David G. Spiller

Pulsatile Stimulation Determines Timing and Specificity of NF-κB-Dependent Transcription

The nuclear factor κB (NF-κB) transcription factor regulates cellular stress responses and the immune response to infection. NF-κB activation results in oscillations in nuclear NF-κB abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor–α at various intervals to mimic pulsatile inflammatory signals. At all pulse intervals that were analyzed, we observed synchronous cycles of NF-κB nuclear translocation. Lower frequency stimulations gave repeated full-amplitude translocations, whereas higher frequency pulses gave reduced translocation, indicating a failure to reset. Deterministic and stochastic mathematical models predicted how negative feedback loops regulate both the resetting of the system and cellular heterogeneity. Altering the stimulation intervals gave different patterns of NF-κB–dependent gene expression, which supports the idea that oscillation frequency has a functional role.

Measurement of single-cell dynamics

Populations of cells are almost always heterogeneous in function and fate. To understand the plasticity of cells, it is vital to measure quantitatively and dynamically the molecular processes that underlie cell-fate decisions in single cells. Early events in cell signalling often occur within seconds of the stimulus, whereas intracellular signalling processes and transcriptional changes can take minutes or hours. By contrast, cell-fate decisions, such as whether a cell divides, differentiates or dies, can take many hours or days. Multiparameter experimental and computational methods that integrate quantitative measurement and mathematical simulation of these noisy and complex processes are required to understand the highly dynamic mechanisms that control cell plasticity and fate.

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 (IC50 = 20 nM), but not by the Fo-ATPase inhibitor, oligomycin (at up to 7 μg/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.

Dynamic Analysis of Stochastic Transcription Cycles

Cycling of gene expression in individual cells is controlled by dynamic chromatin remodeling.

Population robustness arising from cellular heterogeneity

Heterogeneity between individual cells is a common feature of dynamic cellular processes, including signaling, transcription, and cell fate; yet the overall tissue level physiological phenotype needs to be carefully controlled to avoid fluctuations. Here we show that in the NF-κB signaling system, the precise timing of a dual-delayed negative feedback motif [involving stochastic transcription of inhibitor κB (IκB)-α and -ε] is optimized to induce heterogeneous timing of NF-κB oscillations between individual cells. We suggest that this dual-delayed negative feedback motif enables NF-κB signaling to generate robust single cell oscillations by reducing sensitivity to key parameter perturbations. Simultaneously, enhanced cell heterogeneity may represent a mechanism that controls the overall coordination and stability of cell population responses by decreasing temporal fluctuations of paracrine signaling. It has often been thought that dynamic biological systems may have evolved to maximize robustness through cell-to-cell coordination and homogeneity. Our analyses suggest in contrast, that this cellular variation might be advantageous and subject to evolutionary selection. Alternative types of therapy could perhaps be designed to modulate this cellular heterogeneity.

Expression of the leptin receptor in rat and human nodose ganglion neurones

There is evidence for interactions between leptin and cholecystokinin in controlling food intake. Since cholecystokinin acts on vagal afferent neurones, we asked whether the leptin receptor was also expressed by these neurones. Primers for different forms of the leptin receptor were used in reverse transcriptase-polymerase chain reaction (RT-PCR) of rat and human nodose ganglia. RT-PCR yielded products corresponding to the long (functional) form as well as short forms of the rat leptin receptor. Moreover, RT-PCR revealed the long form of the leptin receptor in a human nodose ganglion. The identities of RT-PCR products were confirmed by sequencing. Primers corresponding to leptin itself did not give RT-PCR products in nodose ganglia. Immunocytochemical studies revealed leptin-receptor immunoreactivity in neuronal cell bodies. Many neurones co-expressed the leptin and cholecystokinin type A receptors, or leptin receptor and cocaine- and amphetamine-related transcript. We conclude that vagal afferent neurones that express the cholecystokinin type A receptor and cocaine- and amphetamine-related transcript, may also express the long form of the leptin receptor providing a neurochemical basis for observations of interactions between cholecystokinin and leptin.

Edible mushroom (Agaricus bisporus) lectin, which reversibly inhibits epithelial cell proliferation, blocks nuclear localization sequence-dependent nuclear protein import

The Galβ1–3GalNAcα (TF antigen)-binding lectin (ABL) from the common edible mushroom (Agaricus bisporus) has a potent anti-proliferative effect without any apparent cytotoxicity. This unusual combination of properties prompted investigation of its mechanism of action. In contrast to soluble lectin, agarose-immobilized, and hence noninternalizable ABL had no effect on proliferation of HT29 colon cancer cells. Electron microscopy of HT29 cells incubated with fluorescein- and gold-conjugated ABL showed internalization of the lectin into endocytotic vesicles and multivesicular bodies. Confocal microscopy showed perinuclear accumulation of fluorescein isothiocyanate-conjugated lectin, which also inhibits HT29 cell proliferation, raising the possibility that the lectin might interfere with nuclear pore function. Transport of heat shock protein 70 into the nucleus in response to heat shock was blocked by preincubation of HT29 cells for 6 h with 40 μg/ml ABL. In digitonin-permeabilized cells, nuclear uptake of bovine albumin conjugated to a nuclear localization sequence (NLS)-containing peptide was also inhibited by a 15-min preincubation with 40–100 μg/ml ABL. In contrast, serum-stimulated nuclear translocation of mitogen-activated protein kinase, which is NLS-independent, was not affected by pretreatment of cells with the lectin. These results suggest that the anti-proliferative effect of ABL is likely to be a consequence of the lectin trafficking to the nuclear periphery, where it blocks NLS-dependent protein uptake into the nucleus.

Fluorescent or not? Size-dependent fluorescence switching for polymer-stabilized gold clusters in the 1.1-1.7 nm size range

The synthesis of fluorescent water-soluble gold nanoparticles by the reduction of a gold salt in the presence of a designed polymer ligand is described, the size and fluorescence of the particles being controlled by the polymer to gold ratio; the most fluorescent nanomaterial has a 3% quantum yield, a 1.1 nm gold core and a 6.9 nm hydrodynamic radius.

Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo

Antisense oligonucleotides are designed to specifically hybridize to a target messenger RNA (mRNA) and interfere with the synthesis of the encoded protein. Uniformly modified oligonucleotides containing N3′–P5′ phosphoramidate linkages exhibit (NP) extremely high-affinity binding to single-stranded RNA, do not induce RNase H activity, and are resistant to cellular nucleases. In the present work, we demonstrate that phosphoramidate oligonucleotides are effective at inhibiting gene expression at the mRNA level, by binding to their complementary target present in the 5′-untranslated region. Their mechanism of action was demonstrated by comparative analysis of three expression systems that differ only by the composition of the oligonucleotide target sequence (HIV-1 polypurine tract or PPT sequence) present just upstream from the AUG codon of the firefly luciferase reporter gene: the experiments have been done on isolated cells using oligonucleotide delivery mediated by cationic molecules or streptolysin O (SLO), and in vivo by oligonucleotide electrotransfer to skeletal muscle. In our experimental system phosphoramidate oligonucleotides act as potent and specific antisense agents by steric blocking of translation initiation; they may prove useful to modulate RNA metabolism while maintaining RNA integrity.

Cathepsin L Digestion of Nanobioconjugates upon Endocytosis

Understanding the dynamic fate and interactions of bioconjugated nanoparticles within living cells and organisms is a prerequisite for their use as in situ sensors or actuators. While recent research has provided indications on the effect of size, shape, and surface properties of nanoparticles on their internalization by living cells, the biochemical fate of the nanoparticles after internalization has been essentially unknown. Here we show that, upon internalization in a wide range of mammalian cells, biological molecules attached to the nanoparticles are degraded within the endosomal compartments through peptide cleavage by the protease cathepsin L. Importantly, using bioinformatics tools, we show that cathepsin L is able to cleave more than a third of the human proteome, indicating that this degradation process is likely to happen to most nanoparticles conjugated with peptides and proteins and cannot be ignored in the design of nanomaterials for intracellular applications. Preservation of the bioconjugates can be achieved by a combination of cathepsin inhibition and endosome disruption.