Nuclear envelope and spindle dynamics when the nucleus moves into the neck of kar9delta mutant cells arrested in S phase. Movie plays at 100x real time and is movie S6 in Proc Natl Acad Sci. 2009. 106: 5147-5152.
S. cerevisiae (MATalpha NUP133-tdimer2::k.l.URA3 kar9delta::hygB ura3-52 lys2-801 leu2-delta1::GFP-TUB1::LEU2 his3-delta200 trp1-delta63) expressing GFP-tubulin (GFP-TUB1, to mark spindle and microtubules, top panel, green in bottom panel merge) and RFP-NUP133 (to mark nuclear envelope, middle panel, red in bottom panel merge) 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 Cobolt Jive laser (561 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 20-s intervals for 10 min and each image in the movie represents a composite of 9 planes separated by 500 nm.
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