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*CIL – Cell Image Library accession number. Please use this to reference an image.

CIL:31918*  Cite 

Localization on the cell surface of Alexa Fluor-488-α factor (left, green in merge) and Sla1-RFP (center; red in merge). Within each white box, α-factor spots first appear and are subsequently joined by Sla1p. The yellow boxes show colocalization of α-factor and Sla1p. These observations establish that cortical actin patches are sites of receptor-mediated α-factor internalization. Total internal reflection fluorescence (TIRF) microscopy was used to visualize A488-α factor to reduce background and Sla1-RFP was visualized by epifluorescence. Interval between frames is 2 s. Fig 1F (single frames) and Movie 3 from Toshima et al.

Technical Details

S. cerevisiae (Mata his3-Δ200 leu2-3, 112 ura3-52 bar1Δ::LEU2 SLA1-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 Sla1-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.

Biological Sources
NCBI Organism Classification
Saccharomyces cerevisiae
Cellular Component
endocytic vesicle
endosome membrane
actin cortical patch
Biological Context
Biological Process
receptor-mediated endocytosis
actin cortical patch assembly
Molecular Function
actin binding
ubiquitin binding
Junko Y. Toshima
Jiro Toshima
Marko Kaksonen
Adam C. Martin
David S. King
David G. Drubin
Proc Natl Acad Sci. 2006. 103: 5793-5798.
Digital Object Identifier (DOI)
Archival Resource Key (ARK)
Grouping This image is part of a group.
Sample Preparation
living tissue
Relation To Intact Cell
whole mounted tissue
Spatial Axis Image Size Pixel Size
X 720px ——
Y 240px ——
Time 2 seconds 24