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Complex pathways in nuclear transport
Cell Biology international
Reports, Vol. 14, Abstracts Supplement
MITOTIC PHOSPHORYLATION AND DISASSEMBLY OF THE NUCLEAR LAMINA BY CDC2 KINASE Erich A Nigg, Mathias Peter, Junichi Nakagawa, Gregory Kitten. Marcel Dome* and Jean-Claude Labbe*, Swiss Ins&e for Experimental Cancer Research, CI-I- 1066 Epalinges, Switxerland, and *CNBS, B.P. 5051, F-34033 Montpellier, France. The nuclear lamina is an inuumediate filament type network underlying the inner nuclear membrane. Lamin isoforms (A- and B-type lamins) show differential expression during vertebrate development, as well as differential subcellular distribution during mitosis. Posttranslational modification of the carboxy-terminus of lamin proteins (isoprenylation, pmteolytic processing and carboxyl-methylation) plays an important role in targetting of newly synthesized lamins to the nuclear envelope. Phosuhorvlation of lamin proteins is believed to cause larnina d&assembly during meiotic and mitotic M-phase, but the M-phase specific lamin kinase has proven difficult to purify. We recently found that cdc2 kinase, a major element implicated in controlling the eukaryotic cell cycle, phosphorylates chicken B-type iamins jn vitro on sites that are specifically phosphorylated during M-phase m. Concomitantly, cdc2 kinase is capable of inducing lamina denolvmerixation upon incubation with isolated nuclei. C&&rsely, although both protein kinases A and C readily nhosnhorvlate lamins, neither M-phase site phosphorylation nor lamin’a disassembly can be induced by theseknxymes. One of the target sites of cdc2 kinase is identified as the serine residue within a motif (SPTR) that is conserved in the N-terminal domain of all lamin proteins. These results lead us to conclude that mitotic disassembly of the nuclear lamina results from t&@ phosphotylation of lamins by cdc2 kinase.
s17
INTRACELLULAR TRANSCRIPT LOCALISATION IN EARLY Drosophila EMBRYOS
s19
David Ish-Horowitz and Ilan Davis. ICRF Developmental Biology U@t, South Parks Road, Oxford OX1 3PS, England. Asymmetric intracellular transcript localisation is a notentiallv nowerful mechanism for specifying sub-cellular protein de&nation. We have investigated thg asymmetric localisation of pair-rule segmentation-gene transcripts within the early Drosophila embryo. The syncytial blastoderm includes a peripheral layer of nuclei that subdivides the cortical cytoplasm into an external “apical” layer and an internal “basal” layer (see Figure). Pair-rule transcripts accumulate predominantly in the thin apical cytoplasm rather than the extensive basal cytoplasm. This destination is gene-specific as other transcripts (e.g. from gap-genes) accumulate elsewhere. cELMEMBB*m
M&;ULE
APICAL
fh,ftz. W NUCLEUS
CYTOPLAS BASAL .;.;,;:,::~:~~~,:.;.;,;:;:;:;:;,.. :::::;.:,:,:,,:.~.~.~.:,,,,.,,,::::: * , : , : , : : : :, yolk : * : . : I : ! : : , . : ,,,,.,.,I:I!.’
GAP mRNAs W, hW
We have used a p-galactosidase reporter gene encoding a basal transcript to define sequences responsible for apical localisation. For three pair-rule genes analysed, even-skipped (eve). fushi farazu and hairv. localisation is deoendent on sequences 3’ of the protein-&ding sequences. A transcript retaining only 125bp of eve 3’sequences is apically localised. We show that transcript destination depends on signals within the RNA. We also araue that asymmetric transcript accumulation is a consequence of vectorial export from the nucleus. We can exclude models that attribute transcript destination to DNA-mediated signals such as chromosomeorientation. Apical localisation within the syncytial blastoderm embryo may restrict lateral diffusion of pair-rule proteins, allowing them to define precise spatial domains.
38
1990
9
POSTMITOTIC REORGANIZATION OF DAUGETBR NDCLEI: TEE ROLE OF NUCLEOCYTOPLASMIC INTERACTIONS
Ricardo Benavente. Institute of Zoology University of Wiirzburg, Wiirzburg, FRG. At the end of so-called open mitosis daughter nuclei reorganize in a process requiring nuclear components provided by the mother cell. In order to gain more insight into this complex process an experimental approach was devised that allows the manipulation of living mitotic cells. Cultured cells in metaphase were microinjected with antibodies to various nuclear components and fixed after different incubation times. The effects were monitored by double-label immunofluorescence and electron microscopy. Using this approach the following sequence of events could be established: First, condensed chromosomes become enclosed by a tightly associated double-layered nuclear membrane which excludes non-selectively all macromolecules not associated with the chromosomes. Second, interphase chromatin organization is then progressively restored by selective pore complex-mediated uptake of nuclear proteins from the cytoplasm, including nuclear lamins. Third, nucleoli reappear. Nucleolar reformation is a relatively late postmitotic event requiring completion of steps one and two as well as transcriptional activation of the rRNA genes. Refs: R Benavente & G Krohne: J Cell Biol 103:1847(1986); R Benavente et al.: J Cell Biol 105:1483(1987); Chromosoma 98:233 (1989); Eur J Cell Biol 50:209(1989); U Scheer & R Benavente: BioEssays 12:14(1990)
s20
COMPLEX PATHWAYS IN NUCLEAR TRANSPORT
David S. Goklfarb and Neil Michaud Department of Biology, Universitv of Rochester. Rochester. NY 14627 USA. The’ transport of ‘macromolecules across the nuclear envelope is ultimately catalyzed by the nuclear pore complex, but cytoplasmic factors can play regulatory roles in the process. Although the nuclear localization signals of many nuclear proteins resemble the archetypal signal found in SV40 large T-antigen, the signals responsible for the nuclear transport of many macromolecules, such as RNAs, are not known. Kinetic evidence indicates at least three nucleophiles, including a protein bearing the T-antigen signal, nucleoplasmin, and U6 snRNP, employ the same signal sequence receptor for import. Significantly, the import of U2 snRNP does not require this factor and must, therefore, occur by an independently regulated pathway. Besides mediating signal-dependent transport, each nuclear pore complex contains an approximately 100 angstrom diameter pore, the nuclear pore, which allows the passive diffusion of inert macromolecules less than about 60 kd. The import of histone Hl (21 kd) serves as a good model for small protein nuclear transport. When tissue cultrue cells are chilled the import of microinjected large signal-containing proteins is arrested while the diffusion of small inert proteins into the nucleus procedes unabated. Surprisingly, the transport of histone Hl and a model small nucleopbilic protein was also arrested by chilling. In sinr titration studies indicated that the transport arrest of Hl in chilled cells is probably mediated by a cytoplasmic receptor. Therefore, even though they are potentially able to diffuse into nuclei, histones and other small signal-containing proteins are complexed by cytoplasmic receptors and imported via a receutor-mediated nrocess which urecludes their diffusion through the nuclear pores. This work is funded by grants from the NM and March of Dimes.
Reports, Vol. 14, Abstracts Supplement
MITOTIC PHOSPHORYLATION AND DISASSEMBLY OF THE NUCLEAR LAMINA BY CDC2 KINASE Erich A Nigg, Mathias Peter, Junichi Nakagawa, Gregory Kitten. Marcel Dome* and Jean-Claude Labbe*, Swiss Ins&e for Experimental Cancer Research, CI-I- 1066 Epalinges, Switxerland, and *CNBS, B.P. 5051, F-34033 Montpellier, France. The nuclear lamina is an inuumediate filament type network underlying the inner nuclear membrane. Lamin isoforms (A- and B-type lamins) show differential expression during vertebrate development, as well as differential subcellular distribution during mitosis. Posttranslational modification of the carboxy-terminus of lamin proteins (isoprenylation, pmteolytic processing and carboxyl-methylation) plays an important role in targetting of newly synthesized lamins to the nuclear envelope. Phosuhorvlation of lamin proteins is believed to cause larnina d&assembly during meiotic and mitotic M-phase, but the M-phase specific lamin kinase has proven difficult to purify. We recently found that cdc2 kinase, a major element implicated in controlling the eukaryotic cell cycle, phosphorylates chicken B-type iamins jn vitro on sites that are specifically phosphorylated during M-phase m. Concomitantly, cdc2 kinase is capable of inducing lamina denolvmerixation upon incubation with isolated nuclei. C&&rsely, although both protein kinases A and C readily nhosnhorvlate lamins, neither M-phase site phosphorylation nor lamin’a disassembly can be induced by theseknxymes. One of the target sites of cdc2 kinase is identified as the serine residue within a motif (SPTR) that is conserved in the N-terminal domain of all lamin proteins. These results lead us to conclude that mitotic disassembly of the nuclear lamina results from t&@ phosphotylation of lamins by cdc2 kinase.
s17
INTRACELLULAR TRANSCRIPT LOCALISATION IN EARLY Drosophila EMBRYOS
s19
David Ish-Horowitz and Ilan Davis. ICRF Developmental Biology U@t, South Parks Road, Oxford OX1 3PS, England. Asymmetric intracellular transcript localisation is a notentiallv nowerful mechanism for specifying sub-cellular protein de&nation. We have investigated thg asymmetric localisation of pair-rule segmentation-gene transcripts within the early Drosophila embryo. The syncytial blastoderm includes a peripheral layer of nuclei that subdivides the cortical cytoplasm into an external “apical” layer and an internal “basal” layer (see Figure). Pair-rule transcripts accumulate predominantly in the thin apical cytoplasm rather than the extensive basal cytoplasm. This destination is gene-specific as other transcripts (e.g. from gap-genes) accumulate elsewhere. cELMEMBB*m
M&;ULE
APICAL
fh,ftz. W NUCLEUS
CYTOPLAS BASAL .;.;,;:,::~:~~~,:.;.;,;:;:;:;:;,.. :::::;.:,:,:,,:.~.~.~.:,,,,.,,,::::: * , : , : , : : : :, yolk : * : . : I : ! : : , . : ,,,,.,.,I:I!.’
GAP mRNAs W, hW
We have used a p-galactosidase reporter gene encoding a basal transcript to define sequences responsible for apical localisation. For three pair-rule genes analysed, even-skipped (eve). fushi farazu and hairv. localisation is deoendent on sequences 3’ of the protein-&ding sequences. A transcript retaining only 125bp of eve 3’sequences is apically localised. We show that transcript destination depends on signals within the RNA. We also araue that asymmetric transcript accumulation is a consequence of vectorial export from the nucleus. We can exclude models that attribute transcript destination to DNA-mediated signals such as chromosomeorientation. Apical localisation within the syncytial blastoderm embryo may restrict lateral diffusion of pair-rule proteins, allowing them to define precise spatial domains.
38
1990
9
POSTMITOTIC REORGANIZATION OF DAUGETBR NDCLEI: TEE ROLE OF NUCLEOCYTOPLASMIC INTERACTIONS
Ricardo Benavente. Institute of Zoology University of Wiirzburg, Wiirzburg, FRG. At the end of so-called open mitosis daughter nuclei reorganize in a process requiring nuclear components provided by the mother cell. In order to gain more insight into this complex process an experimental approach was devised that allows the manipulation of living mitotic cells. Cultured cells in metaphase were microinjected with antibodies to various nuclear components and fixed after different incubation times. The effects were monitored by double-label immunofluorescence and electron microscopy. Using this approach the following sequence of events could be established: First, condensed chromosomes become enclosed by a tightly associated double-layered nuclear membrane which excludes non-selectively all macromolecules not associated with the chromosomes. Second, interphase chromatin organization is then progressively restored by selective pore complex-mediated uptake of nuclear proteins from the cytoplasm, including nuclear lamins. Third, nucleoli reappear. Nucleolar reformation is a relatively late postmitotic event requiring completion of steps one and two as well as transcriptional activation of the rRNA genes. Refs: R Benavente & G Krohne: J Cell Biol 103:1847(1986); R Benavente et al.: J Cell Biol 105:1483(1987); Chromosoma 98:233 (1989); Eur J Cell Biol 50:209(1989); U Scheer & R Benavente: BioEssays 12:14(1990)
s20
COMPLEX PATHWAYS IN NUCLEAR TRANSPORT
David S. Goklfarb and Neil Michaud Department of Biology, Universitv of Rochester. Rochester. NY 14627 USA. The’ transport of ‘macromolecules across the nuclear envelope is ultimately catalyzed by the nuclear pore complex, but cytoplasmic factors can play regulatory roles in the process. Although the nuclear localization signals of many nuclear proteins resemble the archetypal signal found in SV40 large T-antigen, the signals responsible for the nuclear transport of many macromolecules, such as RNAs, are not known. Kinetic evidence indicates at least three nucleophiles, including a protein bearing the T-antigen signal, nucleoplasmin, and U6 snRNP, employ the same signal sequence receptor for import. Significantly, the import of U2 snRNP does not require this factor and must, therefore, occur by an independently regulated pathway. Besides mediating signal-dependent transport, each nuclear pore complex contains an approximately 100 angstrom diameter pore, the nuclear pore, which allows the passive diffusion of inert macromolecules less than about 60 kd. The import of histone Hl (21 kd) serves as a good model for small protein nuclear transport. When tissue cultrue cells are chilled the import of microinjected large signal-containing proteins is arrested while the diffusion of small inert proteins into the nucleus procedes unabated. Surprisingly, the transport of histone Hl and a model small nucleopbilic protein was also arrested by chilling. In sinr titration studies indicated that the transport arrest of Hl in chilled cells is probably mediated by a cytoplasmic receptor. Therefore, even though they are potentially able to diffuse into nuclei, histones and other small signal-containing proteins are complexed by cytoplasmic receptors and imported via a receutor-mediated nrocess which urecludes their diffusion through the nuclear pores. This work is funded by grants from the NM and March of Dimes.