Descripción: By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single molecule level, we demonstrate simultaneous multicolor imaging with low crosstalk and down to 15 nm spatial resolution using only two detection color channels. The applicability of the method to biological specimens is demonstrated on mammalian cells. The combination of far-field fluorescence nanoscopy with the recording of a single switchable molecular species at a time opens up a new class of functional imaging techniques. © 2008 American Chemical Society.

Descripción: Previous studies of musical creativity suggest that this process involves multi-regional intra and interhemispheric interactions, particularly in the prefrontal cortex. However, the activity of the prefrontal cortex and that of the parieto-temporal regions, seems to depend on the domains of creativity that are evaluated and the task that is performed. In the field of music, only few studies have investigated the brain process of a creative task and none of them have investigated the effect of the level of creativity on the recruit networks. In this work we used magnetic resonance imaging to explore these issues by comparing the brain activities of subjects with higher creative abilities to those with lesser abilities, while the subjects improvised on different rhythmic fragments. We evaluated the products the subjects created during the fMRI scan using two musical parameters: fluidity and flexibility, and classified the subjects according to their punctuation. We examined the relation between brain activity and creativity level. Subjects with higher abilities generated their own creations based on modifications of the original rhythm with little adhesion to it. They showed activation in prefrontal regions of both hemispheres and the right insula. Subjects with lower abilities made only partial changes to the original musical patterns. In these subjects, activation was only observed in left unimodal areas. We demonstrated that the activations of prefrontal and paralimbic areas, such as the insula, are related to creativity level, which is related to a widespread integration of networks that are mainly associated with cognitive, motivational and emotional processes. © 2013 Villarreal et al.

Descripción: Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3′ splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ~20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA. © 2012 Schor et al.

Descripción: Background: Olfactory systems create representations of the chemical world in the animal brain. Recordings of odour-evoked activity in the primary olfactory centres of vertebrates and insects have suggested similar rules for odour processing, in particular through spatial organization of chemical information in their functional units, the glomeruli. Similarity between odour representations can be extracted from across-glomerulus patterns in a wide range of species, from insects to vertebrates, but comparison of odour similarity in such diverse taxa has not been addressed. In the present study, we asked how 11 aliphatic odorants previously tested in honeybees and rats are represented in the antennal lobe of the ant Camponotus fellah, a social insect that relies on olfaction for food search and social communication.Results: Using calcium imaging of specifically-stained second-order neurons, we show that these odours induce specific activity patterns in the ant antennal lobe. Using multidimensional analysis, we show that clustering of odours is similar in ants, bees and rats. Moreover, odour similarity is highly correlated in all three species.Conclusion: This suggests the existence of similar coding rules in the neural olfactory spaces of species among which evolutionary divergence happened hundreds of million years ago. © 2010 Dupuy et al; licensee BioMed Central Ltd.

Descripción: Confocal microscopy images revealed that the tetratricopeptide repeat motif (TPR) domain immunophilin FKBP51 shows colocalization with the specific mitochondrial marker Mito-Tracker. Signal specificity was tested with different antibodies and by FKBP51 knockdown. This unexpected subcellular localization of FKBP51 was confirmed by colocalization studies with other mitochondrial proteins, biochemical fractionation, and electron microscopy imaging. Interestingly, FKBP51 forms complexes in mitochondria with the glucocorticoid receptor and the Hsp90/Hsp70-based chaperone heterocomplex. Although Hsp90 inhibitors favor FKBP51 translocation from mitochondria to the nucleus in a reversible manner, TPR domain-deficient mutants of FKBP51 are constitutively nuclear and fully excluded from mitochondria, suggesting that a functional TPR domain is required for its mitochondrial localization. FKBP51 overexpression protects cells against oxidative stress, whereas FKBP51 knockdown makes them more sensitive to injury. In summary, this is the first demonstration that FKBP51 is a major mitochondrial factor that undergoes nuclear-mitochondrial shuttling, an observation that may be related to antiapoptotic mechanisms triggered during the stress response. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: We assessed the intracellular association states of the Parkinson's disease related protein α-synuclein (AS) in living cells by transfection with a functional recombinant mutant protein (AS-C4) bearing a tetracysteine tag binding the fluorogenic biarsenical ligands FlAsH and ReAsH, The aggregation states of AS-C4 were assessed by in situ microscopy of molecular translational mobility with FRAP (fluorescence recovery after photobleaching) and of local molecular density with confocal fluorescence anisotropy (CFA). FRAP recovery was quantitative and rapid in regions of free protein, whereas AS in larger aggregates was>80% immobile. A small 16% recovery characterized by an apparent diffusion constant of 0.03-0.04 μm 2/s was attributed to the dynamics of smaller, associated forms of AS-C4 and the exchange of mobile species with the larger immobile aggregates. By CFA, the larger aggregates exhibited high brightness and very low anisotropy, consistent with homoFRET between closely packed AS, for which a Förster distance (R o) of 5.3 nm was calculated. Other bright regions had high anisotropy values, close to that of monomeric AS, and indicative of membrane-associated protein with both low mobility and low degree of association. The anisotropy-fluorescence intensity correlations also revealed regions of free protein or of small aggregates, undetectable by conventional fluorescence imaging alone. The combined strategy (FRAP+CFA) provides a highly sensitive means for elucidating both the dynamics and structural features of protein aggregates and other intracellular complexes in living cells, and can be extended to other amyloid systems and to drug screening protocols. © 2011 Roberti et al.

Descripción: The ASR (for ABA/water stress/ripening) protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family) in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS) in the primary sequence. However, here we prove that such an "NLS" of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE. © 2012 Ricardi et al.