Localization of Alexa Fluor-594-α-factor-labeled endosomes (red), Abp1-GFP- (green) and Abp140-3GFP- (green) labeled endocytic vesicles. It was possible to distinguish endocytic vesicles from actin cables, even though both were labeled with GFP. By labeling endocytic vesicles, actin cables, and early endosomes, we observed that endosomes associated with actin cables move toward endocytic vesicles, which were also associated with the cables. In the boxed area, endosome appears to move along an actin cable to an endocytic vesicle (Abp1p patch). Interval between frames is 1 s. Fig 5C and Movie 10 from Toshima et al.
S. cerevisiae (Mata his3-Δ200 leu2-3, 112 ura3-52 bar1Δ::LEU2 ABP1-GFP::HIS3 ABP140-3GFP::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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