Yeast permeabilized cell assay for actin assembly at cell cortex
Buffers
Wash buffer (3 ml)
 300µl 10X U buffer
 30µl 100mM PMSF (1mM final)
 3µl 1000X PI
 ddH2O to 3.0 ml
Buffer #1 (500µl)
 50µl 10X U buffer
 25µl saponin (0.01g saponin in
1.0ml ddH2O) (0.5mg/ml final)
 5µl 100mM PMSF (1mM final)
 0.5µl 1000X PI
 420µl ddH2O
Buffer #2 (500µl)
 50µl 10X U buffer
 5µl 100mM ATP (1mM final)
 5µl 100mM PMSF (1mM final)
 0.5µl 1000X PI
 440µl ddH2O
Buffer #3 (500µl)
 50µl 10X U buffer
 5µl 100mM ATP (1mM final)
 70µl 37% formaldehyde (5%
final)
 375µl ddH2O
Cell preparation
1. Grow up 10ml cells overnight to OD 600 = 0.15-0.2
2. Achieve G1 arrest by adding 4µl of 5mg/ml alpha factor. Allow cells to arrest for
2-3 hours at 25°C. Check for G1 arrest visually before proceeding (majority of
cells should be unbudded or shmooed).
3. Release cells from arrest by spinning in centrifuge, washing twice with 10ml fresh
media and resuspending in 10ml fresh media.
4. Grow cells out to small budded stage (30-75 min. depending on growth rate of
strain). This step is done at 25°C for WT and null strains or at a semi-permissive
temperature for ts strains. When initially characterizing a strain, try growing out at
different temperatures.
5. When cells are small-budded (determine microscopically), spin down at 2,000rpm
for 2 min. at room temperature.
6. Wash pellet immediately in 1ml of COLD buffer consisting of 0.5X U buffer (1X
U buffer = 50mM KHEPES pH 7.5, 100mM KCl, 3mM MgCl2, 1mM EGTA) and
0.5X SD medium.
7. Spin for 8 seconds in microfuge, remove supernatant and freeze pellet in liquid
N2.
8. Thaw pellet at room temp, resuspend in 100µl cold U buffer + 10% glycerol and
divide cell suspension into aliquots (10-20µl). Re-freeze in liquid N2.
9. Store cells at –80°C NO LONGER THAN 1 MONTH.
Nucleation assay
1. Remove aliquot of rhodamine-actin from –80°C freezer the night before
experiment and allow to thaw at 4°C (on ice) o/n. In the morning, spin in TLA100
at 80K for 20 min. Supernatant can be stored at 4°C for up to a week until used.
Dilute in G buffer immediately before experimental use. When reusing actin on
days following initial 80K spin, spin at 14K in cold room clinical for 10 min.,
then dilute.
2. Prepare 500µl of Buffer #1 on ice, then thaw permeabilized cell aliquot on ice.
Add 2µl of cells to 18µl Buffer 1, mix gently by tapping with finger and incubate
at room temp for 30 min.
3. Pellet cells in microfuge for 8 sec., wash with 100µl of COLD wash buffer.
Resuspend pellet in 18µl COLD Buffer 2 [NOTE: if incubating cells with protein
extract or purified protein, resuspend pellet in 10µl 2X Buffer #2, then add 10µl
control buffer (whatever protein is in) to control and 10µl of protein extract
(diluted to desired concentration) to experimental cells and incubate for 10-15
min. before next step.]
4. Add 2µl of Rd-actin (diluted 1:2 in G-buffer). Incubate reaction for 10-15 min. in
dark at room temp. [You can use 1µl of 10mg/ml fluorescein dextran as
permeability marker.]
5. Pellet cells in microfuge 8 sec. And resuspend pellet in 19µl COLD Buffer #3.
Incubate RT in dark for 20 min.
6. Pellet cells in microfuge, wash cells 2X in 100µl COLD Wash Buffer, resuspend
in 20µl Wash Buffer and pipet onto polyL-lysine coated slide. Incubate 5 min. in
dark.
7. Aspirate off excess cells, wash wells 3X with Wash Buffer, add a drop of
mounting solution, cover with coverslip and seal with nail polish.
8. Count the number of small-budded cells with a nucleus that show polarized Rdactin nucleating in bud and/or compare fluorescent intensity between bud and
mother utilizing Northern Exposure software.