No abstract

The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression.

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis 1 , and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation 2-5 and degeneration 6 .The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron 7,8 . Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape 4,9-11 . We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13 .In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea ( Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape. Video LinkThe video component of this article can be found at http://www.jove.com/video/3662/ Protocol Preparation of reagentsNotes before beginning: Dissection and immunohistochemical staining are carried out in a magnetic chamber and the larva is pinned down using specially shaped insect pins. Detailed instructions on the construction of a magnetic chamber, and preparation of these pins can be found in related references 14,15 . In brief, a 1x1cm square hole is cut into a magnetic sheet and a coverslip affixed to the rear of the sheet to make a small chamber. The sides of the chamber are sealed with epoxy glue; after this glue has set the chamber is washed several times with 70% ethanol before use. Dissection insect pins are prepared by bending to the required shape and then glued onto a metal tab 14,15 . Alternatively to a metal tab, we have used an inverted flat-head steel drawing pin with a handle made from a cut-off yellow tip. Use of this magnetic chamber arrangement allows close control over pin positioning and tissue stretching during dissection.

Huntington's disease is an autosomal dominant neurodegenerative disorder that is caused by abnormal expansion of a polyglutamine tract in the huntingtin protein, resulting in intracellular aggregate formation and neurodegeneration. How neuronal cells are affected by such a polyglutamine tract expansion remains obscure. To dissect the ways in which polyglutamine expansion can cause neural dysfunction, the authors generated Drosophila transgenic strains expressing either a nuclear targeted or cytoplasmic form of pathogenic (NHtt-152Q(NLS), NHtt-152Q), or nonpathogenic (NHtt-18Q(NLS), NHtt-18Q) N-terminal human huntingtin. These proteins were expressed in the dendritic arborization neurons of the larval peripheral nervous system and their effects on neuronal survival, morphology, and larval locomotion were examined. The authors found that NHtt-152Q(NLS) larvae had altered dendrite morphology and larval locomotion, whereas NHtt-152Q, NHtt-18Q(NLS), and NHtt-18Q larvae did not. Furthermore, the authors examined the physiological defect underlying this disrupted larval locomotion in detail by recording spontaneous ongoing segmental nerve activity. NHtt-152Q(NLS) larvae displayed uncoordinated activity between anterior and posterior segments. Moreover, anterior segments had shorter bursts and longer interburst intervals in NHtt-152Q(NLS) larvae than in NHtt-18Q(NLS) larvae, whereas posterior segments had longer bursts and shorter interburst intervals. These results suggest that the pathogenic protein disrupts neuron function without inducing cell death, and describe how this dysfunction leads to a locomotor defect. These results also suggest that sensory inputs are necessary for the coordination of anterior and posterior body parts during locomotion. From these analyses the authors show that examination of motor behaviors in the Drosophila larvae is a powerful new model to dissect non-cell-lethal mechanisms of mutant Htt toxicity.