Fig. 2.

Sec4 localizes to a apical spot distinct from the crescent of the exocyst components. (A) A hyphal cell expressing GFP-Sec4 was imaged at 90 min after induction from a stationary-phase yeast culture. The left image shows the merge of the differential interference contrast (DIC) and GFP channels at low magnification, and the right image shows an enlargement of the tip, with the DIC false colored red to allow the GFP image to be clearly seen. Scale bars: left, 5 μm; right, 1 μm. (B) Montage showing the image from individual Z-stacks of GFP-Sec4 and Exo84-YFP. Images were taken with 0.2-μm vertical separation. Exposure and processing of both sets of images were identical. Scale bars, 1 μm. (C) Strains expressing Exo70-YFP and GFP-Sec4 were imaged using a Zeiss LSM Meta 510 laser scanning confocal microscope. GFP-Sec4 is false colored red in the merged image, in which the tip has been enlarged to facilitate visualization of the different pattern of localization. The 514-nm HFT laser was used to excite YFP, and the 488 HFT laser was used for GFP. The emission was gated to 500 to 530 nm for GFP images and 530 to 570 for YFP images. To test for bleedthrough, images were recorded using single YFP- and GFP-tagged strains using these gates. Bleedthrough of YFP signal with GFP settings was effectively excluded, but some weak fluorescence was apparent from GFP with YFP settings. However, this was so low compared to the genuine YFP signal that the localization of Exo70-YFP could be easily visualized and is different from that of GFP-Sec4. Scale bars: left and center, 1 μm; right, 0.5 μm.