Effects of heat shock on the distribution and expression levels of nuclear proteins in HeLa S3 cells.

Cumulating evidence has led to the idea that nuclear functions such as DNA replication, RNA transcription, RNA splicing and nucleocytoplasmic transport are facilitated by a proteinaceous architectural framework within the nuclear compartment and at the nuclear envelope. In the present study, we have used immunofluorescence microscopy and quantitative Western blotting to compare the distribution and expression levels of several nuclear proteins during the response of HeLa S3 cells to both mild and severe hyperthermia. Cells were exposed to mild (42 degrees C) or severe (45 degrees C) hyperthermia treatment for 90 min and left to recover at 37 degrees C for 1-25 h. The cell response was monitored immediately after the heat stress and at different time intervals during the recovery period. Our observations indicate that inner nuclear membrane proteins, LAP2beta and emerin, as well as major components of the nuclear lamina, lamins A/C and lamin B1, maintain an overall normal distribution at the nuclear periphery throughout the cell response to mild or severe hyperthermia. The response was nevertheless characterized by significant changes in the expression levels of emerin following recovery from a mild stress and of lamin B1 after recovery from a severe stress. Our results also provide evidence that the organization of functional domains within the nuclear interior such as nucleoli and splicing speckles differs between cells responding to a mild or a severe stress. Mild hyperthermia was accompanied by a significant decrease in the expression level of the nucleolar protein 2H12 whereas severe hyperthermia was characterized by a reduction in the expression of the nucleocytoplasmic shuttling protein 2A7. Our data underline the complexity of nuclear function/structure relationships and the needs for a better understanding of protein-protein interactions within the nuclear compartment.

Evaluation of mammalian cell-free systems of nuclear disassembly and assembly.

Mammalian cell-free systems are very useful for the biochemical and structural study of nuclear disassembly and assembly. Through experimental manipulations, the role of specific proteins in these processes can be studied. Recently, we intended to examine the involvement of integral and peripheral inner nuclear membrane proteins in nuclear disassembly and assembly. However, we could not achieve proper disassembly when isolated interphase HeLa nuclei were exposed to mitotic soluble extracts obtained from the same cell line and containing cyclin B1. Homogenates of synchronized mitotic HeLa cells left to reassemble their nuclei generated incomplete nuclear envelopes on chromatin masses. Digitonin-permeabilized mitotic cells also assembled incomplete nuclei, generating a lot of cytoplasmic inclusions of inner nuclear membrane proteins as an intermediate. These results were therefore used as a basis for a critical evaluation of mammalian cell-free systems. We present here evidence that cell synchronization itself can interfere with the progress of nuclear assembly, possibly by causing aberrant nuclear disassembly and/or by inducing the formation of an abnormal number of mitotic spindles.

The lamina-associated polypeptide 2 (LAP2) genes of zebrafish and chicken: no LAP2alpha isoform is synthesised by non-mammalian vertebrates.

The mammalian lamina-associated polypeptide 2 (LAP2) gene encodes six isoforms (LAP2alpha, beta, delta, epsilon, gamma, zeta) that are synthesised from alternatively spliced mRNAs. The mammalian LAP2alpha is one of the predominant isoforms and localised in the nucleoplasm whereas LAP2beta, delta, epsilon, and gamma are integral membrane proteins of the inner nuclear membrane. We have analysed the LAP2 gene structure of the zebrafish Danio rerio as an attractive lower vertebrate model organism. The zebrafish LAP2 (ZLAP2) gene without regulatory sequences spans approximately 19 kb of genomic DNA. It contains 15 exons that encode the isoforms ZLAP2beta, gamma, and omega which are localised in the inner nuclear membrane. By radiation hybrid mapping, we have located the gene onto linkage group 4 between EST markers fc01g04 (213.97cR) and fb49f01 (215.69cR). The identification of a chicken genomic clone comprising the complete coding region of the avian LAP2 gene enabled us to compare the LAP2 gene structure amongst vertebrates. In contrast to the mammalian LAP2 gene, the zebrafish and the chicken sequences do not encode for an alpha-isoform. In parallel we searched for an alpha-isoform in birds using polyclonal and monoclonal LAP2 antibodies specific for the common evolutionary conserved aminoterminal domain present in all isoforms. We detected LAP2beta as the predominant isoform but no LAP2alpha in tissues of 10-day-old chicken embryos and cultured chicken fibroblasts thus confirming the genomic analysis. The comparison of each zebrafish and chicken LAP2 exon with the corresponding exons of the human LAP2 gene demonstrates that the degree of identity at the amino acid level is much higher between the human and chicken than between the human and zebrafish sequences. By Blast search with the nucleotide and amino acid sequences of the human LAP2alpha, we did not find any significant homologies in databases of the zebrafish and chicken sequences. Our data suggest that LAP2alpha is a novelty of mammals.

The lamina-associated polypeptide 2 (LAP2) isoforms beta, gamma and omega of zebrafish: developmental expression and behavior during the cell cycle.

Zebrafish lamina-associated polypeptides 2 (ZLAP2) beta, gamma and omega have in common an N-terminal region with a LEM domain, and in the C-terminal half of the molecule a lamina binding domain and a membrane spanning sequence. The maternally synthesized omega is the largest isoform and the only LAP2 present in the rapidly dividing embryonic cells up to the gastrula stage. ZLAP2omega levels decrease during development, concomitant with the increase of the somatic isoforms ZLAP2beta and gamma. In somatic zebrafish cells ZLAP2gamma is the predominant isoform, whereas only small amounts of ZLAP2beta are present. During early embryonic development, ZLAP2omega becomes associated with mitotic chromosomes before anaphase. The surface of these chromosomes is decorated with vesicles, and each chromosome assembles its own nuclear envelope at the end of mitosis (karyomere formation). Ectopically expressed ZLAP2omega-green fluorescent protein (GFP) fusion protein targets vesicles to mitotic chromosomes in Xenopus A6 cells, suggesting that ZLAP2omega is involved in karyomere formation during early zebrafish development. When ZLAP2beta and gamma were expressed as GFP fusion proteins in Xenopus A6 cells, the beta- but not the gamma-isoform was found in association with mitotic chromosomes, and ZLAP2beta-containing chromosomes were decorated with vesicles. Further analysis of ZLAP2-GFP fusion proteins containing only distinct domains of the ZLAP2 isoforms revealed that the common N-terminal region in conjunction with beta- or omega-specific sequences mediate binding to mitotic chromosomes in vivo.

Lamina-associated polypeptide 2 (LAP2) expression in fish and amphibians.

Somatic and germinal cells of 15 fish and 33 amphibian species were examined by SDS-PAGE followed by immunoblotting to determine the expression of LAP2 (lamina-associated polypeptide 2). LAP2 expression in frogs, salamanders and fish does not vary with the mode of reproduction. In fish and frog cells, a rim-like LAP2 positive region was detected around the nucleus by indirect immunofluorescence microscopy. The cell distribution and expression patterns of LAP2 in fish, frogs and salamanders are comparable with those found in Xenopus and zebrafish. The mammalian somatic cell pattern, which may also occur in gymnophione amphibians, includes LAP2alpha, beta and gamma as major isoforms, whereas LAP2alpha does not occur in cells of fish, frogs and salamanders. In fish, LAP2gamma is the major isoform of somatic cells, suggesting that LAP2gamma may be ancestral. However, in the rainbow trout, as in frogs and salamanders, LAP2beta was the major somatic isoform. Fish and frog sperm only express low molecular weight polypeptides. In contrast, fish and frog oocytes express an oocyte-specific LAP2 isoform of high molecular weight. In the toad Bufo marinus this isoform becomes upregulated in pre-vitellogenic oocytes of 150-200 microm in diameter. The absence of LAP2alpha and the differential expression of LAP2 isoforms in somatic and germ cells, as found in fish and frogs, may be ancestral vertebrate characters. In spite of differences in developmental time, the LAP2 isoforms of somatic cells are upregulated during gastrulation, suggesting that LAP2 may be implicated in the early development of fish and frog.