Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer

In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of the ATP binding cassette family of transport proteins, is found in the plasma membrane of animal cells where it is involved in the ATP hydrolysis driven export of hydrophobic molecules. When expressed in the plasma membrane of cancer cells, the transport activity of Pgp can lead to the failure of chemotherapy by excluding the mostly hydrophobic drugs from the interior of the cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples MgATP binding and hydrolysis to translocation of drug molecules across the lipid bilayer is poorly understood. Using site directed mutagenesis, we have introduced cysteine residues for fluorescence labeling into different regions of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp molecules showed fluctuating FRET efficiencies during drug stimulated ATP hydrolysis suggesting that the NBDs undergo significant movements during catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is applied to minimize FRET artifacts and to select the appropriate molecules. The data show that Pgp is a highly dynamic enzyme that appears to fluctuate between at least two major conformations during steady state turnover.Comment: 10 pages, 7 figure

Behavioural lateralisation in reindeer

Reindeer (Rangifer tarandus) kept in corrals or otherwise forced to clump typically start milling in response to stressing events. This behaviour is generally considered to have an antipredator effect. An inquiry on herd behaviour, to which 35 Norwegian reindeer husbandry districts responded, showed that 32 experienced that corralled rein¬deer consistently circled leftwards, whereas the remaining three reported consistently rightward circling. Regular monitoring of a reindeer herd in central Norway over a two-year period (1993-94), and experimental studies on a fraction of the same herd, revealed the following traits. Free-ranging reindeer showed no right- or left-turning preference during grazing or browsing, but when the reindeer were driven into corrals or forced to clump in the open they invariably rotated leftwards. The circling of corralled reindeer was triggered at an average group size of 20 to 25 animals, apparently independently of the age and sex of the animals. When they dug craters in the snow to reach food, the reindeer used their left foreleg significantly more often than their right. In 23 out of 35 reindeer, the right hemisphere of the brain was heavier than the left. However, in the sample as a whole, the weights of the left and right hemispheres did not differ significantly. Lateralised behaviour in reindeer is thought to be determined by natural and stress induced asymmetries in brain structure and hormonal activity. In addition, learning is probably important for passing on the behaviour between herd members and generations. Differences in lateralised behaviour between nearby herds are thought to be related primarily to different exposure to stress and learning, whereas genetical and environmental fac¬tors (e.g. diet), age structure and sex ratio are probably more important for explaining differences between distant pop¬ulations

The evolution of brain lateralization: A game theoretical analysis of population structure

In recent years, it has become apparent that behavioural and brain lateralization is the rule rather than the exception among vertebrates. The study of lateralization has been so far the province of neurology and neuropsychology. We show how such research can be integrated with evolutionary biology to more fully understand lateralization. In particular, we address the fact that, within a species, left- and right-type individuals are often in a definite proportion different from 1/2 (e.g., hand use in humans). We argue that traditional explanations of brain lateralization (that it may avoid costly duplication of neural circuitry and reduce interference between functions) cannot account for this fact, because increased individual efficiency is unre- lated to the frequency of left- and right-type individuals in a population. A further puzzle is that, if a majority of individuals are of the same type, individual behaviour becomes more predictable to other organisms. Here we show that alignment of the direction of behavioural asymmetries in a population can arise as an evolutionarily stable strategy (ESS), when individually asymmetrical organisms must coordinate their behaviour with that of other asymmetrical organisms. Thus, brain and behavioural lateralization, as we know it in humans and other vertebrates, may have evolved under basically "social" selection pressures

Regulatory assembly of the vacuolar proton pump VOV1-ATPase in yeast cells by FLIM-FRET

We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for transport processes as well as for pH and Ca2+ homoeostasis in the cell. Activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.Comment: 8 pages, 3 figure

Visualizing mitochondrial FoF1-ATP synthase as the target of the immunomodulatory drug Bz-423

Targeting the mitochondrial enzyme FoF1-ATP synthase and modulating its catalytic activities with small molecules is a promising new approach for treatment of autoimmune diseases. The immuno-modulatory compound Bz-423 is such a drug that binds to subunit OSCP of the mitochondrial FoF1-ATP synthase and induces apoptosis via increased reactive oxygen production in coupled, actively respiring mitochondria. Here we review the experimental progress to reveal the binding of Bz-423 to the mitochondrial target and discuss how subunit rotation of FoF1-ATP synthase is affected by Bz-423. Briefly, we report how F\"orster resonance energy transfer (FRET) can be employed to colocalize the enzyme and the fluorescently tagged Bz-423 within the mitochondria of living cells with nanometer resolution.Comment: 10 pages, 2 figure

Brain Asymmetry: Towards an Asymmetrical Neurovisceral Integration

This review was supported by the group of the University of Jaen "Neuroendocrinology and Nutrition" BIO-221.Despite the ancestral evidence of an asymmetry in motor predominance, going through the inspiring discoveries of Broca and Wernicke on the localization of language processing, continuing with the subsequent noise coinciding with the study of brain function in commissurotomized patients—and the subsequent avalanche of data on the asymmetric distribution of multiple types of neurotransmitters in physiological and pathological conditions—even today, the functional significance of brain asymmetry is still unknown. Currently, multiple evidence suggests that functional asymmetries must have a neurochemical substrate and that brain asymmetry is not a static concept but rather a dynamic one, with intra- and inter-hemispheric interactions between its various processes, and that it is modifiable depending on changing endogenous and environmental conditions. Furthermore, based on the concept of neurovisceral integration in the overall functioning of an organism, some evidence has emerged suggesting that this integration could be organized asymmetrically, using the autonomic nervous system as a bidirectional communication pathway, whose performance would also be asymmetric. However, the functional significance of this distribution, as well as the evolutionary advantage of an asymmetric nervous organization, is still unknown.University of Jaen "Neuroendocrinology and Nutrition" BIO-22