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J C Courvalin

Publications and source records attributed to J C Courvalin.

At least 19 recordsLinked to original sources
PubMed2001

Immunolocalization of HP1 proteins in metaphasic mammalian chromosomes.

HP1 proteins are conserved non histone chromosomal proteins involved in the epigenetic repression of transcription. Three HP1 proteins, HP1alpha, HP1beta and HP1gamma are expressed in mammalian cells. Polyclonal antibodies directed against peptides specific for HP1alpha and HP1gamma were elicited in rabbits, affinity purified, then used to localize both proteins on spreads of unfixed metaphasic chromosomes. We show here, by conventional and confocal microscopy, that both proteins are localized at centromeres and pericentromeres, and that HP1gamma is also present on euchromatic sites of chromosome arms.

Centromere↗
PubMed2001

Dynamics of the nuclear envelope at mitosis and during apoptosis.

The nuclear envelope is a highly dynamic structure that reversibly disassembles and reforms at mitosis. The nuclear envelope also breaks down--irreversibly--during apoptosis, a process essential for development and tissue homeostasis. Analyses of fixed cells, time-lapse, imaging studies of live cells and the development of powerful cell-free extracts derived from gametes or mammalian somatic cells have provided insights on the fate of nuclear envelope proteins during mitosis and apoptosis, and on the mechanisms behind nuclear envelope modifications in these processes. In this review, we discuss evidence leading to our understanding of the dynamics of the nuclear envelope alterations at mitosis and during apoptosis. We also present novel imaging and genetic approaches to the study of nuclear envelope dynamics and function.

Animals↗
PubMed2001

Expression and functional analysis of three isoforms of human heterochromatin-associated protein HP1 in Drosophila.

Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal protein associated with pericentromeric heterochromatin in Drosophila. HP1-like proteins have also been found associated with heterochromatin in human cells. The goal of this study was to determine whether proteins of the structurally conserved human HP1 family exhibit conserved heterochromatin targeting and silencing properties in Drosophila. We established transgenic lines of Drosophila melanogaster expressing each of the three human HP1 proteins, HP1Hsalpha, HP1HSbeta, and HP1Hsgamma, under the Hsp70 heat shock promoter. We show that all three isoforms of human HP1 are stably expressed in Drosophila and are associated with heterochromatin in Drosophila chromosomes. Like Drosophila HP1, all three human HP1 proteins are delocalized by an HP1-POLYCOMB chimeric protein, implying that both human HP1 and Drosophila HP1 interact in a common protein complex, and that at least some aspects of heterochromatin structure are highly conserved throughout the evolution of eukaryotes. Ectopic expression of two of the three human HP1 family proteins significantly enhances heterochromatic silencing in Drosophila.

Amino Acid Sequence↗
PubMed2001

Biochemical characterization of nuclear pore complex protein gp210 oligomers.

The membrane-spanning glycoprotein gp210 is a major component of the nuclear pore complex. This nucleoporin contains a large cisternal N-terminal domain, a short C-terminal cytoplasmic tail, and a single transmembrane segment. We show here that dimers of native gp210 can be isolated from cell extracts by immunoprecipitation, and from purified rat liver nuclear envelopes by velocity sedimentation and gel filtration. Cross-linking of proteins in isolated membranes prior to solubilization dramatically increases the proportion of dimers. The dimers are SDS-resistant, as previously observed for some integral membrane proteins of cis-Golgi and plasma membrane proteins, including glycophorin A. Larger oligomers of gp210 can also be obtained by gel filtration and denaturing electrophoresis, but unlike the dimers are dissociated by reduction and heating in the presence of SDS. We propose that gp210 is organized into the pore membrane as a large array of gp210 dimers that may constitute a luminal submembranous protein skeleton.

Animals↗
PubMed2001

Nuclear envelope disorganization in fibroblasts from lipodystrophic patients with heterozygous R482Q/W mutations in the lamin A/C gene.

Dunnigan-type familial partial lipodystrophy (FPLD), characterized by an abnormal body fat redistribution with insulin resistance, is caused by missense heterozygous mutations in A-type lamins (lamins A and C). A- and B-type lamins are ubiquitous intermediate filament proteins that polymerize at the inner face of the nuclear envelope. We have analyzed primary cultures of skin fibroblasts from three patients harboring R482Q or R482W mutations. These cells were euploid and able to cycle and divide. A subpopulation of these cells had abnormal blebbing nuclei with A-type lamins forming a peripheral meshwork, which was frequently disorganized. Inner nuclear membrane protein emerin, an A-type lamin-binding protein, strictly colocalized with this abnormal meshwork. Cells from lipodystrophic patients often had other nuclear envelope defects, mainly consisting of nuclear envelope herniations that were deficient in B-type lamins, nuclear pore complexes, lamina-associated protein 2 beta, and chromatin. The mechanical properties of nuclear envelopes were altered, as judged from the extensive deformations observed in nuclei from heat-shocked cells, and from the low stringency of extraction of their components. These structural nuclear alterations were caused by the lamins A/C mutations, as the same changes were introduced in human control fibroblasts by ectopic expression of R482W mutated lamin A.

Adult↗
PubMed2000

Inner nuclear membrane protein LBR preferentially interacts with DNA secondary structures and nucleosomal linker.

The lamin B receptor (LBR) is an integral protein of inner nuclear membrane whose nucleoplasmic amino-terminal domain contributes to the attachment of the membrane to chromatin. Here we analyzed the interactions of a recombinant GST protein containing the amino-terminal domain of the protein with in vitro reconstituted nucleosomes and short DNA fragments. Data show that the LBR amino-terminal domain (AT) binds linker DNA but does not interact with the nucleosome core. Titration and competition studies revealed that the interaction between LBR AT and DNA is saturable, of high affinity (K(D) approximately 4 nM), independent of DNA sequence, and enhanced by DNA curvature and supercoiling. In this respect, LBR amino-terminal domain binding to nucleosomes is similar to that of histone H1 and non histone proteins HMG1/2 which both bind preferentially to linker DNA and present a significant affinity for DNA secondary structures.

DNA↗
PubMed2000

Sorting nuclear membrane proteins at mitosis.

The nuclear envelope (NE) breaks down reversibly and reassembles at mitosis. Two models of mitotic nuclear membrane disassembly and reformation have emerged from studies of NE dynamics in somatic cells and egg extracts. One model suggests that nuclear membranes fragment reversibly by vesiculation, producing NE-derived vesicles separate from the endoplasmic reticulum. The second model proposes that nuclear membranes vanish by diffusion of their integral proteins through a continuous endoplasmic reticulum. Here, we discuss critically the grounds for the elaboration of these apparently mutually exclusive views. Our conclusions favour a model in which nuclear membranes do not vesiculate during mitosis.

Animals↗
PubMed2000

HP1gamma associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes.

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein, first identified in Drosophila, that plays a dose-dependent role in gene silencing. Three orthologs, HP1alpha, HP1beta, and HP1gamma, have been characterized in mammals. While HP1alpha and HP1beta have been unambiguously localized in heterochromatin by immunocytochemical methods, HP1gamma has been found either exclusively associated with euchromatin or present in both euchromatin and heterochromatin. Here, using an antibody directed against a peptide epitope at the carboxyl-terminal end of the molecule, we localize HP1gamma in both euchromatin and heterochromatin compartments of interphase nuclei, as well as in the pericentromeric chromatin and arms of mitotic chromosomes of 3T3 cells. This dual location was also observed in nuclei expressing HP1gamma as a fusion protein with green fluorescent protein. In contrast, when the distribution of HP1gamma was analyzed with antibodies directed against an amino-terminal epitope, the protein was detectable in euchromatin and not in heterochromatin, except for transient heterochromatin staining during the late S phase, when the heterochromatin undergoes replication. These data suggest that the controversial immunolocalization of HP1gamma in chromatin is due to the use of antibodies directed against topologically distinct epitopes, those present at the amino-terminal end of the molecule being selectively masked in nonreplicative heterochromatin.

3T3 Cells↗
PubMed1999

The BAH (bromo-adjacent homology) domain: a link between DNA methylation, replication and transcriptional regulation.

Using sensitive methods of sequence analysis including hydrophobic cluster analysis, we report here a hitherto undescribed family of modules, the BAH (bromo-adjacent homology) family, which includes proteins such as eukaryotic DNA (cytosine-5) methyltransferases, the origin recognition complex 1 (Orc1) proteins, as well as several proteins involved in transcriptional regulation. The BAH domain appears to act as a protein-protein interaction module specialized in gene silencing, as suggested for example by its interaction within yeast Orc1p with the silent information regulator Sir1p. The BAH module might therefore play an important role by linking DNA methylation, replication and transcriptional regulation.

Amino Acid Sequence↗
PubMed1999

Localization and phosphorylation of HP1 proteins during the cell cycle in mammalian cells.

Mammalian heterochromatin proteins 1 (HP1alpha, HP1beta, and HP1gamma) are nonhistone proteins that interact in vitro with a set of proteins that play a role in chromatin silencing, transcription, and chromatin remodeling. Using antibodies specific for each HP1 isoform, we showed that they segregate in distinct nuclear domains of human HeLa cells. By contrast, in mouse 3T3 interphase cells, HP1alpha and HP1beta are strictly colocalized. In mitotic HeLa cells, all of HP1alpha and a fraction of HP1beta and HP1gamma remain associated with chromosomes. Immunostaining of spread HeLa chromosomes showed that HP1alpha is mainly localized on centromeres as shown previously for HP1beta, while HP1gamma is distributed on discrete sites on the arms of chromosomes. Biochemical analysis showed that HP1alpha and HP1gamma are phosphorylated throughout the cell cycle, although more extensively in mitosis than in interphase, while HP1beta apparently remains unphosphorylated. Therefore, despite their extensive sequence conservation, mammalian HP1 isoforms differ widely in their nuclear localization, mitotic distribution and cell cycle-related phosphorylation. Thus, subtle differences in primary sequence and in posttranslational modifications may promote their targeting at different chromatin sites, generating pleiotropic effects.

Amino Acid Sequence↗
PubMed1999

Caspase-dependent proteolysis of integral and peripheral proteins of nuclear membranes and nuclear pore complex proteins during apoptosis.

We have studied the fate of the nuclear envelope (NE) in different human cells committed to apoptosis by different chemical agents. Using a battery of antibodies against marker proteins of the three domains of the nuclear envelope, namely lamin B (LB) for the lamina, transmembrane proteins LBR and LAP2 for the inner nuclear membrane, and nucleoporins p62, Nup153 and gp210 for the nuclear pore complexes (NPCs), we observed a selective and conserved cleavage of LB, LAP2 and Nup153. In lymphoid cells, the rate of cleavage of these markers was independent of the apoptosis inducing agent, actinomycin D or etoposide, and more rapid than in attached epithelial cells. While lamin B is cleaved by caspase 6, the protease responsible for the cleavage of LAP2 and Nup153 was probably caspase 3, since (1) cleavage of both proteins was specifically prevented by in vivo addition of caspase 3 inhibitor Ac-DEVD-CHO and (2) consensus sites for these caspases are present in both proteins. As LB, LAP2 and Nup153 are exposed at the inner face of the nuclear envelope and all interact with chromatin, we suggest that their cleavage allows both the detachment of NE from chromatin and the clustering of NPCs in the plane of the membrane, two conserved morphological features of apoptosis observed in this study.

Apoptosis↗
PubMed1998

LBR, a chromatin and lamin binding protein from the inner nuclear membrane, is proteolyzed at late stages of apoptosis.

Chromatin condensation and apposition to the nuclear envelope is an important feature of the execution phase of apoptosis. During this process, lamin proteins that are located between the inner nuclear membrane and heterochromatin are proteolyzed by the apoptosis-specific protease caspase 6. We have investigated the fate of nuclear membranes during apoptosis by studying the lamin B receptor (LBR), a transmembrane protein of the inner nuclear membrane. LBR interacts through its nucleoplasmic amino-terminal domain with both heterochromatin and B-type lamins, and is phosphorylated throughout the cell cycle, but on different sites in interphase and mitosis. We report here that: (i) the amino-terminal domain of LBR is specifically cleaved during apoptosis to generate an approximately 20 kDa soluble fragment; (ii) the cleavage of LBR is a late event of apoptosis and occurs subsequent to lamin B cleavage; (iii) the phosphorylation of LBR during apoptosis is similar to that occurring in interphase. As the association of condensed chromatin with the inner nuclear membrane persists until the late stages of apoptosis, we suggest that the chromatin binding protein LBR plays a major role in maintaining this association.

Animals↗
PubMed1997

Protein kinase C-mediated interphase lamin B phosphorylation and solubilization.

Disassembly of the sperm nuclear envelope at fertilization is one of the earliest events in the development of the male pronucleus. We report that nuclear lamina disassembly in interphase sea urchin egg cytosol is a result of lamin B phosphorylation mediated by protein kinase C (PKC). Lamin B of permeabilized sea urchin sperm nuclei incubated in fertilized egg G1 phase cytosolic extract is phosphorylated within 1 min of incubation and solubilized prior to sperm chromatin decondensation. Phosphorylation is Ca2+-dependent. It is reversibly inhibited by the PKC-specific inhibitor chelerythrine, a PKC pseudosubstrate inhibitor peptide, and a PKC substrate peptide, but not by inhibitors of PKA, p34(cdc2) or calmodulin kinase II. Phosphorylation is inhibited by immunodepletion of cytosolic PKC and restored by addition of purified rat brain PKC. Sperm lamin B is a substrate for rat brain PKC in vitro, resulting in lamin B solubilization. Two-dimensional phosphopeptide maps of lamin B phosphorylated by the cytosolic kinase and by purified rat PKC are virtually identical. These data suggest that PKC is the major kinase required for interphase disassembly of the sperm lamina.

Animals↗
PubMed1997

Hydrophobic cluster analysis reveals a third chromodomain in the Tetrahymena Pdd1p protein of the chromo superfamily.

The protein Pdd1p (for programmed DNA degradation) links heterochromatin assembly and DNA elimination in Tetrahymena and has recently been identified as a new member of the chromo superfamily that contains two chromodomains. Using the sensitive bidimensional Hydrophobic Cluster Analysis (HCA) method, we have identified a third, highly divergent chromodomain in the Pdd1p sequence. Based on similarity to chromo shadow domains that mediate protein-protein interactions, this newly identified chromodomain may direct the binding of Pdd1p to other proteins. These findings suggest that Pdd1p may be the prototypical member of a new class in the chromo superfamily as all other members contain only one or two chromodomains. The results also demonstrate the power of HCA in identifying relationships which are not detected by conventional methods of sequence analysis.

Amino Acid Sequence↗
PubMed1997

Domain-specific interactions of human HP1-type chromodomain proteins and inner nuclear membrane protein LBR.

HP1-type chromodomain proteins self-associate as well as interact with the inner nuclear membrane protein LBR (lamin B receptor) and transcriptional coactivators TIF1alpha and TIF1beta. The domains of these proteins that mediate their various interactions have not been entirely defined. HP1-type proteins are predicted by hydrophobic cluster analysis to consist of two homologous but distinct globular domains, corresponding to the chromodomain and chromo shadow domain, separated by a hinge region. We show here that the chromo shadow domain mediates the self-associations of HP1-type proteins and is also necessary for binding to LBR both in vitro and in the yeast two-hybrid assay. Hydrophobic cluster analysis also predicts that the nucleoplasmic amino-terminal portion of LBR contains two globular domains separated by a hinge region. The interactions of the LBR domains with an HP1-type protein were also analyzed by the yeast two-hybrid and in vitro binding assays, which showed that a portion of the second globular domain is necessary for binding. The modular domain organization of HP1-type proteins and LBR can explain some of the diverse protein-protein interactions at the chromatin-lamina-membrane interface of the nuclear envelope.

Amino Acid Sequence↗
PubMed1997

Domain-specific disassembly and reassembly of nuclear membranes during mitosis.

The nuclear envelope contains three distinct membrane domains, the outer membrane, the inner membrane, and the pore membrane, that reversibly vesiculate in mitosis. We previously suggested from single-labeling immunofluorescence microscopy analysis of mitotic cells in culture that mitotic vesiculation of the nuclear membranes may proceed in a domain-specific manner (Chaudhary and Courvalin, J. Cell. Biol. 122, 295-306, 1993). In the present study, we add biochemical support to this hypothesis by sorting domain-specific mitotic vesicles. Antibodies directed against the lamin B receptor, a marker of the inner membrane, and glycoprotein gp210, a marker of the pore membrane, were used to isolate by affinity two populations of mitotic vesicles that were selectively enriched in each of these markers. These two vesicle populations were of different size distribution; the pore membrane-derived vesicles were smaller (80% being < or = 200 nm) than the inner membrane-derived vesicles (80% > or = 200 nm). Double-labeling immunofluorescence microscopy analysis of mitotic cells in culture showed that the time course and topology of disassembly and reassembly of inner and pore membrane domains were different, confirming that domain-specific vesicles are generated during mitosis. In these studies, protein LAP2/thymopoietin beta, another marker of the inner nuclear membrane, was segregating as lamin B receptor, suggesting that both proteins were contained in the same mitotic vesicles.

Animals↗