Computational protocol: Lrrk regulates the dynamic profile of dendritic Golgi outposts through the golgin Lava lamp

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Protocol publication

[…] Live imaging was conducted with modification of a previously described procedure (). Early third-instar larvae were mounted with double-sided tape between two coverslips to be incorporated into an imaging chamber for anesthetization with a short pulse of desflurane (Suprane; Boxter) that sedates larvae for 10 min (). Immersol W (Carl Zeiss) fills between coverslip and lens for imaging at room temperature. Images were acquired via confocal microscope (LSM710 inverted; Carl Zeiss) under a 40× objective lens (NA 1.2, C-Apochromat 40×/1.2 W Corr M27; Carl Zeiss) through the software Zen 2010 (Carl Zeiss). Series of images for ManII-GFP in distal dendritic regions of class IV da neurons were taken with 20-s intervals within 8–10 min. Dynamic ManII-GFP puncta in consecutive images were tracked down by the segmented line tool, and punctum displacement was quantified by ImageJ (version 1.48; National Institutes of Health). In a given field of successive images, ManII-GFP puncta that moved <0.3 µm were considered as stationary ones. Videos of ManII-GFP and YFP-Lrrk were generated by the straighten tool in ImageJ for presentation. YFP-Lrrk and other subcellular compartments such as Rab4-mRFP, GFP-Rab5, mCherry-Rab7, GFP-LAMP, or mito-GFP puncta in da neurons of anesthetized third-instar larvae were imaged with the same mounting procedure and imaging setup. Live imaging of mCD8-GFP–marked dendrites and YFP-Lrrk puncta in awakening third-instar larvae was performed by confocal microscopy (LSM710) with an image captured every 3 to 5 min. Data analysis and statistics were performed via Excel (Microsoft), SigmaPlot (ver. 11.0; Systat Software Inc.), and Matlab (MathWorks) software. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Drosophila melanogaster, Homo sapiens
Diseases Parkinson Disease