Wei, Tee Chee
(2020)
Biochemical characterisation of dystrophin in sensory dendrite and epithelial cells of drosophila melanogaster.
Masters thesis, Universiti Sains Malaysia.
Abstract
Contact-mediated self-avoidance/repulsion restricts dendrites of sensory
neuron in a 2D space. Defect in dendrite-extracellular matrix (ECM) adhesion
disrupts the confinement and results in self-crossing of dendrites in a 3D space. In
addition, these complex and diverse patterns of sensory neurons innervate the
epidermis and muscle. However, the mechanisms governing dendrite patterning are
still poorly understood. The objectives of this study are to uncover and characterise a
novel gene in epithelial cells regulating dendritic morphology of sensory neurons in
Drosophila third instar larvae. First of all, the genetic background of selected
Drosophila mutant lines: adenomatous polyposis coli, dystroglycan and dystrophin
was standardized by backcrossing to the wild type strain and then identified by using
non-lethal PCR genotyping method from Drosophila wings. In this study, dystrophin
in epithelial cells was found underlying the dendritic morphology of sensory neurons
in Drosophila third instar larvae. Mutations or RNAi knockdown of dystrophin in
epidermal cells were led to an increase in dendritic self-crossing of sensory neurons.
In contrast, normal phenotype of dendrite morphology was exhibited upon RNAi
knockdown or transgenic expression of dystrophin in neurons. Surprisingly,
transgenic expression of dystrophin Dp186 in epidermal cells exacerbated its mutant
phenotype, instead of rescuing it. Analysis of dendrite-ECM adhesion with highresolution
confocal microscopy and fluorescent-labelled markers in third instar larva
corroborated that the reduction of dystrophin in epidermal cells promoted dendrites
to be detached from the ECM. Computational analysis predicted the majority of
Dystrophin isoforms contained microtubule-binding domain, which was responsible
for direct binding of microtubules. Immunofluorescence of DmD8 cells and larval
epidermal cells of Drosophila observed a colocalization of Dystrophin Dp186 and
microtubules, but devoid of microtubule-binding domain of Dystrophin Dp186 did
not associate with microtubules. Here, in vivo results were inconclusive due to
technical problems: inclusion body formation by overexpression of Dystrophin
Dp186 and the limited resolution power of confocal microscopy on microtubules.
However, in vitro assay confirmed that bacterial produced Dystrophin Dp186 binds
microtubules directly through its microtubule-binding domain. In future, further indepth
study is required to elucidate the role of Dystrophin in coordinating sensory
dendrite arborization.
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