Technical Reference #1913

1913.

Targeted localization of Inn1; Cyk3 and Chs2 by the mitotic-exit network regulates cytokinesis in budding yeast Franz Meitinger; Boryana Petrova; Ilde Mancini Lombardi; Daniela Trinca Bertazzi; Birgit Hub; Hanswalter Zentgraf and Gislene Pereira, DKFZ-ZMBH Alliance, Journal of Cell Science, 123(1913), (2010)
Link To Paper

Keywords:
Cyk3; Inn1; Budding yeast; Cytokinesis; Mitotic-exit network

Materials & Methods:
Microscopic techniques- For fluorescence microscopy 1.3 l of each cell culture was spotted onto microscope slides and immediately inspected. Cells were fixed with 70% ethanol and resuspended in PBS containing 1 g/ml 4 6-diamino-2-phenylindole (DAPI Sigma) whenever DNA visualization was needed. Cells carrying GFP or Cherry fusion proteins were fixed in 4% formaldehyde for 10-30 minutes before inspection. For actin staining 1 volume of 3.7% formaldehyde was immediately added to 10 volumes of cell culture. After an incubation of 10 minutes cells were resuspended in fresh 3.7% formaldehyde solution in PBS and incubated for 60 minutes. Actin was stained with 2 M Rhodamine-phalloidin (Invitrogen). For live-cell imaging cells were adhered onto glass-bottomed Petri dishes (MatTek) using concanavalin A-Type IV (Sigma) as described (Caydasi and Pereira 2009). A Zeiss Axiophot microscope equipped with a 100 NA 1.45 A Plan-Fluor oil-immersion objective (Zeiss) a Cascade 1K CCD camera (Photometrics) and MetaMorph software (Universal Imaging Corp.) was used for all experiments. Quantification of fluorescence intensity was performed using ImageJ software (NIH Bethesda). The mean pixel intensities of bud-neck signals were determined using the plane with the strongest signal. Background signals were measured in the near proximity of the bud neck and subtracted from bud-neck measurements.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
Yeast cells