Co-imaging extrinsic, intrinsic and effector caspase activity by fluorescence anisotropy microscopy.

In order to overcome intercellular variability and thereby effectively assess signal propagation in biological networks it is imperative to simultaneously quantify multiple biological observables in single living cells. While fluorescent biosensors have been the tool of choice to monitor the dynamics of protein interaction and enzymatic activity, co-measuring more than two of them has proven challenging. In this work, we designed three spectrally separated anisotropy-based Förster Resonant Energy Transfer (FRET) biosensors to overcome this difficulty. We demonstrate this principle by monitoring the activation of extrinsic, intrinsic and effector caspases upon apoptotic stimulus. Together with modelling and simulations we show that time of maximum activity for each caspase can be derived from the anisotropy of the corresponding biosensor. Such measurements correlate relative activation times and refine existing models of biological signalling networks, providing valuable insight into signal propagation.

Chemically induced photoswitching of fluorescent probes--a general concept for super-resolution microscopy.

We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.

Oxygen delivery by blood determines the maximal VO2 and work rate during whole body exercise in humans: in silico studies.

It has been proposed by Saltin (J Exp Biol 115: 345-354, 1985) that oxygen delivery by blood is limiting for maximal work and oxygen consumption in humans during whole body exercise but not during single-muscle exercise. To test this prediction quantitatively, we developed a static (steady-state) computer model of oxygen transport to and within human skeletal muscle during single-muscle (quadriceps) exercise and whole body (cycling) exercise. The main system fluxes, namely cardiac output and oxygen consumption by muscle, are described as a function of the "primary" parameter: work rate. The model is broadly validated by comparison of computer simulations with various experimental data. In silico studies show that, when all other parameters and system properties are kept constant, an increase in the working muscle mass from 2.5 kg (single quadriceps) to 15 kg (two legs) causes, at some critical work intensity, a drop in oxygen concentration in muscle cells to (very near) zero, and therefore oxygen supply by blood limits maximal oxygen consumption and oxidative ATP production. Therefore, the maximal oxygen consumption per muscle mass is significantly higher during single-muscle exercise than during whole body exercise. The effect is brought about by a distribution of a limited amount of oxygen transported by blood in a greater working muscle mass during whole body exercise.

Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies.

It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production.

Theoretical studies on the regulation of anaerobic glycolysis and its influence on oxidative phosphorylation in skeletal muscle.

It is shown, using the computer model of glycolysis in skeletal muscle developed recently by Lambeth and Kushmerick (Ann. Biomed. Bioenerg, 30 (2001) 19-34) incorporated into the computer model of oxidative phosphorylation developed by Korzeniewski et al. (Biophys. Chem. 83 (2001) 19-34) that the regulation of glycolysis by ADP, AMP and P(i) is decidedly insufficient to explain the large increase in the glycolytic flux during transition from rest to intensive exercise in intact skeletal muscle. Computer simulations based on a simple kinetic description of the glycolytic ATP and H(+) production strongly suggests that glycolysis must be directly activated during muscle contraction. They also demonstrate that the inhibition of glycolysis by H(+) is needed to explain the transient activation of this pathway at the onset of exercise as well as the duration time and extent of the initial alkalization after the onset of exercise. Finally, it is shown that ATP supply from anaerobic glycolysis slows down the VO(2) kinetics during rest-to-work transition.