Live cell dishes

Live cell microscopy of RH Tir1-3xFLAG BCC4-mAID-3xMyc parasites expressing YFP-MORN1 was done using a Zeiss LSM880 with Airyscan unit in the Boston College Imaging Core in consultation with Bret Judson. Parasite dynamics were recorded using the “Airyscan fast” settings, in an incubation chamber set to 37 °C. Parasites were grown overnight under standard culture conditions in 3 ml live cell dishes (MatTek). On the next day, culture medium was replaced with live cell imaging medium (DMEM without phenol red, 20 mM HEPES pH 7.4, 1% FBS, Penicillin/Streptomycin, and Fungizone). To induce protein degradation, parasites were treated with 500 µM IAA (in 100% ethanol) 2 hrs before imaging started. The resulting data were deconvolved using standard Airyscan settings and movies processed with FIJI software.

hTERT-RPE-1 cells were grown on live-cell dishes (0.17-mm-thick coverglass; MatTek) and transiently transfected with Cep126-GFP using Lipofectamine 2000 (Life Technologies, Paisley, UK), according to the manufacturer instructions. Twenty-four hours after transfection, the cells were imaged using a Perkin Elmer UltraVIEW ERS 6FE confocal system spinning disk confocal microscope. Imaging and photobleaching were carried out using a 14 mW argon laser at 488 nm, with a Photokinesis device for region-of-interest photobleaching. Live cells were imaged in DMEM without phenol red, supplemented with 0.1 g/l sodium carbonate and 30 mM HEPES, pH 7.4. Frames were acquired at 1-s intervals. The dynamics of photobleaching were determined by fitting curves to a single exponential using GraphPad 4.02 (Prism). The data in Figure3C were pseudocoloured according to time point using a Temporal-Color Coder ImageJ plugin developed by Kota Miura (EMBL) and available within the Fiji implementation of ImageJ.