Heinrich Huber

Systems analysis of effector caspase activation and its control by X-linked inhibitor of apoptosis protein

Activation of effector caspases is a final step during apoptosis. Single‐cell imaging studies have demonstrated that this process may occur as a rapid, all‐or‐none response, triggering a complete substrate cleavage within 15 min. Based on biochemical data from HeLa cells, we have developed a computational model of apoptosome‐dependent caspase activation that was sufficient to remodel the rapid kinetics of effector caspase activation observed in vivo. Sensitivity analyses predicted a critical role for caspase‐3‐dependent feedback signalling and the X‐linked‐inhibitor‐of‐apoptosis‐protein (XIAP), but a less prominent role for the XIAP antagonist Smac. Single‐cell experiments employing a caspase fluorescence resonance energy transfer substrate verified these model predictions qualitatively and quantitatively. XIAP was predicted to control this all‐or‐none response, with concentrations as high as 0.15 μM enabling, but concentrations >0.30 μM significantly blocking substrate cleavage. Overexpression of XIAP within these threshold concentrations produced cells showing slow effector caspase activation and submaximal substrate cleavage. Our study supports the hypothesis that high levels of XIAP control caspase activation and substrate cleavage, and may promote apoptosis resistance and sublethal caspase activation in vivo.

Deposition of ferroelectric PZT thin films by planar multi-target sputtering

Abstract A planar multi-target sputtering system was used to deposit ferroelectric lead zirconate titanate (PZT) films. At substrate temperatures of about 450°C “in-situ” deposition of single phase perovskite PZT was obtained. By investigation with SEM and TEM films exhibited a columnar structure with crystallites of about 200 nm in size. The permittivity of the films varied from 450-600. The films exhibited hysteresis loops, remanent polarization of 15-20 μC/cm2 and coercive field strength of 70-85 kV/cm.

Intracellular signaling dynamics during apoptosis execution in the presence or absence of X-linked-inhibitor-of-apoptosis-protein.

X-linked-inhibitor-of-apoptosis-protein (XIAP) is the most potent intracellular inhibitor of caspases-9, -3 and -7. While highly elevated XIAP levels reduce the apoptotic response to various stimuli, the potency of physiological XIAP expression to control caspase activation and the consequences of XIAP deficiency on apoptosis execution remain controversial. We therefore analyzed parental and XIAP-deficient DLD-1 and HCT-116 colon cancer cells by employing fluorescence-based single-cell imaging of mitochondrial permeabilisation and effector caspase activation. Our results demonstrate that physiological XIAP expression maintains a transient off-state for effector caspase activation following mitochondrial permeabilisation. Loss of XIAP expression instead accelerated the caspase activation response, but did not enhance the measured caspase activity. Apoptosis execution kinetics were independent of activating the intrinsic or extrinsic pathway by either staurosporine or TRAIL, and corresponded to computational systems analyses of caspase activation dynamics. We confirmed a protective role of XIAP upstream of mitochondrial permeabilisation during TRAIL-induced apoptosis, however, once mitochondria permeabilised ultimately no cell could escape effector caspase activation, regardless of potential cell-to-cell variability within the populations or the presence of XIAP. Our study provides comprehensive kinetic and mechanistic insight into the rapid molecular dynamics during apoptosis execution in the presence or absence of physiological XIAP expression.

Diffusion is capable of translating anisotropic apoptosis initiation into a homogeneous execution of cell death

BackgroundApoptosis is an essential cell death process throughout the entire life span of all metazoans and its deregulation in humans has been implicated in many proliferative and degenerative diseases. Mitochondrial outer membrane permeabilisation (MOMP) and activation of effector caspases are key processes during apoptosis signalling. MOMP can be subject to spatial coordination in human cancer cells, resulting in intracellular waves of cytochrome-c release. To investigate the consequences of these spatial anisotropies in mitochondrial permeabilisation on subsequent effector caspase activation, we devised a mathematical reaction-diffusion model building on a set of partial differential equations.ResultsReaction-diffusion modelling suggested that even if strong spatial anisotropies existed during mitochondrial cytochrome c release, these would be eliminated by free diffusion of the cytosolic proteins that instantiate the apoptosis execution network. Experimentally, rapid sampling of mitochondrial permeabilisation and effector caspase activity in individual HeLa cervical cancer cells confirmed predictions of the reaction-diffusion model and demonstrated that the signalling network of apoptosis execution could efficiently translate spatial anisotropies in mitochondrial permeabilisation into a homogeneous effector caspase response throughout the cytosol. Further systems modelling suggested that a more than 10,000-fold impaired diffusivity would be required to maintain spatial anisotropies as observed during mitochondrial permeabilisation until the time effector caspases become activated.ConclusionsMulti-protein diffusion efficiently contributes to eliminating spatial asynchronies which are present during the initiation of apoptosis execution and thereby ensures homogeneous apoptosis execution throughout the entire cell body. For previously reported biological scenarios in which effector caspase activity was shown to be targeted selectively to specific subcellular regions additional mechanisms must exist that limit or spatially coordinate caspase activation and/or protect diffusing soluble caspase substrates from unwanted proteolysis.

Pyroelectric properties of lead titanate thin films deposited on pt-coated si wafers by multi-target sputtering

Abstract The deposition of single phase perovskite lead titanate thin films by planar multi-target sputtering on silicon substrates covered with a thin Pt bottom electrode has been investigated. The films exhibited nearly random crystallographic orientation. The films are self polarized, that means they exhibited pyroelectric currents without poling treatment. After additional furnace annealing and poling the pyroelectric coefficient was 1.7.10−4 C/m2K. The annealing also reduced the dielectric loss tan δ from 0.05 to 0.01. The temperature measurement for calculation of the pyroelectric coefficient was performed by measuring the resistance of the bottom and top electrode via the van der Pauw method and calibration with a thermo chuck.

Ferroelectric Pb (Zr, Ti)O3 thin films prepared by planar multi-target sputtering

Lead zirconate titanate films are deposited using a planar multi-target sputtering system. This system consists of three metallic targets (Zr, Pb, Ti) and a rotating substrate holding pallet achieving a layer-by-layer growth of the material. Substrates used in this study were oxidised (100) Si wafers with thin sputtered Pt layer. At substrate temperatures of about 450°C “in situ” (i.e. without post-deposition annealing) deposition of single phase perovskite PZT was obtained. Deposition rate is 3.5 nm/min. At substrate temperatures of more than 500°C the layers are poor in lead. ZrTiO4 was identified by x-ray diffraction. The dielectric constant and losses of the PZT films varied from 400-500 and from 0.008-0.015 respectively. The films exhibited a hysteresis loop, remanent polarization measured was 7 μC/cm2 and coercive field strength 7.5*106 V/m.

TOXI-SIM-A simulation tool for the analysis of mitochondrial and plasma membrane potentials

Changes in the electrochemical gradients across biological membranes are excellent indicators of pathophysiological processes, drug action, or drug toxicity. Our previous studies have utilized the potentiometric probe tetramethylrhodamine methyl ester (TMRM) to characterize changes in mitochondrial function by monitoring alterations in the mitochondrial membrane potential (Deltapsi(m)) over time during glutamate excitotoxicity. However, fluorescently charged dyes such as TMRM respond to changes in both Deltapsi(m) and the plasma membrane (Deltapsi(p)) potentials making whole cell fluorescence data difficult to interpret. Here we have implemented a mathematical model that exploits the Nernstian behaviour of TMRM and uses automated Newton based root-finding fitting (TOXI-SIM) to model changes in TMRM fluorescence from multiple cells simultaneously, providing output on changes in Deltapsi(m) and Deltapsi(p) over time. Based on Ca(2+) responses, TOXI-SIM allows for an accurate modelling of TMRM traces for different injury paradigms (necrosis, apoptosis, tolerance). TOXI-SIM is provided as a user friendly public web service for trace analysis, with an additional online data base provided for the storage and retrieval of experimental traces (http://systemsbiology.rcsi.ie/tmrm/index.html).

www.rnaworkbench.com: A new program for analyzing RNA interference

RNA interference (RNAi) has become an important tool to study and utilize gene silencing by introducing short interfering RNA (siRNA). In order to predict the most efficient siRNAs, a new software tool, RNA Workbench (RNAWB), has been designed and is freely available (after registration) on http://www.rnaworkbench.com. In addition to the standard selection rules, RNAWB includes the possibility of statistical analyses of the applied selection rules (criteria). The role of RNA secondary structures in the RNA interference process as well as the application of sequence rules are discussed to show the applicability of the software.

Sputtered PZT films for ferroelectric devices

Abstract The article reviews the sputter deposition of ferroelectric thin films in the lead zirconate-lead titanate (PZT) family of compositions. Five different sputtering geometries to deposit PZT, lead titanate and lanthanum doped lead titanate either from ceramic or metallic targets are described. In addition to this selection of different sputtering geometries the films can be processed either by the so called “one step” process or by a “two step” process. In the one step process “in-situ” growth of the perovskite phase at elevated substrate temperatures is achieved, whereas in the two step process the film is deposited at moderate temperatures in an amorphous or partly crystalline form and then transformed to the perovskite phase by an additional annealing step. Planar multi target sputtering of PZT and lead titanate is discussed in more detail.