Life Sciences Research for Lifelong Health

Publications flow-cytometry

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The spindle assembly checkpoint works like a rheostat rather than a toggle switch.
Collin P,Nashchekina O,Walker R,Pines J

The spindle assembly checkpoint (SAC) is essential in mammalian mitosis to ensure the equal segregation of sister chromatids. The SAC generates a mitotic checkpoint complex (MCC) to prevent the anaphase-promoting complex/cyclosome (APC/C) from targeting key mitotic regulators for destruction until all of the chromosomes have attached to the mitotic apparatus. A single unattached kinetochore can delay anaphase for several hours, but how it is able to block the APC/C throughout the cell is not understood. Present concepts of the SAC posit that either it exhibits an all-or-nothing response or there is a minimum threshold sufficient to block the APC/C (ref. 7). Here, we have used gene targeting to measure SAC activity, and find that it does not have an all-or-nothing response. Instead, the strength of the SAC depends on the amount of MAD2 recruited to kinetochores and on the amount of MCC formed. Furthermore, we show that different drugs activate the SAC to different extents, which may be relevant to their efficacy in chemotherapy.

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Nature cell biology, 15, 1476-4679, 1378-85, 2013

PMID: 24096242

Germline potential of parthenogenetic haploid mouse embryonic stem cells.
Leeb M,Walker R,Mansfield B,Nichols J,Smith A,Wutz A

Haploid embryonic stem cells (ESCs) have recently been derived from parthenogenetic mouse embryos and offer new possibilities for genetic screens. The ability of haploid ESCs to give rise to a wide range of differentiated cell types in the embryo and in vitro has been demonstrated. However, it has remained unclear whether haploid ESCs can contribute to the germline. Here, we show that parthenogenetic haploid ESCs at high passage have robust germline competence enabling the production of transgenic mouse strains from genetically modified haploid ESCs. We also show that differentiation of haploid ESCs in the embryo correlates with the gain of a diploid karyotype and that diploidisation is the result of endoreduplication and not cell fusion. By contrast, we find that a haploid karyotype is maintained when differentiation to an extra-embryonic fate is forced by induction of Gata6.

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Development (Cambridge, England), 139, 1477-9129, 3301-5, 2012

PMID: 22912412

A high-throughput, flow cytometry-based method to quantify DNA-end resection in mammalian cells.
Forment JV,Walker RV,Jackson SP

Replication protein A (RPA) is an essential trimeric protein complex that binds to single-stranded DNA (ssDNA) in eukaryotic cells and is involved in various aspects of cellular DNA metabolism, including replication and repair. Although RPA is ubiquitously expressed throughout the cell cycle, it localizes to DNA replication forks during S phase, and is recruited to sites of DNA damage when regions of ssDNA are exposed. During DNA double-strand break (DSB) repair by homologous recombination (HR), RPA recruitment to DNA damage sites depends on a process termed DNA-end resection. Consequently, RPA recruitment to sub-nuclear regions bearing DSBs has been used as readout for resection and for ongoing HR. Quantification of RPA recruitment by immunofluorescence-based microscopy techniques is time consuming and requires extensive image analysis of relatively small populations of cells. Here, we present a high-throughput flow-cytometry method that allows the use of RPA staining to measure cell proliferation and DNA-damage repair by HR in an unprecedented, unbiased and quantitative manner.

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Cytometry. Part A : the journal of the International Society for Analytical Cytology, 81, 1552-4930, 922-8, 2012

PMID: 22893507

A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells.
Ng K,Daigle N,Bancaud A,Ohhata T,Humphreys P,Walker R,Ellenberg J,Wutz A

In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formation of a repressive chromatin domain. To understand localization of Xist over one X chromosome we aimed to develop a system for investigating Xist in living cells. Here we report successful visualization of transgenically expressed MS2-tagged Xist in mouse embryonic stem cells. Imaging of Xist during an entire cell cycle shows that Xist spreads from a single point to a steady state when the chromosome is covered with a constant amount of Xist. Photobleaching experiments of the established Xist cluster indicate that chromosome-bound Xist is dynamic and turns over on the fully Xist covered chromosome. It appears that in interphase the loss of bound Xist and newly produced Xist are in equilibrium. We also show that the turnover of bound Xist requires transcription, and Xist binding becomes stable when transcription is inhibited. Our data reveal a strategy for visualizing Xist and indicate that spreading over the chromosome might involve dynamic binding and displacement.

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Molecular biology of the cell, 22, 1939-4586, 2634-45, 2011

PMID: 21613549

Oct4 and LIF/Stat3 additively induce Krüppel factors to sustain embryonic stem cell self-renewal.
Hall J,Guo G,Wray J,Eyres I,Nichols J,Grotewold L,Morfopoulou S,Humphreys P,Mansfield W,Walker R,Tomlinson S,Smith A

Embryonic stem cell (ESC) pluripotency is dependent on an intrinsic gene regulatory network centered on Oct4. Propagation of the pluripotent state is stimulated by the cytokine leukemia inhibitory factor (LIF) acting through the transcriptional regulator Stat3. Here, we show that this extrinsic stimulus converges with the intrinsic circuitry in Krüppel-factor activation. Oct4 primarily induces Klf2 while LIF/Stat3 selectively enhances Klf4 expression. Overexpression of either factor reduces LIF dependence, but with quantitative and qualitative differences. Unlike Klf4, Klf2 increases ESC clonogenicity, maintains undifferentiated ESCs in the genetic absence of Stat3, and confers resistance to BMP-induced differentiation. ESCs expanded with Klf2 remain capable of contributing to adult chimeras. Postimplantation-embryo-derived EpiSCs lack both Klf2 and Klf4 and expression of either can reinstate naive pluripotency. These findings indicate that Oct4 and Stat3 intersect in directing expression of Klf transcriptional regulators with overlapping properties that additively reinforce ground-state ESC pluripotency, identity, and self-renewal.

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Cell stem cell, 5, 1875-9777, 597-609, 2009

PMID: 19951688

Immunoglobulin aggregation leading to Russell body formation is prevented by the antibody light chain.
D Corcos, MJ Osborn, LS Matheson, F Santos, X Zou, JA Smith, G Morgan, A Hutchings, M Hamon, D Oxley, M Brüggemann

Russell bodies (RBs) are intracellular inclusions filled with protein aggregates. In diverse lymphoid disorders these occur as immunoglobulin (Ig) deposits, accumulating in abnormal plasma or Mott cells. In heavy-chain deposition disease truncated antibody heavy-chains (HCs) are found, which bear a resemblance to diverse polypeptides produced in Ig light-chain (LC)-deficient (L(-/-)) mice. In L(-/-) animals, the known functions of LC, providing part of the antigen-binding site of an antibody and securing progression of B-cell development, may not be required. Here, we show a novel function of LC in preventing antibody aggregation. L(-/-) mice produce truncated HC naturally, constant region (C)gamma and Calpha lack C(H)1, and Cmicro is without C(H)1 or C(H)1 and C(H)2. Most plasma cells found in these mice are CD138(+) Mott cells, filled with RBs, formed by aggregation of HCs of different isotypes. The importance of LC in preventing HC aggregation is evident in knock-in mice, expressing Cmicro without C(H)1 and C(H)2, which only develop an abundance of RBs when LC is absent. These results reveal that preventing antibody aggregation is a major function of LC, important for understanding the physiology of heavy-chain deposition disease, and in general recognizing the mechanisms, which initiate protein conformational diseases.

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Blood, 115, 2, 282-8, 2010

PMID: 19822901
DOI: 10.1182/blood-2009-07-234864

Light chain-deficient mice produce novel multimeric heavy-chain-only IgA by faulty class switching.
LS Matheson, MJ Osborn, JA Smith, D Corcos, M Hamon, R Chaouaf, J Coadwell, G Morgan, D Oxley, M Brüggemann

Recently, we identified that diverse heavy chain (H-chain)-only IgG is spontaneously produced in light chain (L-chain)-deficient mice (L(-/-) with silenced kappa and lambda loci) despite a block in B cell development. In murine H-chain IgG, the first Cgamma exon, C(H)1, is removed after DNA rearrangement and secreted polypeptides are comparable with camelid-type H-chain IgG. Here we show that L(-/-) mice generate a novel class of H-chain Ig with covalently linked alpha chains, not identified in any other healthy mammal. Surprisingly, diverse H-chain-only IgA can be released from B cells at levels similar to conventional IgA and is found in serum and sometimes in milk and saliva. Surface IgA without L-chain is expressed in B220(+) spleen cells, which exhibited a novel B cell receptor, suggesting that associated conventional differentiation events occur. To facilitate the cellular transport and release of H-chain-only IgA, chaperoning via BiP association seems to be prevented as only alpha chains lacking C(H)1 are released from the cell. This appears to be accomplished by imprecise class-switch recombination (CSR) from Smu into the alpha constant region, which removes all or part of the Calpha1 exon at the genomic level.

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International immunology, 21, 8, 957-66, 2009

PMID: 19561045
DOI: 10.1093/intimm/dxp062

Transcription of productive and nonproductive VDJ-recombined alleles after IgH allelic exclusion.
J Daly, S Licence, A Nanou, G Morgan, IL Mårtensson

The process of allelic exclusion ensures that each B cell expresses a B-cell receptor encoded by only one of its Ig heavy (IgH) and light (IgL) chain alleles. Although its precise mechanism is unknown, recruitment of the nonfunctional IgH allele to centromeric heterochromatin correlates with the establishment of allelic exclusion. Similarly, recruitment in activated splenic B cells correlates with cell division. In the latter, the recruited IgH allele was reported to be transcriptionally silent. However, it is not known whether monoallelic recruitment during establishment of allelic exclusion correlates with transcriptional silencing. To investigate this, we assessed the transcriptional status of both IgH alleles in single primary cells over the course of B-cell development, using RNA fluorescence in situ hybridization. Before allelic exclusion both alleles are transcribed. Thereafter, in pre-BII and subsequent developmental stages both functional and nonfunctional VDJ- and DJ-transcription is observed. Thus, after the establishment of IgH allelic exclusion, monoallelic recruitment to heterochromatin does not silence VDJ- or DJ-transcription, but serves another purpose.

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The EMBO journal, 26, 19, 4273-82, 2007

PMID: 17805345
DOI: 10.1038/sj.emboj.7601846

A natural hypomorphic variant of the apoptosis regulator Gimap4/IAN1.
C Carter, C Dion, S Schnell, WJ Coadwell, M Graham, L Hepburn, G Morgan, A Hutchings, JC Pascall, H Jacobs, JR Miller, GW Butcher

The Gimap/IAN family of GTPases has been implicated in the regulation of cell survival, particularly in lymphomyeloid cells. Prosurvival and prodeath properties have been described for different family members. We generated novel serological reagents to study the expression in rats of the prodeath family member Gimap4 (IAN1), which is sharply up-regulated at or soon after the stage of T cell-positive selection in the thymus. During these investigations we were surprised to discover a severe deficiency of Gimap4 expression in the inbred Brown Norway (BN) rat. Genetic analysis linked this trait to the Gimap gene cluster on rat chromosome 4, the probable cause being an AT dinucleotide insertion in the BN Gimap4 allele (AT(+)). This allele encodes a truncated form of Gimap4 that is missing 21 carboxyl-terminal residues relative to wild type. The low protein expression associated with this allele appears to have a posttranscriptional cause, because mRNA expression was apparently normal. Spontaneous and induced apoptosis of BN and wild-type T cells was analyzed in vitro and compared with the recently described mouse Gimap4 knockout. This revealed a "delayed" apoptosis phenotype similar to but less marked than that of the knockout. The Gimap4 AT(+) allele found in BN was shown to be rare in inbred rat strains. Nevertheless, when wild rat DNA samples were studied the AT(+) allele was found at a high overall frequency ( approximately 30%). This suggests an adaptive significance for this hypomorphic allele.

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Journal of immunology (Baltimore, Md. : 1950), 179, 3, 1784-95, 2007

PMID: 17641045

Open Access

Intervertebral disc cell-mediated mesenchymal stem cell differentiation.
Richardson SM,Walker RV,Parker S,Rhodes NP,Hunt JA,Freemont AJ,Hoyland JA

Low back pain is one of the largest health problems in the Western world today, and intervertebral disc degeneration has been identified as a main cause. Currently, treatments are symptomatic, but cell-based tissue engineering methods are realistic alternatives for tissue regeneration. However, the major problem for these strategies is the generation of a suitable population of cells. Adult bone marrow-derived mesenchymal stem cells (MSCs) are undifferentiated, multipotent cells that have the ability to differentiate into a number of cell types, including the chondrocyte-like cells found within the nucleus pulposus (NP) of the intervertebral disc; however, no method exists to differentiate these cells in an accessible monolayer environment. We have conducted coculture experiments to determine whether cells from the human NP can initiate the differentiation of human MSCs with or without cell-cell contact. Fluorescent labeling of the stem cell population and high-speed cell sorting after coculture with cell-cell contact allowed examination of individual cell populations. Real-time quantitative polymerase chain reaction showed significant increases in NP marker genes in stem cells when cells were cocultured with contact for 7 days, and this change was regulated by cell ratio. No significant change in NP marker gene expression in either NP cells or stem cells was observed when cells were cultured without contact, regardless of cell ratio. Thus, we have shown that human NP and MSC coculture with contact is a viable method for generating a large population of differentiated cells that could be used in cell-based tissue engineering therapies for regeneration of the degenerate intervertebral disc.

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Stem cells (Dayton, Ohio), 24, 1066-5099, 707-16, 2006

PMID: 16223853

Expression of the Ian family of putative GTPases during T cell development and description of an Ian with three sets of GTP/GDP-binding motifs.
C Dion, C Carter, L Hepburn, WJ Coadwell, G Morgan, M Graham, N Pugh, G Anderson, GW Butcher, JR Miller

Reports suggest that two members of the novel immune-associated nucleotide (Ian) GTPase family, Ian1 and Ian5, play roles in T cell development. We performed real-time PCR analysis of the expression of Ian genes of the rat during T cell maturation, in macrophages and in cell lines. We found that all of the genes were expressed at relatively low levels at the early double-negative thymocyte stage but were expressed more strongly at later cell stages. Our study also revealed the fact that the previously reported Ian9, Ian10 and Ian11 genes are, instead, parts of a single gene for which we retain the name Ian9, potentially encoding a GTPase with a highly unusual triplicated structure. Antisera were developed against both Ian1 and Ian9. We established that Ian9 is produced as an approximately 75-kDa protein in both T cells and thymocytes. We observed that levels of both Ian1 and Ian9 proteins are profoundly reduced in T cells from lymphopenic rats as compared with wild-type rats. It was demonstrated that thymocytes and B cells from lymphopenic rats (Ian5 null) did not show enhanced sensitivity to gamma-irradiation-induced apoptosis.

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International immunology, 17, 9, 1257-68, 2005

PMID: 16103028
DOI: 10.1093/intimm/dxh302

Open Access