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Richard Wetherbee - One of the best experts on this subject based on the ideXlab platform.
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Cytoskeletal dynamics of Apedinella radians (pedinellophyceae). III: Post-division development, maintenance of cell symmetry, and the re-establishment of interphase morphology
Protoplasma, 1993Co-Authors: Anthony Koutoulis, Richard WetherbeeAbstract:The dynamics of the cytoskeletal proteins Centrin, actin, and tubulin were investigated during post-division development in the radially symmetrical phytoflagellateApedinella radians (Pedinellophyceae). Each daughter cell inherits a triangular arrangement of Centrin filamentous bundles that develops, during post-division, into the six-pointed star configuration observed at interphase. This coincides with developmental processes including plaque duplication and migration, chloroplast division and migration, and spine-scale deployment. Centrin filamentous bundles appear to be involved in maintaining radial symmetry throughout the cell cycle and re-establishing interphase morphology. Actin filamentous bundles, prominent at interphase, depolymerize just prior to mitosis and do not reform until late post-division, indicating they are not involved in maintaining cell symmetry during cell division. Although the precise dynamics of microtubular triads and their associated cylindrical caps has not been determined, they may work in concert with Centrin filamentous bundles in re-establishing interphase morphology. Three Centrin, or Centrin-like, components inA. radians appear to coordinate independent architectural events during the cell cycle. The nature of the three Centrin components is discussed and compared to the flagellar roots/pericentriolar material of the eukaryotic centrosome.
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Cytoskeletal dynamics ofApedinella radians (Pedinellophyceae) II. Cell division and maintenance of cell polarity and symmetry
Protoplasma, 1993Co-Authors: Anthony Koutoulis, Richard WetherbeeAbstract:The dynamics of the cytoskeletal proteins Centrin, actin, and tubulin were followed during cell division in the unicellular phytoflagellate Apedinella radians (Pedinellophyceae). Three Centrin, or Centrin-like, components appear to coordinate independent developmental events during cell division. The first component, basal body Centrin, maintains a physical link between basal bodies and the anterior nuclear membrane. Basal body Centrin divides in two at metaphase, and each portion segregates with two basal bodies at anaphase. As the positioning of basal bodies defines the anterior region of the cell, basal body Centrin appears to play a role in maintaining cell polarity throughout the cell cycle. The second Centrin component consists of an array of filamentous bundles arranged as a six-pointed star. During cell division, the star undergoes a conformational change resulting in two distinct Centrin triangles, one distributed to each daughter cell, suggesting that Centrin filamentous bundles are involved in maintaining cell (radial) symmetry. The third Centrin component is transient and associates with the spindle poles, emerging prior to mitosis and remaining until late anaphase/early telophase. Spindle pole Centrin establishes temporary horizontal bipolarity, thereby establishing the spindle axis. Unlike Centrin filamentous bundles, actin filamentous bundles depolymerize prior to mitosis, indicating they do not influence cell symmetry during cell division. Mitosis is described for the first time in a pedinellid and features a closed spindle, the absence of rhizoplasts and a persistent spindle.
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Cytoskeletal dynamics ofApedinella radians (Pedinellophyceae) III. Post-division development, maintenance of cell symmetry, and the re-establishment of interphase morphology
Protoplasma, 1993Co-Authors: Anthony Koutoulis, Richard WetherbeeAbstract:The dynamics of the cytoskeletal proteins Centrin, actin, and tubulin were investigated during post-division development in the radially symmetrical phytoflagellate Apedinella radians (Pedinellophyceae). Each daughter cell inherits a triangular arrangement of Centrin filamentous bundles that develops, during post-division, into the six-pointed star configuration observed at interphase. This coincides with developmental processes including plaque duplication and migration, chloroplast division and migration, and spine-scale deployment. Centrin filamentous bundles appear to be involved in maintaining radial symmetry throughout the cell cycle and re-establishing interphase morphology. Actin filamentous bundles, prominent at interphase, depolymerize just prior to mitosis and do not reform until late post-division, indicating they are not involved in maintaining cell symmetry during cell division. Although the precise dynamics of microtubular triads and their associated cylindrical caps has not been determined, they may work in concert with Centrin filamentous bundles in re-establishing interphase morphology. Three Centrin, or Centrin-like, components in A. radians appear to coordinate independent architectural events during the cell cycle. The nature of the three Centrin components is discussed and compared to the flagellar roots/pericentriolar material of the eukaryotic centrosome.
John V Kilmartin - One of the best experts on this subject based on the ideXlab platform.
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Structural role of Sfi1p–Centrin filaments in budding yeast spindle pole body duplication
The Journal of cell biology, 2006Co-Authors: Alan M Sandercock, Paul T Conduit, Carol V Robinson, Roger Williams, John V KilmartinAbstract:Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae Centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p–Centrin complexes containing several repeats show Sfi1p as an α helix with Centrins wrapped around each repeat and similar Centrin–Centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p–Centrin complex with 15 Sfi1 repeats and 15 Centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous α helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p–Centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly.
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structural role of sfi1p Centrin filaments in budding yeast spindle pole body duplication
Journal of Cell Biology, 2006Co-Authors: Alan M Sandercock, Paul T Conduit, Carol V Robinson, Roger Williams, John V KilmartinAbstract:Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae Centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p–Centrin complexes containing several repeats show Sfi1p as an α helix with Centrins wrapped around each repeat and similar Centrin–Centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p–Centrin complex with 15 Sfi1 repeats and 15 Centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous α helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p–Centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly.
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sfi1p has conserved Centrin binding sites and an essential function in budding yeast spindle pole body duplication
Journal of Cell Biology, 2003Co-Authors: John V KilmartinAbstract:Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae Centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds Centrin in the repeat region, showing that this Centrin-binding motif is conserved.
Jeffrey L. Salisbury - One of the best experts on this subject based on the ideXlab platform.
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Control of Centrin Stability by Aurora A
PloS one, 2011Co-Authors: Kara B. Lukasiewicz, Jeffrey L. Salisbury, Tammy M. Greenwood, Vivian Negron, Amy K Bruzek, Wilma L. LingleAbstract:Aurora A is an oncogenic serine/threonine kinase which can cause cell transformation and centrosome amplification when over-expressed. Human breast tumors show excess Aurora A and phospho-Centrin in amplified centrosomes. Here, we show that Aurora A mediates the phosphorylation of and localizes with Centrin at the centrosome, with both proteins reaching maximum abundance from prophase through metaphase, followed by their precipitous loss in late stages of mitosis. Over-expression of Aurora A results in excess phospho-Centrin and centrosome amplification. In contrast, centrosome amplification is not seen in cells over-expressing Aurora A in the presence of a recombinant Centrin mutant lacking the serine phosphorylation site at residue 170. Expression of a kinase dead Aurora A results in a decrease in mitotic index and abrogation of Centrin phosphorylation. Finally, a recombinant Centrin mutation that mimics Centrin phosphorylation increases Centrin's stability against APC/C-mediated proteasomal degradation. Taken together, these results suggest that the stability of Centrin is regulated in part by Aurora A, and that excess phosphorylated Centrin may promote centrosome amplification in cancer.
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the structure of the human Centrin 2 xeroderma pigmentosum group c protein complex
Journal of Biological Chemistry, 2006Co-Authors: James R Thompson, Zachary C Ryan, Jeffrey L. Salisbury, Rajiv KumarAbstract:Abstract Human Centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human Centrin-2 and to develop an understanding of molecular interactions between Centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length Centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of Centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered α-helical linker. A stretch of the amino-terminal domain unique to Centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to Centrin-2 also interact to form an α-helical coiled-coil. The interface between Centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for Centrin.
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Centrin gene disruption impairs stage specific basal body duplication and cell cycle progression in leishmania
Journal of Biological Chemistry, 2004Co-Authors: Angamuthu Selvapandiyan, Jeffrey L. Salisbury, Robert Duncan, Alain Debrabant, G. Sreenivas, Poonam Salotra, Jacqueline Muller, Hira L. NakhasiAbstract:Centrin is a calcium-binding cytoskeletal protein involved in the duplication of centrosomes in higher eukaryotes. To explore the role of Centrin in the protozoan parasite Leishmania, we created Leishmania deficient in the Centrin gene (LdCEN). Remarkably, Centrin null mutants (LdCEN(-/-)) showed selective growth arrest as axenic amastigotes but not as promastigotes. Flow cytometry analysis confirmed that the mutant axenic amastigotes have a cell cycle arrest at the G(2)/M stage. The axenic amastigotes also showed failure of basal body duplication and failure of cytokinesis resulting in multinucleated "large" cells. Increased terminal deoxy uridine triphosphate nick end labeling positivity was observed in Centrin mutant axenic amastigotes compared with wild type cells, suggesting the activation of a programmed cell death pathway. Growth of LdCEN(-/-) amastigotes in infected macrophages in vitro was inhibited and also resulted in large multinucleated parasites. Normal basal body duplication and cell division in the LdCEN knockout promastigote is unique and surprising. Further, this is the first report where disruption of a Centrin gene displays stage-specific/cell type-specific failure in cell division in a eukaryote. The Centrin null mutant defective in amastigote growth could be useful as a vaccine candidate against leishmaniasis.
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gfp Centrin as a marker for centriole dynamics in the human breast cancer cell line mcf 7
Italian journal of anatomy and embryology, 2001Co-Authors: A B Dassoro, F Stivala, Susan L Barrett, G Ferrigno, Jeffrey L. SalisburyAbstract:Centrosome duplication plays an important role in genomic stability through bipolar spindle formation and equal chromosome segregation during mitosis. Defects in centrosome duplication and centrosome amplification correlate with aggressive tumors and aneuploidy. Cyclin-dependent cell cycle regulators play a key role in signaling centrosome duplication and the tumor suppressor genes p53, BRCA1 and BRCA2 are suspected to function at mitotic checkpoints that monitor centrosome duplication. The relationship between loss of hormone dependence in breast cancer, and signaling of centrosome duplication in tumor progression is not known. We have developed a MCF-7 cell line expressing GFP-Centrin that allows direct visualization of centriole duplication during the cell cycle in living cells. GFP-Centrin is expressed and selectively incorporated into the structure of both centrioles making them clearly visible in living cells. Our studies demonstrate three important aspects of recombinant GFP-Centrin incorporation into centrioles. 1) GFP-Centrin transfected cells grow normally in culture and show no adverse effect associated with GFP-Centrin expression; 2) newly duplicated centrioles incorporate Centrin during their genesis; and 3) GFP-Centrin incorporation into centrioles does not grossly affect cell cycle progression, or centrosome function.
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Centriole and Centrin degeneration during mouse spermiogenesis.
Cell motility and the cytoskeleton, 1999Co-Authors: G. Manandhar, Jeffrey L. Salisbury, Calvin Simerly, Gerald SchattenAbstract:Centrosome reduction during mouse spermiogenesis has been studied by immunofluorescent microscopy using antiCentrin antibody (20H5) and TEM. Centrin is detected as two spots in round spermatids, corresponding to a pair of centrioles. In elongating spermatids, Centrin spots colocalize with the centrioles in the neck region, while the perinuclear ring from which manchette microtubules arise, does not label with the antibody 20H5. The proximal centriole of the elongating spermatids develops a prominent adjunct, which assembles an aster of microtubules. TEM studies after immunogold labeling revealed that Centrin is associated with the distal and the proximal centrioles, but not with the adjunct. Centrin labeling in the neck region diminishes after spermiation stage, although it is not completely lost from all testicular sperm. Mature epididymal sperm do not display Centrin labeling. Mouse sperm lose both distal and proximal centrioles at maturity. Loss of Centrin staining appears to correlate with the degeneration of centrioles during mouse spermiogenesis. Cell Motil. Cytoskeleton 43:137–144, 1999. © 1999 Wiley-Liss, Inc.
Janine Beisson - One of the best experts on this subject based on the ideXlab platform.
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Centrin diversity and basal body patterning across evolution: new insights from Paramecium
Biology Open, 2017Co-Authors: Anne Aubusson-fleury, Guillaume Balavoine, Michel Lemullois, Khaled Bouhouche, Janine Beisson, France KollAbstract:First discovered in unicellular eukaryotes, Centrins play crucial roles in basal body duplication and anchoring mechanisms. While the evolutionary status of the founding members of the family, Centrin2/Vfl2 and Centrin3/cdc31 has long been investigated, the evolutionary origin of other members of the family has received less attention. Using a phylogeny of ciliate Centrins, we identify two other Centrin families, the ciliary Centrins and the Centrins present in the contractile filaments (ICL Centrins). In this paper, we carry on the functional analysis of still not well known Centrins, the ICL1e subfamily identified in Paramecium, and show their requirement for correct basal body anchoring through interactions with Centrin2 and Centrin3. Using Paramecium as well as an Eukaryote-wide sampling of Centrins from completely sequenced genomes, we revisited the evolutionary story of Centrins. Their phylogeny shows that the Centrins associated with the ciliate contractile filaments are widespread in eukaryotic lineages and could be as ancient as Centrin2 and Centrin3.
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Functional diversification of Centrins and cell morphological complexity.
Journal of Cell Science, 2008Co-Authors: Delphine Gogendeau, Catherine Klotz, Olivier Arnaiz, Agata Malinowska, Michal Dadlez, Nicole Garreau De Loubresse, Françoise Ruiz, Janine BeissonAbstract:In addition to their key role in the duplication of microtubule organising centres (MTOCs), Centrins are major constituents of diverse MTOC-associated contractile arrays. A Centrin partner, Sfi1p, has been characterised in yeast as a large protein carrying multiple Centrin-binding sites, suggesting a model for Centrin-mediated Ca2+-induced contractility and for the duplication of MTOCs. In vivo validation of this model has been obtained in Paramecium, which possesses an extended contractile array - the infraciliary lattice (ICL) - essentially composed of Centrins and a huge Sfi1p-like protein, PtCenBP1p, which is essential for ICL assembly and contractility. The high molecular diversity revealed here by the proteomic analysis of the ICL, including ten subfamilies of Centrins and two subfamilies of Sf1p-like proteins, led us to address the question of the functional redundancy, either between the Centrin-binding proteins or between the Centrin subfamilies. We show that all are essential for ICL biogenesis. The two Centrin-binding protein subfamilies and nine of the Centrin subfamilies are ICL specific and play a role in its molecular and supramolecular architecture. The tenth and most conserved Centrin subfamily is present at three cortical locations (ICL, basal bodies and contractile vacuole pores) and might play a role in coordinating duplication and positioning of cortical organelles.
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Centrin deficiency in paramecium affects the geometry of basal body duplication
Current Biology, 2005Co-Authors: Françoise Ruiz, Nicole Garreau De Loubresse, Catherine Klotz, Janine BeissonAbstract:Summary Background Ciliary or flagellar basal bodies and centrioles share the same architecture and remarkable property of duplicating once per cell cycle. Duplication is known to proceed by budding of the daugther organelle close to and at right angles to the mother structure, but the molecular basis of this geometry remains unknown. Among the handful of proteins implicated in basal-body/centriole duplication, Centrins seem required in all eukaryotes tested, but their mode of action is not clear. We have investigated Centrin function in Paramecium , whose cortical organization allows detection of any spatial or temporal alteration in the pattern of basal-body duplication. Results We have characterized two pairs of genes, PtCEN2a and PtCEN2b as well as PtCEN3a and PtCEN3b , orthologs of HsCEN2 and HsCEN3 , respectively. GFP tags revealed different localization for the two pairs of gene products, at basal bodies or on basal-body-associated filamentous arrays, respectively. Centrin depletion induced by RNAi caused mislocalization of the neoformed basal bodies: abnormal site of budding (PtCen2ap) or absence of separation between mother and daughter organelles (PtCen3ap). Over successive divisions, new basal bodies continued to be assembled, but internalization of the mispositionned basal bodies led to a progressive decrease in the number of cortical basal bodies. Conclusions Our observations show that Centrins (1) are required to define the site and polarities of duplication and to sever the mother-daughter links and (2) play no triggering or instrumental role in assembly. Our data underscore the biological importance of the geometry of the duplication process.
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genetic evidence for a role of Centrin associated proteins in the organization and dynamics of the infraciliary lattice in paramecium
Cytoskeleton, 1997Co-Authors: Catherine Klotz, Nicole Garreau De Loubresse, Françoise Ruiz, Janine BeissonAbstract:Within the superfamily of “EF-hand Ca2+-modulated proteins,” Centrins constitute a family of cytoskeletal proteins that are highly conserved from lower eukaryotes to man. Their cytoskeletal specialization is manifest in their capacity to form filamentous contractile arrays of various shapes and functions and by their association with microtubule organizing centres (MTOCs). While the latter property has been conserved throughout the evolution of eukaryotes, Centrin-based contractile structures are only found in protists where they form arrays of widely diverse organization and function. In the ciliate Paramecium tetraurelia, three Centrin genes have been characterized, which may be part of a larger Centrin gene family [Madeddu et al., 1996: Eur. J. Biochem. 238:121–128]. The products of these genes were originally identified as components of the infraciliary lattice, a contractile cytoskeletal network [Garreau de Loubresse et al., 1991: Biol. Cell 71:217–225]. We show here that Centrins are localized not only in this lattice but also in basal bodies and in the cord, a filamentous structure associated with the oral apparatus. We demonstrate that in the infraciliary lattice, but not in basal bodies, Centrins are associated with high-molecular-weight proteins (ca. 350 kD). Their role in the biogenesis of the infraciliary lattice is documented by cytological and biochemical properties of the mutant “demaille” (dem1) characterized by altered Centrin-associated proteins and abnormal organization and dynamics of the infraciliary lattice. Cell. Motil. Cytoskeleton 38:172–186, 1997. © 1997 Wiley-Liss, Inc.
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Characterization of Centrin Genes in Paramecium
European journal of biochemistry, 1996Co-Authors: Luisa Madeddu, Catherine Klotz, Jean-pierre Le Caer, Janine BeissonAbstract:Centrins are highly conserved, ubiquitous cytoskeletal components which belong to the EF-hand superfamily of Ca2+-modulated proteins. We report here the molecular characterization of new members of the Centrin family, Paramecium Centrins. Previous studies described the organization of the infraciliary lattice (ICL), the innermost cortical cytoskeletal network of Paramecium, and showed that it was composed of a set of low-molecular-mass, Ca2+-binding polypeptides [Garreau de Loubresse, N., Klotz, C., Vigues, B., Rutin, J. & Beisson, J. (1991) Biol. Cell 71, 217–225]. In this paper we show that these polypeptides are recognized by specific anti-Centrin polyclonal antibodies. Their microsequences revealed four distinct N-termini. For one of them, ICL1, N-terminal and internal peptide sequences were used for PCR amplification and cloning of a DNA fragment containing a complete Centrin coding sequence. The deduced amino acid sequence presents about 50% identity with those of Centrins from other species. Further molecular analysis allowed us to identify two additional closely related, co-expressed ICL1 genes, providing the first example of a Centrin multigenic family in a microorganism.
Elmar Schiebel - One of the best experts on this subject based on the ideXlab platform.
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yeast Centrin cdc31 is linked to the nuclear mrna export machinery
Nature Cell Biology, 2004Co-Authors: Tamas Fischer, Elmar Schiebel, Susana Rodrigueznavarro, Gislene Pereira, Attila Racz, Ed HurtAbstract:Centrins are calmodulin-like proteins that function in the duplication of microtubule-organizing centres. Here we describe a new function of the yeast Centrin Cdc31. We show that overproduction of a sequence, termed CID, in the carboxy-terminal domain of the nuclear export factor Sac3 titrates Cdc31, causing a dominant-lethal phenotype and a block in spindle pole body (SPB) duplication. Under normal conditions, the CID motif recruits Cdc31 and Sus1 (a subunit of the SAGA transcription complex) to the Sac3–Thp1 complex, which functions in mRNA export together with specific nucleoporins at the nuclear basket. A previously reported cdc31 temperature-sensitive allele, which is neither defective in SPB duplication nor Kic1 kinase activation, induces mRNA export defects. Thus, Cdc31 has an unexpected link to the mRNA export machinery.
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Identification of a new mammalian Centrin gene, more closely related to Saccharomyces cerevisiae CDC31 gene
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Sandrine Middendorp, Elmar Schiebel, Anne Paoletti, Michel BornensAbstract:Among the numerous Centrin isoforms identified by two-dimensional gel electrophoresis in human cells, an acidic and slow-migrating isoform is particularly enriched in a centrosome fraction. We report here that this isoform specifically reacts with antibodies raised against Saccharomyces cerevisiae Cdc31p and is present, as other Centrin isoforms, in the distal lumen of centrioles. It is encoded by a new Centrin gene, which we propose to name HsCEN3 (Homo sapiens Centrin gene 3). This gene is more closely related to the yeast CDC31 gene, and shares less identity with algae Centrin than HsCEN1 and HsCEN2. A murine CDC31-related gene was also found that shows 98% identity and 100% similarity with HsCEN3, demonstrating a higher interspecies conservation than the murine Centrin gene MmCEN1 (Mus musculus Centrin gene 1) with either HsCEN1, or HsCEN2. Finally, immunological data suggest that a CDC31-related gene could exist in amphibians and echinoderms as well. All together, our data suggest the existence of two divergent protein subfamilies in the current Centrin family, which might be involved in distinct centrosome-associated functions. The possible implication of this new mammalian Centrin gene in centrosome duplication is discussed.
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binding of Centrins and yeast calmodulin to synthetic peptides corresponding to binding sites in the spindle pole body components kar1p and spc110p
Journal of Biological Chemistry, 1996Co-Authors: Birgitta M. Geier, Hans Wiech, Elmar SchiebelAbstract:Abstract Centrins contain four potential Ca2+ binding sites, known as EF-hands, and have essential functions in centrosome duplication and filament contraction. Here we report that Centrins from yeast, green algae, and humans bound with high affinity to a peptide of the yeast centrosomal component Kar1p. Interestingly, Centrin binding was regulated by physiological relevant changes in [Ca2+], and this Ca2+ dependence was influenced by acidic amino acids within the Kar1p peptide, which also prevented efficient binding of the related yeast calmodulin. However, a hybrid protein with the third and fourth EF-hands from the yeast Centrin Cdc31p and the amino-terminal half from yeast calmodulin behaved more like Cdc31p, indicating that the carboxyl-terminal half of Cdc31p influences binding specificity. Besides Kar1p, Centrins bound to a yeast calmodulin binding site, explaining the dosage-dependent suppression of a calmodulin mutant by CDC31. Consistent with an essential role of Ca2+ for Centrin functions, mutations in the first or the fourth EF-hands of Cdc31p, impairing the Ca2+-induced conformational change of Cdc31p, resulted in nonfunctional proteins in vivo. Our results suggest that Centrins are involved in Ca2+ signaling, likely by influencing the properties of target proteins in response to changes in [Ca2+].
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Characterization of Green Alga, Yeast, and Human Centrins SPECIFIC SUBDOMAIN FEATURES DETERMINE FUNCTIONAL DIVERSITY
The Journal of biological chemistry, 1996Co-Authors: Hans Wiech, Jutta Steinkotter, Michael Melkonian, Birgitta M. Geier, Thilo Paschke, Anne Spang, Katrin Grein, Elmar SchiebelAbstract:Centrins are a subfamily within the superfamily of Ca2+-modulated proteins that play a fundamental role in centrosome duplication and contraction of Centrin-based fiber systems. We examined the individual molecular properties of yeast, green alga, and human Centrins. Circular dichroism spectroscopy revealed a divergent influence of Ca2+ binding on the alpha-helical content of these proteins. Ca2+-free Centrins were elongated in shape as determined by size exclusion chromatography. The presence of Ca2+ and binding peptide resulted in more spherical shaped Centrins. In contrast to yeast calmodulin, Centrins formed multimers in the Ca2+-bound state. This oligomerization was significantly reduced in the absence of Ca2+ and in the presence of binding peptide. The Ca2+-dependent polymerization of the green alga Scherffelia dubia Centrin (SdCen) resulted in a filamentous network. This molecular property was mainly dependent on the amino-terminal subdomain and the peptide-binding site of SdCen. Finally, we analyzed whether SdCen and Cdc31p-SdCen hybrid proteins functionally substitute for the Saccharomyces cerevisiae Centrin Cdc31p. Only hybrid proteins containing the amino-terminal subdomain or the third EF-hand of SdCen and the other subdomains from Cdc31p were functional in vivo.
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In search of a function for Centrins
Trends in cell biology, 1995Co-Authors: Elmar Schiebel, Michel BornensAbstract:Spindle pole bodies, basal bodies and centrosomes are morphologically quite different structures that nevertheless perform similar microtubule-organizing functions in diverse cell types. The recent discoveries that both Centrins and gamma-tubulin are common components of these structures suggest a molecular basis for their common functions. The role of Centrins is just beginning to be investigated. These filament-associated proteins bind Ca2+. The filaments contract at least in certain circumstances by an ATP-independent mechanism. However, yeast Centrin is clearly involved in the duplication of the spindle pole body. A common molecular mechanism may underlie these two apparently different functions.