Microtubule detachment from the spindle pole body in kar9delta nip100deltaCAP-gly double mutant cells arrested in S phase. Movie plays at 20x real time and is movie S8 in Proc Natl Acad Sci. 2009. 106: 5147-5152.
S. cerevisiae (MATa NIP100deltaCAP-Gly DYN1-3GFP::TRP1 kar9delta::hygB ura3-52 lys2-801 leu2-delta1::GFP-TUB1::LEU2 his3-delta200 trp1-delta63) expressing GFP-tubulin (GFP-TUB1) and dynein heavy chain tagged with 3GFP (DYN1-3GFP) were arrested in hydroxyurea and mounted on agarose pads for microscopy. Images were captured on an Olympus Bmax-60F microscope equipped with a 1.35NA 100× UPlanApo objective, spinning disc Confocal Scanner Unit (CSU10), Picarro Cyan laser (488 nm), and a Stanford Photonics XR-Mega10 ICCD camera, by using QED software (Media Cybernetics). Image analysis was performed by using ImageJ. Timelapse images were captured at 10-s intervals for 15 min and each image in the movie represents a composite of 9 planes separated by 500 nm.
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