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Biomedical subjects

William C Earnshaw

Publications and source records attributed to William C Earnshaw.

At least 19 recordsLinked to original sources
PubMed2007

Autoantibodies against the chromosomal passenger protein INCENP found in a patient with Graham Little-Piccardi-Lassueur syndrome.

BACKGROUND: Graham Little - Piccardi - Lassueur (GLPL) syndrome is a rare dermatosis characterized by scarring alopecia, loss of pubic and axillary hair, and progressive development of variously located follicular papules. We report a first case ever of an autoimmune response in a patient suffering from GLPL syndrome. METHODS: Immunofluorescence and immunoblot analysis were used in a variety of cell cultures including human, monkey, hamster, mouse and bovine cells to analyze the presence of autoantibodies in a GLPL patient. RESULTS: The autoimmune serum showed a pattern of centromere and spindle microtubule staining resembling that of the chromosomal passenger protein complex. By using a complex of proteins expressed in baculovirus, immunoblot analysis demonstrated that the INCENP protein is a major autoantigen in this patient with GLPL syndrome. CONCLUSION: An autoimmune response in GLPL syndrome is reported against the INCENP centromere protein. The occasional development of autoimmunity in GLPL patients could serve as a test in continuing efforts to detect this disease and for a more directed therapy based on the autoantigen response.

Journal Article↗
PubMed2006

Synthesis of novel caspase inhibitors for characterization of the active caspase proteome in vitro and in vivo.

Caspases are cysteine proteases that are essential for cytokine maturation and apoptosis. To facilitate the dissection of caspase function in vitro and in vivo, we have synthesized irreversible caspase inhibitors with biotin attached via linker arms of various lengths (12a-d) and a 2,4-dinitrophenyl labeled inhibitor (13). Affinity labeling of apoptotic extracts followed by blotting reveals that these affinity probes detect active caspases. Using the strong affinity of avidin for biotin, we have isolated affinity-labeled caspase 6 from apoptotic cytosolic extracts of cells overexpressing procaspase 6 by treatment with 12c, which contains biotin attached to the N(epsilon)-lysine of the inhibitor by a 22.5 A linker arm, followed by affinity purification on monomeric avidin-sepharose beads. Compound 13 has proven sufficiently cell permeable to rescue cells from apoptotic execution. These novel caspase inhibitors should provide powerful probes for the study of the active caspase proteome during apoptosis both in vitro and in vivo.

Affinity Labels↗
PubMed2006

Condensin and Repo-Man-PP1 co-operate in the regulation of chromosome architecture during mitosis.

The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex in certain experimental systems, but not in many others. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure. Here, live-cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 revealed that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the targeting subunit Repo-Man from recruiting protein phosphatase 1 (PP1) to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man-PP1 during anaphase. This activity, provisionally termed 'regulator of chromosome architecture' (RCA), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.

Adenosine Triphosphatases↗
PubMed2006

Mammalian CLASP1 and CLASP2 cooperate to ensure mitotic fidelity by regulating spindle and kinetochore function.

CLASPs are widely conserved microtubule plus-end-tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells. Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent aneuploidy in mammals.

Aneuploidy↗
PubMed2006

Condensin I interacts with the PARP-1-XRCC1 complex and functions in DNA single-strand break repair.

Condensins are essential protein complexes critical for mitotic chromosome organization. Little is known about the function of condensins during interphase, particularly in mammalian cells. Here we report the interphase-specific interaction between condensin I and the DNA nick-sensor poly(ADP-ribose) polymerase 1 (PARP-1). We show that the association between condensin I, PARP-1, and the base excision repair (BER) factor XRCC1 increases dramatically upon single-strand break damage (SSB) induction. Damage-specific association of condensin I with the BER factors flap endonuclease 1 (FEN-1) and DNA polymerase delta/epsilon was also observed, suggesting that condensin I is recruited to interact with BER factors at damage sites. Consistent with this, DNA damage rapidly stimulates the chromatin association of PARP-1, condensin I, and XRCC1. Furthermore, depletion of condensin in vivo compromises SSB but not double-strand break (DSB) repair. Our results identify a SSB-specific response of condensin I through PARP-1 and demonstrate a role for condensin in SSB repair.

Adenosine Triphosphatases↗
PubMed2006

Drosophila Incenp is required for cytokinesis and asymmetric cell division during development of the nervous system.

The chromosomal passenger protein complex has emerged as a key player in mitosis, with important roles in chromatin modifications, kinetochore-microtubule interactions, chromosome bi-orientation and stability of the bipolar spindle, mitotic checkpoint function, assembly of the central spindle and cytokinesis. The inner centromere protein (Incenp; a subunit of this complex) is thought to regulate the Aurora B kinase and target it to its substrates. To explore the roles of the passenger complex in a developing multicellular organism, we have performed a genetic screen looking for new alleles and interactors of Drosophila Incenp. We have isolated a new null allele of Incenp that has allowed us for the first time to study the functions of the chromosomal passengers during development. Homozygous incenp(EC3747) embryos show absence of phosphorylation of histone H3 in mitosis, failure of cytokinesis and polyploidy, and defects in peripheral nervous system development. These defects are consistent with depletion of Aurora B kinase activity. In addition, the segregation of the cell-fate determinant Prospero in asymmetric neuroblast division is abnormal, suggesting a role for the chromosomal passenger complex in the regulation of this process.

Animals↗
PubMed2006

INCENP and Aurora B promote meiotic sister chromatid cohesion through localization of the Shugoshin MEI-S332 in Drosophila.

The chromosomal passenger complex protein INCENP is required in mitosis for chromosome condensation, spindle attachment and function, and cytokinesis. Here, we show that INCENP has an essential function in the specialized behavior of centromeres in meiosis. Mutations affecting Drosophila incenp profoundly affect chromosome segregation in both meiosis I and II, due, at least in part, to premature sister chromatid separation in meiosis I. INCENP binds to the cohesion protector protein MEI-S332, which is also an excellent in vitro substrate for Aurora B kinase. A MEI-S332 mutant that is only poorly phosphorylated by Aurora B is defective in localization to centromeres. These results implicate the chromosomal passenger complex in directly regulating MEI-S332 localization and, therefore, the control of sister chromatid cohesion in meiosis.

Animals↗
PubMed2005

Vascular smooth muscle cell polyploidization involves changes in chromosome passenger proteins and an endomitotic cell cycle.

Vascular smooth muscle cell polyploidization occurs during normal development and is enhanced under physiologic stress, but the mechanism of this cell cycle has not been explored. We show via time-lapse video imaging and immunofluorescence analyses that primary vascular smooth muscle cells (VSMC) undergo an endomitotic-type cell cycle, including a normal progression through part of mitosis. Mononuclear polyploid cells are generated by defects in sister chromatid separation and/or segregation, and cellular binucleation occurs by reversal of cytokinesis. To obtain further leads to regulators involved, we examined the chromosomal passenger proteins, Aurora B, inner centromere protein and Survivin, and concluded that Aurora B and inner centromere protein are normally colocalized in centromeres, the midzone, and the midbody during mitosis. Survivin, however, is dim and diffused; it does not colocalize with either Aurora B or inner centromere protein in VSMC, which could account for defects in sister chromatid separation and/or segregation and reversal of cytokinesis. In accordance with the reported dependency of Aurora B activity on Survivin, the Aurora B substrate, vimentin, is not phosphorylated during cytokinesis. Finally, the data show that ectopically expressed Survivin inhibits polyploidization in vascular smooth muscle cells. Hence, aberrant chromosome passenger protein activity and endomitosis are associated with VSMC polyploidization.

Animals↗
PubMed2005

Shugoshin: a centromeric guardian senses tension.

To ensure accurate chromosome segregation during mitosis, the spindle checkpoint monitors chromosome alignment on the mitotic spindle. Indjeian and colleagues have investigated the precise role of the shugoshin 1 protein (Sgo1p) in this process in budding yeast. The Sgo proteins were originally identified as highly conserved proteins that protect cohesion at centromeres during the first meiotic division. Together with other recent findings, the study highlighted here has identified Sgo1 as a component that informs the mitotic spindle checkpoint when spindle tension is perturbed. This discovery has provided a molecular link between sister chromatid cohesion and tension-sensing at the kinetochore-microtubule interface.

Animals↗
PubMed2005

Trashing the genome: the role of nucleases during apoptosis.

Two classes of nucleases degrade the cellular DNA during apoptosis. Cell-autonomous nucleases cleave DNA within the dying cell. They are not essential for apoptotic cell death or the life of the organism, but they might affect the efficiency of the process. By contrast, waste-management nucleases are essential for the life of the organism. In post-engulfment DNA degradation, the DNA of apoptotic cells is destroyed in lysosomes of the cells that have phagocytosed the corpses. Waste-management nucleases also destroy DNA that is released into the extracellular compartment. Here, we describe the complex group of nucleases that are involved in DNA destruction during apoptotic cell death.

Apoptosis↗
PubMed2004

Novel components of human mitotic chromosomes identified by proteomic analysis of the chromosome scaffold fraction.

Chromosomal nonhistone proteins have important roles in mitotic chromosome formation and dynamics. In order to identify novel abundant proteins with a potential involvement in these processes, we initiated a proteomic screen of the chromosome scaffold fraction. This screen identified 79 proteins, 30 of which had not previously been described as components of mitotic chromosomes. Furthermore, half of these proteins had no documented function. We analyzed the cell-cycle dependent distribution of three uncharacterized proteins by expressing them as green fluorescent protein (GFP) fusions and showed that they associate with mitotic chromosomes in vivo. One of the proteins, nuclear protein p30, is a novel component of the inner centromere. Over-expression experiments indicated that p30 may have an active role in the formation of centromeric heterochromatin.

Cell Cycle↗
PubMed2004

The dynamic kinetochore-microtubule interface.

The kinetochore is a control module that both powers and regulates chromosome segregation in mitosis and meiosis. The kinetochore-microtubule interface is remarkably fluid, with the microtubules growing and shrinking at their point of attachment to the kinetochore. Furthermore, the kinetochore itself is highly dynamic, its makeup changing as cells enter mitosis and as it encounters microtubules. Active kinetochores have yet to be isolated or reconstituted, and so the structure remains enigmatic. Nonetheless, recent advances in genetic, bioinformatic and imaging technology mean we are now beginning to understand how kinetochores assemble, bind to microtubules and release them when the connections made are inappropriate, and also how they influence microtubule behaviour. Recent work has begun to elucidate a pathway of kinetochore assembly in animal cells; the work has revealed that many kinetochore components are highly dynamic and that some cycle between kinetochores and spindle poles along microtubules. Further studies of the kinetochore-microtubule interface are illuminating: (1) the role of the Ndc80 complex and components of the Ran-GTPase system in microtubule attachment, force generation and microtubule-dependent inactivation of kinetochore spindle checkpoint activity; (2) the role of chromosomal passenger proteins in the correction of kinetochore attachment errors; and (3) the function of microtubule plus-end tracking proteins, motor depolymerases and other proteins in kinetochore movement on microtubules and movement coupled to microtubule poleward flux.

Animals↗
PubMed2004

Centrosome amplification induced by DNA damage occurs during a prolonged G2 phase and involves ATM.

Centrosomes are the principal microtubule organising centres in somatic cells. Abnormal centrosome number is common in tumours and occurs after gamma-irradiation and in cells with mutations in DNA repair genes. To investigate how DNA damage causes centrosome amplification, we examined cells that conditionally lack the Rad51 recombinase and thereby incur high levels of spontaneous DNA damage. Rad51-deficient cells arrested in G2 phase and formed supernumerary functional centrosomes, as assessed by light and serial section electron microscopy. This centrosome amplification occurred without an additional DNA replication round and was not the result of cytokinesis failure. G2-to-M checkpoint over-ride by caffeine or wortmannin treatment strongly reduced DNA damage-induced centrosome amplification. Radiation-induced centrosome amplification was potentiated by Rad54 disruption. Gene targeting of ATM reduced, but did not abrogate, centrosome amplification induced by DNA damage in both the Rad51 and Rad54 knockout models, demonstrating ATM-dependent and -independent components of DNA damage-inducible G2-phase centrosome amplification. Our data suggest DNA damage-induced centrosome amplification as a mechanism for ensuring death of cells that evade the DNA damage or spindle assembly checkpoints.

Androstadienes↗
PubMed2004

Chromosomal passengers: the four-dimensional regulation of mitotic events.

Chromosomal passengers are proteins that are involved in coordinating the chromosomal and cytoskeletal events of mitosis. The passengers are present in cells as a complex with at least four members: Aurora B, a protein kinase; inner centromeric protein, an activation and targeting subunit; Survivin (function unknown) and Borealin (function also unknown). The kinase is activated at the onset of mitosis, at least partly accomplished by regulation of the levels of its constituents. As mitosis progresses, the kinase complex moves to a highly choreographed series of locations in the mitotic cell, activating key substrates at precise locations and specific times. Functions that require chromosomal passenger activity include chromatin modification (phosphorylation of histone H3), correction of kinetochore attachment errors, aspects of the spindle assembly checkpoint, assembly of a stable bipolar spindle and the completion of cytokinesis. The chromosomal passenger complex provides an essential mechanism for mitotic regulation.

Animals↗
PubMed2004

Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation.

A number of proteins accumulate in the anaphase spindle midzone, but the interaction and precise role of these proteins in midzone organization remain obscure. Here, we found that the microtubule-bundling protein PRC1 bound separately to the three motor proteins, KIF4, MKLP1 and CENP-E, but not to the chromosomal passenger proteins. In KIF4-deficient cells, the central spindle was disorganized, and all midzone-associated proteins including PRC1 failed to concentrate at the midline, instead being dispersed along the loosened microtubule bundles of the central spindle. This suggests that KIF4 is essential for the organization of central spindles and for midzone formation. In PRC1-deficient cells, no midzone was formed, KIF4 and CENP-E did not localize to the disconnected half-spindle, and MKLP1 and chromosomal passenger proteins localized to discrete subdomains near microtubule plus ends in the half-spindle. Thus, PRC1 is required for interaction of the two half-spindles and for localization of KIF4 and CENP-E. These results suggest that KIF4 and its binding partner PRC1 play essential roles in the organization of central spindles and midzone formation.

Anaphase↗
PubMed2004

Borealin: a novel chromosomal passenger required for stability of the bipolar mitotic spindle.

The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore-spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B-INCENP subcomplex.

Amino Acid Sequence↗