Cytochalasin is known to inhibit cytoplasmic streaming rapidly in characean cells ... streaming inhibition are now shown to disrupt bundle assembly and, over ...
J. Cell Sci. 85, 21-32 (1986)
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Printed in Great Britain © The Company of Biologists Limited 1986
GROWTH AND REGROWTH OF ACTIN BUNDLES IN CHARA: BUNDLE ASSEMBLY BY MECHANISMS DIFFERING IN SENSITIVITY TO CYTOCHALASIN RICHARD E. WILLIAMSON AND URSULA A. HURLEY Department of Developmental Biology, Research School of Biological Sciences, The Australian National University, PO Box 475, Canberra City, ACT2601, Australia
SUMMARY Cytochalasin is known to inhibit cytoplasmic streaming rapidly in characean cells without disassembling their actin bundles. Lower cytochalasin concentrations than those needed for streaming inhibition are now shown to disrupt bundle assembly and, over longer periods, assembled bundles. After local wounding, cytochalasin limited bundle regeneration to the production of polygons and straight, discontinuous bundles that rarely connected to bundles outside the wound. The regenerated bundles supported only scattered organelle movements, whereas long, oriented bundles of control cells were connected to those outside the wound and supported bulk endoplasmic streaming. Unwounded Chara plants cultured for up to 2 weeks in 1 ^M-cytochalasin maintained normal bundle orientation and rapid cytoplasmic streaming, but the mean number of bundles per file of chloroplasts fell from 5'2 in controls to 2-0 in growing cells and 3-4 in nongrowing cells. These structural effects seem more likely than the streaming inhibition to reflect cytochalasin's in vitro effect of blocking extension at the barbed but not the pointed end of F-actin. In particular, cytochalasin inhibited the extension into the wound of bundles in which only the barbed ends of filaments would be exposed. However, short lengths of isolated bundles grew within the wound and bundle growth in the intact cell continued, albeit in modified form. It is suggested that these examples of continuing bundle growth involve cytochalasin-resistant mechanisms that are not wholly dependent on barbed-end filament growth.
INTRODUCTION
Cytoplasmic streaming in characean algae involves subcortical bundles of unipolar actin filaments (Williamson, 1975; Palevitz & Hepler, 1975; Kersey et al. 1976). Groups of bundles lie beneath each chloroplast file, anchored at the boundary between the streaming endoplasm and the surrounding sleeve of stationary cortical cytoplasm. Streaming is inhibited by cytochalasin B (Wessels et al. 1971; Williamson, 1972, 1975; Bradley, 1973; Bostrom & Walker, 1976; Nagai & Kamiya, 1977; Kuroda & Kamiya, 1981; Nothnagel et al. 1981), a fungal metabolite inhibiting many actin-based processes. Cytochalasin may affect actin in several ways, but at low concentrations it binds in vitro to the barbed ends of actin filaments where G-actin is added most rapidly (MacLean-Fletcher & Pollard, 1980; Pollard & Mooseker, 1981). At equilibrium in the presence of cytochalasin, short filaments coexist with an increased concentration of G-actin (Hartwig & Stossel, 1979; Tellam & Frieden, 1982). The actin cytoskeleton is usually severely disrupted when animal cells are treated with cytochalasin Key words: actin, Chara, immunofluorescence, cytochalasin.
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R. E. Williamson and U. A. Hurley
and the mechanism of its action is not always clear, although valuable attempts have been made to relate the concentration dependence of different in vivo effects to the concentration dependence of different in vitro effects on actin (Yahara et al. 1982). The uncertainty is even greater with the characean algae, where alterations to the actin bundles in the presence of cytochalasin were not detected by light or electron microscopy (Wessels et al. 1971; Bradley, 1973; Williamson, 1972, 1975). (Binding of a fluorescent phallotoxin was reduced in cells that had been internally perfused with cytochalasin but, since the effect occurred more slowly than streaming inhibition (Nothnagel et al. 1981), its relevance to that inhibition is doubtful.) Such stability to cytochalasin, however, is not general in plant cells (Blatt et al. 1980; Hoch & Staples, 1983; Parthasarathy, 1985; Perdue & Parthasarathy, 1985; Witztum & Parthasarathy, 1985). The actin cytoskeleton of the mature characean cell differs from that of most other cells in not undergoing major developmental-, environmental- or cell-cycle-related changes during the cell's life of many months. Such stability might be associated with mechanisms restricting the exchange of G-actin at filament ends, mechanisms that might also offer some stability towards cytochalasin. We therefore studied cytochalasin's effects on Chara over longer periods than previously used and in two situations where actin bundles would be changing. The first was the regrowth of actin bundles following their local destruction (Kamitsubo, 1972; Williamson et al. 1984) and the second was the growth of actin bundles required to maintain continuous, nearly longitudinal bundles in extending cells. We found that both existing and growing actin bundles are affected by concentrations of cytochalasin that do not inhibit streaming along assembled bundles.
MATERIALS AND METHODS
Plant material Chara corallina was grown in a glasshouse and in the laboratory with room and window light. Plastic bins (75 1) with a 3 cm layer of soil were used for glasshouse cultures; 201 glass aquaria with 1 cm of agar (Bacto-Agar, Difco Laboratories) were used in the laboratory. The nutrient solution (broadly similar to that used previously; Williamson, 1975) contained (mM): CaCl2, 0-1; MgSO 4 , 0-1; Na 2 CO 3 , 0-2; NH 4 C1, 0-04; NaCl, 0-5; KC1, 0-1; morpholinopropane sulphonic acid, 0-5; together with ( / I M ) : K H 2 P O 4 , 0-86; FeCl 3 , 2-48; nitrilotriacetic acid, 10-5; ZnCl 2 , 0-74; MnCl 2 , 2 - l x K T 2 ; CoCl 2 , 155X10" 2 ; CuCl 2 , 2-99X10" 2 ; Na 2 B 4 O 7 , 1-98; Na 2 MoO 4 , 0-49; p H 7 0 . Cultures in soil were started with plants collected locally (mainly from Lake Ginnindera, Belconnen, ACT), cultures in agar with 20-30 apical cuttings from soil-cultured plants. The cutting comprised the growing point with (normally) one expanded internode (>20 mm long) that was pushed into agar cooled to just above its gelling temperature (=30°C).
Experimental To determine the effects of cytochalasin B (Sigma Chemical Co.) on the numbers of actin bundles in Chara internodal cells, a 10 mM solution in dimethyl sulphoxide (DMSO) was added to an agar culture to give a final cytochalasin concentration of 1 fiM and a final DMSO concentration of 0-01 % (v/v). Controls received an equal volume of DMSO. The additions were made roughly 2 weeks after planting at a time of rapid growth. The length of each macroscopic internodal cell on every plant was measured with a ruler at intervals of one to several days. This gave the growth history of every cell until harvested for immunofluorescence.
Cytochalasin
and Chara actin bundles
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The effects of cell length and the presence or absence of cell growth on actin bundle number were studied in similar experiments, except that no additions were made to the media and all cells were harvested 21 or 22 days after planting to minimize any changes due to culture age. Chloroplast-free 'windows' were prepared (Wiliamson et al. 1984) in internodal cells (=5 cm long) grown in agar-based cultures and cytochalasin B (1 or 10f
