A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the beta-19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (Delta-F/F) approximate to 10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for Delta-F agreed well with those for epibatidine-induced currents, but were shifted approximate to 100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the alpha-gamma-binding site than to the alpha-delta site and (ii) ACh binds with reverse-site selectivity, these data suggest that Delta-F monitors an event linked to binding specifically at the alpha-delta-subunit interface. In experiments with flash-applied agonists, the earliest detectable Delta-F occurs within milliseconds, i.e., during activation. At low [ACh] (less than or equal to 10 muM), a phase of Delta-F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from Delta-F is complete before the slowest phase of recovery from desensitization (time constant approximate to 250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the alpha-delta-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes

Scaling property of the critical hopping parameters for the Bose-Hubbard model

Recently precise results for the boundary between the Mott insulator phase and the superfluid phase of the homogeneous Bose-Hubbard model have become available for arbitrary integer filling factor g and any lattice dimension d > 1. We use these data for demonstrating that the critical hopping parameters obey a scaling relationship which allows one to map results for different g onto each other. Unexpectedly, the mean-field result captures the dependence of the exact critical parameters on the filling factor almost fully. We also present an approximation formula which describes the critical parameters for d > 1 and any g with high accuracy.Comment: 5 pages, 5 figures. to appear in EPJ

Nuclear target search at the single molecule level: protein interactions define the exploration landscape

Gene regulation relies on highly mobile transcription factors (TFs) exploring the nucleoplasm in search of their targets. Our view of the nucleus has evolved from that of an isotropic and homogenous reactor to that of a highly organized yet very dynamic organelle. However important questions remain on how these regulatory factors explore the nuclear environment in search of their DNA or protein targets, and how their exploration strategy affects the kinetics of transcriptional regulation. We implemented a single-molecule tracking assay to determine the TFs dynamics using photoactivatable tags in human cells. We investigated the mobility of several nuclear proteins, including the transcription factor c-Myc and the elongation factor P-TEFb. We found that, while their diffusion speed was comparable, these proteins largely differed in terms of their exploration geometry. We discovered that c-Myc is a global explorer diffusing in the nucleus without spatial constraints. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment, constrained by a fractal nuclear architecture. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. We also measured the mobility of a P-TEFb mutant in which the interaction with the CTD of the RNA Pol II was truncated. In this case, the single-molecule experiments suggested a global exploration of the P-TEFb mutant, consistent with free diffusion. Our observations are in line with a model in which the exploration geometry of TFs is constrained by their interactions and not by exclusion properties. Our findings have strong implications on how proteins react in the nucleus and how their function can be regulated in space and time

Loop structure of the lowest Bloch band for a Bose-Einstein condensate

We investigate analytically and numerically Bloch waves for a Bose--Einstein condensate in a sinusoidal external potential. At low densities the dependence of the energy on the quasimomentum is similar to that for a single particle, but at densities greater than a critical one the lowest band becomes triple-valued near the boundary of the first Brillouin zone and develops the structure characteristic of the swallow-tail catastrophe. We comment on the experimental consequences of this behavior.Comment: 4 pages, 7 figure

Mapping adaptation of barley to droughted environments

Identifying barley genomic regions influencing the response of yield and its components to water deficits will aid in our understanding of the genetics of drought tolerance and the development of more drought tolerant cultivars. We assembled a population of 192 genotypes that represented landraces, old, and contemporary cultivars sampling key regions around the Mediterranean basin and the rest of Europe. The population was genotyped with a stratified set of 50 genomic and EST derived molecular markers, 49 of which were Simple Sequence Repeats (SSRs), which revealed an underlying population sub-structure that corresponded closely to the geographic regions in which the genotypes were grown. A more dense whole genome scan was generated by using Diversity Array Technology (DArT®) to generate 1130 biallelic markers for the population. The population was grown at two contrasting sites in each of seven Mediterranean countries for harvest 2004 and 2005 and grain yield data collected. Mean yield levels ranged from 0.3 to 6.2 t/ha, with highly significant genetic variation in low-yielding environments. Associations of yield with barley genomic regions were then detected by combining the DArT marker data with the yield data in mixed model analyses for the individual trials, followed by multiple regression of yield on markers to identify a multi-locus subset of significant markers/QTLs. QTLs exhibiting a pre-defined consistency across environments were detected in bins 4, 6, 6 and 7 on barley chromosomes 3H, 4H, 5H and 7H respectivel

From Single-Molecule Interactions to Population-Level Dynamics: Understanding the Complex Organization of RNA Pol II in the Nucleus of Living Cells

Transcription involves a complex exchange within a reservoir of proteins in the nucleoplasm, and the specific recruitment of individual proteins at specific gene loci. However, understanding the spatial distribution of individual proteins and the temporal behavior in the nucleus of living cells remains challenging. Using 3D super-resolution fluorescence microscopy and cluster analysis, we observe that the distribution of RNA Polymerase II (Pol II) cluster sizes, measured as the number of polymerases per cluster, follows a −3/2 power law. Radial dependent analysis of the spatial distribution of Pol II also shows scale-invariance, consistent with a so-called self-organized criticality in a fractal geometry of dimension ∼2.7. These results suggest a diffusion-based mechanism whereby, via transient interactions, massive recruitment and dismissal of pol II molecules can occur at specific loci in the nucleoplasm. Kinetic measurements using single-molecule detection in live cells reveal Pol II binding dynamics within minutes. Serum-induced transcription increased Pol II binding kinetics in live cells by an order of magnitude. Together, these results provide a comprehensive view of the spatio-temporal organization of Pol II in the nucleus: from the global population distribution, to single molecule recruitment at specific loci in live cells. This comprehensive single-cell approach can be adopted for other proteins beside RNA Pol II, for real-time quantification of protein organization in vivo, with single-molecule sensitivity

A mapping approach to synchronization in the "Zajfman trap": stability conditions and the synchronization mechanism

We present a two particle model to explain the mechanism that stabilizes a bunch of positively charged ions in an "ion trap resonator" [Pedersen etal, Phys. Rev. Lett. 87 (2001) 055001]. The model decomposes the motion of the two ions into two mappings for the free motion in different parts of the trap and one for a compressing momentum kick. The ions' interaction is modelled by a time delay, which then changes the balance between adjacent momentum kicks. Through these mappings we identify the microscopic process that is responsible for synchronization and give the conditions for that regime.Comment: 12 pages, 9 figures; submitted to Phys Rev