A Coronin7 Homolog with Functions in Actin-driven Processes
2010; Elsevier BV; Volume: 285; Issue: 12 Linguagem: Inglês
10.1074/jbc.m109.083725
ISSN1083-351X
AutoresMaria C. Shina, Can Ünal, Ludwig Eichinger, Annette Müller‐Taubenberger, Michael Schleicher, Michael Steinert, Angelika A. Noegel,
Tópico(s)Cellular transport and secretion
ResumoDictyostelium discoideum Coronin7 (DdCRN7) together with human Coronin7 (CRN7) and Pod-1 of Drosophila melanogaster and Caenorhabditis elegans belong to the coronin family of WD-repeat domain-containing proteins. Coronin7 proteins are characterized by two WD-repeat domains that presumably fold into two β-propeller structures. DdCRN7 shares highest homology with human CRN7, a protein with roles in membrane trafficking. DdCRN7 is present in the cytosol and accumulates in cell surface projections during movement and phago- and pinocytosis. Cells lacking CRN7 have altered chemotaxis and phagocytosis. Furthermore, loss of CRN7 affects the infection process by the pathogen Legionella pneumophila and allows a more efficient internalization of bacteria. To provide a mechanism for CNR7 action, we studied actin-related aspects. We could show that CRN7 binds directly to F-actin and protects actin filaments from depolymerization. CRN7 also associated with F-actin in vivo. It was present in the Triton X-100-insoluble cytoskeleton, colocalized with F-actin, and its distribution was sensitive to drugs affecting the actin cytoskeleton. We propose that the CRN7 role in chemotaxis and phagocytosis is through its effect on the actin cytoskeleton. Dictyostelium discoideum Coronin7 (DdCRN7) together with human Coronin7 (CRN7) and Pod-1 of Drosophila melanogaster and Caenorhabditis elegans belong to the coronin family of WD-repeat domain-containing proteins. Coronin7 proteins are characterized by two WD-repeat domains that presumably fold into two β-propeller structures. DdCRN7 shares highest homology with human CRN7, a protein with roles in membrane trafficking. DdCRN7 is present in the cytosol and accumulates in cell surface projections during movement and phago- and pinocytosis. Cells lacking CRN7 have altered chemotaxis and phagocytosis. Furthermore, loss of CRN7 affects the infection process by the pathogen Legionella pneumophila and allows a more efficient internalization of bacteria. To provide a mechanism for CNR7 action, we studied actin-related aspects. We could show that CRN7 binds directly to F-actin and protects actin filaments from depolymerization. CRN7 also associated with F-actin in vivo. It was present in the Triton X-100-insoluble cytoskeleton, colocalized with F-actin, and its distribution was sensitive to drugs affecting the actin cytoskeleton. We propose that the CRN7 role in chemotaxis and phagocytosis is through its effect on the actin cytoskeleton. IntroductionCoronins are highly conserved proteins of the WD-repeat domain superfamily with representatives in all eukaryotes with the exception of plants and distant protists. Structurally they are characterized by WD (tryptophan-aspartic acid) repeats that form a seven-bladed β-propeller as in the β-subunit of heterotrimeric G-proteins (1.Appleton B.A. Wu P. Wiesmann C. Structure. 2006; 14: 87-96Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) followed by C-terminal extensions and a coiled coil domain that mediates oligomerization (2.Goode B.L. Wong J.J. Butty A.C. Peter M. McCormack A.L. Yates J.R. Drubin D.G. Barnes G. J. Cell Biol. 1999; 144: 83-98Crossref PubMed Scopus (189) Google Scholar). The propeller structures differ in the residues exposed on the surface of the propeller, making them suitable interaction sites for different proteins.Coronins have been associated with the control of actin-dependent processes, F-actin assembly, and the regulation of the Arp2/3 complex but also with signal transduction processes, transcriptional regulation, and regulation of vesicular trafficking. The first coronin (corA) was isolated from Dictyostelium discoideum as a major co-purifying protein in a preparation of contracted actomyosin. F-actin association and its sequence relation to the β-subunit of heterotrimeric G-proteins implicated a role for coronin as an important regulator of the actin cytoskeleton. Subsequently, the analysis of coronin-deficient cells revealed roles in cell division, cell migration, and phagocytosis (3.de Hostos E.L. Bradtke B. Lottspeich F. Guggenheim R. Gerisch G. EMBO J. 1991; 10: 4097-4104Crossref PubMed Scopus (242) Google Scholar, 4.de Hostos E.L. Rehfuess C. Bradtke B. Waddell D.R. Albrecht R. Murphy J. Gerisch G. J. Cell Biol. 1993; 120: 163-173Crossref PubMed Scopus (205) Google Scholar, 5.Maniak M. Rauchenberger R. Albrecht R. Murphy J. Gerisch G. Cell. 1995; 83: 915-924Abstract Full Text PDF PubMed Scopus (307) Google Scholar). CoroninA is also involved in the uptake of pathogenic bacteria such as Legionella pneumophila and Mycobacterium marinum (6.Fajardo M. Schleicher M. Noegel A. Bozzaro S. Killinger S. Heuner K. Hacker J. Steinert M. Microbiology. 2004; 150: 2825-2835Crossref PubMed Scopus (57) Google Scholar, 7.Solomon J.M. Rupper A. Cardelli J.A. Isberg R.R. Infect. Immun. 2000; 68: 2939-2947Crossref PubMed Scopus (177) Google Scholar, 8.Solomon J.M. Leung G.S. Isberg R.R. Infect. Immun. 2003; 71: 3578-3586Crossref PubMed Scopus (101) Google Scholar).In addition to coronin proteins composed of one WD-repeat domain, most eukaryotes express a long form of coronin proteins designated Pod or CRN7 2The abbreviations used are: CRN7coronin7GFPgreen fluorescent proteinGSTglutathione S-transferasem.o.i.multiplicity of infectionPSTproline-, serine-, and threonine-richTRITCtetramethylrhodamine isothiocyanatePIPES1,4-piperazinediethanesulfonic acidmAbmonoclonal antibody. that contain two coronin repeats in tandem. They presumably adopt a structure similar to the one of Caenorhabditis elegans actin-interacting protein 1 (Aip1) and Saccharomyces cerevisiae Aip1 that contains two WD-repeat domains (9.Mohri K. Vorobiev S. Fedorov A.A. Almo S.C. Ono S. J. Biol. Chem. 2004; 279: 31697-31707Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 10.Voegtli W.C. Madrona A.Y. Wilson D.K. J. Biol. Chem. 2003; 278: 34373-34379Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). All long coronins lack the C-terminal coiled coil segments; instead, they contain short acidic domains.Pod proteins have been studied in Drosophila melanogaster, C. elegans, and man. The name Pod stands for polarization-defective and was derived from C. elegans mutants that suffer from a mislocalization of Par proteins important for establishing polarity in the one cell stage. C. elegans Pod-1 is polarized along the anterior-posterior axis of the one cell embryo and provides a link to F-actin. Embryos lacking Pod-1 have also physiological defects like osmotic sensitivity, abnormal endocytic vesicles, membrane protrusions, and thinner eggshells (11.Rappleye C.A. Paredez A.R. Smith C.W. McDonald K.L. Aroian R.V. Genes Dev. 1999; 13: 2838-2851Crossref PubMed Scopus (109) Google Scholar, 12.Tagawa A. Rappleye C.A. Aroian R.V. Dev. Biol. 2001; 233: 412-424Crossref PubMed Scopus (33) Google Scholar). D. melanogaster Pod-1 mutants show misguidance of growth cones in the nervous system of the fly where Pod-1 is predominantly present. Furthermore, it cross-links actin filaments and microtubules in vitro (13.Rothenberg M.E. Rogers S.L. Vale R.D. Jan L.Y. Jan Y.N. Neuron. 2003; 39: 779-791Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).Whereas Pod proteins in lower eukaryotes participate primarily in cytoskeleton-associated processes, the mammalian homolog associates with Golgi membranes, is responsible for Golgi morphology, and affects trafficking processes as revealed by small interfering RNA knockdown. CRN7 co-precipitates with the clathrin adaptor AP-1 through a tyrosine-based motif (Tyr-288) that interacts with the μ1-subunit of AP-1. For targeting to membranes, tyrosine phosphorylation of CRN7 is required, which most likely depends on src kinase, because the broad-range tyrosine kinase inhibitor genistein and the specific Src inhibitor SU6656 led to a redistribution of CRN7 to the cytosol (14.Rybakin V. Stumpf M. Schulze A. Majoul I.V. Noegel A.A. Hasse A. FEBS Lett. 2004; 573: 161-167Crossref PubMed Scopus (49) Google Scholar, 15.Rybakin V. Gounko N.V. Späte K. Höning S. Majoul I.V. Duden R. Noegel A.A. J. Biol. Chem. 2006; 281: 31070-31078Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 16.Rybakin V. Rastetter R.H. Stumpf M. Uetrecht A.C. Bear J.E. Noegel A.A. Clemen C.S. Cell. Mol. Life Sci. 2008; 65: 2419-2430Crossref PubMed Scopus (14) Google Scholar).We used D. discoideum to study the role of CRN7 in a lower eukaryote and to reveal the primordial function of CRN7 proteins. D. discoideum is a single-celled amoeba that lives on the soil feeding on bacteria and yeast. It is a professional phagocyte using chemotaxis to find its prey and phagocytosis to take up its food source. It shares these properties with neutrophils and has been used as a convenient model system to explore the underlying mechanisms (17.Jin T. Xu X. Fang J. Isik N. Yan J. Brzostowski J.A. Hereld D. Immunol. Res. 2009; 43: 118-127Crossref PubMed Scopus (43) Google Scholar, 18.Stephens L. Milne L. Hawkins P. Curr. Biol. 2008; 18: R485-R494Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Chemotaxis is also used to organize multicellular development. From an evolutionary point of view Dictyostelium deviated from the animal fungal lineage after the plant animal split (19.Eichinger L. Pachebat J.A. Glöckner G. Rajandream M.A. Sucgang R. Berriman M. Song J. Olsen R. Szafranski K. Xu Q. Tunggal B. Kummerfeld S. Madera M. Konfortov B.A. Rivero F. Bankier A.T. Lehmann R. Hamlin N. Davies R. Gaudet P. Fey P. Pilcher K. Chen G. Saunders D. Sodergren E. Davis P. Kerhornou A. Nie X. Hall N. Anjard C. Hemphill L. Bason N. Farbrother P. Desany B. Just E. Morio T. Rost R. Churcher C. Cooper J. Haydock S. van Driessche N. Cronin A. Goodhead I. Muzny D. Mourier T. Pain A. Lu M. Harper D. Lindsay R. Hauser H. James K. Quiles M. Madan Babu M. Saito T. Buchrieser C. Wardroper A. Felder M. Thangavelu M. Johnson D. Knights A. Loulseged H. Mungall K. Oliver K. Price C. Quail M.A. Urushihara H. Hernandez J. Rabbinowitsch E. Steffen D. Sanders M. Ma J. Kohara Y. Sharp S. Simmonds M. Spiegler S. Tivey A. Sugano S. White B. Walker D. Woodward J. Winckler T. Tanaka Y. Shaulsky G. Schleicher M. Weinstock G. Rosenthal A. Cox E.C. Chisholm R.L. Gibbs R. Loomis W.F. Platzer M. Kay R.R. Williams J. Dear P.H. Noegel A.A. Barrell B. Kuspa A. Nature. 2005; 435: 43-57Crossref PubMed Scopus (999) Google Scholar). However, despite this early divergence, it shares many more genes with animals than do fungi. Throughout the text we will use the recently proposed nomenclature for coronin proteins (20.Morgan R.O. Fernandez M.P. Subcell. Biochem. 2008; 48: 41-55Crossref PubMed Scopus (20) Google Scholar).DISCUSSIONCoronin7 proteins seem to have acquired different roles during evolution. For the C. elegans and D. melanogaster proteins, a function in cell polarity has been identified presumably mediated through its role in organizing specific aspects of the actin cytoskeleton, whereas human CRN7 mainly functions in trafficking processes in the Golgi apparatus (14.Rybakin V. Stumpf M. Schulze A. Majoul I.V. Noegel A.A. Hasse A. FEBS Lett. 2004; 573: 161-167Crossref PubMed Scopus (49) Google Scholar). Our studies in D. discoideum for the first time also address an F-actin interaction of a Coronin7 protein in vitro in detail.Cell fractionation studies of Dictyostelium lysates supported this interaction. They revealed that the majority of CRN7 is present in the cytosolic fraction as a monomer (data not shown). When we separated cytoskeletal from soluble fractions, CRN7 distributed between both fractions, and subcellular localization studies of endogenous and GFP-tagged CRN7 indicated a co-localization with the actin cytoskeleton and in particular with F-actin-rich structures that are formed around pino- and phagosomes. We identified the first WD-repeat domain as being responsible for this distribution using GFP-tagged fusion proteins. The results suggested the presence of an F-actin binding region that was confirmed by in vitro studies. The second WD-repeat domain did not participate in the F-actin association. For human CRN1 an actin binding site has been mapped to the first WD repeat, where a surface-exposed arginine residue at position 30 appears to be crucial as the F-actin binding capability in vitro is strongly reduced upon mutation of this arginine residue to an aspartate (50.Cai L. Makhov A.M. Bear J.E. J. Cell Sci. 2007; 120: 1779-1790Crossref PubMed Scopus (84) Google Scholar). This residue and its surroundings are well conserved in mammalian and invertebrate small coronins. In DdCRN7 the arginine residue is replaced by an asparagine at position 26 with the surrounding sequence from amino acids 17 to 40 being well conserved. In Drosophila, C. elegans, and mammalian CRN7 this sequence is not conserved. The second WD-repeat domain of DdCRN7 does not harbor a region of homology. However, the suggestion that this amino acid stretch represents the F-actin binding site has to be taken with caution as recent studies indicate that a more extended region of CRN1 is in contact with actin molecules in the filament (49.Galkin V.E. Orlova A. Brieher W. Kueh H.Y. Mitchison T.J. Egelman E.H. J. Mol. Biol. 2008; 376: 607-613Crossref PubMed Scopus (43) Google Scholar).Disruption of the corB gene resulted in several phenotypic alterations. Although the corB− cells displayed no significant alterations in motility and polarization when we analyzed their behavior in a cAMP gradient, pseudopod extension initially occurred in a more random fashion. Instable pseudopods may result from a less stable actin network. Our findings that CRN7 associates with the Triton-insoluble cytoskeleton after a cAMP stimulus support such a notion. We propose that CRN7 is involved in actin dynamics during formation and extension of pseudopods and temporally associates with filaments that are formed and stabilizes them.When we followed GFP-CRN7 in vivo we noticed a highly dynamic behavior during cell migration where the protein constantly relocated to the leading pseudopod. A similar dynamic behavior was observed during uptake of yeast particles and Legionella. In both instances CRN7 accumulated at the forming phagocytic cup and then quickly dispersed into the cytosol. An involvement of CRN7 in these processes was confirmed when we carried out quantitative analysis of uptake in corB− cells. Loss of CRN7 led to an increased uptake of yeast cells and Legionella, whereas uptake of latex beads was reduced. Growth rates on E. coli B/r and Klebsiella were not affected, pointing to an unimpaired uptake of these bacterial species.Data from previous work show that different mechanisms are employed during uptake of particles that differ in size and surface properties and that functionally independent binding sites for bacteria and other particles like yeast or latex beads exist on the cell surface of D. discoideum (51.Thilo L. Vogel G. Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 1015-1019Crossref PubMed Scopus (78) Google Scholar, 52.Vogel G. Thilo L. Schwarz H. Steinhart R. J. Cell Biol. 1980; 86: 456-465Crossref PubMed Scopus (125) Google Scholar). For example, E. coli cells lacking glucose residues on their surface were not recognized by a presumably lectin-type receptor and were not taken up (52.Vogel G. Thilo L. Schwarz H. Steinhart R. J. Cell Biol. 1980; 86: 456-465Crossref PubMed Scopus (125) Google Scholar). Mutants in Phg1p, a nine-transmembrane protein, caused a defect in phagocytosis of latex beads and E. coli but not of K. aerogenes (53.Cornillon S. Pech E. Benghezal M. Ravanel K. Gaynor E. Letourneur F. Brückert F. Cosson P. J. Biol. Chem. 2000; 275: 34287-34292Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), and a SadA mutant, which lacks another nine-transmembrane cell adhesion protein, did not take up latex beads (54.Fey P. Stephens S. Titus M.A. Chisholm R.L. J. Cell Biol. 2002; 159: 1109-1119Crossref PubMed Scopus (91) Google Scholar). These reports show that differential adhesion will affect uptake of particles.In these mutants the phagocytosis defect is frequently associated with reduced cell substrate adhesion, whereas in the CRN7-deficient cells we observed an increased adhesion to plastic surfaces and a decreased uptake of latex beads, which also have a hydrophilic surface. It is difficult to reconcile these results; however, the CRN7 effects might be due to its regulation of the actin cytoskeleton. Mutants with increased surface adhesion have not been tested for phagocytosis (55.Jeon T.J. Lee D.J. Merlot S. Weeks G. Firtel R.A. J. Cell Biol. 2007; 176: 1021-1033Crossref PubMed Scopus (90) Google Scholar).Several mutants with defects in cytoskeletal proteins also have altered adhesion properties (56.Niewöhner J. Weber I. Maniak M. Müller-Taubenberger A. Gerisch G. J. Cell Biol. 1997; 138: 349-361Crossref PubMed Scopus (125) Google Scholar). We can envision that alterations in the cortical actin cytoskeleton brought about by a loss of CRN7 will affect the composition of the plasma membrane, thereby affecting adhesion. We also conclude from our in vivo analysis that CRN7 is actively involved in the uptake of particles, forming a coat that then quickly disassembles once the particle is ingested. Interestingly, in yeast and L. pneumophila uptake CRN7 suppresses the phagocytosis process, whereas for ingestion of E. coli or Klebsiella it is not a relevant factor.Mechanisms that regulate the phagocytosis process play an important role in the host defense against invading microorganisms, and several studies have addressed the relationship between coronins and the intracellular survival of pathogens (57.Yan M. Collins R.F. Grinstein S. Trimble W.S. Mol. Biol. Cell. 2005; 16: 3077-3087Crossref PubMed Scopus (71) Google Scholar). TACO, the CRN4 homolog in mice, was found to be important for the lysosomal delivery of phagosomes containing Mycobacterium bovis. Here TACO remained associated with phagosomes containing live M. bovis, preventing phagosomes from fusing with lysosomes and allowing the mycobacteria to survive (58.Ferrari G. Langen H. Naito M. Pieters J. Cell. 1999; 97: 435-447Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 59.Jayachandran R. Gatfield J. Massner J. Albrecht I. Zanolari B. Pieters J. Mol. Biol. Cell. 2008; 19: 1241-1251Crossref PubMed Scopus (48) Google Scholar). Also D. discoideum coronin (GFP-CRN12) was observed at the phagocytic cup upon internalization of L. pneumophila but was lost 60 s after internalization (8.Solomon J.M. Leung G.S. Isberg R.R. Infect. Immun. 2003; 71: 3578-3586Crossref PubMed Scopus (101) Google Scholar).Up to now no long coronin has been implicated in innate immunity against natural pathogens as all previous studies involved exclusively small coronin proteins. We show that GFP-CRN7-overexpressing cells and corB− cells display a strong converse phenotype upon infection with L. pneumophila. Whereas GFP-CRN7-expressing cells display a reduced internalization and an increased replication rate of L pneumophila, corB− is more susceptible to internalization but restrains the replication rate to the one of Ax2. Additionally, internalization of L. pneumophila in corB− is comparable with the increase of the phagocytosis rate in experiments with TRITC-labeled yeast cells. We propose that CRN7 plays a role in regulating internalization of these particles in a repressive manner. Our experiments reveal that the loss of CRN7 renders Dictyostelium more susceptible to L. pneumophila infection, making this protein an important component of the innate immunity system of Dictyostelium.The mechanism for the CRN7 role in uptake processes and in chemotaxis is most likely provided by its impact on the dynamics of the actin cytoskeleton. CRN7 normally will prevent depolymerization of filaments and might allow formation and stabilization of distinct F-actin structures that are built, for example, at a phagosome or in an extending pseudopod.Comparing the activities of the long coronins, we conclude that CRN7 proteins from lower eukaryotes function in actin-regulated processes. In the case of Drosophila Pod-1, an additional function by cross-linking actin filaments and microtubules has been acquired, and for C. elegans CRN7 (POD-1), a role in regulation of trafficking processes by a so far unknown mechanism has been shown. The mammalian CRN7 has diverged farthest in terms of function. It is both a cytosolic and a membrane-associated protein and has roles in membrane trafficking. It will be interesting to see whether it also acts in innate immunity and whether its huge cytosolic pool might interact with F-actin as well. IntroductionCoronins are highly conserved proteins of the WD-repeat domain superfamily with representatives in all eukaryotes with the exception of plants and distant protists. Structurally they are characterized by WD (tryptophan-aspartic acid) repeats that form a seven-bladed β-propeller as in the β-subunit of heterotrimeric G-proteins (1.Appleton B.A. Wu P. Wiesmann C. Structure. 2006; 14: 87-96Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) followed by C-terminal extensions and a coiled coil domain that mediates oligomerization (2.Goode B.L. Wong J.J. Butty A.C. Peter M. McCormack A.L. Yates J.R. Drubin D.G. Barnes G. J. Cell Biol. 1999; 144: 83-98Crossref PubMed Scopus (189) Google Scholar). The propeller structures differ in the residues exposed on the surface of the propeller, making them suitable interaction sites for different proteins.Coronins have been associated with the control of actin-dependent processes, F-actin assembly, and the regulation of the Arp2/3 complex but also with signal transduction processes, transcriptional regulation, and regulation of vesicular trafficking. The first coronin (corA) was isolated from Dictyostelium discoideum as a major co-purifying protein in a preparation of contracted actomyosin. F-actin association and its sequence relation to the β-subunit of heterotrimeric G-proteins implicated a role for coronin as an important regulator of the actin cytoskeleton. Subsequently, the analysis of coronin-deficient cells revealed roles in cell division, cell migration, and phagocytosis (3.de Hostos E.L. Bradtke B. Lottspeich F. Guggenheim R. Gerisch G. EMBO J. 1991; 10: 4097-4104Crossref PubMed Scopus (242) Google Scholar, 4.de Hostos E.L. Rehfuess C. Bradtke B. Waddell D.R. Albrecht R. Murphy J. Gerisch G. J. Cell Biol. 1993; 120: 163-173Crossref PubMed Scopus (205) Google Scholar, 5.Maniak M. Rauchenberger R. Albrecht R. Murphy J. Gerisch G. Cell. 1995; 83: 915-924Abstract Full Text PDF PubMed Scopus (307) Google Scholar). CoroninA is also involved in the uptake of pathogenic bacteria such as Legionella pneumophila and Mycobacterium marinum (6.Fajardo M. Schleicher M. Noegel A. Bozzaro S. Killinger S. Heuner K. Hacker J. Steinert M. Microbiology. 2004; 150: 2825-2835Crossref PubMed Scopus (57) Google Scholar, 7.Solomon J.M. Rupper A. Cardelli J.A. Isberg R.R. Infect. Immun. 2000; 68: 2939-2947Crossref PubMed Scopus (177) Google Scholar, 8.Solomon J.M. Leung G.S. Isberg R.R. Infect. Immun. 2003; 71: 3578-3586Crossref PubMed Scopus (101) Google Scholar).In addition to coronin proteins composed of one WD-repeat domain, most eukaryotes express a long form of coronin proteins designated Pod or CRN7 2The abbreviations used are: CRN7coronin7GFPgreen fluorescent proteinGSTglutathione S-transferasem.o.i.multiplicity of infectionPSTproline-, serine-, and threonine-richTRITCtetramethylrhodamine isothiocyanatePIPES1,4-piperazinediethanesulfonic acidmAbmonoclonal antibody. that contain two coronin repeats in tandem. They presumably adopt a structure similar to the one of Caenorhabditis elegans actin-interacting protein 1 (Aip1) and Saccharomyces cerevisiae Aip1 that contains two WD-repeat domains (9.Mohri K. Vorobiev S. Fedorov A.A. Almo S.C. Ono S. J. Biol. Chem. 2004; 279: 31697-31707Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 10.Voegtli W.C. Madrona A.Y. Wilson D.K. J. Biol. Chem. 2003; 278: 34373-34379Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). All long coronins lack the C-terminal coiled coil segments; instead, they contain short acidic domains.Pod proteins have been studied in Drosophila melanogaster, C. elegans, and man. The name Pod stands for polarization-defective and was derived from C. elegans mutants that suffer from a mislocalization of Par proteins important for establishing polarity in the one cell stage. C. elegans Pod-1 is polarized along the anterior-posterior axis of the one cell embryo and provides a link to F-actin. Embryos lacking Pod-1 have also physiological defects like osmotic sensitivity, abnormal endocytic vesicles, membrane protrusions, and thinner eggshells (11.Rappleye C.A. Paredez A.R. Smith C.W. McDonald K.L. Aroian R.V. Genes Dev. 1999; 13: 2838-2851Crossref PubMed Scopus (109) Google Scholar, 12.Tagawa A. Rappleye C.A. Aroian R.V. Dev. Biol. 2001; 233: 412-424Crossref PubMed Scopus (33) Google Scholar). D. melanogaster Pod-1 mutants show misguidance of growth cones in the nervous system of the fly where Pod-1 is predominantly present. Furthermore, it cross-links actin filaments and microtubules in vitro (13.Rothenberg M.E. Rogers S.L. Vale R.D. Jan L.Y. Jan Y.N. Neuron. 2003; 39: 779-791Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).Whereas Pod proteins in lower eukaryotes participate primarily in cytoskeleton-associated processes, the mammalian homolog associates with Golgi membranes, is responsible for Golgi morphology, and affects trafficking processes as revealed by small interfering RNA knockdown. CRN7 co-precipitates with the clathrin adaptor AP-1 through a tyrosine-based motif (Tyr-288) that interacts with the μ1-subunit of AP-1. For targeting to membranes, tyrosine phosphorylation of CRN7 is required, which most likely depends on src kinase, because the broad-range tyrosine kinase inhibitor genistein and the specific Src inhibitor SU6656 led to a redistribution of CRN7 to the cytosol (14.Rybakin V. Stumpf M. Schulze A. Majoul I.V. Noegel A.A. Hasse A. FEBS Lett. 2004; 573: 161-167Crossref PubMed Scopus (49) Google Scholar, 15.Rybakin V. Gounko N.V. Späte K. Höning S. Majoul I.V. Duden R. Noegel A.A. J. Biol. Chem. 2006; 281: 31070-31078Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 16.Rybakin V. Rastetter R.H. Stumpf M. Uetrecht A.C. Bear J.E. Noegel A.A. Clemen C.S. Cell. Mol. Life Sci. 2008; 65: 2419-2430Crossref PubMed Scopus (14) Google Scholar).We used D. discoideum to study the role of CRN7 in a lower eukaryote and to reveal the primordial function of CRN7 proteins. D. discoideum is a single-celled amoeba that lives on the soil feeding on bacteria and yeast. It is a professional phagocyte using chemotaxis to find its prey and phagocytosis to take up its food source. It shares these properties with neutrophils and has been used as a convenient model system to explore the underlying mechanisms (17.Jin T. Xu X. Fang J. Isik N. Yan J. Brzostowski J.A. Hereld D. Immunol. Res. 2009; 43: 118-127Crossref PubMed Scopus (43) Google Scholar, 18.Stephens L. Milne L. Hawkins P. Curr. Biol. 2008; 18: R485-R494Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Chemotaxis is also used to organize multicellular development. From an evolutionary point of view Dictyostelium deviated from the animal fungal lineage after the plant animal split (19.Eichinger L. Pachebat J.A. Glöckner G. Rajandream M.A. Sucgang R. Berriman M. Song J. Olsen R. Szafranski K. Xu Q. Tunggal B. Kummerfeld S. Madera M. Konfortov B.A. Rivero F. Bankier A.T. Lehmann R. Hamlin N. Davies R. Gaudet P. Fey P. Pilcher K. Chen G. Saunders D. Sodergren E. Davis P. Kerhornou A. Nie X. Hall N. Anjard C. Hemphill L. Bason N. Farbrother P. Desany B. Just E. Morio T. Rost R. Churcher C. Cooper J. Haydock S. van Driessche N. Cronin A. Goodhead I. Muzny D. Mourier T. Pain A. Lu M. Harper D. Lindsay R. Hauser H. James K. Quiles M. Madan Babu M. Saito T. Buchrieser C. Wardroper A. Felder M. Thangavelu M. Johnson D. Knights A. Loulseged H. Mungall K. Oliver K. Price C. Quail M.A. Urushihara H. Hernandez J. Rabbinowitsch E. Steffen D. Sanders M. Ma J. Kohara Y. Sharp S. Simmonds M. Spiegler S. Tivey A. Sugano S. White B. Walker D. Woodward J. Winckler T. Tanaka Y. Shaulsky G. Schleicher M. Weinstock G. Rosenthal A. Cox E.C. Chisholm R.L. Gibbs R. Loomis W.F. Platzer M. Kay R.R. Williams J. Dear P.H. Noegel A.A. Barrell B. Kuspa A. Nature. 2005; 435: 43-57Crossref PubMed Scopus (999) Google Scholar). However, despite this early divergence, it shares many more genes with animals than do fungi. Throughout the text we will use the recently proposed nomenclature for coronin proteins (20.Morgan R.O. Fernandez M.P. Subcell. Biochem. 2008; 48: 41-55Crossref PubMed Scopus (20) Google Scholar).
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