Journal of cell science. Supplement最新文献

The eukaryotic cell nucleus is a highly dynamic organelle. This is illustrated most dramatically during mitosis, when the nuclear envelope breaks down, the nuclear lamina disassembles, chromosomes condense, and a microtubule-based spindle apparatus distributes sister chromatids to the dividing daughter cells. Many of these dramatic changes in nuclear architecture and microtubule organization are controlled by phosphorylation and dephosphorylation events. Whereas the cardinal role of cyclin-dependent kinases (CDKs) in the regulation of mitosis is well established, there is now clear evidence for the requirement of additional mitotic protein kinases. Studies into the regulation of CDKs and other mitotic kinases have revealed that these enzymes undergo cell cycle dependent changes in subcellular distribution, suggesting that localization may contribute to regulating their activities. This article describes some recent findings relating to the nucleocytoplasmic translocation of CDK/cyclin complexes at the onset of mitosis. In addition, it summarizes recent information on two novel human protein kinases which have been implicated in the control of mitotic progression.

Apoptotic cell death is characterized by a dramatic morphological transformation during which apparently healthy cells suddenly initiate a comprehensive program of motility changes and degradative activities that culminates in disassembly of the cell into membrane-enclosed vesicles. The mechanism of the cellular changes during this spectacular execution phase of apoptosis is just now yielding to biochemical analysis. In our laboratory, we have applied a novel in vitro system to the study of these events. In this system, nuclei isolated from healthy cells undergo the characteristic changes of apoptosis rapidly and synchronously. Using this system we have identified the first substrates for interleukin-1 beta-converting enzyme (ICE)-like proteinases during apoptotic execution. One of these, the nuclear enzyme poly (ADP-ribose) polymerase is cleaved very early in the apoptotic process. A second class of proteins, the nuclear lamins, is cleaved later in the pathway. Lamin cleavage requires a second ICE-related proteinase, and is essential for the complete dissolution of nuclei into apoptotic bodies. Studies with our cell-free extracts reveal that the various proteinases and nucleases that operate during the execution phase of apoptosis do so largely in independent parallel biochemical pathways. However, all of these pathways require the action of ICE-related proteinases for their initiation.

Recent experiments suggest that active polymerases are concentrated in large structures, 'factories', within eukaryotic nuclei. Data concerning the structure of these factories is reviewed.

We have been studying the evolution and function of DNA methylation in vertebrate animals using three related approaches. The first is to further characterise proteins that bind to methylated DNA. Such proteins can be viewed as 'receptors' of the methyl-CpG 'ligand' that mediate downstream consequences of DNA modification. The second approach involves CpG islands. These patches of non-methylated DNA coincide with most gene promoters, but their origin and functional significance have only recently become the subject of intensive study. The third approach is to trace the evolution of DNA methylation. Genomic methylation patterns of vertebrates are strikingly different from those of invertebrates. By studying methylation in animals that diverged from common ancestors near to the invertebrate/vertebrate boundary, we will assess the possibility that changes in DNA methylation contributed causally to the evolution of the complex vertebrate lineage.

Base excision-repair, which is required for correction of spontaneous hydrolytic and oxidative damage to DNA as well as lesions inflicted by alkylating agents, is a relatively well understood repair pathway. Mammalian factors involved in this pathway are reviewed, with emphasis on current uncertainties. Most DNA replication and repair enzymes in mammalian cell nuclei, e.g. DNA polymerases alpha, beta, delta, and epsilon, have direct counterparts in yeast. In contrast, the abundant enzymes in mammalian cell nuclei that bind and are activated specifically by DNA strand interruptions, poly(ADP-ribose) polymerase and DNA-dependent protein kinase, have not been detected in yeast; nor has p53, which is elevated in response to DNA strand breaks. We have found a family of four distinct DNA ligases in human cell nuclei, whereas only a single DNA ligase has been detected in yeast. It would appear that the cellular responses to DNA strand breaks may differ markedly between higher and lower eukaryotes.

Yeast chromosomes may lack the linker histone H1 (normally required to compact 10 nm beads-on-a-string fiber into the 30 nm fiber) and there is no cytological evidence for higher order fiber structure but they do contain regions which correspond to euchromatin and heterochromatin of higher eukaryotes. Both euchromatin and heterochromatin contain nucleosomal particles (composed of two molecules each of H2A, H2B, H3 and H4), however histones have been shown to regulate genes in these regions in quite different ways. The mechanisms by which such regulation occurs are the topic of this paper.

In Drosophila, over 50 genes have been identified in which loss-of-function mutations lead to excess cell proliferation in the embryo, in the central nervous system, imaginal discs or hematopoietic organs of the larva, or in the adult gonads. Twenty-two of these genes have been cloned and characterized at the molecular level, and nine of them show clear homology to mammalian genes. Most of these mammalian genes had not been previously implicated in cell proliferation control. Overgrowth in some of the mutants involves conversion to a cell type that, in normal development, shows more cell proliferation than the original cell type. Thus the neurogenic mutants, including Notch, show conversion of epidermal cells to neuroblasts, leading to the 'neurogenic' phenotype of excess nervous tissue. The ovarian tumor mutants show conversion of the female germ line to a cell type resembling the male germ line, which undergoes more proliferation than the female germ line. Mutations of the fat locus cause hyperplastic overgrowth of imaginal discs, in which the epithelial structure is largely intact. The predicted fat protein product is a giant relative of cadherins, supporting indications from human cancer that cadherins play an important role in tumor suppression. Mutations in the lethal(2)giant larvae and lethal(1)discs large genes cause neoplastic overgrowth of imaginal discs as well as the larval brain. The dlg gene encodes a membrane-associated guanylate kinase homolog that is localized at septate junctions between epithelial cells. This protein is a member of a family of homologs that also includes two proteins found at mammalian tight junctions (ZO-1 and ZO-2) and a protein found at mammalian synaptic junctions (PSD-95/SAP90). Genes in which mutations cause blood cell overproduction include aberrant immune response-8, which encodes the RpS6 ribosomal protein and hopscotch, which encodes a putative non-receptor protein tyrosine kinase. The gene products identified by ovarian tumor mutants do not show clear amino acid sequence homology to known proteins. Drosophila provides an opportunity to rapidly identify and characterize tumor suppressor genes, many of which have mammalian homologs that might also be involved in cell proliferation control and tumor suppression.

Wilms' tumor has served as an example of Knudson's two-hit hypothesis of recessive tumor genes, but the genetics has proven to be surprisingly complex. WT1, a tumor suppressor gene on 11p13, is mutated in only a small fraction of Wilms' tumors, and a second chromosomal region, 11p15, harbors a second Wilms' tumor gene also involved in other cancers. In addition, loss of genomic imprinting, or parental origin-specific gene expression of at least two genes, appears to be an early step in Wilms' tumorigenesis and common cancers. Finally, genes on other chromosomes also play a role. I propose a model of Wilms' tumorigenesis in which multiple genetic alterations act within a specific developmental context, accounting for the epidemiological and pathological heterogeneity of Wilms' tumor, as well as the tissue specificity of the tumor types arising from alterations in these genes.

Actin reorganization is an early response to many extracellular factors. In Swiss 3T3 fibroblasts, the Ras-related GTP-binding proteins Rho and Rac act as key signal transducers in these responses: Rho is required for growth factor-induced formation of stress fibres and focal adhesions, whereas membrane ruffling is regulated by Rac proteins. Several proteins that act as GTPase activating proteins (GAPs) for Rho-related proteins have been identified, and these could act either as targets or down-regulators of Rho or Rac in cells. In vitro, the GAP domain of p190 has a striking preference for Rho as a substrate, and when microinjected into Swiss 3T3 cells it inhibits stress fibre formation but not membrane ruffling induced by growth factors. BcrGAP acts on Rac but not Rho in vitro, and specifically inhibits membrane ruffling in vivo. Finally, RhoGAP acts preferentially on the Rho-related protein G25K/Cdc42Hs in vitro, but can inhibit Rho-mediated responses in vivo. These results suggest that p190, Bcr and RhoGAP play specific roles in signalling pathways through different Rho family members. The mechanisms underlying Rho-regulated stress fibre formation have been investigated further by analysing the role of other signals known to be activated by lysophosphatidic acid (LPA). Neither activation of PK-C, increased intracellular Ca2+, decreased cAMP levels or Ras activation appear to mediate stress fibre formation. However, LPA stimulates tyrosine phosphorylation of a number of proteins, including the focal adhesion kinase, pp125FAK, and genistein, a tyrosine kinase inhibitor, prevents this increase in tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct point mutations in the RET proto-oncogene are the cause of the inherited multiple endocrine neoplasia type 2 syndromes (MEN 2), and the congenital gut disorder Hirschsprung disease. The site and type of these mutations suggests that they have differing effects on the activity of the receptor tyrosine kinase encoded by RET. The normal function of the RET receptor tyrosine kinase has yet to be determined. However, this has been investigated by the inactivation of the RET gene in transgenic mice. The developmental abnormalities apparent in these mice, together with the observation that the major tissues affected in MEN 2 and Hirschsprung disease have a common origin in the embryonal neural crest, suggest that RET encodes a receptor for a developmental regulator involved in the genesis of a variety of neural crest derivatives, and in the organogenesis of the kidney.