Mobilität von eGFP-Oligomeren in lebenden Zellkernen

Bewegungen von Partikeln in Zellkernen können durch die Viskosität, aktiven Transport oder die Anwesenheit von Hindernissen, wie zum Beispiel dem Chromatinnetzwerk, beeinflusst werden. In dieser Arbeit wurde untersucht, ob die Mobilität von kleinen Proteinen einer Größe zwischen 27 und 108 kDa von der Chromatindichte beeinflusst wird. Die Diffusion von inerten fluoreszierenden Proteinen wurde in Zellkernen von lebenden Zellen mit Fluoreszenzkorrelationsspektroskopie (FCS) an einem zwei-Farben-Detektions-system untersucht. Zunächst werden der Neuaufbau und die Optimierung des Messsystems, sowie die Entwicklung einer neuen, patentierten Messkammer für mikroskopische Beobachtungen und Messungen (CHAMO) beschrieben. Im Anschluss werden Experimente vorgestellt, die FCS-spezifische Artefakte in lebenden Zellen untersuchen, sowie Strategien, um diese zu vermeiden. Insbesondere werden Artefakte besprochen, die aus der Auswahl der Fluorophore zur Kalibrierung des Fokusvolumens sowie aus der Temperatur und der Aufnahmebedingungen bei den Messungen der Fluoreszenzfluktuationen resultieren. Nachdem die optimalen Aufnahmebedingungen definiert wurden, zeigen die Ergebnisse der Experimente, dass die Mobilität von eGFP in Zellkernen lebender Zellen signifikant variiert, aber nicht mit der Chromatindichte korreliert. Die intranukleare diffusive Mobilität hängt stark von der Proteingröße ab: in einer Serie von eGFP-Oligomeren, die als inerte Sonde verwendet wurden, nimmt der Diffusionskoeffizient vom Monomer zum Tetramer viel stärker ab als für freie Moleküle in wässriger Lösung. Dennoch bleibt das gesamte intranukleare Chromatinnetzwerk für kleine Proteine bis zu einer Größe von dem eGFP-Tetramer frei erreichbar, ungeachtet der Chromatindichte oder der Zelllinie. Diffusionsmessungen in freier Lösung deuten auf eine stäbchenförmige Struktur für die eGFP-Oligomere. Diese Annahme beruht auf den gemessenen Diffusionskoeffizienten und theoretischen Rechnungen für die Diffusion zweier möglicher Geometriemodelle. Schließlich werden erstmalig Karten der Diffusion von Proteinen in Nuklei lebender Zellen, sowie von gesamten lebenden Zellen vorgestellt. Diese Kartierung unterstützt die Ergebnisse der Einzelpunktmessungen: die diffusive Mobilität von kleinen eGFP Proteinen zeigt starke Variationen in einem gegebenen Zellkern, jedoch ohne Korrelation zur Chromatindichte

The ventral habenulae of zebrafish develop in prosomere 2 dependent on Tcf7l2 function

BACKGROUND: The conserved habenular neural circuit relays cognitive information from the forebrain into the ventral mid- and hindbrain. In zebrafish, the bilaterally formed habenulae in the dorsal diencephalon are made up of the asymmetric dorsal and symmetric ventral habenular nuclei, which are homologous to the medial and lateral nuclei respectively, in mammals. These structures have been implicated in various behaviors related to the serotonergic/dopaminergic neurotransmitter system. The dorsal habenulae develop adjacent to the medially positioned pineal complex. Their precursors differentiate into two main neuronal subpopulations which differ in size across brain hemispheres as signals from left-sided parapineal cells influence their differentiation program. Unlike the dorsal habenulae and despite their importance, the ventral habenulae have been poorly studied. It is not known which genetic programs underlie their development and why they are formed symmetrically, unlike the dorsal habenulae. A main reason for this lack of knowledge is that the vHb origin has remained elusive to date. RESULTS: To address these questions, we applied long-term 2-photon microscopy time-lapse analysis of habenular neural circuit development combined with depth color coding in a transgenic line, labeling all main components of the network. Additional laser ablations and cell tracking experiments using the photoconvertible PSmOrange system in GFP transgenic fish show that the ventral habenulae develop in prosomere 2, posterior and lateral to the dorsal habenulae in the dorsal thalamus. Mutant analysis demonstrates that the ventral habenular nuclei only develop in the presence of functional Tcf7l2, a downstream modulator of the Wnt signaling cascade. Consistently, photoconverted thalamic tcf7l2(exl/exl) mutant cells do not contribute to habenula formation. CONCLUSIONS: We show in vivo that dorsal and ventral habenulae develop in different regions of prosomere 2. In the process of ventral habenula formation, functional tcf7l2 gene activity is required and in its absence, ventral habenular neurons do not develop. Influenced by signals from parapineal cells, dorsal habenular neurons differentiate at a time at which ventral habenular cells are still on their way towards their final destination. Thus, our finding may provide a simple explanation as to why only neuronal populations of the dorsal habenulae differ in size across brain hemispheres

Decomposing degenerate graphs into locally irregular subgraphs

International audienceA (undirected) graph is locally irregular if no two of its adjacent vertices have the same degree. A decomposition of a graph G into k locally irregular subgraphs is a partition E_1,...,E_k of E(G) into k parts each of which induces a locally irregular subgraph. Not all graphs decompose into locally irregular subgraphs; however, it was conjectured that, whenever a graph does, it should admit such a decomposition into at most three locally irregular subgraphs. This conjecture was verified for a few graph classes in recent years.This work is dedicated to the decomposability of degenerate graphs with low degeneracy. Our main result is that decomposable k-degenerate graphs decompose into at most 3k+1 locally irregular subgraphs, which improves on previous results whenever k≤9. We improve this result further for some specific classes of degenerate graphs, such as bipartite cacti, k-trees, and planar graphs. Although our results provide only little progress towards the leading conjecture above, the main contribution of this work is rather the decomposition schemes and methods we introduce to prove these results

Habenula Circuit Development: Past, Present, and Future

The habenular neural circuit is attracting increasing attention from researchers in fields as diverse as neuroscience, medicine, behavior, development, and evolution. Recent studies have revealed that this part of the limbic system in the dorsal diencephalon is involved in reward, addiction, and other behaviors and its impairment is associated with various neurological conditions and diseases. Since the initial description of the dorsal diencephalic conduction system (DDC) with the habenulae in its center at the end of the nineteenth century, increasingly sophisticated techniques have resolved much of its anatomy and have shown that these pathways relay information from different parts of the forebrain to the tegmentum, midbrain, and hindbrain. The first part of this review gives a brief historical overview on how the improving experimental approaches have allowed the stepwise uncovering much of the architecture of the habenula circuit as we know it today. Our brain distributes tasks differentially between left and right and it has become a paradigm that this functional lateralization is a universal feature of vertebrates. Moreover, task dependent differential brain activities have been linked to anatomical differences across the left–right axis in humans. A good way to further explore this fundamental issue will be to study the functional consequences of subtle changes in neural network formation, which requires that we fully understand DDC system development. As the habenular circuit is evolutionarily highly conserved, researchers have the option to perform such difficult experiments in more experimentally amenable vertebrate systems. Indeed, research in the last decade has shown that the zebrafish is well suited for the study of DDC system development and the phenomenon of functional lateralization. We will critically discuss the advantages of the zebrafish model, available techniques, and others that are needed to fully understand habenular circuit development

Mapping eGFP Oligomer Mobility in Living Cell Nuclei

Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers

Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications

Protein Diffusion in Mammalian Cell Cytoplasm

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS