The association of actin and tubulin with plasma membranes: characterization using inside-out vesicles formed by Brij 58

Most processes of eukaryotic cells depend on the cortical cytoskeleton (CS), a protein filament structure associated to the plasma membrane (PM). With animal cells, much information has been collected on the mechanisms behind CS-PM interactions, but for plant cells the CS-PM links are poorly characterized. To allow investigations on these links, isolated PM from cauliflower were here treated with Brij 58, a detergent that causes the PM vesicles to turn inside-out (cytoplasmic side-out), thereby exposing the CS components. When actin and tubulin co-pelleted with inside-out PM were separated using sucrose gradient centrifugation, actin and tubulin were recovered with PM-marker activities, supporting intact links between these CS proteins and the Brij-treated PM. Inside-out PM was also treated with different media to learn more about the CS-PM interaction. Extensive dialysis against a low ionic strength medium released actin but not tubulin from these PM, while dialysis against 0.7 M NaCl had no effect. Neither 50 mM DTT, 10 mM CaCl2 nor 2 M NaCl had any effect on the release of either actin or tubulin from PM, but actin was completely released with 6 M urea or 0.6 M KI. Tubulin was also released by urea but not by KI. Incubation of PM in sodium carbonate at increasing pH led to a total release of actin at pH 10, of α-tubulin at pH 11 and of β-tubulin at pH 11.4. In many respects, these characteristics agree with reported findings using e.g., fluorescence microscopy with protoplast ghosts, suggesting that inside-out vesicles obtained with Brij 58 can be used in investigations aimed at understanding the role of the cortical CS in regulating PM-bound components.