Localization on the cell surface of Alexa Fluor-488-α factor (left, green in merge) and Ede1-RFP (center; red in merge). The Ede1p spots in the white boxes appear first and are then joined by α factor. The yellow boxes show colocalization of α factor and Ede1p. Thus, endocytic sites form before α-factor recruitment. Total internal reflection fluorescence (TIRF) microscopy was used to visualize A488-α factor to reduce background and Ede1p was visualized by epifluorescence. Interval between frames is 2 s. Fig 1G (single frames) and Movie 4 from Toshima et al.
S. cerevisiae (Mata his3-Δ200 leu2-3, 112 ura3-52 bar1Δ::LEU2 EDE1-RFP::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-α 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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