Purification of intact nuclear lamina and identification of novel laminlike proteins in Raji, a cell line devoid of lamins A and C.

Research on the structure of the nuclear lamina and the nuclear matrix of cells devoid of lamins A and C has been hampered by the fact that intact residual nuclear structures are difficult to isolate from such cells. In this paper, we show that some extraction parameters, such as buffer composition and the nature of the detergent used to remove nuclear membranes, are critical for achieving isolation of whole nuclear residual structures from the lymphoblastic cell line Raji, used as a model for cells without lamins A and C. Electron microscopic analysis shows that the nuclear lamina of Raji cells is formed by a network of intermediate-size filaments interrupted with circular discontinuities. Both lamins B1 and B2, and lamin D/E, are present in this structure. In addition, a group of 45-kDa proteins or intermediate filament protein--reacting proteins (IFA-RPs), located uniquely in the lamina, were found to exhibit the same immunological and chemical characteristics as lamins. Although they behave like nuclear lamins, microsequencing analysis of the IFA-RPs has revealed no homology with known lamins. These IFA-RPs may contribute to the formation of the nuclear lamina filament network in the absence of lamins A and C.

Structure of the gene coding for the mouse TRiC-P5 subunit of the cytosolic chaperonin TRiC.

TRiC is a cytoplasmic chaperonin involved in actin and tubulin folding. It is formed by six to nine different but related proteins of 52 to 65 kDa arranged in two hetero-oligomeric rings. We have cloned the gene coding for the mouse TRiC-P5 subunit (also called CCT gamma) using a XbaI-DraIII fragment of the mTRiC5 cDNA. The mouse genome contains one TRiC5 gene and one TRiC5 pseudogene located on chromosomes 3F and 5B, respectively. The 2-kb transcript of TRiC5 is encoded by 14 exons distributed within 25 kb of genomic DNA. The largest exon is 312 bp and the smallest exon is 51 bp. We have used primer extension to demonstrate multiple transcription start points for the TRiC5 gene. This is consistent with the lack of any obvious TATA box upstream of the transcription start points.

The cytosolic chaperonin subunit TRiC-P5 begins to be expressed at the two-cell stage in mouse embryos.

The cytosolic chaperonin TRiC is a large protein complex involved in the folding of newly synthesized actin and tubulin. The fertilization of the mouse oocyte is followed by a remodelling of the actin and tubulin filaments. The TRiC subunit TCP1 is expressed only from the 4-cell stage on, even though actin and tubulin are synthesized in the previous stages. We investigated the onset of synthesis of another subunit, TRiC-P5, during early mouse embryogenesis. We report that TRiC-P5 is synthesized at the 2-cell stage in an alpha-amanitin sensitive manner. Thus, it is expressed before TCP1 and is one of the first proteins to be synthesized after zygotic genome activation.

Neuron specificity of the neurofilament light promoter in transgenic mice requires the presence of DNA unwinding elements.

Three reporter genes, the chloramphenicol acetyltransferase (CAT), the lacZ, and the intronless NF-L DNA, were used to test the activity of the proximal promoter region (-292 bp) of the human neurofilament light (hNF-L) gene in transgenic mice. Surprisingly, the hNF-L/CAT construct was highly sensitive to position effect, and its expression was found at low levels in several tissues of adult transgenic mice (Beaudet, L., Charron, G., Houle, D., Tretjakoff, I. Peterson, A., and Julien, J.-P. (1992) Gene (Amst.) 116, 205-214). In contrast, the hNF-L/lacZ or the hNF-L/intronless constructs were expressed exclusively in the nervous system during embryonic development and in adult animals. The DNA sequences analysis of the different reporter genes revealed the presence of matrix attachment regions (MARs) within the 3'-untranslated regions of all three transgenes. DNA unwinding elements were found within the MARs of lacZ and hNF-L gene constructs but not in the CAT gene construct. When this element was removed from the lacZ construct, expression of the hNF-L/lacZ transgene became susceptible to position effect and was no longer tissue-specific. These results indicate that DNA unwinding elements are essential for position effect independence conferred by MARs to the hNF-L basal promoter.

TRiC-P5, a novel TCP1-related protein, is localized in the cytoplasm and in the nuclear matrix.

We have recently reported the cloning of a novel protein, TRiC-P5, with significant homology with protein 1 of the t-complex (TCP1). In the present study, the cellular localization of TRiC-P5 in Raji cells has been determined using an antiserum raised against a 18.5 kDa fusion protein. Results from cell fractionation and immunoblot studies indicate that TRiC-P5 is mainly localized in the cytoplasm. In addition, a significant part of TRiC-P5 is also found in the nucleus where it is attached to the nuclear matrix, a complex filament network involved in essential cellular functions such as DNA replication, and RNA transcription and maturation. Immunofluorescence experiments using the anti-TRiC-P5 antibodies confirm these results. We also provide evidence that, in the cytoplasm, TRiC-P5 is part of a large protein complex, most probably the TCP1-ring complex (TRiC), a hetero-oligomeric ring complex that plays a role of molecular chaperone in the folding of actin and tubulin.

Assignment of the human homologue of the mTRiC-P5 gene (TRIC5) to band 1q23 by fluorescence in situ hybridization.

The TCP1 ring complex (TRiC) is a molecular chaperone involved in actin and tubulin folding. Little is known about the components of this complex. The first component identified was TCP1, a protein coded by a gene in the t-complex locus on mouse chromosome 17. This locus is involved in several embryonic defects, male sterility, and the transmission ratio distortion. In humans, the t-complex genes map to chromosome 6. Other components of TRiC are thought to be TCP1-related proteins. Recently, a mouse cDNA coding for one of these proteins has been cloned and named mTRiC-P5. Here we report the cloning of a partial human cDNA clone, homologous to mTRiC-P5, and its chromosome localization by fluorescence in situ hybridization. The human TRiC-P5 gene (TRIC5) maps to human chromosome 1q23, a region known to be a preferential chromosomal breakpoint involved in leukemia. Therefore, even if TCP1 and TRiC-P5 are related proteins and are found in the same protein complex, they are not coded by syntenic genes in humans.

cDNA encoding a novel TCP1-related protein.

We report the cloning of a mouse cDNA encoding a novel protein which has significant homology with the t-complex protein 1b (TCP1b). In addition, this protein has high sequence identity with tryptic peptides from the bovine P5 subunit of the TCP1-ring complex. We named this novel protein mTRiC-P5 for mouse TCP1-Ring Complex Protein #5. Results indicate that mTRiC-P5 is a new member of the TCP1-TF55 family and is part of the TCP1-ring complex.

Presence of a novel nuclear lamina protein in Raji cells.

In mammalian tissues, the nuclear lamina is composed of the major lamins A, B, and C, and minor lamins D/E. Although lamin B is present in all cell types, lamins A and C are absent from embryonic cells and most undifferentiated cells from hematopoietic lineage. We have investigated the nuclear lamina protein composition of the Raji cell line, lymphoblast-like cells established from a Burkitt lymphoma patient. Lamins A and C were confirmed absent by immunodetection and Northern blot analysis. Besides lamins B and D/E, a protein migrating around 71 kilodaltons was recognized by a serum directed against the nuclear lamina of BHK-21 fibroblasts. Cellular localization by sequential extraction established this 71-kilodalton protein as an exclusive component of the nuclear lamina fraction. These results indicate that the nuclear lamina has a more complex composition than previously thought to be the case for cells devoid of lamins A and C.

Nuclear matrix protein mitotin messenger RNA is expressed at constant levels during the cell cycle.

During the course of an investigation on nuclear matrix protein cDNAs, we have isolated a cDNA clone hybridizing with the messenger RNA encoding mitotin. Mitotin is a 125 kDa/pI 6.5 nuclear matrix protein present in proliferating but not in resting cells. This protein was shown to have a marked increase and characteristic redistribution in G2/M phase of the cell cycle. In this report, using synchronized Raji and WISH cells, we demonstrate that mitotin messenger RNA is expressed at the same level throughout the cell cycle.

Expression of nuclear matrix proteins in rat liver tissue.

We have studied the synthesis of nuclear matrix proteins as it occurs in the rat liver. To investigate their kinetics in tissue, nuclear matrix proteins were prepared from liver of rats injected with radioactive methionine. Synthesis of lamins was not observed in quiescent hepatocytes although they were the principal proteins of this subcellular fraction, suggesting that lamins are very stable in the liver. When hepatocytes were stimulated to divide by partial hepatectomy, only synthesis of lamin B was initiated. Many proteins not visible on Coomassie blue-stained gels were detectable by autoradiography. In the nuclear matrix extracts of quiescent hepatocytes, one of the most prominently labeled ones was a protein of 70 kDa. After hepatectomy, an additional protein of 62 kDa was detectable. These proteins were visible 1 h after the injection of radioactivity, but were no longer observed in nuclear matrices prepared 24 h after injection. These experiments indicate that in addition to lamins, two nuclear matrix proteins are present in the rat liver that were not detected previously, perhaps because of their rapid turnover.

Differential transport and integration into the nuclear lamina for lamins A, B, and C.

Lamins A, B, and C are the major proteins of the mammalian nuclear lamina and have been well studied in BHK-21 cells. Using in vivo labelling, cell fractionation, and immunoprecipitation, we have found that lamins have different patterns of nuclear transport and solubility. Newly synthesized lamin A is translocated to the nucleus faster than lamin C or B. It is the most tightly bound lamin and cannot be extracted from the lamina by nonionic detergent or high-salt buffers. Lamins B and C migrate more slowly to the nucleus. Partitioning between cytoskeleton and detergent-soluble fractions shows that integration of lamins B and C is not completed before a 1-h chase. For lamin C this process is dependent upon protein synthesis and can be inhibited with cycloheximide. Even though lamins A and C are almost identical, lamin C is never firmly bound to the lamina and can be partially solubilized upon high-salt treatment.

A 165 kd protein of the herpes simplex virion shares a common epitope with the regulatory protein, ICP4.

We have investigated the possibility that immediate-early (IE) protein ICP4 could be a part of herpes simplex virus type 1 (HSV-1) virion particle. Immunodetection with a monoclonal antibody against ICP4 reveals that a component of the virion, migrating at 165 kd, shares a common epitope with this immediate-early protein. Immunolocalization studies on purified virions indicate that the antigen can be detected only in virions without membranes, and is located outside the capsid, most probably in the tegument. Ultrastructural localizations on HSV-1 infected BHK cells extracted with a nonionic detergent confirm that the protein immunoreacting with anti-ICP4 is present in virions.

Synthesis of nuclear lamins in BHK-21 cells synchronized with aphidicolin.

Lamins A, B and C are the major proteins of mammalian nuclear lamina and have been well studied in BHK-21 cells. By synchronizing BHK-21 cells with aphidicolin, a potent inhibitor of DNA alpha-polymerase, we were able to detect a differential pattern of synthesis for nuclear lamins during the cell cycle. Lamin B starts to be synthesized only in S phase up to mitosis while synthesis of lamins A and C remain stable throughout the cell cycle. The precursor of lamin A see its half-life increase from a reported 63 min in interphase cells to 103 min in G2/M cells.

A novel autoantibody causing a peripheral fluorescent antinuclear antibody pattern is specific for nuclear pore complexes.

We characterized serum from a patient with polymyositis, and found that it produced a peripheral (rim) fluorescent antinuclear antibody pattern on rat liver substrate. Indirect immunofluorescence analysis revealed a punctate pattern at the nuclear surface of PtK2, BHK-21, and HEp-2 cells. This pattern was still present after sequential extraction in situ with non-ionic detergent, DNase, RNase, and high ionic strength buffer (2M NaCl). Immunogold electron microscopic localization was specific for nuclear pore complexes. By immunoblot analysis, the antigens were polypeptides of 200 kd and 130 kd that were enriched in the nuclear fraction.

DNA-binding proteins of herpes simplex virus type 1-infected BHK cell nuclear matrices.

The nuclear matrix is involved in the replicative cycle of herpes simplex virus type 1 (HSV-1) and in at least some cases viral DNA has been shown to be closely associated with this structure. In this communication, we report the presence of five DNA-binding proteins in the nuclear matrix of HSV-1-infected BHK cells. These proteins (p114, p89, p77, p37 and p29) were detected by probing with 32P-labelled HSV DNA after Western blotting of nuclear matrix proteins. Three were identified as virion components: p89 as VP12, p77 as VP13 and p37 as the capsid protein VP22a. These polypeptides were detected in cells and nuclei and found to be associated with the nuclear matrix late during the lytic cycle, long after the onset of viral DNA replication. The nuclear matrix-binding capacity of VP22a depended on viral DNA replication, since after DNA polymerase inhibition it was still synthesized and transported into the nucleus but was no longer associated with the nuclear matrix. After inhibition of viral DNA synthesis, VP13 was no longer found in cells, nuclei or nuclear matrices. These results suggest a possible involvement in anchoring viral progeny DNA to the nuclear matrix.

Role of the nuclear matrix in adenovirus maturation.

The nuclear matrix has been implicated in several important cellular processes. In this paper, we investigate the role of the nuclear matrix in adenovirus type 2 assembly. Electron microscopic examination of nuclear matrices isolated from adenovirus infected Hep-2 cells clearly reveals that late in the lytic cycle, adenovirus capsids are intimately associated with the nuclear matrix. SDS-PAGE analysis showed that the viral core polypeptides V, PVII and 11 kDa were enriched in the nuclear matrix fraction. After a 3 h chase period a constant high ratio of PVII to VII prevailed in the nuclear matrix suggesting that mostly young virions and viral cores are bound to this structure. Most of the virus maturation endoproteinase activity co-purified with the nuclear matrix and the data suggest that the enzyme may be released from fragile young virions or assembly intermediates. Together these experiments suggest that the nuclear matrix is the site of adenovirus assembly and that mature virions may be released from the matrix by the viral endoproteinase.

Detection in BHK cells of a precursor form for lamin A.

Lamins are structural proteins found in the fibrous lamina underlining the nuclear envelope. In vitro translation of polyadenylated RNA or polysomes followed by immunoprecipitation with a serum raised against BHK nuclear matrix proteins showed that lamin A (72 kD) is synthesized as a high molecular weight precursor (74 kD) (Laliberté et al., J Cell Biol 98 (1984) 980) [23]. We have thus investigated the presence in BHK cells of this putative precursor by in vivo labelling with [35S]methionine and immunoprecipitation of lamin proteins. Short labelling times, ranging from 5 to 60 min reveal the presence of the 74 kD protein. Pulse-chase experiments indicate that the half-life of the precursor is about 60 min. On two-dimensional gel, the 74 kD protein is resolved in a cluster of isovariants between pH 7.4 and 6.6, which are generally slightly more alkaline than their counterparts in lamin A. These results indicate that lamin A is synthesized as a precursor of 74 kD; the long half-life further suggests that pre-lamin A might accumulate in some sort of cellular pool before undergoing post-transcriptional modification(s) to give the mature form of lamin A.

Nuclear matrix modifications at different stages of infection by herpes simplex virus type 1.

In BHK-21 cells infected with herpes simplex virus type 1 many virus-induced proteins were found attached to the nuclear matrix. To understand the role of this cell fraction during virogenesis, matrix-associated proteins were analysed at different stages of infection. All the immediate-early protein species were bound to the nuclear matrix and their association with this structure was stable. During the first few hours of infection, the pattern of virus-induced proteins attached to the nuclear matrix remained identical, indicating that polypeptides from the early group are not associated with this cell fraction. Among the late proteins, which are generally structural proteins, 60% of the nuclear proteins were tightly bound to the nuclear matrix. This suggests that the nuclear matrix is involved in at least two different events during virogenesis, regulation of viral infection and assembly of viral capsids.

In situ localization of the major capsid protein during lytic infection by herpes simplex virus.

The intracellular localization of the major capsid protein (ICP5) of herpes simplex virus was studied during virogenesis. Except for a brief period at the onset of synthesis, this protein was found almost exclusively inside the nucleus. Its localization was not at random since 80% was tightly bound to the nuclear matrix as early as 4 h after infection. Discrete modifications of the fluorescence pattern occurred in an orderly fashion during the progression of the infection. Immunoelectron microscopic studies using Protein A-gold labelling demonstrated that this protein is synthesized on cytoskeleton-bound polyribosomes and accumulates in the central part of the nucleus where formation of viral capsids occurs; no gold particles were found in association with the peripheral chromatin or with the nucleolus.