Internalization of Alexa 594-a-factor. When added to cells, A594-α-factor (and A488-α-factor, data not shown) was first seen in internal endocytic compartments by 5 min. By 10 min, A594-α-factor began to concentrate in the vacuole and in bright structures that often were proximal to the vacuole. By 20 min, the α-factor was mostly in the vacuole. Interval between frames is 4 s. Corresponds to Fig 1B (single frames) and Movie 1 from Toshima et al.
S. cerevisiae (Mata his3-Δ200 leu2-3, 112 ura3-52 bar1Δ::LEU2) 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 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) and rhodamine/Texas-red filter.
|Spatial Axis||Image Size||Pixel Size|