Transferring and culturing an architecturally intact layer of cells from animal tissue on membrane substrates.
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Animals↗
Biomedical subjects
J B Rattner
Publications and source records attributed to J B Rattner.
Histone acetyltransferase is associated with the nuclear matrix.
Only a small fraction of the adult chicken erythrocyte histones is involved in dynamic acetylation. We have reported previously that the rapidly acetylated and deacetylated H4 histones are primarily associated with the transcriptionally active DNA-enriched chromatin fragments that remain attached to the residual nuclear material following micrococcal nuclease digestion and chromatin solubilization. Furthermore, this nuclear fraction contained most of the histone deacetylase activity. In this study we show that the bulk of the nuclear histone acetyltransferase activity is located with the insoluble residual nuclear material. We demonstrate that in vitro the enzymes associated with the residual nuclear material catalyze reversible acetylation when the endogenous histones of the nuclear skeleton-bound chromatin fragments are used as substrate. Nuclear matrices isolated from adult chicken immature erythrocyte and trout liver nuclei had 60-76% of the nuclear histone acetyltransferase activity. Procedures that solubilized the internal nuclear matrix also resulted in the release of the enzyme from the nuclear matrix. Together, our observations suggest that histone acetyltransferase and deacetylase are associated with the internal nuclear matrix, and one of the functions of these enzymes may be to mediate a dynamic attachment between transcriptionally active chromatin and the nuclear matrix.
Acetates↗
CENP-F is a .ca 400 kDa kinetochore protein that exhibits a cell-cycle dependent localization.
We have identified a novel .ca 400 kDa cell-cycle dependent kinetochore associated protein in human cells, designated CENP-F, using human autoimmune serum. Immunofluorescence staining using the native serum, affinity purified antibodies, or antibodies raised against a cloned portion of CENP-F first reveals CENP-F homogeneously distributed throughout the nucleus of HeLa cells in the G2 stage of the cell cycle. Progression into prophase is accompanied by the localization of CENP-F to all the kinetochore regions of the karyotype. Kinetochore association is maintained throughout metaphase, but at the onset of anaphase CENP-F is no longer detected in association with the kinetochore but is found at the spindle mid-zone. By telophase, it is concentrated into a narrow band on either side of the midbody. Studies of the interaction of CENP-F with the kinetochore indicate that this protein associates with the kinetochore independent of tubulin and dissociation is dependent on events connected with the onset of anaphase. Nuclease digestion studies and immunoelectron-microscopy indicate that CENP-F is localized to the kinetochore plates and specifically to the outer surface of the outer kinetochore plate. The distribution of CENP-F closely parallels that of another high molecular weight kinetochore associated protein, CENP-E. Comparative studies indicate that there are antibodies in the CENP-F reactive autoimmune serum that recognize determinants present in the central helical rod domain of CENP-E. Immune depletion experiments confirm that CENP-F exhibits the distribution pattern in cells that was seen with the native autoimmune serum.
Animals↗
Centrosome repositioning immediately following karyokinesis and prior to cytokinesis.
The behaviour of the centrosome immediately following cell division in tissue culture cells has been investigated. We find that following karyokinesis, but preceding cytokinesis, sister centrosomes relocate from the spindle poles to a position adjacent to the intercellular bridge. This repositioning is accompanied by the appearance of a microtubule bundle that extends from the poleward region of the cell to the centrosome and increases in length as the centrosome approaches the intercellular bridge. Disruption of this bundle with colcemid interrupts centrosome repositioning. In contrast, centrosome repositioning persists in late mitotic cells grown in the presence of cytochalasin D. However, the position of the microtubule-centrosome complex within the cell is randomized suggesting that the path, but not the process, of centrosome repositioning is dependent on an intact actin filament network. This study points out, for the first time, that the complex migration of the centrosome preceding mitosis is paralleled by an equally complex set of events following cell division. We suggest that post-mitotic centrosome repositioning may play a role in ensuring that daughter cells have equal but opposite polarity and may reflect an interrelationship between the establishment of the interphase cytoskeleton and the completion of cytokinesis.
Anaphase↗
Mapping DNA within the mammalian kinetochore.
The location of the cis-acting DNA sequences that direct the assembly of the mammalian kinetochore is not known. A variety of circumstantial evidence, however, has led to the widespread belief that they are present throughout the kinetochore including the kinetochore outer plate. To investigate this question directly, we have used two independent methods to localize DNA in and around the mammalian kinetochore. Both methods fail to reveal DNA in the outer kinetochore plate, finding instead that the outer-most detectable DNA in the centromere is located in the inner kinetochore plate. Our results imply that the outer kinetochore plate is primarily a proteinaceous structure. It is thus unlikely that fibers observed in the outer plate correspond to chromatin, as previously assumed. Our observations suggest that current models of kinetochore structure may need to be reconsidered.
Animals↗
Mapping the mammalian intercellular bridge.
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Anaphase↗
Integrating chromosome structure with function.
Historically, the metaphase chromosome and its architecture has been viewed as the ultimate representation of a non-functional inactive chromatin state. Recent studies of centromere (kinetochore) function in concert with studies of the placement and function of several classes of chromosomal proteins now call for a reevaluation of this view. In this article a model of chromosome structure with functional correlates is presented. Evidence for the existence of a functional chromosomal region, the "Surface Domain" is discussed.
Animals↗
MSA-36: a chromosomal and mitotic spindle-associated protein.
We have identified a novel M(r) 36,000 protein (MSA-36) that has a complex cell cycle dependent distribution. This protein is first detected in interphase nuclei just prior to the onset of chromosome condensation. MSA-36 is found along condensing chromosomes and is a component of the centromere through metaphase. At anaphase, this protein is no longer detected in association with the chromosomes but appears at the forming stembodies and subsequently within the intercellular bridge at either side of the midbody. At the completion of cell division, the amount of MSA-36 in the bridge appears to decline concurrent with the appearance of this protein briefly within the reforming nucleus. To investigate whether MSA-36 is an active component of the chromosome or a passive passenger protein, we studied the behaviour of this protein in cells exhibiting premature chromosome condensation and in cells during and following recovery from mitotic arrest. These studies suggest that MSA-36 is not essential for a variety of major chromosome-associated events.
Autoantigens↗
Enolase is present at the centrosome of HeLa cells.
Antibodies raised against the C-terminus and N-terminus region of gamma gamma enolase, as well as a polyclonal antibody raised against bovine brain gamma gamma enolase, were used to study the distribution of this glycolytic enzyme during the cell cycle in HeLa cells. Enolase was found to be present throughout the cytoplasm of both interphase and dividing cells. In addition, a portion of cellular enolase was detected at the centrosome throughout the cell cycle. The capacity of glycolytic enzymes to play a structural as well as a glycolytic role suggests that the presence of enolase at the centrosome may be correlated with the organization of both the interphase cytoskeleton and the mitotic spindle.
Amino Acid Sequence↗
Kinetochore formation and behaviour following premature chromosome condensation.
The potential for interphase centromeres to support kinetochore formation following premature chromosome condensation (PCC) has been investigated. We show that the centromere remains competent to initiate kinetochore formation throughout the cell cycle. PCC-kinetochores display a typical trilaminar morphology, associate with microtubules and show movement towards the centrosome. Indirect immunofluorescence studies illustrate that the centromere/kinetochore region of prematurely condensed chromosomes associates with proteins that are normally found within this region in both a cell cycle-dependent and an independent manner.
Centromere↗
Autoantibodies to the centrosome (centriole) react with determinants present in the glycolytic enzyme enolase.
Autoantibodies to cellular Ag are found in the sera of patients with systemic rheumatic diseases. Identification and characterization of the reactive autoantigens has helped clinicians to define subsets of rheumatic diseases and has assisted biologists in defining the function within the cell of these molecules. We have studied autoantibodies from patients that react with the centrosome (centriole) region of the cell. We found by immunoblotting techniques that these antibodies react with a 48-kDa protein. Additional immunoblotting and affinity purification studies indicate that the Ag may be the glycolytic enzyme enolase.
Autoantibodies↗
Identification of novel single-stranded d(TC)n binding proteins in several mammalian species.
A group of single-stranded d(TC)n specific binding proteins has been detected in the nuclear extracts of several mammalian species that included mouse, human, African green monkey, chimpanzee, and Chinese muntjac. Southwestern analysis of 500 mM KCI nuclear extracts has shown that these proteins cluster in a similar size range, 55.5 to 57 kD. An additional 54 kD band was present for the three primate species examined. The single-stranded d(TC)n binding activity was confirmed with bandshift assay. Specific double-stranded binding activity for duplex d(TC)n.d(GA)n or single-stranded d(GA)n was not detected. The conservation of size distribution and d(TC)n-binding activity across the species examined indicates that this class of single-stranded binding proteins may have an important biological function in vivo.
Animals↗
The structure of the mammalian centromere.
The mammalian centromere is a multifunctional chromosomal domain with a complexity that is reflected in its higher order structure, DNA sequence organization and protein composition. The centromere plays a major role during cell division where it functions as the site for the integration of the chromosome with the mitotic spindle, the site of the mechanochemical motor responsible for the movement of chromosomes and the major and last point of interaction between sister chromatids. Recent studies have focused on characterizing the components of the centromere and establishing their relationship to its function. The following brief review summarizes some selected aspects of this recent work.
Animals↗
Studies of mitotic and centromeric abnormalities in Roberts syndrome: implications for a defect in the mitotic mechanism.
Roberts syndrome is an inherited human condition that is of particular interest because separation of centromeres and constitutive heterochromatin is observed in metaphase chromosomes. In this study we investigated the frequency of other cytological abnormalities in three Roberts syndrome patients. Our findings when taken with previous cytological reports emphasize that there are other features that are equally characteristic of Roberts syndrome: (1) aneuploidy with random chromosome loss and (2) micronuclei and/or nuclear lobulations of 8%-24% of interphase cells. We observed abnormal chromosome movement involving one or all the chromosomes during anaphase. Evidence is presented suggesting that aneuploidy, micronuclei and abnormal nuclear morphology are a direct result of lagging chromosomes. The cytological features documented for Roberts syndrome indicate that this is a human mitotic mutant.
Abnormalities, Multiple↗
hsp70 is localized to the centrosome of dividing HeLa cells.
Monoclonal antibodies specific for inducible and constitutive members of the hsp70 family have been used to investigate the distribution of these proteins during the cell cycle of HeLa cells with special reference to mitosis. Indirect immunofluorescence studies illustrate that a portion of the constitutive form, hsp73, is localized to the centrosome during cell division. In addition a subset of the inducible form, hsp72, collects at the centrosome of dividing cells following heat shock. These observations suggest that members of the hsp70 family are cell cycle specific components of the centrosome in HeLa cells and may play an important role in the function of this microtubule organizing center.
Antibodies, Monoclonal↗
The use of autoantibodies in the study of nuclear and chromosomal organization.
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Animals↗
p34cdc2 kinase is localized to distinct domains within the mitotic apparatus.
Antibodies to both the C-terminal and the N-terminal regions of the 34 kd serine-threonine specific protein kinase, p34cdc2, were used to study the distribution of this protein in dividing cells and isolated chromosomes of the Indian muntjac. p34cdc2 was found to be present throughout the cytoplasm of dividing cells. In addition, a portion of cellular p34cdc2 was localized to the centrosome, kinetochore, and intercellular bridge and along kinetochore-to-pole microtubules during cell division. Tubulin-denuded metaphase kinetochores retained their association with p34cdc2. The detection of p34cdc2 within a variety of domains of the mitotic apparatus, in addition to the previous reported association with the centrosome [Bailly et al., EMBO J. 8:3985-3995, 1989; Raibowol et al., Cell 57:393-401, 1989] suggests that p34cdc2 may play a role in events associated with anaphases A and B as well as with the transition between interphase and mitosis.
Amino Acid Sequence↗
The chromosomal distribution of the major and minor satellite is not conserved in the genus Mus.
The cytological distribution of the major and minor satellite first identified in Mus musculus was studied in the karyotypes of three related subspecies and two other species of the genus Mus. Both the major and minor satellite showed species dependent hybridization patterns. The major satellite is confined to the centromere region in M. musculus and related subspecies. However, in M spretus and M. caroli, the chromosomal arm regions contain this sequence class. In contrast the minor satellite is found at the kinetochore region in M. musculus and related subspecies but is distributed throughout the entire centromeric domain in M. spretus and appears to be excluded from the chromosomes of M. caroli. There is an apparent correlation between the chromosomal location of these satellites and their phylogenetic relationship. Determination of the biological roles of the major and minor satellites from M. musculus must take into account their differential chromosomal distribution in other Mus species.
Animals↗