during capping of lymphocyte surface molecules

Intermediate filaments (IF) constitute a major cytoplasmic filamentous network of higher eukaryotic cells that is distinct from actin and myosin microfilaments or microtubules. Although structurally similar, these filaments are formed by chemically and antigenically different proteins. Vimentin is the major IF polypeptide of mesenchymal cells and cultured non-mesenchymal cell lines. Recently, we have characterized a monoclonal IgM antibody from a patient with Waldenstr?m's macroglobulinaemia which is directed against vimentin. Using this monoclonal antibody, we have shown by direct immunofluorescence that intermediate filaments of human B and T lymphocytes consist of vimentin. In cells exposed to colcemid, the intermediate filaments retracted into a juxtanuclear aggregate ('coli') characteristic of vimentin filaments. As most components of the cytoskeleton, especially actin and myosin, have been implicated in the capping phenomenon, we investigated the effect of capping of either beta 2-microglobulin or membrane immunoglobulins on the organization of the intermediate filament network. We report that capping of these surface molecules induced the redistribution of vimentin just beneath the cap. When colcemid-treated cells were allowed to cap, the location of the cap always coincided with the coil, suggesting that the anchorage point of intermediate filaments is situated within the uropod.     

The RickA protein of Rickettsia conorii activates the Arp2/3 complex

Actin polymerization, the main driving force for cell locomotion, is also used by the bacteria Listeria and Shigella and vaccinia virus for intracellular and intercellular movements. Seminal studies have shown the key function of the Arp2/3 complex in nucleating actin and generating a branched array of actin filaments during membrane extension and pathogen movement. Arp2/3 requires activation by proteins such as the WASP-family proteins or ActA of Listeria. We previously reported that actin tails of Rickettsia conorii, another intracellular bacterium, unlike those of Listeria, Shigella or vaccinia, are made of long unbranched actin filaments apparently devoid of Arp2/3 (ref. 4). Here we identify a R. conorii surface protein, RickA, that activates Arp2/3 in vitro, although less efficiently than ActA. In infected cells, Arp2/3 is detected on the rickettsial surface but not in actin tails. When expressed in mammalian cells and targeted to the membrane, RickA induces filopodia. Thus RickA-induced actin polymerization, by generating long actin filaments reminiscent of those present in filopodia, has potential as a tool for studying filopodia formation.     

Effects of nitrogen ion implantation on lily pollen germination and the distribution of the actin cytoskeleton during pollen germination

The effects of low energy nitrogen ion implantation on lily (Lilium davidii Duch.) pollen germination and the distribution of the actin cytoskeleton during pollen germination have been studied. Preliminary results showed that the ratio of pollen germination increased from (16.0± 1.6)% to (27.0±2.1)% when implanted with nitrogen ions by 100 keV and a dose of 10¹³ ions/cm². Further experiments were performed by staining the actin filaments in pollen with rhodamine-phalloidin and detected by using laser confocol microscopy. After hydration for 10 h, the actin filaments in ion implanted pollen grains tended to form thick bundles oriented in parallel or ring shape at the germinal furrow, indicating that the effect of nitrogen ion implantation on the germination of pollen might be mediated by reorganization of the actin cytoskeleton.     

Regulation of non-muscle myosin assembly by calmodulin-dependent light chain kinase

The presence of actin and myosin in non-muscle cells suggests that they may be involved in a wide range of cellular contractile activities. The generally accepted view is that interaction between actin and myosin in these cells and in vertebrate smooth muscle, is regulated by the level of phosphorylation of the 20,000-molecular weight (MW) light chain. In the absence of calcium, this light chain is not phosphorylated and the myosin cannot interact with actin. Calcium activates a specific calmodulin-dependent kinase which phosphorylates the light chain, initiating actin-myosin interaction. Although most studies on the role of phosphorylation have concentration on the regulation of actin-activated myosin Mg-ATPase activity, phosphorylation of the light chain also seems to control the assembly of smooth muscle myosin into filaments. Using purified smooth muscle light chain kinase, we have confirmed this observation. We report here studies of myosins isolated from the two non-muscle sources, thymus cells and platelets. We observed that these myosins are assembled into filaments at physiological ionic strength and Mg-ATP concentrations, only when the 20,000-MW light chain is phosphorylated.     

Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate

The actin-binding protein gelsolin requires micromolar concentrations of calcium ions to sever actin filaments, to potentiate its binding to the end of the filament and to promote the polymerization of monomeric actin into filaments. Because transient increases in both intracellular [Ca2+] and actin polymerization accompany the cellular response to certain stimuli, it has been suggested that gelsolin regulates the reversible assembly of actin filaments that accompanies such cellular activations. But other evidence suggests that these activities do not need increased cytoplasmic [Ca2+] and that once actin-gelsolin complexes form in the presence of Ca2+ in vitro, removal of free Ca2+ causes dissociation of only one of two bound actin monomers from gelsolin and the resultant binary complexes cannot sever actin filaments. The finding that cellular gelsolin-actin complexes can be dissociated suggests that a Ca2+-independent regulation of gelsolin also occurs. Here we show that, like the dissociation of profilin-actin complexes, phosphatidylinositol 4,5-bisphosphate, which undergoes rapid turnover during cell stimulation, strongly inhibits the actin filament-severing properties of gelsolin, inhibits less strongly the nucleating ability of this protein and restores the potential for filament-severing activity to gelsolin-actin complexes.     

Actin microfilament dynamics in locomoting cells

The dynamic behaviour of actin filaments has been directly observed in living, motile cells using fluorescence photoactivation. In goldfish epithelial keratocytes, the actin microfilaments in the lamellipodium remain approximately fixed relative to the substrate as the cell moves over them, regardless of cell speed. The rate of turnover of actin subunits in the lamellipodium is remarkably rapid. Cell movement is directly and tightly coupled to the formation of new actin filaments at the leading edge.     

含果蝇肌动蛋白基因的重组棉铃虫核型多角体病毒的构建

本实验利用昆虫杆状病毒表达载体，将果蝇肌动蛋白基因5Cactin克隆入杆状病毒表达载体，以棉铃虫单核衣壳核多角体病毒为亲本病毒，在脂质体介导下共转染棉铃虫细胞，空斑纯化得到重组病毒，以深入研究棉铃虫病毒在入侵、复制及装配过程中，宿主肌动蛋白的动态变化对病毒复制的作用与影响。     

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.