Description

Kre2-GFP is not efficiently maintained in the Golgi apparatus when Pik1-mediated PtdIns4P synthesis (the Golgi-localized PtdIns 4-kinase in yeast is composed of the Pik1 catalytic subunit and a myristoylated calcium-binding protein, Frq1) ceases, implicating Pik1 signaling in retrograde trafficking of Golgi residents. In this image, Kre2-GFP is expressed in the isogenic control strain for the frq1-1 mutant, shifted to restrictive temperature of 37C for 1h. Cells grown in liquid medium were mounted in growth medium and 3D image stacks were collected at 0.4-µm z increments on a DeltaVision workstation (Applied Precision) based on an inverted microscope (IX-70; Olympus) using a 100× NA 1.4 oil immersion lens. Images were captured at 23C with a 12-bit CCD camera (CoolSnap HQ; Photometrics) and deconvolved using the iterative-constrained algorithm (Agard, 1984) and the measured point spread function. One image from the approximate center of z stack is shown in Fig4D 37C FRQ1 panel in J Cell Biol. 187: 967-975. 2009. Images in Fig 4D include CIL#s 13460, 13461, 13462, 13463, 13464, 13465, 13466, 13467.