Timelapse movie of GFP-labeled microtubules in kar9delta cells (arrested in S-phase) combined with a deletion of the CAP-Gly domain of Nip100. The number of cells in which the spindle moved from the mother cell compartment through the neck was decreased in the CAP-Gly mutant. However, in CAP-Gly mutant cells where the spindle did move through the neck, the frequency of transits of the spindle, back-and-forth through the neck was similar to wild-type cells (compare with Movies S1 and S3, CIL#s 23599, 23604). Movie plays at 50x real time and is Movie S4 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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