Summary
Isolated plasma membrane vesicles purified by aqueous polymer two-phase partitioning were used as a model system for studies on the membrane-associated (cortical) cytoskeleton in plants. Actin, as identified by immunoblotting, was found to be specifically attached to plasma membrane vesicles from cauliflower (Brassica oleracea L.). The actin was not washed off as the vesicles were turned inside-out, indicative of a fairly strong attachment. Triton X-100 extraction of plasma membrane vesicles resulted in an insoluble and hence pelletable fraction where actin could be found together with several other proteins. Our results show that the cortical cytoskeleton is to some extent co-purified with the plasma membrane, and we believe that well defined, inside-out and right-side-out plasma membrane vesicles can be used to study the structure and dynamics of the plant cortical cytoskeleton.
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Abbreviations
- ATP:
-
adenosine 5′-triphosphate
- BCIP:
-
5-bromo-4-chloro-3-indolyl phosphate
- BSA:
-
bovine serum albumin
- CCD:
-
counter-current distribution
- DTT:
-
dithiothreitol
- EDTA:
-
ethylene-diamine-tetraacetic acid
- EGTA:
-
ethylene glycol-bis(β-aminoethyl ether)
- GSII:
-
1,3-β-glucan synthase
- HEPES:
-
N-[2-hydroxyethyl]-piperazine-N′-[2-ethane sulfonic acid]
- MES:
-
2-(N-morpholino)ethane sulfonic acid
- NBT:
-
p-nitro blue tetrazolium chloride
- IDP:
-
inosine 5′-diphosphate
- PAGE:
-
polyacrylamide gel electrophoresis
- PBS:
-
phosphate buffered saline
- PIPES:
-
piperazine-N,N′-bis(2-ethane-sulfonic acid)
- PPB:
-
potassium phosphate buffer
- PM:
-
plasma membrane
- PMSF:
-
phenylmethylsulfonyl fluoride
- PVDF:
-
polyvinylidene difluoride
- PVPP:
-
polyvinylpolypyrrolidone
- SDS:
-
sodium dodecyl sulfate
- TBS:
-
Tris-buffered saline
- TTBS:
-
Tris-buffered saline with Tween 20
- Tris:
-
tris(hydroxymethyl) aminomethane
References
Abe S, Ito Y, Davies E (1992) Co-sedimentation of actin, tubulin and membranes in the cytoskeleton fractions from peas and mouse 3T3 cells. J Exp Bot 43: 941–949
Askerlund P (1990) Redox processes of plant plasma membranes. Studies with isolated plasma membrane vesicles. PhD Thesis, University of Lund, Lund, Sweden
—, Larsson C, Widell S (1988) Localizaton of donor and acceptor sites of dehydrogenase activities using inside-out and right-sideout plasma membrane vesicles from plants. FEBS Lett 239: 23–28
Baginski ES, Foa PP, Zak B (1967) Determination of phosphate: study of labile organic phosphate interference. Clin Chim Acta 15: 155–158
Bearden JC Jr (1978) Quantification of submicrogram quantities of protein by an improved protein-dye binding assay. Biochim Biophys Acta 533: 525–529
Berridge MJ, Irvine RF (1989) Inositol phosphates and cell signalling. Nature 341: 197–205
Drøbak BK (1991) Plant signal perception and transduction: the role of the phosphoinositide system. Essays Biochem 26: 27–37
Faraday CD, Spanswick R (1993) Evidence for a membrane skeleton in higher plants. A spectrin-like polypeptide co-isolates with rice root plasma membranes. FEBS Lett 318: 313–316
Fredrikson K, Larsson C (1989) Activation of 1,3-β-glucan synthase by Ca2 +, spermine and cellobiose. Localization of activator sites using inside-out plasma membrane vesicles. Physiol Plant 77: 196–201
Haupt W, Scheuerlein R (1990) Chloroplast movement. Plant Cell Environ 13: 595–614
Hepler PK, Wayne RO (1985) Calcium and plant development. Annu Rev Plant Physiol 36: 397–439
Kropf DL, Berge SK, Quatrano RS (1989) Actin localization duringFucus embryogenesis. Plant Cell 1: 191–200
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685
Larsson C (1985) Plasma membranes. In: Linskens HF, Jackson JF (eds) Cell components. Springer, Berlin Heidelberg New York Tokyo, pp 85–104 (Modern methods of plant analysis, new series, vol 1)
—, Kjellbom P, Widell S, Lundborg T (1984) Sidedness of plant plasma membrane vesicles purified by partitioning in aqueous two-phase systems. FEBS Lett 171: 271–276
—, Widell S, Kjellbom P (1987) Preparation of high-purity plasma membranes. Methods Enzymol 148: 558–568
Lassing I, Lindberg U (1985) Specific interactions between phosphatidylinositol 4,5-bisphosphate and profilactin. Nature 314: 472–474
Lundborg T, Widell S, Larsson C (1981) Distribution of ATPases in wheat root membranes separated by phase partition. Physiol Plant 52: 89–95
Mescher MF, Jose MJL, Balk SP (1981) Actin containing matrix associated with the plasma membrane of murine tumour and lymphoid cells. Nature 289: 139–144
Niggli V, Jenni V (1989) Actin-associated proteins in human neutrophils: identification and reorganization upon cell activation. Eur J Cell Biol 49: 366–372
Palmgren MG, Sommarin M (1989) Lysophosphatidylcholine stimulates ATP dependent proton accumulation in isolated root plasma membrane vesicles. Plant Physiol 90: 1009–1014
—, Askerlund P, Fredrikson K, Widell S, Sommarin M, Larsson C (1990 a) Sealed inside-out and right-side-out plasma membrane vesicles. Plant Physiol 92: 871–880
—, Sommarin M, Ulvskov P, Larsson C (1990 b) Effect of detergents on the proton ATPase activity of inside-out and right-side-out plant plasma membrane vesicles. Biochim Biophys Acta 1021: 133–140
Payrastre B, van Bergen EN, Henegouwen PMP, Breton M, den Hartigh JC, Plantavid M, Verkleij AJ, Boonstra J (1991) Phosphoinositide kinase, diacylglycerol kinase, and phospholipase C activities associated to the cytoskeleton: effect of epidermal growth factor. J Cell Biol 115: 121–128
Sandelius AS, Morré DJ (1990) Plasma membrane isolation. In: Larsson C, Møller IM (eds) The plant plasma membrane. Springer, Berlin Heidelberg New York Tokyo, pp 44–75
Schliwa M, Blerkom J (1990) Structural interaction of cytoskeletal components. J Cell Biol 90: 222–235
Seitz K (1979) Cytoplasmic streaming and cyclosis of chloroplasts. In: Haupt W, Feinleib ME (eds) Physiology of movements. Springer, Berlin Heidelberg New York, pp 150–169 [Pirson A, Zimmermann MH (eds) Encyclopedia of plant physiology, new series, vol 7]
Shariff A, Luna EJ (1992) Diacylglycerol-stimulated formation of actin nucleation sites at plasma membranes. Science 256: 245–247
Sommarin M, Sandelius AS (1988) Phosphatidylinositol and phosphatidylinositol phosphate kinase in plant plasma membranes. Biochim Biophys Acta 958: 268–278
Spudich JA, Watt S (1971) The regulation of rabbit skeletal muscle contraction. J Biol Chem 246: 4866–4871
Traas JA (1990) The plasma membrane-associated cytoskeleton. In: Larsson C, Møller IM (eds) The plant plasma membrane. Springer, Berlin Heidelberg New York Tokyo, pp 269–292
Tan Z, Boss W (1992) Association of phosphatidylinositol kinase, phosphatidylinositol monophosphate kinase and diacylglycerol kinase with the cytoskeleton and F-actin fractions of carrot (Daucus carota L.) cells grown in suspension culture. Plant Physiol 100: 2116–2120
Widell S, Larsson C (1990) A critical evaluation of markers used in plasma membrane purification. In: Larsson C, Møller IM (eds) The plant plasma membrane. Springer, Berlin Heidelberg New York Tokyo, pp 16–43
—, Sommarin M (1991) Purification of endoplasmic reticulum from wheat roots and shoots (Triticum aestivum). Comparison of their blue light-sensitive redox components with those of highly purified plasma membrane. Physiol Plant 82: 9–18
Williamson RE, McCurdy DW, Hurley UA, Perkin JL (1987) Actin ofChara giant internodal cells. A single isoform in the subcortical filament bundles and a larger, immunologically related protein in the chloroplasts. Plant Physiol 85: 268–272
Xu P, Lloyd CW, Staiger CJ, Drøbak BK (1992) Association of phosphatidylinositol 4-kinase with the plant cytoskeleton. Plant Cell 4: 941–951
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Sonesson, A., Widell, S. Cytoskeleton components of inside-out and right-side-out plasma membrane vesicles from plants. Protoplasma 177, 45–52 (1993). https://doi.org/10.1007/BF01403398
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DOI: https://doi.org/10.1007/BF01403398