Computational protocol: Kinetochore-localized BUB-1/BUB-3 complex promotes anaphase onset in C. elegans

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Protocol publication

[…] For imaging of one- and two-cell embryos, hermaphrodite adult worms were dissected into M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl, and 1 mM MgSO4•7H2O), and embryos were transferred to 2% agarose pads positioned on a microscope slide and covered with an 18 × 18 nm coverslip.Imaging of strains expressing GFP::H2B;γ-tubulin::GFP was performed on a deconvolution microscope (DeltaVision; Applied Precision/GE Healthcare) controlled by a softWoRx workstation (DeltaVision; Applied Precision/GE Healthcare) equipped with a charge-coupled device camera (CoolSNAP; Roper Scientific) with 5 × 2 µm z stacks, 2 × 2 binning, and a 60× 1.3 NA U-Plan-Apochromat objective lens (Olympus) at 10-s intervals and 100-ms exposure at 18°C. Acquired sequences were processed and analyzed using ImageJ (Fiji) and MetaMorph software (Molecular Devices). Pole tracking was performed by clicking on the center of each spindle pole and measuring the distance between them from NEBD onwards. NEBD was scored as the frame where free histone signal in the nucleus equilibrates with the cytoplasm, which is just before abrupt chromosome movement starts. Anaphase onset was scored as the first frame with visible separation of sister chromatids. Lagging chromatin was scored as visible threads of GFP::H2b signal between separating chromatid masses.For all other strains, images were acquired on a spinning disc confocal system (Revolution XD Confocal System; Andor Technology) controlled by iQ software (Andor Technology) and a spinning disk confocal scanner unit (CSU-10; Yokogawa Electric Corporation) mounted on an inverted microscope (TE2000-E; Nikon) equipped with 100× or 60× 1.4 NA Plan-Apochromat lenses, and outfitted with an electron multiplication back-thinned charged-coupled device camera (iXon; Andor Technology) at 20°C.To monitor BUB-1::GFP localization in the KNL-1::mCh mutants, a 5 × 2-µm z series, with 1 × 1 binning, was acquired every 20 s, with 200-ms and 300-ms exposure times for GFP and mCherry, respectively. For the separase sensor assay, mCh::H2B was monitored by acquiring 5 × 2 µm z-series, with 2 × 2 binning, every 20 s with 200-ms exposure time from NEBD to chromosome alignment. Then images for mCh::H2B and the GFP::Sensor were acquired as a 5 × 2-µm z series every 5 s with 200-ms exposure for GFP and for mCherry. To monitor GFP::HCP-1 localization, a 5 × 2-µm z series, with 1 × 1 binning, was acquired every 20 s, with 50-ms exposure for GFP and 300-ms exposure for mCherry. To monitor GFP::BUB-3 localization, a 5 × 2-µm z series, with 1 × 1 binning, was acquired every 20 s, with 200-ms exposure for GFP and 200-ms exposure for mCherry.To quantify fluorescence, sequences were analyzed using ImageJ (Fiji). Z stacks were projected, a rectangular box was drawn around the entire chromosome set for each frame, and the integrated intensity in the box was recorded. Then the box was expanded by 5 pixels on each side, and the integrated intensity was measured. The signal and area difference between the expanded box and the original box were used to calculate the average background signal per pixel. The integrated chromosomal GFP intensity in the original box was then calculated by subtracting the background signal.All p-values were calculated using unpaired t tests in GraphPad Prism (GraphPad Software). […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Organisms Caenorhabditis elegans