CiteULike: jimmithy imaging

Analysis of the hypoxia-sensing pathway in Drosophila melanogaster.

N Arquier — 2006-10-23T20:07:10-00:00

Biochem J, Vol. 393, No. Pt 2. (15 January 2006), pp. 471-480.

The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1alpha (hypoxia-inducible factor-1 alpha subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1alpha to the ubiquitin/proteasome pathway. HIF-1alpha thus accumulates and promotes gene transcription. In the present study, first we provide direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1alpha and Similar, the Drosophila homologue of HIF-1alpha. The enzyme promotes human and Drosophila [(35)S]VHL binding to GST (glutathione S-transferase)-ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD-GFP), we analysed the hypoxic cascade in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD-GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia.

Progress in Functional Neuroanatomy: Precise Automatic Geometric Reconstruction of Neuronal Morphology From Confocal Image Stacks

JF Evers — 2006-10-23T20:01:34-00:00

J Neurophysiol, Vol. 93, No. 4. (1 April 2005), pp. 2331-2342.

Dendritic architecture provides the structural substrate for myriads of input and output synapses in the brain and for the integration of presynaptic inputs. Understanding mechanisms of evolution and development of neuronal shape and its respective function is thus a formidable problem in neuroscience. A fundamental prerequisite for finding answers is a precise quantitative analysis of neuronal structure in situ and in vivo. Therefore we have developed a tool set for automatic geometric reconstruction of neuronal architecture from stacks of confocal images. It provides exact midlines, diameters, surfaces, volumes, and branch point locations and allows analysis of labeled molecule distribution along neuronal surfaces as well as direct export into modeling software. We show the high accuracy of geometric reconstruction and the analysis of putative input synapse distribution throughout entire dendritic trees from in situ light microscopy preparations as a possible application. The binary version of the reconstruction module is downloadable at no cost. 10.1152/jn.00761.2004

In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses.

W Supatto — 2005-01-27T03:55:46-00:00

Proc Natl Acad Sci U S A, Vol. 102, No. 4. (25 January 2005), pp. 1047-1052.

The complex biomechanical events associated with embryo development are investigated in vivo, by using femtosecond laser pulse-induced ablation combined with multimodal nonlinear microscopy. We demonstrate controlled intravital ablations preserving local cytoskeleton dynamics and resulting in the modulation of specific morphogenetic movements in nonmutant Drosophila embryos. A quantitative description of complex movements is obtained both in GFP-expressing systems by using whole-embryo two-photon microscopy and in unlabeled nontransgenic embryos by using third harmonic generation microscopy. This methodology provides insight into the issue of mechano-sensitive gene expression by revealing the correlation of in vivo tissue deformation patterns with Twist protein expression in stomodeal cells at gastrulation.

Ultrastructure of Insect and Spider Cocoon Silks.

Osnat Hakimi — 2006-10-23T19:50:27-00:00

Biomacromolecules, Vol. 7, No. 10. (9 October 2006), pp. 2901-2908.

Despite much interest in the extraordinary mechanical properties of silks, the structure of native silk fibers is still not fully understood. In the present study, the morphology, topography, and organization of insect and spider cocoon silks were investigated using a range of imaging methods. Field emission scanning electron microscopy was used to observe transverse and longitude structures in silk fibers subjected to tensile fracturing, freeze fracturing, or polishing. In addition, ultrathin sections of silk brins embedded in resin were examined using transmission electron microscopy. Finally, dry silk brins were examined by confocal microscopy. The results confirmed the existence of well-oriented bundles of nanofibrils in all the silks examined and gave an indication of a hierarchical construction of the brin. Observed separation of the microfibrils in fractured brins suggests that the multifibrillar structure of the silk fiber contributes to toughness by allowing dissipation of energy in the controlled propagation of cracks.

Whole insect and mammalian embryo imaging with confocal microscopy: Morphology and apoptosis.

Robert M Zucker — 2006-10-23T19:47:36-00:00

Cytometry A, Vol. 69A, No. 11. (18 October 2006), pp. 1143-1152.

BACKGROUND:: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, which both correlate to apoptosis activity. LT has been shown to be an indicator of apoptotic cell death which is correlated to other standard apoptotic assays. METHODS:: The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Mosquitoes were fixed with MEOH and stained with propidium iodide. Next the tissues were dehydrated with MEOH and cleared with BABB. RESULTS:: Tissues as thick as 500 mum can be visualized after clearing with BABB. LT staining revealed apoptotic regions in mammalian limbs, fetuses, and embryos. Morphological observation of insect tissue consisted of combining autofluorescence with either nucleic acid staining (either propidium iodide or ethidium bromide). CONCLUSIONS:: The use of BABB matches the RI of the tissue within the suspending medium. It helps in increasing the penetration of laser light in a confocal microscope by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissues. LT is a probe that stains the acid regions of tissues and cells and has been correlated to apoptosis. Morphological features of a tissue or organism (embryo, mosquito larvae) can be elucidated by fixation aldehydes, autofluorescence, and red-emitting probes. This sample preparation procedure with optimization of confocal laser scanning microscopy allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were approximately 500-mum thick. (c) 2006 International Society for Analytical Cytology.