Biochemical characterisation of dystrophin in sensory dendrite and epithelial cells of drosophila melanogaster
Year: 2020
Type: dissertation
Abstract: Contact-mediated self-avoidance/repulsion restricts dendrites of sensory \nneuron in a 2D space. Defect in dendrite-extracellular matrix (ECM) adhesion \ndisrupts the confinement and results in self-crossing of dendrites in a 3D space. In \naddition, these complex and diverse patterns of sensory neurons innervate the \nepidermis and muscle. However, the mechanisms governing dendrite patterning are \nstill poorly understood. The objectives of this study are to uncover and characterise a \nnovel gene in epithelial cells regulating dendritic morphology of sensory neurons in \nDrosophila third instar larvae. First of all, the genetic background of selected \nDrosophila mutant lines: adenomatous polyposis coli, dystroglycan and dystrophin \nwas standardized by backcrossing to the wild type strain and then identified by using \nnon-lethal PCR genotyping method from Drosophila wings. In this study, dystrophin \nin epithelial cells was found underlying the dendritic morphology of sensory neurons \nin Drosophila third instar larvae. Mutations or RNAi knockdown of dystrophin in \nepidermal cells were led to an increase in dendritic self-crossing of sensory neurons. \nIn contrast, normal phenotype of dendrite morphology was exhibited upon RNAi \nknockdown or transgenic expression of dystrophin in neurons. Surprisingly, \ntransgenic expression of dystrophin Dp186 in epidermal cells exacerbated its mutant \nphenotype, instead of rescuing it. Analysis of dendrite-ECM adhesion with highresolution \nconfocal microscopy and fluorescent-labelled markers in third instar larva \ncorroborated that the reduction of dystrophin in epidermal cells promoted dendrites \nto be detached from the ECM. Computational analysis predicted the majority of \nDystrophin isoforms contained microtubule-binding domain, which was responsible \nfor direct binding of microtubules. Immunofluorescence of DmD8 cells and larval \nepidermal cells of Drosophila observed a colocalization of Dystrophin Dp186 and \nmicrotubules, but devoid of microtubule-binding domain of Dystrophin Dp186 did \nnot associate with microtubules. Here, in vivo results were inconclusive due to \ntechnical problems: inclusion body formation by overexpression of Dystrophin \nDp186 and the limited resolution power of confocal microscopy on microtubules. \nHowever, in vitro assay confirmed that bacterial produced Dystrophin Dp186 binds \nmicrotubules directly through its microtubule-binding domain. In future, further indepth \nstudy is required to elucidate the role of Dystrophin in coordinating sensory \ndendrite arborization.
Author: Tee Chee Wei
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3183771828