Frog oocytes to unveil the structure and supramolecular organization of human transport proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

Sleep stage II contributes to the consolidation of declarative memories

Various studies suggest that non-rapid eye movement (NREM) sleep, especially slow-wave sleep (SWS), is vital to the consolidation of declarative memories. However, sleep stage 2 (S2), which is the other NREM sleep stage besides SWS, has gained only little attention. The current study investigated whether S2 during an afternoon nap contributes to the consolidation of declarative memories. Participants learned associations between faces and cities prior to a brief nap. A cued recall test was administered before and following the nap. Spindle, delta and slow oscillation activity was recorded during S2 in the nap following learning and in a control nap. Increases in spindle activity, delta activity, and slow oscillation activity in S2 in the nap following learning compared to the control nap were associated with enhanced retention of face-city associations. Furthermore, spindles tended to occur more frequently during up-states than down-states within slow oscillations during S2 following learning versus S2 of the control nap. These findings suggest that spindles, delta waves, and slow oscillations might promote memory consolidation not only during SWS, as shown earlier, but also during S2.

Subcolumnar Dendritic and Axonal Organization of Spiny Stellate and Star Pyramid Neurons within a Barrel in Rat Somatosensory Cortex

Excitatory neurons at the level of cortical layer 4 in the rodent somatosensory barrel field often display a strong eccentricity in comparison with layer 4 neurons in other cortical regions. In rat, dendritic symmetry of the 2 main excitatory neuronal classes, spiny stellate and star pyramid neurons (SSNs and SPNs), was quantified by an asymmetry index, the dendrite-free angle. We carefully measured shrinkage and analyzed its influence on morphological parameters. SSNs had mostly eccentric morphology, whereas SPNs were nearly radially symmetric. Most asymmetric neurons were located near the barrel border. The axonal projections, analyzed at the level of layer 4, were mostly restricted to a single barrel except for those of 3 interbarrel projection neurons. Comparing voxel representations of dendrites and axon collaterals of the same neuron revealed a close overlap of dendritic and axonal fields, more pronounced in SSNs versus SPNs and considerably stronger in spiny L4 neurons versus extragranular pyramidal cells. These observations suggest that within a barrel dendrites and axons of individual excitatory cells are organized in subcolumns that may confer receptive field properties such as directional selectivity to higher layers, whereas the interbarrel projections challenge our view of barrels as completely independent processors of thalamic input.

Time-dependent hierarchical organization of spatial working memory: a transcranial magnetic stimulation study

The performance of memory-guided saccades with two different delays (3 and 30 s of memorization) was studied in seven healthy subjects. Double-pulse transcranial magnetic stimulation (dTMS) with an interstimulus interval of 100 ms was applied over the right dorsolateral prefrontal cortex (DLPFC) early (1 s after target presentation) and late (28 s after target presentation). Early stimulation significantly increased in both delays the percentage of error in amplitude (PEA) of contralateral memory-guided saccades compared to the control experiment without stimulation. dTMS applied late in the delay had no significant effect on PEA. Furthermore, we found a significantly smaller effect of early stimulation in the long-delay paradigm. These results suggest a time-dependent hierarchical organization of the spatial working memory with a functional dominance of DLPFC during the early memorization, independent from the memorization delay. For a long memorization delay, however, working memory seems to have an additional, DLPFC-independent component.

Supramolecular Organization of Heptapyrenotide Oligomers—An in Depth Investigation by Molecular Dynamics Simulations

We present a molecular modeling study based on ab initio and classical molecular dynamics calculations, for the investigation of the tridimensional structure and supramolecular assembly formation of heptapyrenotide oligomers in water solution. Our calculations show that free oligomers self-assemble in helical structures characterized by an inner core formed by π- stacked pyrene units, and external grooves formed by the linker moieties. The coiling of the linkers has high ordering, dominated by hydrogen-bond interactions among the phosphate and amide groups. Our models support a mechanism of longitudinal supramolecular oligomerization based on interstrand pyrene intercalation. Only a minimal number of pyrene units intercalate at one end, favoring formation of very extended longitudinal chains, as also detected by AFM experiment. Our results provide a structural explanation of the mechanism of chirality amplification in 1:1 mixtures of standard heptapyrenotides and modified oligomers with covalently linked deoxycytidine, based on selective molecular recognition and binding of the nucleotide to the groove of the left-wound helix.