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.     

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.     

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.     

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.     

Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants

Most mitochondrial proteins are encoded by nuclear genes and are synthesized as precursors containing a presequence at the N terminus. In yeast and in mammalian cells, the function of the presequence in mitochondrial targeting has been revealed by chimaeric gene studies. Fusion of a mitochondrial presequence to a foreign protein coding sequence enables the protein to be imported into mitochondria in vitro as well as in vivo. Whether plant mitochondrial presequences function in the same way has been unknown. We have previously isolated and characterized a nuclear gene (atp2-1) from Nicotiana plumbaginifolia that encodes the beta-subunit of the mitochondrial ATP synthase. We have constructed a chimaeric gene comprising a putative atp2-1 presequence fused to the bacterial chloramphenicol acetyltransferase (CAT) coding sequence and introduced it into the tobacco genome. We report here that a segment of 90 amino acids of the N terminus of the beta-subunit precursor is sufficient for the specific targeting of the CAT protein to mitochondria in transgenic plants. Our results demonstrate a high specificity for organelle targeting in plant cells.     

New yeast actin-like gene required late in the cell cycle.

Actin, a major cytoskeletal component of all eukaryotic cells, is one of the most highly conserved proteins. It is involved in various cellular processes such as motility, cytoplasmic streaming, chromosome segregation and cytokinesis. The actin from the yeast Saccharomyces cerevisiae, encoded by the essential ACT1 gene, is 89% identical to mouse cytoplasmic actin and is involved in the organization and polarized growth of the cell surface. We report here the characterization of ACT2, a previously undescribed yeast split gene encoding a putative protein (391 amino acids, relative molecular mass (Mr) 44,073) that is 47% identical to yeast actin. The requirement of the ACT2 gene for vegetative growth of yeast cells and the existence of related genes in other eukaryotes indicate an important and conserved role for these actin-like proteins. Superimposition of the Act2 polypeptide onto the three-dimensional structure of known actins reveals that most of the divergence occurred in loops involved in actin polymerization, DNase I and myosin binding, leaving the core domain mainly unaffected. To our knowledge, the Act2 protein from S. cerevisiae is the first highly divergent actin molecule described. Structural and physiological data suggest that the Act2 protein might have an important role in cytoskeletal reorganization during the cell cycle.     

Requirement of yeast fimbrin for actin organization and morphogenesis in vivo

The SAC6 gene was found by suppression of a yeast actin mutation. Its protein product, Sac6p (previously referred to as ABP67), was independently isolated by actin-filament affinity chromatography and colocalizes with actin in vivo. Thus Sac6p binds to actin in vitro, and functionally associates with actin structures involved in the development and maintenance of cell polarity in vivo. We report here that Sac6p is an actin-filament bundling protein 43% identical in amino-acid sequence to the vertebrate bundling protein fimbrin. This yeast fimbrin homologue contains two putative actin-binding regions homologous to domains of dystrophin, beta-spectrin, filamin, actin-gelation protein and alpha-actinin. Mutants lacking Sac6p do not form normal actin structures and are defective in morphogenesis. These findings demonstrate an in vivo role for the well-documented biochemical interaction between fimbrin and actin.     

Reconstitution of actin-based motility of Listeria and Shigella using pure proteins.

Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, alpha-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.     

Structural insights into yeast septin organization from polarized fluorescence microscopy

Septins are polymerizing GTPases that function in cortical organization and cell division. In Saccharomyces cerevisiae they localize at the isthmus between the mother and the daughter cells, where they undergo a transition from a non-dynamic hourglass-shaped assembly to two separate rings, at the onset of cytokinesis. Septins form filaments as pure protein and in vivo, but the filament organization within the hourglass and ring structures is controversial. Here, we use polarized fluorescence microscopy of orientationally constrained green fluorescent protein to determine septin filament organization and dynamics in living yeast. We found that the hourglass is made of filaments aligned along the yeast bud neck. During the transition from hourglass to rings the filaments rotate through 90 degrees in the membrane plane and become circumferential. These data resolve a long-standing controversy in the field and provide strong evidence that septins have a mechanical function in cell division.     

Abolition of actin-bundling by phosphorylation of human erythrocyte protein 4.9

Protein 4.9, first identified as a component of the human erythrocyte membrane skeleton, binds to and bundles actin filaments. Protein 4.9 is a substrate for various kinases, including a cyclic AMP(cAMP)-dependent one, in vivo and in vitro. We show here that phosphorylation of protein 4.9 by the catalytic subunit of cAMP-dependent protein kinase reversibly abolishes its actin-bundling activity, but phosphorylation by protein kinase C has no such effect. A quantitative immunoassay showed that human erythrocytes contain 43,000 trimers of protein 4.9 per cell, which is equivalent to one trimer for each actin oligomer in these red blood cells. As analogues of protein 4.9 have been identified together with analogues of other erythroid skeletal proteins in non-erythroid tissues of numerous vertebrates, phosphorylation and dephosphorylation of protein 4.9 may be the basis for a mechanism that regulates actin bundling in many cells.     

A test for intron function in the yeast actin gene

Many eukaryotic genes contain intervening sequences (IVS), but the rationale for their existence remains a mystery. Previous studies done in our laboratory demonstrated that the intron in a yeast tRNATyr gene, SUP6, does have a function. We used the same approach to determine the role of introns in nuclear genes encoding messenger RNAs. A single actin gene with one intron exists in Saccharomyces cerevisiae. The level of actin in yeast appears to be crucial to viability: either too much or too little actin inhibits growth. Therefore, small effects on synthesis of actin protein resulting from the removal of the actin gene intron would be expected to cause measurable changes in cell growth. In the present study, an intron-deleted actin gene was constructed in vitro and was used to replace the single resident actin gene in a haploid strain. Analysis of the cells carrying the intron-deleted actin gene shows that the intervening sequence is not essential for actin gene expression.     

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.     

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.     

细胞不对称分裂机制--芽殖酵母接合型不对称转换

芽列酵母的母细胞与子细胞呈不对称接合型转换，其原因是只有母细胞可表达编码核酸内切酶的基因HO，使相反接合型的缄默基因转位到活动位点取代了原来的接合型基因。HO的不对称表达是因在细胞分裂的末期至G1早期，子细胞核中存在有Ashlp转录抑制因子。Ashlp的不对称分布是由其mRNA的定向转运而实现的：ASH1 mRNA在有丝分裂期被转录出之后，通过接头蛋白She2p和She3p与肌球蛋白Myo4p结合成核糖核蛋白颗粒，经肌动蛋白纤维转运到子细胞远端皮层而锚定并翻译。     

Regulation of glutamate receptor binding by the cytoskeletal protein fodrin

The erythrocyte cytoskeleton, which consists primarily of a meshwork of spectrin and actin, controls cell shape and the disposition of proteins within the membrane. Proteins similar to spectrin have recently been found in diverse cells and tissues, and it is possible that they mediate the capping of cell-surface receptors, although this has not been demonstrated directly. In neurones, the spectrin-like protein fodrin lines the cortical cytoplasm and may link actin filaments to the membrane. Fodrin has been hypothesized to regulate the number of receptor binding sites on neuronal membranes for the putative neurotransmitter L-glutamate. Micromolar calcium concentrations activate the thiol protease calpain I, induce fodrin degradation and more than double the density of glutamate binding sites; these effects are all blocked by thiol protease inhibitors. We have now used specific antibodies to examine further the role of fodrin proteolysis in regulating glutamate receptors. We report that fodrin antibodies block the fodrin degradation and increase in glutamate binding normally induced by calcium, and so provide direct evidence for control of membrane receptors by a non-erythroid spectrin.     

Expression of a cotton profilin gene GhPFN1 is associated with fiber cell elongation

Profilin is an important actin-binding protein involved in regulating the organization of actin filaments. A cotton profilin gene (GhPFN1) that shares 71% identity to profilin1 of Arabidopsis in its amino acid sequence was isolated. Semi-quantitative RT-PCR showed that GhPFN1 was expressed preferentially in the developing cotton fibers and reached the highest level at the fast elongation stage. The function of GhPFN1 in vivo was analyzed using the S. pombe system, and results suggested that GhPFN1 plays a role in fiber cell elongation.     

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.     

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.     

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.