PubMed Health⌕ Search

Biomedical subjects

W C Earnshaw

Publications and source records attributed to W C Earnshaw.

At least 19 recordsLinked to original sources
PubMed1992

CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate.

We have isolated and characterized a set of overlapping cDNA clones that encode the human centromere autoantigen centromere protein C (CENP-C). The identity of these clones has been established using several criteria. First, they were shown to encode a polypeptide that migrates at the expected position for CENP-C on SDS-polyacrylamide gel electrophoresis. Second, we have demonstrated that this polypeptide shares at least two epitopes with human CENP-C. Polyclonal antibodies were raised to fusion proteins encoded by nonoverlapping regions of the cDNA clones. These antibodies were shown to recognize a protein at a position appropriate for CENP-C on immunoblots of human chromosomal proteins. In addition, we used indirect immunofluorescence to demonstrate that these antibodies recognize centromeres of HeLa chromosomes in the expected pattern for CENP-C. Localization of CENP-C by immunoelectron microscopy reveals that this protein is a component of the inner kinetochore plate.

Amino Acid Sequence↗
PubMed1992

Centromere and kinetochore structure.

Recent studies have begun to yield some insight into the structural and regulatory components of centromeres, and new assays have been developed that promise to be of use in advancing our understanding of centromere structure and function. In the budding yeast Saccharomyces cerevisiae new proteins that are required for centromere function have been identified and an in vitro microtubule-binding assay that should assist in dissecting the process of centromere microtubule attachment has been developed. The centromere-specific DNA sequences in the fission yeast Schizosaccharomyces pombe have been identified and partially characterized. In addition, several mammalian centromere proteins have been further characterized, and localization and inhibition studies suggest roles for these proteins in the regulation and assembly of a functional kinetochore.

Animals↗
PubMed1992

Identification of a subdomain of CENP-B that is necessary and sufficient for localization to the human centromere.

We have combined in vivo and in vitro approaches to investigate the function of CENP-B, a major protein of human centromeric heterochromatin. Expression of epitope-tagged deletion derivatives of CENP-B in HeLa cells revealed that a single domain less than 158 residues from the amino terminus of the protein is sufficient to localize CENP-B to centromeres. Centromere localization was abolished if as few as 28 amino acids were removed from the amino terminus of CENP-B. The centromere localization signal of CENP-B can function in an autonomous fashion, relocating a fused bacterial enzyme to centromeres. The centromere localization domain of CENP-B specifically binds in vitro to a subset of alpha-satellite DNA monomers. These results suggest that the primary mechanism for localization of CENP-B to centromeres involves the recognition of a DNA sequence found at centromeres. Analysis of the distribution of this sequence in alpha-satellite DNA suggests that CENP-B binding may have profound effects on chromatin structure at centromeres.

Autoantigens↗
PubMed1991

Disruption of centromere assembly during interphase inhibits kinetochore morphogenesis and function in mitosis.

The relationship between the kinetochore and the centromeric heterochromatin that surrounds it is unknown. Anti-centromere autoantibodies (ACAs) that recognize antigens found in the heterochromatin beneath the kinetochore disrupt mitotic events when microinjected into human cells. We show here that ACAs interfere with two different stages of centromere assembly during interphase, resulting in abnormal kinetochore structures during mitosis. Antibody injection prior to late G2 results in the subsequent failure to assemble a trilaminar kinetochore. Such chromosomes bind microtubules but are incapable of movement. Antibody disruption of events during G2 produces unstable kinetochores that prevent the normal transition into anaphase. These experiments present a novel way to examine events in the pathway of kinetochore assembly that occur during interphase, at a time when this structure cannot be visualized directly.

Autoantibodies↗
PubMed1991

CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAb177) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, approximately 250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAb177 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAb177 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.

Anaphase↗
PubMed1991

Chromosomal passengers: toward an integrated view of mitosis.

The major events of mitosis have traditionally been considered to represent two distinct pathways and have been studied by two separate groups of workers. The chromosomal events (chromosome condensation and sister chromatid disjunction) have been the principal focus for one group, while the cytoskeletal events (nuclear envelope breakdown, chromosomal movements, cytokinesis) have been the focus for the other. This historical division is epitomized by the view of many cell biologists, which was aptly caught by Mazia's comparison of the role of the chromosome arms in mitosis to that of "the corpse at the funeral" which "provide a reason for the proceedings but do not take an active part in them" (Mazia 1961). More recent studies have demonstrated that the role of the chromosomes in mitotic movements is somewhat more active than this. That the kinetochore may play an important role in chromosome movements has long been suspected (see early references in Mazia 1961) but was only proven rather recently (Brinkley et al. 1988; Gorbsky et al. 1987; Nicklas 1989). This has led to a burst of recent interest in all aspects of kinetochore structure and function. Our studies have led us to ask whether chromosomes may play an even more extensive role in the events of mitosis. We suggest here that in addition to their active role in movements, the chromosome may make important structural contributions to the anaphase spindle and cleavage furrow, which are normally thought of as "cytoskeletal" functions. These structural contributions may be made by members of a new class of "chromosomal passenger" proteins that use the chromosomes as a means of conveyance so that they are correctly positioned at the metaphase plate to carry out their nonchromosomal functions during anaphase and the subsequent mitotic events.

Chromosomes↗
PubMed1991

Compartmentalization within the nucleus: discovery of a novel subnuclear region.

Antibodies to a set of structurally related autoantigens (p23-25) bind to a previously uncharacterized, large structural domain in the nucleus of a variety of human cell types. This subnuclear domain is visible by phase contrast alone as a region of decreased density after several different fixation protocols. The morphology of this region changes dramatically during the cell cycle and we have given it the name PIKA (for polymorphic interphase karyosomal association) based on preliminary evidence that the PIKA proteins may be associated with chromatin. The function of the PIKA is not yet known, but our immunolocalization data indicate that it is unlikely to be associated with regions of ongoing DNA replication, heterogeneous nuclear RNA storage, or mRNA processing. The discovery of the PIKA provides evidence supporting an emerging model of nuclear structure. It now appears that the nucleus is organized into distinct domains which include not only the nucleolus, but also previously unidentified regions such as the PIKAs. Furthermore, structural rearrangements undergone by the nucleolus and the PIKAs may be indicative of a broad tendency for nuclear organization to change in a cell cycle-specific fashion.

Animals↗
PubMed1991

Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation.

The INCENPs are two polypeptides of 135 x 10(3) and 150 x 10(3) Mr that enter mitosis as tightly bound chromosomal proteins, but subsequently leave the chromosomes altogether and become associated with the central spindle and cell cortex at the contractile ring. In the experiments reported here we have used confocal microscopy and immunoelectron microscopy to provide a detailed picture of the intracellular location of these proteins during mitosis. The experiments have not only revealed a number of new details concerning the properties of the INCENPs in mitosis, but have revealed a number of novel aspects of the mitotic process itself. The first of these is the existence of a sequential pathway of structural changes in the chromosomes that occurs during metaphase. This pathway is revealed by the existence of four distinct INCENP staining patterns in mitotic cells. In 'early' and 'early/mid' metaphase, the INCENPs gradually become concentrated at the centromeres, forming a ring at the center of the metaphase plate. During 'mid/late' metaphase they exit from the chromosomes, so that by late metaphase they are found solely in streaks that traverse the plate parallel to the spindle axis. The streaks probably correspond to INCENPs closely associated with microtubule bundles, perhaps as part of the stem body material. Examination of transverse optical sections of the spindle interzone during early anaphase reveals an unexpectedly high degree of order. The INCENP antigens are localized on fibers that are organized into a hollow ring 8 microns in diameter and approximately 4 microns beneath the cell cortex. Measurement of cellular dimensions in the confocal microscope reveals that the maximum diameter of early anaphase cells lies across the spindle equator, so that when the cleavage furrow forms, it does so around the maximum circumference of the cell. During anaphase, a subpopulation of the INCENP antigen becomes localized to the cortex where the furrow will subsequently form. This occurs prior to any other evidence of furrowing. Thus, binding of the INCENPs to this region may represent an early step in furrow formation. Together, these results suggest that the INCENPs may represent a new class of 'chromosomal passenger' proteins that are carried to the spindle equator by the chromosomes and subsequently perform a cytoskeletal role following their release from the chromosomes at the metaphase:anaphase transition.

Anaphase↗
PubMed1991

Idiotypic analysis of human anti-topoisomerase I autoantibodies.

Anti-topoisomerase I autoantibodies (anti-topo I) are associated with proximal scleroderma and are of prognostic significance in patients with Raynaud's phenomenon. Polyclonal anti-idiotypic sera were raised against affinity-purified anti-topo I from 2 patients with scleroderma (EM, SG) and 1 healthy individual (NM). All 3 anti-topo I preparations expressed immunodominant private Ids in or near the antigen binding site of the autoantibody. Further analysis of Id-EM showed isotypic restriction to IgG and a stable Id-expression over the course of 9 years. Id-SG and Id-NM were expressed on IgG and on IgA. The idiotypic character of anti-topo I closely resembles that of anti-centromere autoantibodies which are associated with the CREST syndrome of scleroderma. The data suggest an antigen-driven process in the origin of autoantibodies in scleroderma.

Antigens↗
PubMed1991

Idiotypic analysis of human anticentromere autoantibodies.

The idiotypes (Ids) of anticentromere antibodies (ACA) have been studied using a fusion protein obtained from cloned cDNA of the major centromere antigen, CENP-B, for isolation of the autoantibodies. IgG-ACA were affinity purified from 4 patient sera and anti-Ids prepared in rabbits. Analysis revealed the existence of two distinct types of immunodominant Ids. One Id is near the antibody combining site and one is framework associated. A longterm longitudinal study of Id expression in a patient who seroconverted from ACA (-) to ACA (+) when she developed Raynaud's phenomenon showed a close correlation between Id expression and ACA titers (r = 0.94). These results may be interpreted as evidence for an autoantigen driven process in the anticentromere immune response.

Adult↗
PubMed1990

The IgG, IgM, and IgA isotypes of anti-topoisomerase I and anticentromere autoantibodies.

We studied the expression of IgG, IgM, and IgA autoantibodies in the anti-topoisomerase I and anticentromere immune responses by enzyme-linked immunosorbent assay, immunoblotting, and immunofluorescence. While IgG autoantibodies were most common, IgA autoantibodies were also frequently found, but IgM autoantibodies were rare. This is the first report of IgA autoantibodies in scleroderma.

Autoantibodies↗
PubMed1990

Use of molecular cloning methods to map the distribution of epitopes on topoisomerase I (Scl-70) recognized by sera of scleroderma patients.

We report the initial molecular characterization of the autoimmune response against DNA topoisomerase I (topo I; Scl-70). Sera from 36 patients with scleroderma and 4 healthy control subjects were studied using 6 subcloned portions of topo I. Twenty-three sera recognized at least 2 independent epitopes on the molecule. Therefore, anti-topo I, like other non-organ-specific autoantibodies characterized to date, is polyclonal and multifocal. The cloned protein should prove suitable for sensitive early detection of anti-topo I in the clinical setting.

Autoantibodies↗
PubMed1990

Autoantibodies to topoisomerase I (Scl-70): analysis by gel diffusion, immunoblot, and enzyme-linked immunosorbent assay.

Anti-topoisomerase I autoantibodies (anti-topo I, anti-Scl-70) are associated with proximal scleroderma and are of prognostic significance in patients with Raynaud's disease. To establish a highly sensitive and specific system for the detection of anti-topo I, we have investigated sera from 409 patients and controls by Ouchterlony gel diffusion, Western immunoblot on chromosome proteins, and solid-phase enzyme-linked immunosorbent assay (ELISA) with purified topoisomerase I as antigen. The ELISA was more sensitive than the gel diffusion technique and was more specific than the Western immunoblot, while the immunoblot may identify additional autoantibodies.

Autoantibodies↗
PubMed1990

Structure of the human centromere at metaphase.

Until recently the centromere was thought to be a relatively homogeneous region of densely packed heterochromatin with a single differentiated domain--the kinetochore--at its surface, representing the point of attachment of the mitotic spindle. We now know that the centromere of higher eukaryotes is composed of several domains that have been identified using antibody probes. Somewhere within the domains are located both the factor(s) that control the disjunction of sister chromatids and the molecular motor responsible for chromosome movement towards the spindle poles.

Centromere↗
PubMed1990

CENP-B: a major human centromere protein located beneath the kinetochore.

The family of three structurally related autoantigens CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD) are the best characterized components of the human centromere, and they have been widely assumed to be components of the kinetochore. Kinetochore components are currently of great interest since this structure, which has long been known to be the site of microtubule attachment to the chromosome, is now believed to be a site of force production for anaphase chromosome movement. In the present study we have mapped the distribution of CENP-B in mitotic chromosomes by immunoelectron microscopy using two monospecific polyclonal antibodies together with a newly developed series of ultra-small 1-nm colloidal gold probes. We were surprised to find that greater than 95% of CENP-B is distributed throughout the centromeric heterochromatin beneath the kinetochore. This strongly supports other emerging evidence that CENP-B is specifically associated with alpha-satellite heterochromatin. Although in certain instances CENP-B can be seen to be concentrated immediately adjacent to the lower surface of the kinetochore, the outer plate remains virtually unlabeled. Similar analysis with a human autoimmune serum that recognizes all three CENP antigens reveals an additional unsuspected feature of kinetochore structure. In addition to recognizing antigens in the centromeric heterochromatin, the autoantiserum recognizes a concentration of antigens lateral to the kinetochore. This difference in staining pattern may reflect the presence of a "collar" of chromatin rich in CENP-C and/or CENP-A encircling the kinetochore plates.

Autoantibodies↗