Gebiss: an ImageJ plugin for the specification of ground truth and the performance evaluation of 3D segmentation algorithms.

Background: Image segmentation is a crucial step in quantitative microscopy that helps to define regions of tissues, cells or subcellular compartments. Depending on the degree of user interactions, segmentation methods can be divided into manual, automated or semi-automated approaches. 3D image stacks usually require automated methods due to their large number of optical sections. However, certain applications benefit from manual or semi-automated approaches. Scenarios include the quantification of 3D images with poor signal-to-noise ratios or the generation of so-called ground truth segmentations that are used to evaluate the accuracy of automated segmentation methods. Results: We have developed Gebiss; an ImageJ plugin for the interactive segmentation, visualisation and quantification of 3D microscopic image stacks. We integrated a variety of existing plugins for threshold-based segmentation and volume visualisation. Conclusions: We demonstrate the application of Gebiss to the segmentation of nuclei in live Drosophila embryos and the quantification of neurodegeneration in Drosophila larval brains. Gebiss was developed as a cross-platform ImageJ plugin and is freely available on the web at http://imaging.bii.a-star.edu.sg/projects/gebiss

The study of Drosophila melanogaster blue cheese (bchs) function in the involvement of autophagy in neurodegenerative disorders

Neurodegenerative disorders are becoming an increasing concern in today's aging society whereby cognitive, postural and memory abilities are impaired in sporadic and familial forms of the disorders. Extensive research over the years has generated insights into the pathophysiological mechanisms of proteins implicated in the etiology of these diseases. A common pathological hallmark is the accumulation of cytotoxic protein aggregates in the brain that accompanies neuronal dysfunction and atrophy. The intracellular protein quality control system consists of three main components: chaperone-mediated folding, the ubiquitin-proteasome system, and autophagy. In this study, it is shown that autophagy function is impaired in Drosophila melanogaster neurodegenerative mutants in the gene blue cheese (bchs), and conversely that augmenting autophagy is sufficient to rescue the degeneration. We show this by rescuing neuronal atrophy through rapamycin feeding and atg7 over-expression. Rapamycin feeding also alleviates the accumulation of ubiquitinated aggregates in neuronal termini of bchs motor neurons. Autophagy inhibitors wortmannin and 3-methyladenine in contrast exacerbate neuronal death in bchs mutants. In the absence of Bchs protein, AtgS-positive and Atg8-positive autophagic vacuoles are not able to be recruited to ubiquitinated aggregates. Colocalization analysis and live imaging on primary neurons show that Bchs dissociates from Rabll and associates with AtgS during Huntingtin Q93 expression. However, there is no effect on the association of Bchs with Atg8 or Spinster, a late endolysosomal marker, in response to the induction of aggrephagy. These findings suggest that Bchs is involved in the early steps of autophagosome formation and recognition of target substrates, in a similar manner as the human homologue Autophagy Linked FYVE (ALFY) protein. Bchs is unlikely to be a motor protein adaptor mediating cargo assembly due to the absence of interaction with dynein or kinesin compared to APP-Iike interacting protein 1 (Aplipl). Therefore, this study demonstrates that Bchs functions as an adaptor bridging the autophagy machinery to aggregate-prone proteins using a neurodegenerative disease model in Drosophila melanogaster.DOCTOR OF PHILOSOPHY (SBS