Methods to Study Centrosomes and Cilia in Drosophila

The deposited item is a book chapter and is part of the series " Methods in Molecular Biology book series ([MIMB, volume 1454]) published by the publisher Humana Press.The deposited book chapter is a pre-print version and hasn't been submitted to peer reviewing.There is no public supplementary material available for this publication.This publication hasn't any creative commons license associated.Centrioles and cilia are highly conserved eukaryotic organelles. Drosophila melanogaster is a powerful genetic and cell biology model organism, extensively used to discover underlying mechanisms of centrosome and cilia biogenesis and function. Defects in centrosomes and cilia reduce fertility and affect different sensory functions, such as proprioception, olfaction, and hearing. The fly possesses a large diversity of ciliary structures and assembly modes, such as motile, immotile, and intraflagellar transport (IFT)-independent or IFT-dependent assembly. Moreover, all the diverse ciliated cells harbor centrioles at the base of the cilia, called basal bodies, making the fly an attractive model to better understand the biology of this organelle. This chapter describes protocols to visualize centrosomes and cilia by fluorescence and electron microscopy.Fundação Portuguesa para a Ciência e Tecnologia grants: (SFRH/BPD/87479/2012, SFRH/BD/52176/2013); EMBO installation grant; ERC starting grant.info:eu-repo/semantics/publishedVersio

Gorab is a Golgi protein required for structure and duplication of Drosophila centrioles.

We demonstrate that a Drosophila Golgi protein, Gorab, is present not only in the trans-Golgi but also in the centriole cartwheel where, complexed to Sas6, it is required for centriole duplication. In addition to centriole defects, flies lacking Gorab are uncoordinated due to defects in sensory cilia, which lose their nine-fold symmetry. We demonstrate the separation of centriole and Golgi functions of Drosophila Gorab in two ways: first, we have created Gorab variants that are unable to localize to trans-Golgi but can still rescue the centriole and cilia defects of gorab null flies; second, we show that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype overcome by mutations preventing Golgi targeting. Our findings suggest that during animal evolution, a Golgi protein has arisen with a second, apparently independent, role in centriole duplication.D.M.G. is grateful for a Wellcome Investigator Award, which supported this work. The study was initiated with support from Cancer Research UK

Regulation of Cilium Length and Intraflagellar Transport

Primary cilia are highly conserved sensory organelles that extend from the surface of almost all vertebrate cells. The importance of cilia is evident from their involvement in many diseases, called ciliopathies. Primary cilia contain a microtubular axoneme that is used as a railway for transport of both structural components and signaling proteins. This transport machinery is called intraflagellar transport (IFT). Cilia are dynamic organelles whose presence on the cell surface, morphology, length and function are highly regulated. It is clear that the IFT machinery plays an important role in this regulation. However, it is not clear how, for example environmental cues or cell fate decisions are relayed to modulate IFT and cilium morphology or function. This chapter presents an overview of molecules that have been shown to regulate cilium length and IFT. Several examples where signaling modulates IFT and cilium function are used to discuss the importance of these systems for the cell and for understanding of the etiology of ciliopathies

Dynamic magnetic resonance imaging: reliability of anatomical landmarks and reference lines used to assess pelvic organ prolapse.

Contains fulltext : 80868.pdf (publisher's version ) (Closed access)The aim of this study was to determine the intra- and interobserver reliability of dynamic magnetic resonance (MR) staging in pelvic organ prolapse patients. In 30 patients with pelvic organ prolapse, dynamic MR images were assessed independently by two observers. Various anatomical landmarks to asses pelvic organ prolapse were used in relation to the pubococcygeal line, H-line, and mid-pubic line. Clinical measurement points were assessed in relation to the mid-pubic line. The intraclass correlation coefficients (ICC) were calculated to describe the intra- and interobserver reliability. Overall, the intra- and interobserver reliability of MR imaging measurements was excellent to good. The pubococcygeal line showed superior reliability (ICC range 0.70-0.99). The reliability of clinical measurement points, however, were only moderate (ICC range 0.20-0.96). The intra- and interobserver reliability of quantitative prolapse staging on dynamic MR imaging were good to excellent. The pubococcygeal line appears the most reliable to use