Localization of Alexa Fluor-594-α factor labeled endosomes (red) and Sla1-GFP labeled endocytic vesicles (green). Note that several vesicles are targeted to the prominent endosome in the (upper) mother cell, whereas the prominent endosome in the (lower) daughter cell appears to move toward the vesicles just as they are being released from the plasma membrane. Total internal reflection fluorescence (TIRF) microscopy was used to visualize A594-α factor to reduce background and Sla1-GFP was visualized by epifluorescence. Interval between frames is 2.8 s. Fig 2A (single frames) and Movie 5 from Toshima et al.
S. cerevisiae (Mata his3-Δ200 leu2-3, 112 ura3-52 bar1Δ::LEU2 SLA1-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. Fluorescence microscopy (used for Abp1-red fluorescent protein) was performed by using an Olympus IX81 microscope equipped with a x100/NA1.4 or a x100/NA 1.45 (Olympus) objective and Orca-ER cooled CCD camera (Hamamatsu). For TIRF illumination (used for Alexa488-alpha factor), the expanded beam (488 nm) of an argon krypton laser (Melles Griot) was used to excite Alexa Fluor-488. The beam was focused at an off-axis position in the back focal plane of the objective.
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