Localization of Abp1-RFP (red; left) and Abp140-3GFP (green; center). By expressing Abp1-mRFP and Abp140–3GFP to visualize endocytic vesicles and actin cables respectively, we observed that approximately 85% of endocytic vesicles associate with actin cables and that these vesicles invariably appeared to form in association with the cables. Boxes identify several patches that appear to form in association with cables. Patches also appear to move on cables. Interval between frames is 1 s. Fig 5A (single frames) and Movie 9 from Toshima et al.
S. cerevisiae (Mata his3-Δ200 leu2-3, 112 ura3-52 bar1Δ::LEU2 bni1-12::URA3 bnr1Δ::KanMX6 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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