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Biomedical subjects

David G Spiller

Publications and source records attributed to David G Spiller.

13 recordsLinked to original sources
PubMed2006

Automated tracking of gene expression in individual cells and cell compartments.

Many intracellular signal transduction processes involve the reversible translocation from the cytoplasm to the nucleus of transcription factors. The advent of fluorescently tagged protein derivatives has revolutionized cell biology, such that it is now possible to follow the location of such protein molecules in individual cells in real time. However, the quantitative analysis of the location of such proteins in microscopic images is very time consuming. We describe CellTracker, a software tool designed for the automated measurement of the cellular location and intensity of fluorescently tagged proteins. CellTracker runs in the MS Windows environment, is freely available (at http://www.dbkgroup.org/celltracker/), and combines automated cell tracking methods with powerful image-processing algorithms that are optimized for these applications. When tested in an application involving the nuclear transcription factor NF-kappaB, CellTracker is competitive in accuracy with the manual human analysis of such images but is more than 20 times faster, even on a small task where human fatigue is not an issue. This will lead to substantial benefits for time-lapse-based high-content screening.

Animals↗
PubMed2006

Encoded microcarriers for high-throughput multiplexed detection.

Since the decoding of the human genome, the quest to obtain more and more molecular information from smaller and smaller samples is intensifying. Today the burden of this challenge is being borne by planar arrays, but the quality of the data provided by this approach is limited by variations in performance between different arrays. Suspension arrays of encoded microspheres provide higher quality data, but the amount of molecular information that can be acquired with them is limited by the number of codes that can be distinguished in the same sample. New methods of preparing encoded particles promise to alleviate this problem, but in the face of a growing number of new technologies it is sometimes difficult to decide which, if any, will succeed. Herein we appraise these new forms of encoded particle critically, and ask if they can deliver the necessary multiplexing power and whether they will perform well in multiplexed assays.

Humans↗
PubMed2005

CLL, but not normal, B cells are dependent on autocrine VEGF and alpha4beta1 integrin for chemokine-induced motility on and through endothelium.

Vascular endothelial cell growth factor (VEGF) is a multifunctional cytokine involved in tumor formation. In chronic lymphocytic leukemia (CLL), it is known that the malignant cells secrete VEGF and possess VEGF receptors. This suggests that an autocrine loop might be important in the pathogenesis of CLL. Here we show that, in patients with lymphadenopathy, autocrine VEGF and alpha(4)beta(1) integrin are involved in the chemokine-dependent motility of CLL cells on and through endothelium-processes important for the invasion of lymphoreticular tissues, a major determinant of disease outcome. In contrast, normal lymphocytes were not dependent on autocrine VEGF or alpha(4)beta(1) for either type of cell movement. Moreover, in contrast to normal B lymphocytes, CLL cells failed to cluster and activate alpha(L)beta(2) in response to chemokines, unless VEGF receptor(s) and alpha(4)beta(1) were also engaged by their respective ligands. This is the first demonstration that autocrine VEGF is involved in CLL-cell motility, and that the alpha(L)beta(2) on the malignant cells is functionally altered compared with that of normal B cells in not undergoing activation in response to chemokine alone. Given the importance of cell motility for tissue invasion, the present results provide a rationale for a trial of VEGF and alpha(4) blockade in patients with CLL who have tissue disease.

B-Lymphocytes↗
PubMed2004

Calcium-dependent regulation of the cell cycle via a novel MAPK--NF-kappaB pathway in Swiss 3T3 cells.

Nuclear factor kappa B (NF-kappaB) has been implicated in the regulation of cell proliferation and transformation. We investigated the role of the serum-induced intracellular calcium increase in the NF-kappaB--dependent cell cycle progression in Swiss 3T3 fibroblasts. Noninvasive photoactivation of a calcium chelator (Diazo-2) was used to specifically disrupt the transient rise in calcium induced by serum stimulation of starved Swiss 3T3 cells. The serum-induced intracellular calcium peak was essential for subsequent NF-kappaB activation (measured by real-time imaging of the dynamic p65 and IkappaBalpha fluorescent fusion proteins), cyclin D1 (CD1) promoter-directed transcription (measured by real-time luminescence imaging of CD1 promoter-directed firefly luciferase activity), and progression to cell division. We further showed that the serum-induced mitogen-activated protein kinase (MAPK) phosphorylation is calcium dependent. Inhibition of the MAPK- but not the PtdIns3K-dependent pathway inhibited NF-kappaB signaling, and further, CD1 transcription and cell cycle progression. These data suggest that a serum-dependent calcium signal regulates the cell cycle via a MAPK--NF-kappaB pathway in Swiss 3T3 cells.

Animals↗
PubMed2004

Density-dependent location and interactions of truncated APC and beta-catenin.

Adenomatous polyposis coli (APC) is a multifunctional tumour suppressor protein, central to development and the mature organism. It is mutated in most cases of colorectal cancer, rendering it ineffective in mediating beta-catenin degradation. We show that localization of full-length APC in colon carcinoma and noncancer cell lines is independent of cell density. However, the location of truncated APC is a function of cell density and in high-density cells truncated APC is predominantly not nuclear. Although the distribution of truncated APC and beta-catenin is closely linked in subconfluent SW480 cells, at high cell density they are not colocalized. We postulated that in this cell line this could be due to an increase in beta-catenin bound to E-cadherin with formation of adherens junctions at high cell density. However, while in coimmunoprecipitation assays we observe an increase in binding between beta-catenin and E-cadherin and a corresponding decrease in binding between beta-catenin and APC at high cell density, we did not observe a strict colocalization of beta-catenin and E-cadherin at the membrane of all cells.

Adenomatous Polyposis Coli Protein↗
PubMed2003

Mammalian NADH diphosphatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein.

The human NUDT12 Nudix hydrolase has been expressed in insect cells from a baculovirus vector as a His-tagged recombinant protein. In vitro, it efficiently hydrolyses NAD(P)H to NMNH and AMP (2',5'-ADP), and diadenosine diphosphate to AMP. It also has activity towards NAD(P)(+), ADP-ribose and diadenosine triphosphate. K (m) values for NADH, NADPH and NAD(+) are 11, 16 and 190 microM and k (cat) values are 11, 16 and 10.5 s(-1) respectively. Thus, like other NADH diphosphatases of the Nudix family, NUDT12 has a marked substrate preference for the reduced nicotinamide nucleotides. Optimal activity was supported by 50 microM Mn(2+) ions in vitro, with 3-fold lower activity at 0.4 mM Mg(2+). Expression of NUDT12 as a C-terminal fusion to green fluorescent protein revealed that it was targeted to peroxisomes by the C-terminal tripeptide PNL acting as a novel type 1 peroxisomal targeting signal. Deletion of PNL resulted in diffuse cellular fluorescence. In addition, C-terminal, but not N-terminal, fusions with or without the PNL signal accumulated in large, unidentified cytoplasmic structures. NUDT12 may act to regulate the concentration of peroxisomal nicotinamide nucleotide cofactors required for oxidative metabolism in this organelle.

Amino Acid Sequence↗
PubMed2003

The digestive food vacuole of the malaria parasite is a dynamic intracellular Ca2+ store.

The acidic food vacuole of Plasmodium falciparum has been the subject of intense scientific investigation in the 40 years since its role in the digestion of host hemoglobin was first suggested. This proposed role has important implications for the complex host-parasite inter-relationship and also for the mode of action of several of the most effective antimalarial drugs. In addition, adaptive changes in the physiology of this organelle are implicated in drug resistance. Here we show that in addition to these functions, the digestive food vacuole of the malaria parasite is a dynamic internal store for free Ca2+, a role hitherto unsuspected. With the aid of live-cell laser scanning confocal imaging, spatiotemporal studies revealed that maintenance of elevated free Ca2+ in the digestive food vacuole (relative to cytosolic levels) is achieved by a thapsigargin (and cyclopiazonic acid)-sensitive Ca2+-pump in cooperation with a H+-dependent Ca2+ transporter. Redistribution of free cytosolic and vacuolar Ca2+ during parasite growth also suggests that vacuolar Ca2+ plays an essential role in parasite morphogenesis. These data imply that the digestive food vacuole of the malaria parasite is functionally akin to the vacuole of plants (tonoplast) and the small electron-dense granules of some parasites (acidocalcisomes) whereby H+-coupled Ca2+ transport is involved in ion transport, Ca2+ homeostasis, and signal transduction. These findings have significant implications for parasite development, antimalarial drug action, and mechanisms of drug resistance.

Animals↗
PubMed2003

NF-kappaB signalling is inhibited by glucocorticoid receptor and STAT6 via distinct mechanisms.

NF-kappaB transcription factors are involved in the cellular response to stress, and are regulated by inhibitor (IkappaB) proteins, which prevent NF-kappaB-mediated transcription by maintaining NF-kappaB in the cytoplasm. Proteins from other pathways are also known to regulate NF-kappaB negatively, notably the glucocorticoid receptor (GR) and IL-4-responsive STAT6. Both pathways were shown to inhibit NF-kappaB-mediated transcription, by expressing either STAT6 or GR and activating the respective pathways. Using fluorescent fusion proteins, we show that GR alters the timing of activated p65 NF-kappaB nuclear occupancy by increasing the export rate of p65 and is independent of whether GR is present as a dimer or monomer. Expression of STAT6 was also shown to alter p65 nuclear occupancy but appeared to affect the import rate and hence the overall maximal level of p65 translocation. Activating STAT6 with IL-4 prior to activating NF-kappaB significantly increased this inhibition. Investigation of IkappaBa showed that activated STAT6 inhibited TNFalpha-mediated IkappaBa phosphorylation and degradation, whereas GR activation did not alter IkappaBalphakinetics. This demonstrates a clear separation of two distinct mechanisms of inhibition by STAT6 and GR upon the NF-kappaB pathway.

Active Transport, Cell Nucleus↗
PubMed2003

The mitochondrial network of human neutrophils: role in chemotaxis, phagocytosis, respiratory burst activation, and commitment to apoptosis.

It is commonly assumed that human neutrophils possess few, if any, functional mitochondria and that they do not depend on these organelles for cell function. We have used the fluorescent mitochondrial indicators, JC-1, MitoTracker Red, and dihydrorhodamine 123 to show that live neutrophils possess a complex mitochondrial network that extends through the cytoplasm. The membrane potential of these mitochondria was rapidly (within 2 min) disrupted by the addition of FCCP (IC(50) = 20 nM), but not by the Fo-ATPase inhibitor, oligomycin (at up to 7 microg/ml). However, inhibition of mitochondrial function with both agents resulted in cell shape changes. Neither activation of the respiratory burst nor phagocytosis of either latex particles or serum-opsonized Staphylococcus aureus was affected by the addition of FCCP or oligomycin. However, FCCP inhibited chemotaxis at concentrations that paralleled disruption of mitochondrial membrane potential. Furthermore, prolonged (2-h) incubation with oligomycin resulted in an impaired ability to activate a respiratory burst and also inhibited chemotaxis. These observations indicate that intact mitochondrial function is required to sustain some neutrophil functions, but not for the rapid initiation of the respiratory burst or phagocytosis. Loss of mitochondrial membrane potential was a very early marker for commitment of neutrophils into apoptosis and preceded the appearance of phosphatidylserine on the cell surface. However, inhibition of mitochondrial function did not accelerate the rate of neutrophil apoptosis. These data shed important insights into the hitherto unrecognized importance of mitochondria in the function of neutrophils during infection and inflammation.

Apoptosis↗
PubMed2002

Dynamic analysis of STAT6 signalling in living cells.

Functional activity of N- and C-terminal fluorescent fusion proteins between STAT6 and EGFP was demonstrated through IL-4-dependent transcriptional activation and nuclear translocation. The N-terminal (EGFP-STAT6) fusion protein appeared to be more active than the C-terminal fusion. In HEK-293 cells both fusion proteins formed fluorescent nuclear foci following IL-4 stimulation, but in HeLa cells nuclear accumulation was homogeneous. Stimulation of the NF-kappaB pathway through TNFalpha treatment, or expression of p65-EGFP fusion protein, repressed both basal STAT6-dependent transcriptional activity and the extent of activation in response to IL-4. This indicates a novel mechanism of inhibition of STAT6 signalling by NF-kappaB activation.

Active Transport, Cell Nucleus↗
PubMed2002

Multi-parameter analysis of the kinetics of NF-kappaB signalling and transcription in single living cells.

Proteins of the NF-kappaB transcription factor family normally reside in the cytoplasm of cells in a complex with IkappaB inhibitor proteins. Stimulation with TNFalpha leads to proteosomal degradation of the IkappaB proteins and nuclear translocation of the NF-kappaB proteins. Expression of p65 and IkappaBalpha fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IkappaBalpha-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression, the half-time of IkappaBalpha-EGFP degradation was reduced and that of p65 translocation was significantly increased when compared with cells expressing the single fluorescent fusion proteins. These results suggest that the ratio of IkappaBalpha and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IkappaBalpha is the true substrate for TNFalpha stimulation in mammalian cells. When cells were treated with the CRM-1-dependent nuclear export inhibitor, leptomycin B (LMB), there was nuclear accumulation of IkappaBalpha-EGFP and p65-dsRed, with IkappaBalpha-EGFP accumulating more rapidly. No NF-kappaB-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IkappaBalpha-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNFalpha, degradation of IkappaBalpha-EGFP was observed in both the cytoplasm and nucleus. A normal transient transcription response was observed in the same cells using luminescence imaging of NF-kappaB-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-kappaB-dependent transcription occur even when nuclear export of NF-kappaB is inhibited. The results provide functional evidence that other factors, such as modification of p65 by phosphorylation, or interaction with other proteins such as transcriptional co-activators/co-repressors, may critically modulate the kinetics of transcription through this signalling pathway.

Active Transport, Cell Nucleus↗
PubMed2002

The pH of the Plasmodium falciparum digestive vacuole: holy grail or dead-end trail?

The maintenance of acidic pH in the digestive vacuole of the malaria parasite is thought to be crucial to the digestion of host cell haemoglobin and the subsequent process of heme detoxification. It may also be important in the mode of action of chloroquine and in the mechanism of resistance to the drug. Obtaining a definitive measurement of digestive vacuole pH has been surprisingly difficult. Some of the techniques for the measurement of pH in acid vesicles are outlined here along with some key aspects that are specific to malaria parasites. The use of acridine orange and dextran-tagged dyes as probes for the measurement of digestive vacuole pH has proved problematic, yet some surprising findings have emerged from work with these compounds.

Acridine Orange↗