Summary
Two different techniques have been adapted forMicrasterias denticulata to depict the actin cytoskeleton of both untreated and inhibitor-treated developing cells: the “quickstaining method”, where the cells are fixed in a mixture of glutaraldehyde and formaldehyde followed by staining with phalloidin without embedding, and the “methacrylate method”, where the cells are also fixed by aldehydes and where the embedding medium is removed prior to incubation with an actin antibody. Both methods produce sufficient preservation and visualization of actin microfilaments (MFs) and confirm earlier observations on the presence of a cortical actin MF network in both the growing and the nongrowing semicell as well as of a basketlike MF arrangement around the migrating nucleus. The results show that a network of actin MFs is essential for the proper development of the young lobes ofM. denticulata. Early developmental stages expanding uniformly at the beginning of growth lack any netlike actin MF arrangement. The actin cytoskeleton in developing cells treated with the actin-targeting agents cytochalasin D and latrunculin B is markedly influenced. Cytochalasin D, which produces the most pronounced effects, causes a breakdown of the network of actin MFs, resulting in bright actin clusters as well as in short and abnormally thick actin fragments particularly in cortical cell regions. In latrunculin B-treated cells remnants of the former actin MF network are still visible, yet most of the actin cytoskeleton appears collapsed and is reduced to short filament pieces. The disturbance of the actin MF system visualized in the present study correlates with the severe morphological and ultrastructural changes occurring in desmid cells as a consequence of both drugs. The dinitroanilin herbicide oryzalin, known to deploymerize cytoplasmic microtubules, causes also an impairment of the actin cytoskeleton inM. denticulata though not sufficient to influence normal cell growth and differentiation.
Similar content being viewed by others
Abbreviations
- CB:
-
cytochalasin B
- CD:
-
cytochalasin D
- DMSO:
-
dimethyl sulfoxide
- FA:
-
formaldehyde
- GA:
-
glutaraldehyde
- LAT-A:
-
latrunculin A
- LAT-B:
-
latrunculin B
- MFs:
-
microfilaments
- MT:
-
microtubule
References
Angerer LM, Angerer RC (1991) Localization of mRNAs by in situ hybridization: functional organization of the nucleus: a laboratory guide. Methods Cell Biol 35: 37–71
Ayscough KR, Stryker J, Pokala N, Sanders M, Crews Ph, Drubin DG (1997) High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin A. J Cell Biol 137: 399–416
Bachewich C, Heath IB (1998) Radial F-actin arrays precede new hypha formation inSaprolegnia: implications for establishing polar growth and regulating tip morphogenesis. J Cell Sci 111: 2005–2016
Baskin TI, Busby CH, Fowke LC, Sammut M, Gubler F (1992) Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa. Planta 187: 405–413
—, Miller DD, Vos JW, Wilson JE, Hepler PK (1996) Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy. J Microsc 182: 149–161
Bubb MR, Senderowicz AMJ, Sausville EA, Duncan KLK, Korn ED (1994) Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. J Biol Chem 269: 14869–14871
Cho SO, Wick SM (1990) Distribution and function of actin in the developing stomatal complex of winter rye (Secale cereale cv. Puma). Protoplasma 157: 154–164
Cooper JA (1987) Effects of cytochalasin and phalloidin on actin. J Cell Biol 105: 1473–1478
Coué M, Brenner SL, Spector I, Korn ED (1987) Inhibition of actin polymerization by latrunculin A. FEBS J 213: 316–318
Doris FP, Steer MW (1996) Effects of fixatives and permeabilisation buffers on pollen tubes: implications for localisation of actin microfilaments using phalloidin staining. Protoplasma 195: 25–36
Grolig F, Weigang-Köhler K, Wagner G (1990) Different extent of F-actin bundling in walled cells and in protoplasts ofMougeotia scalaris. Protoplasma 157: 225–230
Gubler F (1989) Immunofiuorescence localization of microtubules in plant root tips embedded in butyl-methyl methacrylate. Cell Biol Int Rep 13: 137–145
Hepler PK (1985) The plant cytoskeleton. In: Robards AW (ed) Botanical microscopy. Oxford University Press, Oxford, pp 233–262
—, Gunning BES (1998) Confocal fluorescence microscopy of plant cells. Protoplasma 201: 121–157
Höftberger M, Lütz-Meindl U (1999) Septum formation in the desmidXanthidium (Chlorophyta): effects of cytochalasin D and latrunculin B suggest the involvement of actin microfilaments. J Phycol 35: 768–777
Holzinger A, Meindl U (1997) Jasplakinolide, a novel actin targeting peptide, inhibits cell growth and induces actin filament polymerization in the green algaMicrasterias. Cell Motil Cytoskeleton 38: 365–372
—, Mittermann I, Laffer S, Valenta R, Meindl U (1997) Microinjection of profilin from different sources into the green algaMicrasterias causes transient inhibition of cell growth. Protoplasma 199: 124–134
—, DeRuijter N, Emons AM, Lütz-Meindl U (1999) Spectrin-like proteins in green algae (Desmidiaceae). Cell Biol Int 23: 335–344
Hush JM, Overall RL (1992) Re-orientation of cortical F-actin is not necessary for wound-induced microtubule re-orientation and cell polarity establishment. Protoplasma 169: 97–106
Kiermayer O (1981) Cytoplasmic basis of morphogenesis inMicrasterias. In: Kiermayer O (ed) Cytomorphogenesis in plants. Springer, Wien New York, pp 147–189 (Cell biology monographs, vol 8)
Lancelle SA, Hepler PK (1988) Cytochalasin-induced ultrastructural alterations inNicotiana pollen tubes. Protoplasma Suppl 2: 65–75
Lehtonen J (1983) Action of cytochalasin B on cytoplasmic streaming systems and morphogenesis inMicrasterias torreyi (Conjugatophyceae). Nord J Bot 3: 521–531
Lillie SH, Brown SS (1987) Artifactual immunofluorescent labelling in yeast, demonstrated by affinity purification of antibody. Yeast 311: 63–70
McCurdy DW, Palevitz BA, Gunning BES (1991) Effects of cytochalasins on actin in dividing root tip cells ofAllium andTriticum: a comparative immunocytochemical study. Cell Motil Cytoskeleton 18: 107–112
Meindl U (1983) Cytoskeletal control of nuclear migration and anchoring in developing cells ofMicrasterias denticulata and the change caused by the anti-microtubular herbicide amiprophosmethyl (APM). Protoplasma 118: 75–90
Meindl U (1992) Cytoskeleton-based nuclear translocation in desmids. In: Menzel D (ed) The cytoskeleton of the algae. CRC Press, Boca Raton, pp 138–147
— (1993)Micrasterias cells as a model system for research on morphogenesis. Microbiol Rev 57: 415–433
—, Kiermayer O (1981) Biologischer Test zur Bestimmung der Antimikrotubuli-Wirkung verschiedener Stoffe mit Hilfe der GrünalgeMicrasterias denticulata. Mikroskopie 38: 325–336
—, Lancelle S, Hepler PK (1992) Vesicle production and fusion during lobe formation inMicrasterias visualized by high-pressure freeze fixation. Protoplasma 170: 104–114
—, Zhang D, Hepler PK (1994) Actin microfilaments are associated with the migrating nucleus and the cell cortex in green algaMicrasterias. J Cell Sci 107: 1929–1934
Noguchi T, Ueda K (1981) Effect of metabolic inhibitors on the formation of cell walls in a green alga,Micrasterias crux melitensis. Plant Cell Physiol 22: 1437–1445
— — (1988) Cortical microtubules and cortical microfilanients in the green algaMicrasterias pinnatifida. Protoplasma 143: 188–192
Peat L, Oliveira L (1994) Organization of the cytoskeleton in the coencytic algaVaucheria longicaulis var.macounii: an experimental study. Protoplasma 177: 95–107
Schlösser UG (1982) Sammlung von Algenkulturen. Ber Deutsch Bot Ges 95: 181–276
Schmid VHR, Meindl U (1992) Microtubules do not control orientation of secondary wall microfibril deposition inMicrasterias. Protoplasma 169: 148–154
Sonobe S, Shibaoka H (1989) Cortical fine actin filaments in higher plant cells visualized by rhodamine-phalloidin after pretreatment withm-maleimidobenzoyl-N-hydroxysuccinimid ester. Protoplasma 148: 80–86
Spector I, Shochet NR, Blasberger D, Kashman Y (1989) Latrun-culins — novel marine macrolides that disrupt microfilament organization and affect cell growth I: comparison with cytochalasin D. Cell Motil Cytoskeleton 13: 127–144
Staiger CH, Lloyd C (1991) The plant cytoskeleton. Curr Opin Cell Biol 3: 33–42
Strachan SD, Hess FD (1983) The biochemical mechanism of action of the dinitroanliline herbicide Oryzalin. Pest Biochem Physiol 20: 141–150
Tang X, Lancelle SA, Hepler PK (1989) Fluorescence microscopic localization of actin in pollen tubes: comparison of actin antibody and phalloidin staining. Cell Motil Cytoskeleton 12: 216–224
Tippit DH, Pickett-Heaps JD (1974) Experimental investigations into morphogenesis inMicrasterias. Protoplasma 81: 271–296
Tominaga M, Morita K, Sonobe S, Yokota E, Shimmen T (1997) Microtubules regulate the organization of actin filaments at the cortical region in root hair cells ofHydrocharis. Protoplasma 199: 83–92
Ueda K, Noguchi T (1988) Microfilament bundles of F-actin and cytomorphogenesis in the green algaMicrasterias crux melitensis. Eur J Cell Biol 46: 61–67
Url T, Höftberger M, Meindl U (1993) Cytochalasin B influences dictyosomal vesicle production and morphogenesis in the desmidEuastrum. J Phycol 29: 667–674
Author information
Authors and Affiliations
Corresponding author
Additional information
Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday
Rights and permissions
About this article
Cite this article
Pflügl-Haill, M., Vidali, L., Vos, J.W. et al. Changes of the actin filament system in the green algaMicrasterias denticulata induced by different cytoskeleton inhibitors. Protoplasma 212, 206–216 (2000). https://doi.org/10.1007/BF01282921
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01282921