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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/4438044
- author
- Sonesson, Anders
LU
and Widell, Susanne
LU
- organization
- publishing date
- 1998
- 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
- John Wiley & Sons Inc.
- external identifiers
-
- scopus:0031854913
- ISSN
- 0031-9317
- DOI
- 10.1034/j.1399-3054.1998.1030308.x
- language
- English
- LU publication?
- yes
- additional info
- Department affilation moved from v1000887 (CED - Centre for Educational Development) to v1000942 (Division for Higher Education Development) on 2016-03-31 08:48:48.
- 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
- 2016-04-04 09:17:20
- date last changed
- 2023-03-20 23:34:34
@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}},
keywords = {{Actin; cortical cytoskeleton; inside-out; plasma membrane vesicles; tubulin}},
language = {{eng}},
number = {{3}},
pages = {{354--362}},
publisher = {{John Wiley & Sons Inc.}},
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}},
doi = {{10.1034/j.1399-3054.1998.1030308.x}},
volume = {{103}},
year = {{1998}},
}