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ACtin assembly in neutrophils: Evidence for calcium—dependent and calcium-independent mechanisms
218
Coiloque "Contr61e M o l t c u l a i r e du C y c l e Cellulaire", Lyon, juillet 1992
T i l E CENTROSOME DUPLICATION CYCLE AND THE CELL DIVISION CYCLE TOURNIER Frtdtric, BAILLY Eric and BORNENS Michel. Centre de Gtnttiqu¢ Moltculaire du CNRS, 91 198 GIF s[Yvette, France. The centrosome-miorotubule complex is a prominent feature of animal cells. Typically, the centrosome ensures a unique organization and a collective behavior of the microtubular network in the interphasie and the mitotic cell and an such acts as a determinant for the organization of the cytoplasm and for the fidelity of cell division. In growth-limited cells, cell mass and growth rate must be monitored and coupled to the M-phase control mechanism. Microtubules are reasonable candidate for structures which participate in monitoring cell mass. The duplication mechanism of the centrosome which involves a discontinuous increase in the number of centriole copies, from two to four, and an increase in the microtubule nucleating capacity, and which encompasses all four phases of the cell cycle, could be the basis for an appropriate mechanism which could monitor time and mass during the cell cycle. An important issue is to understand what controls the centrosome cycle. Is it entirely driven by the same biochemieai clock which governs most of the nuclear and cytoplasmic events, or is it governed by independent mechanisms7 In the second case, the centrosome duplication cycle could function as an additional pace-maker which would interact with the M-phase clock for the cell cycle to ensure correct timing for the cell division. Results obtained with two independent experimental approaches will be discussed, one dealing with the duplication of heterologous centrosomes in Xenopus eggs and the design of a cell free extract to study this process(I-3), and the other one with the centrosomai localization of part of the mitotic kinase subunits in human growth-limited cells(4,5). All together, the results favor the view that the centrosome dupie~tion cycle is independent from the mitotic clock and that the hecessary biochemical coupling between the two pathways involves direct interaction of the mitotic kinase with the centrosome. Other data(6) suggest that late events of the centrosome cycle
may provide signals for the activation of the mitotic activity('/).
Julia Morales~ Patrick Cormier, Robert Poulhe, Odila Minella, Odile Mulner-Lorillon et Robert Bellt. Targets of MPF during meiotic cell division. Using meiotic maturation el Xenopus oocytes as a model, we have characterized two substrates of the protein kinase activity (p34 cdc2) of M-phase lacier (MPF), a universal factor involved in the regulation of cell division control. The lirst subslrate of MPF is a biochemically well known protein kinase, casein kinase II, which happens to be phosphorylated on its regulatory subunit, and consequently activated. This result provides biochemical explanation to the burst in protein phosphorylation associated with MPF. Furthermore, its provides some clues to the analysis of the pleiotropic effects el MPF. The second substrale has been identified to the ~ subunit (p47) of a macromolecular complex known to be implicated in peptide chain elongation, called elongation lacier 1 (EF 1). This p47 protein is phosphorylated by MPF during the course el meiotic cell division. We have cloned and sequenced the cOb!A encoding tot the p47 protein using expression library, screened with purified antibodies. We have identified the phospho-acceplor in the p47 protein to 23OThreonine, using bidimensional analysis of paptides matching the cDNA-deduced protein sequence of the clone encoding [or the protein. We have also cloned and sequenced the cDNA encoding for two other components of the complex containing p47 protein (EF 113 and 6 ). We demonstrate the structural sequence homology between the ~ and 8 proteins which are bolh guanine nucleotide exchange proteins. Our results provide for the first time a direct link between MPF and regulation of protein synthesis which occurs during meiosis.
(1) TOURNIER F, E KARSENTI and M BORNENS (lgBg). Dev. Biol. 136,321-32g. (2) TOURNIER F, S KOMESLI, M PAINTRAND, O JOB and M BORNENS (1991). J. Cell Biol. 113, 1361-136g. (3) TOURNIER F, M CYRKLAFF, E KARSENTI and M BORNENS (1991). Prec. Nat. Acad. Sci. US. 8B, gg29-gg33. (4) BAILLY E. M DOREE, P NURSE and MBORNENS (tgBg).EMBO J. 13, 3895-39@5. 15) BAILLY E, J PINES, T HUNTER and M BORNENS (1992). J. Cell Sc1.,101,529-545. (6) MANIOTIS A and M SCHLIWA (1991). Cell, 67, 495-504. (7) BAILLY E and M BORNENS (1992). Nature, 355. 300-301.
CONTROL OF THE CELL CYCLE AND DNA REPLICATION DURING EARLY EMBRYONIC DEYELOPMENT.
~
, VRIZ Sophie, HYRIEN Ofivier, LEMAIT?,E JeanMarc, LEI:BOVICI blichel, and PRIOLEAU Marie.N0~e. lnstitet Jacqu~ Monod, Unit~ d'Embryologie Mol~cu/ake, Tour 43, 2 place Jussieu, 75251 Paris Ce.dex 05 - Fran.::e. We have an~.lyzeAthe subccl]ular loc~l!7~r{~x of proteim involved in
the control ofDNA repfication and gene exp~,sion during the cell cycle. Under conditionsof growin.g,PCHA, D N A p o l y m e ~ ¢-myc, los, sad hsp72 were detected in the nucleus.Serum starvationez~rimeats show that all these proteins except hsp72 leave the nucleus for the
cytoplasm, and with differing kinetics. During serum stiuulation these proteins relocalize into the nucleus with :.he in~,er~ kin~cs. Tae localization of PCNA and c-mvc has been also maalyzed Curing ti;e early development of Xenopas laevis when rapid and synchronized cycles of division occur. A strong correlation be;ween S phase and &stinct zad specific nucle~xdistributio,-s of these two proteins was observe~ The localization of ~plication ofigkts in the ribosomal DNA was also analyzed. Plasmids carrying different parts of the ribosomal DNA sequence were allowed to replicate either in vitro in egg extrac! or in vivo alter microinjection into unfertilized eggs. Replication in the normal chrom0semal conte~t was also examined during early devdopment h all cases replicati~ baitiate and terminate at multiple locations, apparently in a random fashion. In v/tro ext~erimcnts air~ing tO analyz~ the correlation between DNA replication sad gene expression wiL'be pteseated.
ACTIN ASSEMBLY
IN NEUTROPHILS
: EVIDENCE
FOR
CALCIUM-DEPENDENT AND CALCIUM-INDEPENDENT MECHARISMS ZAFFRAN Yona, LEPIDI Hubert, MEGE Jean-Louis, BONGRAND Pierre and CAPO Christian.
Labo~a:toLa¢ d'3mmunoiog, Le , Htpi..Za,Lde Sa.i.nZe-~a,zg.uea~¢ 13277 fC4R.5EYtl_.£ Cedex 9. In human neutrophils, the conversion of globular actin (G-actin) in filamentous actin (F-actin) is essential for the morphological and motile events induced by chemotactic agents, such as the formyl peptide FMLP. Intracellular free calcium may play a pivotal role in the actin assembly or disassembly. First, numerous actin-binding proteins are directly activated by calcium. Second, chemotactic factorinduced calcium rise is involved in oxidative metabolism and degranulation of neutrophils. Cytosolic calcium was measured by using the fluorescent probe flue-3. BAPTA/AM was used to buffer or to chelate intracellular calcium. The cell content in F-actin was analyzed in flow cytometry by right angle light scatter measurement or by staining neutrophils with bodipy phallacidin. The F-actin content significantly increased in neutrophils incubated in calcium-free medium, or calcium-depleted neutrophils, as compared to cells incubated in calciumcontaining medium, or calcium-buffered cel]s. Cytochalasin B inhibited the increase in F-actin observed in calciumfree conditions, suggestin E that polymerization of actin occurs through the barbed end of microfilaments. The polymerization and the subsequent depolymerization of actin in neutrophils stimulated by FMLP were neither affected by the extracellular calcium nor the modulation of cytosolic calcium, demonstrating that two different mechanisms (i.e. calcium-dependent and calcium-independent) account for actin assembly in neutrophils. Moreover, these both mechanisms were additive and affected the barbed end of microfilaments. We suggest that calcium lowering of neutrophils allows the elongation of preformed filaments and chemoattractant rises tile number of actin filaments in s calcium-dependent way.
Coiloque "Contr61e M o l t c u l a i r e du C y c l e Cellulaire", Lyon, juillet 1992
T i l E CENTROSOME DUPLICATION CYCLE AND THE CELL DIVISION CYCLE TOURNIER Frtdtric, BAILLY Eric and BORNENS Michel. Centre de Gtnttiqu¢ Moltculaire du CNRS, 91 198 GIF s[Yvette, France. The centrosome-miorotubule complex is a prominent feature of animal cells. Typically, the centrosome ensures a unique organization and a collective behavior of the microtubular network in the interphasie and the mitotic cell and an such acts as a determinant for the organization of the cytoplasm and for the fidelity of cell division. In growth-limited cells, cell mass and growth rate must be monitored and coupled to the M-phase control mechanism. Microtubules are reasonable candidate for structures which participate in monitoring cell mass. The duplication mechanism of the centrosome which involves a discontinuous increase in the number of centriole copies, from two to four, and an increase in the microtubule nucleating capacity, and which encompasses all four phases of the cell cycle, could be the basis for an appropriate mechanism which could monitor time and mass during the cell cycle. An important issue is to understand what controls the centrosome cycle. Is it entirely driven by the same biochemieai clock which governs most of the nuclear and cytoplasmic events, or is it governed by independent mechanisms7 In the second case, the centrosome duplication cycle could function as an additional pace-maker which would interact with the M-phase clock for the cell cycle to ensure correct timing for the cell division. Results obtained with two independent experimental approaches will be discussed, one dealing with the duplication of heterologous centrosomes in Xenopus eggs and the design of a cell free extract to study this process(I-3), and the other one with the centrosomai localization of part of the mitotic kinase subunits in human growth-limited cells(4,5). All together, the results favor the view that the centrosome dupie~tion cycle is independent from the mitotic clock and that the hecessary biochemical coupling between the two pathways involves direct interaction of the mitotic kinase with the centrosome. Other data(6) suggest that late events of the centrosome cycle
may provide signals for the activation of the mitotic activity('/).
Julia Morales~ Patrick Cormier, Robert Poulhe, Odila Minella, Odile Mulner-Lorillon et Robert Bellt. Targets of MPF during meiotic cell division. Using meiotic maturation el Xenopus oocytes as a model, we have characterized two substrates of the protein kinase activity (p34 cdc2) of M-phase lacier (MPF), a universal factor involved in the regulation of cell division control. The lirst subslrate of MPF is a biochemically well known protein kinase, casein kinase II, which happens to be phosphorylated on its regulatory subunit, and consequently activated. This result provides biochemical explanation to the burst in protein phosphorylation associated with MPF. Furthermore, its provides some clues to the analysis of the pleiotropic effects el MPF. The second substrale has been identified to the ~ subunit (p47) of a macromolecular complex known to be implicated in peptide chain elongation, called elongation lacier 1 (EF 1). This p47 protein is phosphorylated by MPF during the course el meiotic cell division. We have cloned and sequenced the cOb!A encoding tot the p47 protein using expression library, screened with purified antibodies. We have identified the phospho-acceplor in the p47 protein to 23OThreonine, using bidimensional analysis of paptides matching the cDNA-deduced protein sequence of the clone encoding [or the protein. We have also cloned and sequenced the cDNA encoding for two other components of the complex containing p47 protein (EF 113 and 6 ). We demonstrate the structural sequence homology between the ~ and 8 proteins which are bolh guanine nucleotide exchange proteins. Our results provide for the first time a direct link between MPF and regulation of protein synthesis which occurs during meiosis.
(1) TOURNIER F, E KARSENTI and M BORNENS (lgBg). Dev. Biol. 136,321-32g. (2) TOURNIER F, S KOMESLI, M PAINTRAND, O JOB and M BORNENS (1991). J. Cell Biol. 113, 1361-136g. (3) TOURNIER F, M CYRKLAFF, E KARSENTI and M BORNENS (1991). Prec. Nat. Acad. Sci. US. 8B, gg29-gg33. (4) BAILLY E. M DOREE, P NURSE and MBORNENS (tgBg).EMBO J. 13, 3895-39@5. 15) BAILLY E, J PINES, T HUNTER and M BORNENS (1992). J. Cell Sc1.,101,529-545. (6) MANIOTIS A and M SCHLIWA (1991). Cell, 67, 495-504. (7) BAILLY E and M BORNENS (1992). Nature, 355. 300-301.
CONTROL OF THE CELL CYCLE AND DNA REPLICATION DURING EARLY EMBRYONIC DEYELOPMENT.
~
, VRIZ Sophie, HYRIEN Ofivier, LEMAIT?,E JeanMarc, LEI:BOVICI blichel, and PRIOLEAU Marie.N0~e. lnstitet Jacqu~ Monod, Unit~ d'Embryologie Mol~cu/ake, Tour 43, 2 place Jussieu, 75251 Paris Ce.dex 05 - Fran.::e. We have an~.lyzeAthe subccl]ular loc~l!7~r{~x of proteim involved in
the control ofDNA repfication and gene exp~,sion during the cell cycle. Under conditionsof growin.g,PCHA, D N A p o l y m e ~ ¢-myc, los, sad hsp72 were detected in the nucleus.Serum starvationez~rimeats show that all these proteins except hsp72 leave the nucleus for the
cytoplasm, and with differing kinetics. During serum stiuulation these proteins relocalize into the nucleus with :.he in~,er~ kin~cs. Tae localization of PCNA and c-mvc has been also maalyzed Curing ti;e early development of Xenopas laevis when rapid and synchronized cycles of division occur. A strong correlation be;ween S phase and &stinct zad specific nucle~xdistributio,-s of these two proteins was observe~ The localization of ~plication ofigkts in the ribosomal DNA was also analyzed. Plasmids carrying different parts of the ribosomal DNA sequence were allowed to replicate either in vitro in egg extrac! or in vivo alter microinjection into unfertilized eggs. Replication in the normal chrom0semal conte~t was also examined during early devdopment h all cases replicati~ baitiate and terminate at multiple locations, apparently in a random fashion. In v/tro ext~erimcnts air~ing tO analyz~ the correlation between DNA replication sad gene expression wiL'be pteseated.
ACTIN ASSEMBLY
IN NEUTROPHILS
: EVIDENCE
FOR
CALCIUM-DEPENDENT AND CALCIUM-INDEPENDENT MECHARISMS ZAFFRAN Yona, LEPIDI Hubert, MEGE Jean-Louis, BONGRAND Pierre and CAPO Christian.
Labo~a:toLa¢ d'3mmunoiog, Le , Htpi..Za,Lde Sa.i.nZe-~a,zg.uea~¢ 13277 fC4R.5EYtl_.£ Cedex 9. In human neutrophils, the conversion of globular actin (G-actin) in filamentous actin (F-actin) is essential for the morphological and motile events induced by chemotactic agents, such as the formyl peptide FMLP. Intracellular free calcium may play a pivotal role in the actin assembly or disassembly. First, numerous actin-binding proteins are directly activated by calcium. Second, chemotactic factorinduced calcium rise is involved in oxidative metabolism and degranulation of neutrophils. Cytosolic calcium was measured by using the fluorescent probe flue-3. BAPTA/AM was used to buffer or to chelate intracellular calcium. The cell content in F-actin was analyzed in flow cytometry by right angle light scatter measurement or by staining neutrophils with bodipy phallacidin. The F-actin content significantly increased in neutrophils incubated in calcium-free medium, or calcium-depleted neutrophils, as compared to cells incubated in calciumcontaining medium, or calcium-buffered cel]s. Cytochalasin B inhibited the increase in F-actin observed in calciumfree conditions, suggestin E that polymerization of actin occurs through the barbed end of microfilaments. The polymerization and the subsequent depolymerization of actin in neutrophils stimulated by FMLP were neither affected by the extracellular calcium nor the modulation of cytosolic calcium, demonstrating that two different mechanisms (i.e. calcium-dependent and calcium-independent) account for actin assembly in neutrophils. Moreover, these both mechanisms were additive and affected the barbed end of microfilaments. We suggest that calcium lowering of neutrophils allows the elongation of preformed filaments and chemoattractant rises tile number of actin filaments in s calcium-dependent way.