Comparison of Multiscale Imaging Methods for Brain Research

A major challenge in neuroscience is how to study structural alterations in the brain. Even small changes in synaptic composition could have severe outcomes for body functions. Many neuropathological diseases are attributable to disorganization of particular synaptic proteins. Yet, to detect and comprehensively describe and evaluate such often rather subtle deviations from the normal physiological status in a detailed and quantitative manner is very challenging. Here, we have compared side-by-side several commercially available light microscopes for their suitability in visualizing synaptic components in larger parts of the brain at low resolution, at extended resolution as well as at super-resolution. Microscopic technologies included stereo, widefield, deconvolution, confocal, and super-resolution set-ups. We also analyzed the impact of adaptive optics, a motorized objective correction collar and CUDA graphics card technology on imaging quality and acquisition speed. Our observations evaluate a basic set of techniques, which allow for multi-color brain imaging from centimeter to nanometer scales. The comparative multi-modal strategy we established can be used as a guide for researchers to select the most appropriate light microscopy method in addressing specific questions in brain research, and we also give insights into recent developments such as optical aberration corrections

TRIP6 functions in brain ciliogenesis

TRIP6, a member of the ZYXIN-family of LIM domain proteins, is a focal adhesion compo- nent. Trip6 deletion in the mouse, reported here, reveals a function in the brain: ependymal and choroid plexus epithelial cells are carrying, unexpectedly, fewer and shorter cilia, are poorly differentiated, and the mice develop hydrocephalus. TRIP6 carries numerous protein interaction domains and its functions require homodimerization. Indeed, TRIP6 disruption in vitro (in a choroid plexus epithelial cell line), via RNAi or inhibition of its homodimerization, confirms its function in ciliogenesis. Using super-resolution microscopy, we demonstrate TRIP6 localization at the pericentriolar material and along the ciliary axoneme. The requirement for homodimerization which doubles its interaction sites, its punctate localiza- tion along the axoneme, and its co-localization with other cilia components suggest a scaf- fold/co-transporter function for TRIP6 in cilia. Thus, this work uncovers an essential role of a LIM-domain protein assembly factor in mammalian ciliogenesis

Composition and Hierarchical Organisation of a Spider Silk

Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies

The promyelocytic leukemia (pml) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology

Spatial Association of Homologous Pericentric Regions in Human Lymphocyte Nuclei during Repair

ABSTRACT Spatial positioning of pericentric chromosome regions in human lymphocyte cell nuclei was investigated during repair after H 2 O 2 /L-histidine treatment. Fifteen to three-hundred minutes after treatment, these regions of chromosomes 1, 15, and X were labeled by fluorescence in situ hybridization. The relative locus distances (LL-distances), the relative distances to the nuclear center (LC-distances), and the locus-nuclear center-locus angles (LCL-angles) were measured in ;5000 nuclei after two-dimensional microscopy. Experimental frequency histograms were compared to control data from untreated stimulated and quiescent (G 0 ) nuclei and to a theoretical two-dimensional projection from random points. Based on the frequency distributions of the LL-distances and the LCL-angles, an increase of closely associated labeled regions was found shortly after repair activation. For longer repair times this effect decreased. After 300 min the frequency distribution of the LL-distances was found to be compatible with the random distance distribution again. The LL-distance frequency histograms for quiescent nuclei did not significantly differ from the theoretical random distribution, although this was the case for the stimulated control of chromosomes 15 and X. It may be inferred that, concerning the distances, homologous pericentric regions appear not to be randomly distributed during S-phase, and are subjected to dynamic processes during replication and repair

Neuronal ROS signaling rather than AMPK/sirtuin-mediated energy sensing links dietary restriction to lifespan extension

Dietary restriction (DR) extends lifespan and promotes metabolic health in evolutionary distinct species. DR is widely believed to promote longevity by causing an energy deficit leading to increased mitochondrial respiration. We here show that inhibitors of mitochondrial complex I promote physical activity, stress resistance as well as lifespan of Caenorhabditis elegans despite normal food uptake, i.e. in the absence of DR. However, complex I inhibition does not further extend lifespan in dietarily restricted nematodes, indicating that impaired complex I activity mimics DR. Promotion of longevity due to complex I inhibition occurs independently of known energy sensors, including DAF-16/FoxO, as well as AAK-2/AMPK and SIR-2.1/sirtuins, or both. Consistent with the concept of mitohormesis, complex I inhibition transiently increases mitochondrial formation of reactive oxygen species (ROS) that activate PMK-1/p38 MAP kinase and SKN-1/NRF-2. Interference with this retrograde redox signal as well as ablation of two redox-sensitive neurons in the head of the worm similarly prevents extension of lifespan. These findings unexpectedly indicate that DR extends organismal lifespan through transient neuronal ROS signaling rather than sensing of energy depletion, providing unexpected pharmacological options to promote exercise capacity and healthspan despite unaltered eating habits

Biochemical composition of outer layers.

<p>Panel A. Oil red staining of fibres. Silk fibres were treated with water (upper filament) or ether extracted (lower filament) followed by oil red staining. Bar corresponds to 2000 nm. Panel B. Concavalin A (Con A) staining of fibres. Fibres gently washed in phosphate buffered saline or vigorously washed in 0.1% Triton X-100 were reacted with biotinylated Con A. The presence of α-methyl mannoside (α-MM), an inhibitor of Con A, is indicated by “I”. Bound Con A was visualised by gold conjugated streptavidin and SEM. The bar equals 500 nm.</p

Solubilisation of polymerised spidroins.

<p>Dragline silk from <i>N. clavipes</i> was incubated with the indicated solvents and the soluble fraction was analysed by gel-electrophoresis followed by Coomassie staining (A) and western blotting employing antibodies S1Rx and enhanced luminescence (ECL) for detection. The molecular weights of marker proteins are indicated in kilo Daltons (kDa).</p

Protein composition of silk layers.

<p>Filter strips obtained by western blotting and loaded with material extracted from the indicated fibre layers were stained with Ponceau S (Pon) or reacted with Concavalin A (ConA), pre-immune serum (PIS), S1Rx, S2Rx and S-pbs. The running position of a 200 kDa marker is indicated by the lines.</p