1,720,984 research outputs found
Evidence for centriolar satellite localization of CDK1 and cyclin B2
No full textCentriolar satellites are 70-100 nm non-membranous particles implicated in the trafficking and folding of many centrosomal proteins, including the products of several disease genes. Centriolar satellites are often focused around the centrosome in interphase and either scattered throughout the cell or mostly undetectable in mitosis. The molecular mechanism underlying their disassembly/dispersal in mitosis remains unknown. Here, we present immunofluorescence microscopy data suggesting that CDK1 and cyclin B2 localise to centriolar satellites. These findings support recent biochemical data suggesting that a major component of centriolar satellites (PCM1) is a CDK1 substrate, and thus point to a role for this kinase in promoting disassembly of centriolar satellites in mitosis
Dataset supporting the University of Southampton Doctoral thesis 'Eosinophilic Inflammation in Chronic Obstructive Pulmonary Disease'
No full textDataset supporting the University of Southampton Doctoral thesis 'Eosinophilic Inflammation in Chronic Obstructive Pulmonary Disease'.
This data consists of area of FITC and CY5 signal calculated from microscopy scans, which is presented as an excel workbook titled "MICA II_cellspermm2 calculations", consisting of 4 sheets; EG2 Image Analysis, IL5R Image Analysis, EG2 cells_mm2 and IL5R cells-mm2.
No special software is needed to view the data. </span
Evidence for reciliation of RPE1 cells in late G1 phase, and ciliary localisation of cyclin B1
No full textThe primary cilium, an organelle that transduces extracellular signals important for development and tissue homeostasis, is typically assembled upon cell cycle exit and disassembled upon cell cycle re-entry. Cilium assembly is thought to be suppressed in cycling cells, however the extent of suppression is not clear. For example, primary cilia are present in certain proliferating cells during development, and a period of reciliation has been reported to occur in late G1 in murine 3T3 cells released from serum starvation-induced quiescence. Human retinal pigmented epithelial (hTERT-RPE1; herein, RPE1) cells are commonly used to investigate pathways regulating cilium disassembly, however the ciliary disassembly profile of these cells remains uncertain. A period of reciliation has not been observed. Here, we analyse the ciliary disassembly profile of RPE1 cells by immunofluorescence microscopy. The results suggest a profile similar to 3T3 cells, including a period of reciliation in late G1 and a second wave of deciliation in S phase. We present evidence that arresting cells in early S phase with hydroxyurea or excess thymidine prevents the second wave of deciliation, and that deciliation is initiated shortly after release from a thymidine block, consistent with coupling to DNA replication. These findings support the often overlooked notion that cilium formation can occur in late G1, and suggest that RPE1 cells could serve as a model system for studying the molecular pathways that direct this process, in addition to those that stimulate cilium disassembly. We also present immunofluorescence data indicating that cyclin B1 localises to primary cilia
Dataset supporting the University of Southampton Doctoral Thesis "The role of non-typeable haemophilus influenzae biofilms in host-microbial and microbial-microbial interactions in chronic obstructive pulmonary disease"
No full textDataset supporting the University of Southampton Doctoral Thesis "The role of non-typeable haemophilus influenzae biofilms in host-microbial and microbial-microbial interactions in chronic obstructive pulmonary disease".
The dataset includes zipped file:Data_-_PURE.zip
The research was funded by AstraZeneca.
To date, this data has not been associated with any publications. Publications are in progress.
This data is embargoed until 24/6/27
this data is under embargo until 24/6/27
This work was undertaken using a BBSRC iCASE PhD
studentship (BB/T508135/1) awarded for JW’s doctoral studies.
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Phospholamban and sarcolipin are maintained in the endoplasmic reticulum by retrieval from the ER-Golgi intermediate compartment
No full textObjectivePhospholamban and sarcolipin are small transmembrane proteins that modulate cardiac contractility through their interaction with the sarcoplasmic reticulum (SR) calcium pumps (SERCAs). We have examined the hypothesis that phospholamban and sarcolipin are maintained in the SR by a process of retrieval from post-SR compartments and the role of their transmembrane domains in targeting.MethodsAntibodies directed against phospholamban and protein markers of the endoplasmic reticulum/Golgi intermediate compartment (ERGIC) and the trans-Golgi were used in fluorescence microscopy studies of cultured human fetal cardiac myocytes. In addition, sarcolipin and phospholamban were tagged at the N-terminus with enhanced-green-fluorescent protein (EGFP) and expressed in COS 7 cells. The EGFP-tagged constructs were localised using fluorescence microscopy and cell fractionation. The length of the transmembrane domains of phospholamban and sarcolipin were extended and the effect on cellular location was also examined.ResultsIn fetal cardiac myocytes phospholamban was located in the SR and the ERGIC, but did not migrate to the trans-Golgi network. Tagged-phospholamban and sarcolipin were located in the endoplasmic reticulum (ER) of COS 7 cells indicating that their targeting was unaffected by the EGFP tag. Significant proportions of the tagged phospholamban and sarcolipin were also located in the ERGIC but not in the trans-Golgi. Increasing the length of the transmembranous domains of EGFP-tagged phospholamban and sarcolipin resulted in their mis-targeting to the plasma membrane.ConclusionsPhospholamban and sarcolipin are maintained in the SR/ER by a process that includes their retrieval from the ERGIC following their passage from the SR/ER into the ERGIC. The transmembrane domains of phospholamban and sarcolipin are involved in the retrieval process
Subcellular localization of ALMS1 supports involvement of centrosome and basal body dysfunction in the pathogenesis of obesity, insulin resistance, and type 2 diabetes
No full textAlström syndrome is a rare autosomal recessive disorder caused by mutations in a novel gene of unknown function, ALMS1. Central features of Alström syndrome include obesity, insulin resistance, and type 2 diabetes, and therefore investigating ALMS1 function stands to offer new insights into the pathogenesis of these common conditions. To begin this process, we have analyzed the subcellular localization and tissue distribution of ALMS1 by immunofluorescence. We show that ALMS1 is widely expressed and localizes to centrosomes and to the base of cilia. Fibroblasts with disrupted ALMS1 assemble primary cilia and microtubule cytoskeletons that appear normal, suggesting that the Alström syndrome phenotype results from impaired function rather than abnormal development. Coupled with recent data on the complex phenotype of Bardet-Biedl syndrome, our findings imply an unexpected central role for basal body and centrosome dysfunction in the pathogenesis of obesity, insulin resistance, and type 2 diabetes. Unraveling the molecular mechanisms underlying the Alström syndrome phenotype will be important in the search for new therapeutic targets for these conditions
Derivation of human embryonic germ cells: an alternative source of pluripotent stem cells
No full textBased on evidence suggesting similarities to human embryonic stem cells, human embryonic germ (hEG) cells have been advocated as an alternative pluripotent stem cell resource but have so far received limited attention. To redress this imbalance, human fetal gonads were collected for the isolation and culture of primordial germ cells at 7-9 weeks postconception. We provide evidence for the derivation, culture, and differentiation of hEG cells in vitro. This evidence includes the expression of markers characteristic of pluripotent cells, the retention of normal XX or XY karyotypes, and the demonstration of pluripotency, as suggested by the expression of markers indicative of differentiation along the three germ lineages (ectoderm, mesoderm, and endoderm) and an associated loss of pluripotent markers. In assessing this differentiation, however, we also demonstrate a hitherto unacknowledged overlap in gene expression profiles between undifferentiated and differentiated cell types, highlighting the difficulty in ascribing cell lineage by gene expression analyses. Furthermore, we draw attention to the problems inherent in the management of these cells in prolonged culture, chiefly the difficulty in preventing spontaneous differentiation, which hinders the isolation of pure, undifferentiated clonal lines. While these data advocate the pursuit of pluripotent hEG cell studies with relevance to early human embryonic development, culture limitations carry implications for their potential applicability to ambitious cell replacement therapies
Human embryonic germ cells for future neuronal replacement therapy
No full textStem cell therapy offers exciting potential for ambitious cellular replacement to treat human (h) disease, such as Parkinson's disease, Alzheimer's disease or even replacement of the cell death that follows thromboembolic stroke. The realisation of these treatments requires cellular resources possessing three essential characteristics: (i) self-renewal, (ii) the ability to differentiate to physiologically normal cell types and (iii) lack of tumourigenicity.Here, we describe work on human embryonic germ cells (hEGCs), a population of cells alongside human embryonic stem cells (hESCs) with the potential to address these issues
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