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

Abstract

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.