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

Sonesson, Anders LU and Widell, Susanne LU (1998) In Physiologia Plantarum 103(3). p.354-362
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Actin, cortical cytoskeleton, inside-out, plasma membrane vesicles, tubulin
in
Physiologia Plantarum
volume
103
issue
3
pages
354 - 362
publisher
Wiley-Blackwell
external identifiers
  • scopus:0031854913
ISSN
0031-9317
DOI
10.1034/j.1399-3054.1998.1030308.x
language
English
LU publication?
yes
id
7b44b2c1-bcf7-421a-b0d7-a2430c24d133 (old id 4438044)
alternative location
http://dx.doi.org/10.1034/j.1399-3054.1998.1030308.x
date added to LUP
2014-05-21 11:47:00
date last changed
2017-01-01 07:46:07
@article{7b44b2c1-bcf7-421a-b0d7-a2430c24d133,
  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.},
  author       = {Sonesson, Anders and Widell, Susanne},
  issn         = {0031-9317},
  keyword      = {Actin,cortical cytoskeleton,inside-out,plasma membrane vesicles,tubulin},
  language     = {eng},
  number       = {3},
  pages        = {354--362},
  publisher    = {Wiley-Blackwell},
  series       = {Physiologia Plantarum},
  title        = {The association of actin and tubulin with plasma membranes: characterization using inside-out vesicles formed by Brij 58},
  url          = {http://dx.doi.org/10.1034/j.1399-3054.1998.1030308.x},
  volume       = {103},
  year         = {1998},
}