The actin system forms a supramolecular, membrane-associated network that serves multiple functions in Dictyostelium cells, including cell motility controlled by chemoattractant, phagocytosis, macropinocytosis, and cytokinesis. In executing these functions the monomeric G-actin polymerizes reversibly, and the actin filaments are assembled into membrane-anchored networks together with other proteins involved in shaping the networks and controlling their dynamics. Most impressive is the speed at which actin-based structures are built, reorganized, or disassembled. GFP-tagged coronin and Arp3, an intrinsic constituent of the Arp2/3 complex, were used as examples of proteins that are recruited to highly dynamic actin-filament networks. By fluorescence recovery after photobleaching (FRAP), average exchange rates of cell-cortex bound coronin were estimated. A nominal value of 5 s for half-maximal incorporation of coronin into the cortex, and a value of 7 s for half-maximal dissociation from cortical binding sites has been obtained. Actin dynamics implies also flow of F-actin from sites of polymerization to sites of depolymerization, i.e. to the tail of a migrating cell, the base of a phagocytic cup, and the cleavage furrow in a mitotic cell. To monitor this flow, in Dictyostelium cells, a GFP-tagged actin-binding fragment of talin was expressed. This fragment (GFP-TalC63) translocates from the front to the tail during cell migration and from the polar regions to the cleavage furrow during mitotic cell division. The intrinsic dynamics of the actin system can be manipulated in vivo by drugs or other probes that act either as inhibitors of actin polymerization or as stabilizers of filamentous actin. In order to investigate structure-function relationships in the actin system, a technique of reliably arresting transient network structures is required. The potential of electron tomography of vitrified cells to visualize actin networks in their native association with membranes is discussed (Bretschneider, 2002).
Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, actin and a homolog of coronin, Crn1p, were co-isolated from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 x 10-9 M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Delta deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Delta cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast (Goode, 1999).
Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. A novel mode of Arp2/3 complex regulation has been identified, mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin (Humphries, 2002).
Establishment of anterior-posterior (a-p) polarity in the Caenorhabditis elegans embryo depends on filamentous (F-) actin. An F-actin-binding protein has been isolated that is enriched in the anterior cortex of the one-cell embryo and is hypothesized to link developmental polarity to the actin cytoskeleton. This protein, POD-1, is a new member of the coronin family of actin-binding proteins. A deletion within the pod-1 gene has been generated. Elimination of POD-1 from early embryos results in a loss of physical and molecular asymmetries along the a-p axis. For example, PAR-1 and PAR-3, which themselves are polarized and required for a-p polarity, are delocalized in pod-1 mutant embryos. However, unlike loss of PAR proteins, loss of POD-1 gives rise to the formation of abnormal cellular structures, namely large vesicles of endocytic origin, membrane protrusions, unstable cell divisions, a defective eggshell, and deposition of extracellular material. It is concluded that, analogous to coronin, POD-1 plays an important role in intracellular trafficking and organizing specific aspects of the actin cytoskeleton. Models are proposed to explain how the role of POD-1 in basic cellular processes could be linked to the generation of polarity along the embryonic a-p axis (Rappleye, 1999).
The asymmetric division of the one-cell Caenorhabditis elegans zygote gives rise to two cells of different size and fate, thereby establishing the animal's a-p axis. A number of genes required for this polarity have been characterized, but many components remain unidentified. A mutation in the pod-1 gene (for polarity and osmotic defective) uniquely perturbs polarity and osmotic protection. A new C. elegans polarity gene identified has been identified from screens for conditional embryonic lethals. Embryos in which this gene has been mutated show a loss of physical and developmental asymmetries in the one-cell embryo, including the mislocalization of PAR and POD-1 proteins required for early polarity. Furthermore, mutant embryos are osmotically sensitive, allowing this gene to be designated pod-2. Thus, pod-2, along with pod-1, defines a new class of C. elegans polarity genes. Genetic analyses indicates that pod-2 functions in the same pathway as pod-1. Temperature-shift studies indicate that pod-2 is required during oogenesis, indicating that aspects of embryonic polarization may precede fertilization. pod-2 mutant embryos also exhibit a unique germline inheritance defect in which germline identity localizes to the wrong spot in the one-cell embryo and is therefore inherited by the wrong cell at the four-cell stage. These data suggest that pod-2 may be required to properly position an a-p polarity cue (Tagawa, 2001).
Coronin is an actin-binding protein, which contains WD (Trp-Asp) repeats and a coiled-coil motif, and plays a role in regulating organization of the actin cytoskeletal network. Coronin localizes to the cell periphery, is involved in lamellipodium extension, and has an implicated role in cytokinesis, cell motility and phagocytosis. Experiments with two different tagged forms of Xenopus coronin (Xcoronin) have shown that Xcoronin forms an oligomer. This oligomer complex is stable, resistant to 2.4 M NaCl, 0.6 M KI or 2 M urea. Physiochemical analysis of endogenous Xcoronin and the protein expressed in COS7 cells or in bacteria has revealed that the oligomer complex is an Xcoronin dimer. A C-terminal coiled-coil motif of Xcoronin is necessary and sufficient for the dimerization. Mutations in the coiled-coil motif generate dimerization deficient mutants of Xcoronin. Moreover, these mutant forms of Xcoronin fail to localize to the cell periphery, suggesting that dimerization is important for the proper subcellular localization of Xcoronin. It is concluded that Xcoronin forms a stable dimer via its C-terminal coiled-coil region. It is proposed that coronin dimerization is necessary for its proper subcellular localization and function (Asano, 2001).
Coronin 3 is a ubiquitously expressed member of the coronin protein family in mammals. In fibroblasts and HEK 293 cells, it is localized both in the cytosol and in the submembranous cytoskeleton, especially at lamellipodia and membrane ruffles. The carboxyl terminus of all coronins contains a coiled coil suggested to mediate dimerization. In contrast to other coronin homologs, the recombinant human coronin 3 carboxyl terminus forms oligomers rather than dimers, and this part is sufficient to bind to and cross-link F-actin in vitro. The carboxyl terminus alone also confers membrane association in vivo, and removal of the coiled coil abolishes membrane localization but not in vitro F-actin binding. Coronin 3 is exclusively extracted as an oligomer from both the cytosol and the membrane fraction. Because oligomerization has not reported for other coronins, it might be a key feature governing coronin 3-specific functions. Cytosolic coronin 3 shows a high degree of phosphorylation, which is likely to regulate the subcellular localization of the protein (Spoerl, 2002).
Coronin is a WD repeat-containing actin-binding protein, which was originally identified in the cellular slime mold Dictyostelium. Coronin-null Dictyostelium cells show defects in cytokinesis, cell motility and phagocytosis. Although the existence of coronin in higher eukaryotes has been reported, its function in vertebrate cells has not been elucidated. A Xenopus homolog of coronin (Xcoronin) has been cloned and its actin-binding properties, subcellular localization and possible functions were examined. Xcoronin consists of 480 amino acids and is 63% identical to human coronin (p57). Bacterially expressed recombinant Xcoronin co-sediments with F-actin in vitro. The WD repeat domain (residues 64-299) alone does not have any affinity for F-actin. Anti-Xcoronin antibodies react specifically with a single 57 kDa protein present in an extract of the Xenopus A6 cell line. Indirect immunofluorescent staining of A6 cells has revealed that Xcoronin is present in the cytoplasm and concentrates in the cell periphery in membrane ruffles. During spreading after replating or wound healing after scratching a confluent monolayer, Xcoronin becomes concentrated in the leading edges of lamellipodia. A GFP-fusion protein of Xcoronin showed a subcellular distribution essentially identical to endogenous Xcoronin. The localization of Xcoronin to the cell periphery is resistant to treatment with 0.1% Triton X-100. The deletion of 63 N-terminal amino acids or of 65 C-terminal amino acids abolishes the localization of Xcoronin to the cell periphery. Xcoronin expressed in 3T3 fibroblasts is concentrated to the leading edges of lamellipodia induced by active Rac. Remarkably, expression of a truncated form of Xcoronin (64-299), but not of full-length Xcoronin, significantly decreases the rate of cell spreading after replating and markedly inhibits lamellipodium extension induced by active Rac. These results suggest that Xcoronin plays an important role in lamellipodium extension and cell spreading (Mashima, 1999).
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