Localization of Alexa Fluor-594-α factor labeled endosomes (red; center) and Abp1-GFP labeled endocytic patches (green; left) in an arp3-D11A cell. Although the patches do not move off the plasma membrane in this mutant, the boxed endosome appears to move to the patches, which appear to fuse with the endosome as it contacts them. Total internal reflection fluorescence (TIRF) microscopy was used to visualize A488-α factor to reduce background and Abp1-DsRed was visualized by epifluorescence. Interval between frames is 6.5 s. Fig 2E (single frames) and Movie 6 from Toshima et al.
S. cerevisiae (Mata his3-Δ200 leu2-3,112 ura3-52 lys2-801 bar1Δ::URA3 arp3-Δ11A:: Leu2 ABP1-GFP::HIS3) were grown to an OD600 of 0.2 in 1.25 ml of YPD, briefly centrifuged, and resuspended in 50 μl of synthetic media (SM) with 1% (wt/vol) BSA and 5 μM Alexa-α-factor. After incubation on ice for 2 h, cells were washed into ice-cold SM containing 1% BSA. Internalization was initiated by the addition of ice-cold SM containing 4% Glucose and amino acids and then transferring cells to a glass slide at room temperature. Simultaneous imaging of red and green fluorescence was performed by using an Olympus IX81 microscope equipped with a ×100/NA 1.45 (Olympus) objective, Orca-ER cooled CCD camera (Hamamatsu), and an image splitter (Dual-View; Optical Insights) that divided the red and green components of the images with a 565-nm dichroic mirror and passed the red component through a 630/50-nm filter and the green component through a 530/30-nm filter.
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