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Plasma membrane/cytoskeleton interactions in plants

Sonesson, Anders LU (1997)
Abstract
The way in which the cortical cytoskeleton associates to the plasma membrane (PM) must be elucidated to understand the structural dynamics of many processes in the plant cell. In the present study, isolated cauliflower (Brassica oleracea L.) PM vesicles were used to characterize membrane/cytoskeleton interactions. Both actin and tubulin were shown to copurify with PM vesicles and this copurification was specific to the PM. In order to expose the PM-associated cytoskeleton, Brij 58 was used to form sealed, inside-out vesicles (cytoplasmic side out). Both actin and tubulin colocalized with Brij 58-treated PM vesicles in sucrose-gradient centrifugation, indicating a lack of disruption of the actin/PM and tubulin/PM associations following... (More)
The way in which the cortical cytoskeleton associates to the plasma membrane (PM) must be elucidated to understand the structural dynamics of many processes in the plant cell. In the present study, isolated cauliflower (Brassica oleracea L.) PM vesicles were used to characterize membrane/cytoskeleton interactions. Both actin and tubulin were shown to copurify with PM vesicles and this copurification was specific to the PM. In order to expose the PM-associated cytoskeleton, Brij 58 was used to form sealed, inside-out vesicles (cytoplasmic side out). Both actin and tubulin colocalized with Brij 58-treated PM vesicles in sucrose-gradient centrifugation, indicating a lack of disruption of the actin/PM and tubulin/PM associations following inside-out vesicle formation. In order to characterize the PM links to actin and tubulin, inside-out PM vesicles were washed in a range of media. Both actin and tubulin co-sedimented with the PM vesicles in the presence of 50 mM DTT, 10 mM CaCl2 and 2 M NaCl (separately). Actin, but not tubulin, was completely released in the presence of 0.6 M KI, and both proteins were released in either 6 M urea or at pH>11.4. The association of actin to the PM was sensitive to extensive dialysis against a low ionic-strength medium. The presence of a pool of a- and b-tubulin with hydrophobic properties, "hydrophobic tubulin", was shown by Triton X-114 fractionation. This hydrophobicity was restricted to PM-associated tubulin and was not seen in soluble tubulin. Hydrophobic tubulin constituted at least 15% of the recovered PM-associated tubulin. Washes in the presence of high concentrations of salt or calcium did not release the hydrophobic tubulin, while high pH (“11.0) did release this fraction. The hydrophobicity of a fraction of the recovered tubulin could reflect a direct or indirect interaction of this tubulin with the PM lipid bilayer or to an integral membrane protein. Hydrophobic tubulin was associated in a salt-sensitive manner to detergent-resistant proteins, likely to be of peripheral/cytoskeletal origin. It is possible that the presence of hydrophobic tubulin in isolated PM reflects the anchoring of the cortical tubulin cytoskeleton to the PM. Further elucidation of the cytoskeleton anchoring mechanism, based on the present study will lead to a better understanding of the structural dynamics of many cellular events. (Less)
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author
supervisor
opponent
  • Dr Cyr, Richard J., Department of Biology, The Pennsylvania State University, University Park, Pennsylvania, USA
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Triton X-114, hydrophobic tubulin, tubulin, actin, cytoskeleton, Brij 58, inside-out, plasma membrane, plant, cauliflower, Physiological biophysics, Växtfysiologi
pages
105 pages
publisher
Section of Plant Physiology, P.O.Box 117, S-221 00 Lund, Sweden,
defense location
Department of Plant Biology, Sölvegatan 35, Lund
defense date
1997-05-27 10:15
external identifiers
  • Other:ISRN: LUNBDS/nbfb-1033/1-105
ISBN
91-628-2529-1
language
English
LU publication?
yes
id
ba6c44b4-82e6-4bdc-91ed-731434de94b8 (old id 29213)
date added to LUP
2007-06-12 16:36:09
date last changed
2016-12-07 11:27:04
@misc{ba6c44b4-82e6-4bdc-91ed-731434de94b8,
  abstract     = {The way in which the cortical cytoskeleton associates to the plasma membrane (PM) must be elucidated to understand the structural dynamics of many processes in the plant cell. In the present study, isolated cauliflower (Brassica oleracea L.) PM vesicles were used to characterize membrane/cytoskeleton interactions. Both actin and tubulin were shown to copurify with PM vesicles and this copurification was specific to the PM. In order to expose the PM-associated cytoskeleton, Brij 58 was used to form sealed, inside-out vesicles (cytoplasmic side out). Both actin and tubulin colocalized with Brij 58-treated PM vesicles in sucrose-gradient centrifugation, indicating a lack of disruption of the actin/PM and tubulin/PM associations following inside-out vesicle formation. In order to characterize the PM links to actin and tubulin, inside-out PM vesicles were washed in a range of media. Both actin and tubulin co-sedimented with the PM vesicles in the presence of 50 mM DTT, 10 mM CaCl2 and 2 M NaCl (separately). Actin, but not tubulin, was completely released in the presence of 0.6 M KI, and both proteins were released in either 6 M urea or at pH>11.4. The association of actin to the PM was sensitive to extensive dialysis against a low ionic-strength medium. The presence of a pool of a- and b-tubulin with hydrophobic properties, "hydrophobic tubulin", was shown by Triton X-114 fractionation. This hydrophobicity was restricted to PM-associated tubulin and was not seen in soluble tubulin. Hydrophobic tubulin constituted at least 15% of the recovered PM-associated tubulin. Washes in the presence of high concentrations of salt or calcium did not release the hydrophobic tubulin, while high pH (“11.0) did release this fraction. The hydrophobicity of a fraction of the recovered tubulin could reflect a direct or indirect interaction of this tubulin with the PM lipid bilayer or to an integral membrane protein. Hydrophobic tubulin was associated in a salt-sensitive manner to detergent-resistant proteins, likely to be of peripheral/cytoskeletal origin. It is possible that the presence of hydrophobic tubulin in isolated PM reflects the anchoring of the cortical tubulin cytoskeleton to the PM. Further elucidation of the cytoskeleton anchoring mechanism, based on the present study will lead to a better understanding of the structural dynamics of many cellular events.},
  author       = {Sonesson, Anders},
  isbn         = {91-628-2529-1},
  keyword      = {Triton X-114,hydrophobic tubulin,tubulin,actin,cytoskeleton,Brij 58,inside-out,plasma membrane,plant,cauliflower,Physiological biophysics,Växtfysiologi},
  language     = {eng},
  pages        = {105},
  publisher    = {ARRAY(0xb83d498)},
  title        = {Plasma membrane/cytoskeleton interactions in plants},
  year         = {1997},
}