Isolation and chromosomal assignment of a novel human gene, CORO1C, homologous to coronin-like actin-binding proteins

We have isolated a gene, termed CORO1C (human coronin-like actin-binding protein 1C), that encodes a new member of the coronin-like family of proteins. The cDNA consists of 3,857 nucleotides, with an open reading frame of 1,422 bp encoding a 474 amino acid protein. The deduced amino acid sequence shared 65% identity with p57 (human coronin-like actin-binding protein), as well as 46% identity with coronin, a protein first isolated from the slime mold Dictyostelium discoideum. Computer analysis predicted that the product of the CORO1C gene would contain five WD repeats in its N-terminal region and a coiled-coil motif in its C-terminal region, both of which are conserved among coronin-like proteins. CORO1C was ubiquitously expressed in all human tissues examined, in contrast to other known coronin-like molecules, each of which is expressed in a tissue-specific manner. Immunocytochemical staining demonstrated that CORO1C was co-localized with F-actin; therefore, the gene product is likely to be important in cytokinesis, motility, and signal transduction, as are the other members of this molecular family. We assigned this novel gene to chromosome 12q24.1 by fluorescence in situ hybridization.     

The coronin family of actin-associated proteins.

Coronin was first isolated from Dictyostelium, but similar proteins have been identified in many species and individual cell types. The coronin-like protein in yeast promotes actin polymerization and also interacts with microtubules. Dictyostelium mutants lacking coronin are impaired in cytokinesis and all actin-mediated processes. Analysis of coronin-GFP (green-fluorescent protein) fusions and knockout mutants shows that coronin participates in the remodelling of the cortical actin cytoskeleton that is responsible for phagocytosis and macropinocytosis. Likewise, in mammalian neutrophils, a coronin-like protein is also associated with the phagocytic apparatus. The diversity of function in this family of actin-associated proteins is just beginning to be explored.     

A neurally enriched coronin-like protein, ClipinC, is a novel candidate for an actin cytoskeleton-cortical membrane-linking protein

Brain-enriched human FC96 protein shows a close sequence similarity to the Dictyostelium actin-binding protein coronin, which has been implicated in cell motility, cytokinesis, and phagocytosis. A phylogenetic tree analysis revealed that FC96 and two other mammalian molecules (p57 and IR10) form a new protein family, the coronin-like protein (Clipin) family; thus hereafter we refer to FC96 as ClipinC. A WD domain and a succeeding alpha-helical region are conserved among coronin and Clipin family members. ClipinC is predominantly expressed in the brain, and discrete areas in the mouse brain were intensely labeled with anti-ClipinC antibodies. ClipinC was also shown to bind directly to F-actin in vitro. Immunocytochemical analysis revealed that ClipinC accumulated at focal adhesions as well as at neurite tips and stress fibers. Furthermore, ClipinC was associated with vinculin, which is a major component of focal contacts. These results indicate that ClipinC is also a component part of the cross-bridge between the actin cytoskeleton and the plasma membrane. These findings and the previously reported function of coronin suggest that ClipinC may play specific roles in the reorganization of neuronal actin structure, a change that has been implicated in both cell motility and growth cone advance.     

Characterization, cloning and immunolocalization of a coronin homologue in Trichomonas vaginalis

On adhesion to host cells the flagellate Trichomonas vaginalis switches to an amoeboid form rich in actin microfilaments. We have undertaken the identification of actin-associated proteins that regulate actin dynamics. A monoclonal antibody 4C12 raised against a cytoskeletal fraction of T. vaginalis labeled a protein doublet at circa 50 kDa. These two bands were recognized by the antibody against Dictyostelium discoideum coronin. During cell extraction and actin polymerization, T. vaginalis coronin cosedimented with F-actin. By two-dimensional gel electrophoresis, the protein doublet was separated into two sets of isoforms covering two Ip zones around 6 and 7. By screening a T. vaginalis library with 4C12, two clones Cor 1 and Cor 2 were isolated. This gene duplicity is a particularity among unicellular organisms examined. The complete sequence of the gene Cor 1 encodes a 435-residue protein with a calculated molecular mass of 48 kDa and Ip of 5.58. The incomplete sequence Cor 2 was very similar but with a more basic calculated Ip than Cor 1 on the same region. T. vaginalis coronin had 50% similarity with the coronin family, possessing the five WD-repeats and a leucine zipper in its C-terminal part. Double immunofluorescence labeling showed that coronin mainly colocalized with actin at the periphery of the adherent amoeboid cells. However, coronin labeling displayed patches within a reticular array. Immunogold electron microscopy confirmed the coronin labeling in the actin-rich microfilamentous fringe beneath the plasma membrane, with accumulation in phagocytic zones and pseudopodial extensions. In T. vaginalis, one of the first emerging lineage of eukaryotes, coronin seems to play an important role in actin dynamics and may be a downstream target of a signaling mechanism for the cytoskeleton reorganization.     

Isolation and characterization of a cDNA encoding a mammalian cathepsin L-like cysteine proteinase from Acanthamoeba healyi

We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoeba healyi OC-3A strain isolated from the brain of a granulomatous amoebic encephalitis patient. A DNA probe for an A. healyi cDNA library screening was amplified by PCR using degenerate oligonucleotide primers designed on the basis of conserved amino acids franking the active sites of cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteinases. Cysteine proteinase gene of A. healyi (AhCP1) was composed of 330 amino acids with signal sequence, a proposed pro-domain and a predicted active site made up of the catalytic residues. Cys25, His159, and Asn175. Deduced amino acid sequence analysis indicates that AhCP1 belong to ERFNIN subfamily of C1 peptidases. By Northern blot analysis, no direct correlation was observed between AhCP1 mRNA expression and virulence of Acanthamoeba, but the gene was expressed at higher level in amoebae isolated from soil than amoeba from clinical samples. These findings raise the possibility that Ahcp1 protein may play a role in protein metabolism and digestion of phagocytosed bacteria or host tissue debris rather than in invasion of amoebae into host tissue.     

Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility

Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis.     

Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.

A soluble actin binding protein of Dictyostelium discoideum cells has been extracted and purified from precipitated actin-myosin complexes. This protein with a relative molecular mass of 55 kDa has been named coronin because of its association with crown-shaped cell surface projections of growth-phase D. discoideum cells. In aggregating cells, which respond most sensitively to the chemoattractant cyclic AMP, coronin is accumulated at the front where surface projections are directed towards a cAMP source. Since these cells can quickly change shape and polarity, it follows that coronin is rapidly reshuffled within the cells during motion and chemotactic orientation. The cDNA derived sequence of coronin indicates a protein of 49 kDa, consisting of an amino-terminal domain with similarities to the beta subunits of G proteins and a carboxy-terminal domain with a high tendency for alpha-helical structure. It is hypothesized that coronin is implicated in the transmission of chemotactic signals from cAMP receptors in the plasma membrane through G proteins to the cortical cytoskeleton, whose structure and activity is locally modulated.     

Architectural dynamics and gene replacement of coronin suggest its role in cytokinesis

Coronin is a ubiquitous actin-binding protein representing a member of proteins portraying a WD-repeat sequence, including the beta-subunits of trimeric G-proteins. Coronin has been suggested to participate in multiple, actin-based physiological activities such as cell movement and cell division. Although the slow growth of coronin deletion mutants has been attributed to a defect in the fluid-phase uptake of nutrients, the exact role of coronin in cytoskeletal organization has not been elucidated. In this study, we examined a role of coronin in cytokinesis by analyzing the effect of coronin deletion on the actin cytoskeleton and its dynamic distribution using a green fluorescent protein (GFP)-coronin fusion protein. We show that GFP-coronin works similarly to natural coronin in vivo and in vitro. In live cells, GFP-coronin was found to accumulate into the cleavage furrow during cytokinesis. The fluorescence pattern suggests its association to the contractile ring throughout cytokinesis. Interestingly, a substantial amount of coronin was also bound to F-actin at the prospective posterior cortex of the daughter cells. We also show that the coronin null cells reveal irregularities in organization of actin and myosin II and divide by a process identical to the traction-mediated cytofission reported in myosin II mutants. Overall, this study suggests that coronin is essential for organizing the normal actin cytoskeleton and plays a significant role in cell division.     

GRAIL (gene related to anergy in lymphocytes) regulates cytoskeletal reorganization through ubiquitination and degradation of Arp2/3 subunit 5 and coronin 1A

Anergy is an important mechanism for the maintenance of peripheral tolerance and avoidance of autoimmunity. The up-regulation of E3 ubiqitin ligases, including GRAIL (gene related to anergy in lymphocytes), is a key event in the induction and preservation of anergy in T cells. However, the mechanisms of GRAIL-mediated anergy induction are still not completely understood. We examined which proteins serve as substrates for GRAIL in anergic T cells. Arp2/3-5 (actin-related protein 2/3 subunit 5) and coronin 1A were polyubiquitinated by GRAIL via Lys-48 and Lys-63 linkages. In anergic T cells and GRAIL-overexpressed T cells, the expression of Arp2/3-5 and coronin 1A was reduced. Furthermore, we demonstrated that GRAIL impaired lamellipodium formation and reduced the accumulation of F-actin at the immunological synapse. GRAIL functions via the ubiquitination and degradation of actin cytoskeleton-associated proteins, in particular Arp2/3-5 and coronin 1A. These data reveal that GRAIL regulates proteins involved in the actin cytoskeletal organization, thereby maintaining the unresponsive state of anergic T cells.     

A member of the Plasmodium falciparum Pf60 multigene family codes for a nuclear protein expressed by readthrough of an internal stop codon

Four large multigene families have been described in Plasmodium falciparum malaria parasites (var, rif, stevor and Pf60). var and rif genes code for erythrocyte surface proteins and undergo clonal antigenic variation. We report here the characterization of the first Pf60 gene. The 6.1 gene is constitutively expressed by all mature blood stages and codes for a protein located within the nucleus. It has a single copy, 7-exon, 5' domain, separated by an internal stop codon from a 3' domain that presents a high homology with var exon II. Double-site immunoassay and P. falciparum transient transfection using the reporter luciferase gene demonstrated translation through the internal ochre codon. The 6.1 N-terminal domain has no homology with any protein described to date. Sequence analysis identified a leucine zipper and a putative nuclear localization signal and showed a high probability for coiled coils. Evidence for N-terminal coiled coil-mediated protein interactions was obtained. This identifies the 6.1 protein as a novel nuclear protein. These data show that the Pf60 and var genes form a superfamily with a common 3' domain, possibly involved in regulating homo- or heteromeric interactions.     

The platelet-derived growth factor-inducible KC gene encodes a secretory protein related to platelet alpha-granule proteins

A full length cDNA clone of the platelet-derived growth factor-inducible KC gene has been isolated, sequenced, and expressed in COS cells. Both sequence analysis and expression studies indicate that KC encodes a secretory protein. Sequence analysis shows that, furthermore, the protein encoded by KC belongs to a growing superfamily of inducible proteins with a common ancestral linkage to the platelet alpha-granule proteins, platelet factor 4, and connective tissue-activating peptide III. A computer-generated phylogenetic tree documents interrelationships between KC and six additional members of this peptide superfamily. The KC gene is, in all probability, the murine homologue of a human gene termed "gro." By extension, the KC protein is the murine counterpart of the protein encoded by the gro gene. The gro protein corresponds to a factor described as "melanoma growth-stimulating activity."     

Coronin 1 trimerization is essential to protect pathogenic mycobacteria within macrophages from lysosomal delivery

Coronin 1 is a member of the evolutionarily conserved coronin protein family. Coronin proteins are characterized by the presence of a central WD repeat and a C-terminal coiled coil that in coronin 1 is responsible for trimerization. Coronin 1 was identified as a host protein protecting intracellularly residing mycobacteria from degradation by activating the Ca²⁺/calcineurin pathway but whether or not trimerization is essential for this function remains unknown. We here show that trimerization is essential to promote mycobacterial survival within macrophages and activate calcineurin. Furthermore, macrophage activation that induces serine-phosphorylation on coronin 1 resulted in coronin 1 monomerization. These results suggest that modulation of coronin 1 oligomerization is an effective way to determine the outcome of a mycobacterial infection in macrophages.     

Intracellular localization and trafficking of serine proteinase AhSub and cysteine proteinase AhCP of Acanthamoeba healyi

Proteinases have been proposed to play important roles in pathogenesis and various biologic actions in Acanthamoeba. Although genetic characteristics of several proteases of Acanthamoeba have been reported, the intracellular localization and trafficking of these enzymes has yet to be studied. In the present study, we analyzed the intracellular localization and trafficking of two proteinases, AhSub and AhCP, of Acanthamoeba healyi by transient transfection. Full-length AhSub-enhanced green fluorescent protein (EGFP) fusion protein was found in intracellular vesicle-like structures of transfected amoebae. Time-lapse photographs confirmed the secretion of the fluorescent material of the vesicle toward the extracellular space. The mutated AhSub, of which the pre or prepro region was deleted, was found to localize diffusely throughout the cytoplasm of the amoeba rather than concentrated in the secretory vesicle. Transfection of the construct containing the pre region only showed the same localization and trafficking of the full-length AhSub. A cysteine proteinase AhCP-EGFP fusion protein showed similar localization in the vesicle-like structure in the amoeba. However, using Lyso Tracker analysis, these vesicular structures of AhCP were confirmed to be lysosomes rather than secretory vesicles. The AhCP construct with a deletion of the prepro region showed a dispersed distribution of fluorescence in the cytoplasm of the cells. These results indicated that AhSub and AhCP would play different roles in Acanthameoba biology and that the pre region of AhSub and pro region of AhCP are important for proper intracellular localization and trafficking of each proteinase.     

Functional Surfaces on the Actin-binding Protein Coronin Revealed by Systematic Mutagenesis

Coronin is a conserved actin-binding protein that co-functions with ADF/cofilin and Arp2/3 complex to govern cellular actin dynamics. Despite emerging roles for coronin in a range of physiological processes and disease states, a detailed understanding of the molecular interactions of coronin with actin and other binding partners has been lacking. Here, we performed a systematic mutational analysis of surfaces on the yeast coronin β-propeller domain, which binds to F-actin and is conserved in all coronin family members. We generated 21 mutant alleles and analyzed their biochemical effects on actin binding and ADF/cofilin activity. Conserved actin-binding residues mapped to a discrete ridge stretching across one side of the β-propeller. Mutants defective in actin binding showed loss of synergy with ADF/cofilin in severing filaments, diminished localization to actin structures in vivo, and loss of coronin overexpression growth defects. In addition, one allele showed normal actin binding but impaired functional interactions with ADF/cofilin. Another allele showed normal actin binding but failed to cause coronin overexpression defects. Together, these results indicate that actin binding is critical for many of the biochemical and cellular functions of coronin and that the β-propeller domain mediates additional functional interactions with ADF/cofilin and possibly other ligands. Conservation of the actin-binding surfaces across distant species and in all three major classes of coronin isoforms suggests that the nature of the coronin-actin association may be similar in other family members.     

Coronin-1 function is required for phagosome formation

Coronin-1 is an actin-associated protein whose function in actin dynamics has remained obscure. All coronin proteins have a variable N-terminal domain, followed by WD repeats and a C-terminal coiled-coil dimerization domain. Transfection of coronin-1-GFP into RAW 264.7 cells revealed that coronin rapidly and transiently associates with the phagosome. To determine if coronin is involved in mammalian phagocytosis we used a dominant-negative approach by expressing only the central WD domains. However, this caused cell rounding and dissociation from the substratum, hampering analysis of their phenotype. We therefore developed TAT-fusion constructs of coronin-1 WD domains to acutely introduce the recombinant protein fragment into live cells. We show that although TAT-WD has no effect on binding of opsonized RBCs to RAW 264.7 cells, receptor clustering or several downstream signaling events, lamellipodial extensions, and actin accumulation at the base of the bound particle were diminished. Furthermore, Arp3 accumulation at the phagosome was impaired after TAT-WD treatment. Interestingly, whereas coronin-1 also accumulates at the sites of actin remodeling associated with Salmonella invasion, TAT-WD had no effect on this process. Together, our data demonstrates that coronin-1 is required for an early step in phagosome formation, consistent with a role in actin polymerization.     

Migration and homeostasis of naive T cells depends on coronin 1-mediated prosurvival signals and not on coronin 1-dependent filamentous actin modulation

Coronins are WD repeat-containing proteins highly conserved in the eukaryotic kingdom implicated in the regulation of F-actin. Mammalian coronin 1, one of the most conserved isoforms expressed in leukocytes, regulates survival of T cells, which has been suggested to be due to its role in preventing F-actin-induced apoptosis. In this study, we come to a different conclusion. We show that coronin 1 does not modulate F-actin and that induction of F-actin failed to induce apoptosis. Instead, coronin 1 was required for providing prosurvival signals, in the absence of which T cells rapidly underwent apoptosis. These results argue against a role for coronin 1 in F-actin-mediated T cell apoptosis and establish coronin 1 as an essential regulator of the balance between prosurvival and proapoptotic signals in naive T cells.     

Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has effects on cell migration

The ampA gene has a role in cell migration in Dictyostelium discoideum. Cells overexpressing AmpA show an increase in cell migration, forming large plaques on bacterial lawns. A second-site suppressor of this ampA-overexpressing phenotype identified a previously uncharacterized gene, ndm, which is described here. The Ndm protein is predicted to contain a coiled-coil BAR-like domain-a domain involved in endocytosis and membrane bending. ndm-knockout and Ndm-monomeric red fluorescent protein-expressing cell lines were used to establish a role for ndm in suppressing endocytosis. An increase in the rate of endocytosis and in the number of endosomes was detected in ndm(-) cells. During migration ndm(-) cells formed numerous endocytic cups instead of the broad lamellipodia structure characteristic of moving cells. A second lamellipodia-based function-cell spreading-was also defective in the ndm(-) cells. The increase in endocytosis and the defect in lamellipodia formation were associated with reduced chemotaxis in ndm(-) cells. Immunofluorescence results and glutathione S-transferase pull-down assays revealed an association of Ndm with coronin and F-actin. The results establish ndm as a gene important in regulating the balance between formation of endocytic cups and lamellipodia structures.