GROWTH AND REGROWTH OF ACTIN BUNDLES IN CHARA: … · 2005-08-25 · maintained normal bundle orientation and rapid cytoplasmic streaming, ... albeit in modified form. It is suggested

J. Cell Sci. 85, 21-32 (1986) 21Printed 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. HURLEYDepartment 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 withoutdisassembling their actin bundles. Lower cytochalasin concentrations than those needed forstreaming inhibition are now shown to disrupt bundle assembly and, over longer periods,assembled bundles. After local wounding, cytochalasin limited bundle regeneration to the pro-duction of polygons and straight, discontinuous bundles that rarely connected to bundles outsidethe 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 bulkendoplasmic streaming. Unwounded Chara plants cultured for up to 2 weeks in 1 ^M-cytochalasinmaintained normal bundle orientation and rapid cytoplasmic streaming, but the mean number ofbundles per file of chloroplasts fell from 5'2 in controls to 2-0 in growing cells and 3-4 in non-growing cells. These structural effects seem more likely than the streaming inhibition to reflectcytochalasin'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 thebarbed ends of filaments would be exposed. However, short lengths of isolated bundles grew withinthe wound and bundle growth in the intact cell continued, albeit in modified form. It is suggestedthat these examples of continuing bundle growth involve cytochalasin-resistant mechanisms thatare not wholly dependent on barbed-end filament growth.

INTRODUCTION

Cytoplasmic streaming in characean algae involves subcortical bundles of uni-polar actin filaments (Williamson, 1975; Palevitz & Hepler, 1975; Kersey et al.1976). Groups of bundles lie beneath each chloroplast file, anchored at the bound-ary between the streaming endoplasm and the surrounding sleeve of stationarycortical 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 metaboliteinhibiting many actin-based processes.

Cytochalasin may affect actin in several ways, but at low concentrations it bindsin vitro to the barbed ends of actin filaments where G-actin is added most rapidly(MacLean-Fletcher & Pollard, 1980; Pollard & Mooseker, 1981). At equilibrium inthe presence of cytochalasin, short filaments coexist with an increased concentrationof G-actin (Hartwig & Stossel, 1979; Tellam & Frieden, 1982). The actin cyto-skeleton is usually severely disrupted when animal cells are treated with cytochalasin

Key words: actin, Chara, immunofluorescence, cytochalasin.

22 R. E. Williamson and U. A. Hurley

and the mechanism of its action is not always clear, although valuable attempts havebeen made to relate the concentration dependence of different in vivo effects to theconcentration dependence of different in vitro effects on actin (Yahara et al. 1982).The uncertainty is even greater with the characean algae, where alterations to theactin bundles in the presence of cytochalasin were not detected by light or electronmicroscopy (Wessels et al. 1971; Bradley, 1973; Williamson, 1972, 1975). (Bindingof a fluorescent phallotoxin was reduced in cells that had been internally perfusedwith cytochalasin but, since the effect occurred more slowly than streaming inhi-bition (Nothnagel et al. 1981), its relevance to that inhibition is doubtful.) Suchstability 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 differsfrom that of most other cells in not undergoing major developmental-, environ-mental- or cell-cycle-related changes during the cell's life of many months. Suchstability might be associated with mechanisms restricting the exchange of G-actin atfilament ends, mechanisms that might also offer some stability towards cytochalasin.We therefore studied cytochalasin's effects on Chara over longer periods thanpreviously used and in two situations where actin bundles would be changing. Thefirst was the regrowth of actin bundles following their local destruction (Kamitsubo,1972; Williamson et al. 1984) and the second was the growth of actin bundlesrequired to maintain continuous, nearly longitudinal bundles in extending cells. Wefound that both existing and growing actin bundles are affected by concentrations ofcytochalasin that do not inhibit streaming along assembled bundles.

MATERIALS AND METHODS

Plant materialChara 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 with1 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; MgSO4,0-1; Na2CO3, 0-2; NH4C1, 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; FeCl3, 2-48; nitrilotriacetic acid, 10-5; ZnCl2, 0-74; MnCl2,2 - lxKT 2 ; CoCl2, 155X10"2; CuCl2, 2-99X10"2; Na2B4O7, 1-98; Na2MoO4, 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. Thecutting comprised the growing point with (normally) one expanded internode (>20 mm long) thatwas pushed into agar cooled to just above its gelling temperature (=30°C).

ExperimentalTo 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 toan agar culture to give a final cytochalasin concentration of 1 fiM and a final DMSO concentration of0-01 % (v/v). Controls received an equal volume of DMSO. The additions were made roughly 2weeks after planting at a time of rapid growth. The length of each macroscopic internodal cell onevery plant was measured with a ruler at intervals of one to several days. This gave the growthhistory of every cell until harvested for immunofluorescence.

Cytochalasin and Chara actin bundles 23

The effects of cell length and the presence or absence of cell growth on actin bundle number werestudied in similar experiments, except that no additions were made to the media and all cells wereharvested 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 cmlong) grown in agar-based cultures and cytochalasin B (1 or 10f<M) or DMSO (0-01 or 0-1 %, v/v)was added shortly afterwards.

ImmunofluorescenceThe preparation techniques have been described (Williamson et al. 1984) and also the mono-

clonal antibodies CC2 and CC6 (Williamson et al. 1986) that bind to the subcortical actin bundles.The number of bundles per file of chloroplasts was counted for each clearly focused file onphotographs taken at 6-12 sites along each cell. Counting was done without knowledge of thetreatment received by the cells and areas around the neutral lines were avoided.

MicroscopyThe techniques for fluorescence microscopy have been described (Williamson et al. 1984).

Differential interference contrast microscopy used video recording (Suzaki & Williamson, 1985). Atemporal filter (Arlunya T F 4000, Vermont, Victoria 3133, Australia) could enhance stationarycortical structures and filter out moving, endoplasmic structures.

RESULTS

Cell cultures

All cells with lengths exceeding the 10 mm needed for perfusion showed typical(Williamson et al. 1986) arrays of bundles at the cortex—endoplasm interface(Fig. 1A). They did not contain detectable bundle ends such as were readily visiblearound and within windows during the early stages of regeneration (see fig. 3,Williamson et al. 1984). The number of bundles per file of chloroplasts showedslight variations with cell length and between growing and older, non-growing cells(Fig. 2). Culturing cells in 1 jUM-cytochalasin reduced the number of bundles perchloroplast file (Fig. 3). This effect, detectable within 24 h, was more pronounced ingrowing than in non-growing cells (Fig. 1B,C; Fig. 3) and could be seen in vivo(Fig. ID), proving that it was not a perfusion artefact. There was no evidence ofdiscontinuities or disorientation in the depleted bundle array. The apparent thick-ness of the remaining bundles was increased whether seen in vivo (Fig. ID), asenhanced brightness by immunofluorescence (Fig. 1C), or by scanning electronmicroscopy (not shown). The number of bundles per chloroplast file was slightlyreduced by DMSO alone as the solvent for the cytochalasin (Fig. 3). Cells depletedof bundles by cytochalasin treatment still streamed actively.

Windowed cells

Bundle regeneration in the presence of DMSO alone was similar to that describedpreviously (Williamson et al. 1984). After 12-24 h of regeneration, immunofluor-escence (Fig. 4) revealed long curving lengths of actin bundles that were particularlyabundant towards the edges of a window where they curved round to join theoppositely polarized arrays formerly separated by the neutral line. The centres of


such windows were occupied by varying amounts of less-ordered bundles. We havepreviously suggested explanations for the origin and orientation of these bundles(Williamson et al. 1984).

Fig. 1. The effect of 1 f<M-cytochalasin B on the actin bundle arrays in agar-culturedChara cells. A. Immunofluorescence view of DMSO-treated control showing =5 bundlesper chloroplast file as seen in untreated controls. Although their red autofluorescence isremoved by a barrier filter, the positions of the light-absorbing chloroplasts are indicatedby the periodic attenuation of bundle fluorescence where the bundles lie beneath them.B. The number of bundles per chloroplast file is slightly reduced by cytochalasin in non-growing cells. C. Bundle depletion is much more pronounced in a growing cell treatedwith cytochalasin. D. The single bundles per chloroplast file produced by cytochalasinare readily visible in vivo. Bars: A,B,C, 20fan; D, lOjum.

2 3 4 5 6 7 1 2 3 4 5 6Cell length (cm)

Fig. 2. The number of actin bundles per chloroplast file in cells of different length.A. Cells that were growing when harvested. B. Cells that had ceased growing whenharvested. The lines are drawn from a least-squares linear regression analysis. Each pointis the mean for a single cell.


Immunofluorescence observations were supplemented by video microscopy ofcells prior to their fixation. The general features of organelle movements in thewindows of control cells allowed more than 12 h for regeneration were readily relatedto the immunofluorescence image: bulk streaming with a velocity comparable to thatoutside the window followed a U-turn at each side of the window where immuno-fluorescence showed long, curving actin bundles (see Fig. 4). Only intermittentmovements of usually single organelles occurred in various directions around thecentral region, where actin bundles were fewer and less ordered. In vivo observationsrevealed few of the bundles that could be demonstrated by immunofluorescence.

Treatment of cells with either 1 fiu or lOjUM-cytochalasin markedly reduced thequantity of regenerated actin bundles and altered their arrangement. Whereascontrols with DMSO alone had an extensive bundle system after 12 h of regeneration(Fig. 4), cytochalasin-treated cells had fewer bundles even after 21 h (Fig. 5A). Thelong, gently curving bundles seen in controls (Fig. 4) were absent. Short lengths ofusually rather straight, thick bundles and variously shaped rings or polygons(Fig. 5A) were prominent to varying degrees in different cells. The regeneratedbundles were rarely connected to bundles outside the window and lacked a pre-dominant orientation. The rings and polygons always enclosed chloroplasts emittingred autofluorescence (Fig. 5B,C) but not all such chloroplasts were thus enclosed.Chloroplasts retaining red autofluorescence must have reached the window afterits creation by irradiation, since this induces white chloroplast autofluorescence(Williamson et al. 1984). They could have originated from the endoplasm ofcharacean algae, which contains a few chloroplasts that usually rotate as they are

CO

- 2 2 4Time (clays)

Fig. 3. Time course showing the effects of 1 /ZM-cytochalasin B and 0-01 % DMSO on thenumber of actin bundles per chloroplast file. Additions were made at day 0. Solid symbolsdenote non-growing cells, open symbols growing cells. (A,A) No additions; ( • , • )DMSO; ( • . O ) cytochalasin. Each point represents the mean (with standard error) of>3 cells.


Fig. 4. Montage of immunofluorescence micrographs showing the bundles regeneratedfor over 12 h in the window of a DMSO-containing control cell. The regenerated bundlesform characteristically curved arrays at either side of the window, while fewer less-oriented bundles regenerate in the central region. The curved arrays follow the path ofpassive endoplasmic flow set up when streaming locally ceases as the window.is formed.They join the original antiparallel arrays of bundles that are running tranversely and areseparated by the neutral line («/). Damaged chloroplasts (ct) persisting at the windowedges appear strongly autofluorescent even with the red-excluding barrier filter thatabsorbs the autofluorescence of undamaged chloroplasts. Bar, 70 jum.

carried in the streaming cytoplasm (see Kamiya, 1962). Very occasionally such achloroplast was indeed seen to become fixed to the cortical cytoplasm in the window,thus halting its rotational and translational movements.

Towards the sides of cytochalasin-treated windows, endoplasm moved en masse ina U-turn across the neutral line. The velocity was slow (=5jums"') compared to thevelocity of streaming outside the window (=50 /Mm s~ ) and comparable to velocitiesof the U-turn flow seen in control windows before they contained regeneratedbundles. In the central regions, such mass flow of endoplasm was absent. Dis-continuous movements of single organelles occurred throughout such windows invarious directions and over limited distances. These movements were seen close tothe cortex—endoplasm boundary but not deeper in the endoplasm. Towards the sides


Fig. 5. Effects of cytochalasin on bundle regeneration. A. Montage of immuno-fluorescence micrographs showing the degree of bundle regeneration after 21 h in thepresence of 1 f<M-cytochalasin B. The position of the neutral line (nl) is again indicated.The regenerated bundles occur as rings and polygons or as short, rather straight lengthsof bundle that only rarely connect with the bundles outside the window. A portion ofthe undamaged array of bundles outside the window is seen on the extreme left.B,C. Immunofluorescence and bright-field micrographs of a small area of the window inA. Chloroplasts lie at the centre of the loops and polygons. Their red autofluorescencedoes not pass the barrier filters showing that they are not part of the original chloroplastpopulation but arrived after the window was produced. Not all chloroplasts havepolygons. Bars: A, 50,um; B,C,


of the window, discontinuous organelle movements in various directions couldoverlie bulk flow of the deeper endoplasm that was following a U-turn pathway. As incontrols, fibres were only occasionally seen where organelles were actively moving.To emphasize that severe inhibition of regeneration occurred without inhibition ofstreaming elsewhere in the cell, the velocity of streaming outside the window justbefore fixation was 55 /jxns~ for the cell shown in Fig. 5A, 93 % of the velocity justbefore cytochalasin was applied.

DISCUSSION

The results will be discussed in two contexts: the action of cytochalasin oncharacean cells and the assembly of characean actin bundles.

Cytochalasin action

Some amendments are required to the theories of how cytochalasin affectscharacean cells. Earlier studies showed that relatively high cytochalasin concen-trations (40-200/IM) inhibited streaming in a few minutes with little if any dis-ruption of the actin bundles (see Introduction). These conclusions remain valid butthe present, longer-term experiments show that actin bundle assembly and organ-ization are sensitive to cytochalasin concentrations that are insufficient to inhibitstreaming. The one instance of severe inhibition by 1 |UM-cytochalasin occurs in thewindow. It may reasonably be attributed to the scarcity of regenerated bundlesrather than to any inhibition of bundle function, since previously assembled bundlescontinue to support rapid streaming outside the window.

Cytochalasin's known effects on actin polymerization can more readily explain theeffects on bundle assembly than the inhibition of streaming. Similar concentrationsproduce the in vivo bundle assembly effect (3=1 /XM) and the in vitro polymerizationeffect ( 2 ^ M ; MacLean-Fletcher & Pollard, 1980; Pollard & Mooseker, 1981),whereas streaming along pre-existing bundles is virtually insensitive to such lowconcentrations (Bradley, 1973; this study). As will be discussed, the effects ofcytochalasin on bundle assembly in various situations differ in ways that may relate toits in vitro effects on actin polymerization.

There remain, however, strong grounds for believing that higher cytochalasinconcentrations inhibit streaming by acting directly on the force-generating actinsystem, even if this does not involve depolymerization. Cytochalasin inhibits motiveforce production by affecting the stationary, cortical cytoplasm rather than thestreaming endoplasm (Nagai & Kamiya, 1977; Kuroda & Kamiya, 1981) and, mostpersuasively, inhibition is rapid and complete in demembranated models whereATP, pH, Ca and other variables are controlled (Williamson, 1975; Nothnagele/ al.1981; Shimmen & Tazawa, 1983). Moreover, cytochalasin rapidly makes the actinbundles insoluble in low salt solutions (Williamson, 1978; Williamson et al. 1985)and, more slowly, decreases their binding of phallotoxin (Nothnagel et al. 1981).Such an idea of two or more in vivo effects with different concentration depen-dencies accords well with the ideas of Yahara et al. (1982). They postulated for


mammalian cells that several in vivo effects with different concentration depen-dencies reflected multiple in vitro effects on actin with similarly disparate concen-tration dependencies.

Bundle assembly

Following the earlier immunofluorescence studies, two origins were proposed forbundles regenerating in windows (Williamson et al. 1984): by growth as extensionsof the ends of upstream bundles (i.e. those delivering endoplasm to the window), andby growth following independent initiation within the windows. The mechanism ofassembly in normally growing cells might be different again since intact cells lack thevisible bundle ends suggested to support growth in the window. Bundle assemblyitself probably involves filament growth, cross-linking and anchorage to the cortex,but many variations can be imagined in the order, subcellular location and detailedmechanism by which these basic steps could generate a bundle.

The effects of cytochalasin further support the existence of more than onemechanism for bundle assembly in characean cells. The extension of the upstreambundles into windows is very efficiently blocked by cytochalasin. This inhibition maydepend on cytochalasin's ability to cap the barbed ends of actin filaments that wouldbe exposed where the upstream bundles were severed. However, other bundles doregenerate in the windows of both control and cytochalasin-treated cells withoutconnections to the bundle endings at the window edge. With cytochalasin, they formeither chloroplast-associated polygons or fibres that generally seem thicker,straighter and less branched than those regenerated in controls. This indicates that asecond mechanism(s) of assembly is operating that is less sensitive to cytochalasinthan that which extends the upstream bundles. The continuation of some bundlegrowth is reasonable since nucleation (Tellam & Frieden, 1982), filament annealing(MacLean-Fletcher & Pollard, 1980) and pointed-end extension (Pollard &Mooseker, 1981) of F-actin all continue in vitro even when cytochalasin blocksbarbed-end monomer addition.

The loops and polygons in cytochalasin-treated windows surround chloroplastswhose red autofluorescence indicates their arrival after the irradiation that createdthe window. Settlement of chloroplasts travelling in the streaming endoplasm wasdirectly observed in a few cases. Specific binding molecules perhaps link thechloroplast envelope to the specialized layer of cortical cytoplasm beneath the plasmamembrane (Williamson, 1985). Since chloroplasts with red autofluorescence incontrol windows are not surrounded by loops or polygons when processed forimmunofluorescence, cytochalasin may promote their formation. However, chloro-plasts travelling in the endoplasm rotate (see Kamiya, 1962), so that they may arrivein the window already carrying an actin loop to propel rotation. Cytochalasin mighttherefore prevent the disassembly of such loops, perhaps by its ability to block theremoval as well as the addition of monomer at the barbed end of actin filaments(Pollard & Mooseker, 1981).

Bundles regenerating in cytochalasin-treated windows are not aligned like those incontrol windows (compare Fig. 4 with 5A). This is surprising since the postulated


orienting agent (passive endoplasmic flow; Williamson et al. 1984) is still present.The composition of the bundles assembled in the presence of cytochalasin may bealtered so that they are less easily deformed and can therefore resist the forcesexperienced when aligned at an angle to the direction of flow. Alternatively, flow maybe sufficiently rapid only immediately adjacent to the upstream bundle ends so thatthe mechanism fails when cytochalasin blocks their extension.

In unwounded cells, the average number of actin bundles per chloroplast fileremains nearly constant as a cell grows from =10 mm to 50 mm. Interruptions in theactin bundles were never seen. While cytochalasin reduced the number of bundlesassociated with each file of chloroplasts, neither bundle continuity nor orientationwas disrupted in the way recorded in the windows. These bundles must also grow bya mechanism that lacks the obligatory dependence on barbed-end extension thatmakes the extension of upstream bundles into the window highly sensitive tocytochalasin. It seems more likely that the actin bundles are stretched and aligned bycell extension, presumably recruiting more actin filaments along their length tomaintain their thickness approximately. (Such stretching and alignment is welldocumented for the wall and for the chloroplasts of the cortical cytoplasm (TaizeJ al.1981; Green, 1964).) Since cytochalasin reduces the number of bundles in non-growing cells, even the structure of these apparently stable bundles is cyto-chalasin-sensitive. It remains to be determined whether cytochalasin displaces theG:F equilibrium towards unpolymerized actin (Tellam & Frieden, 1982) and whatcauses the thickening of individual bundles.

Thus we consider that the present results strengthen our previous conclusion(Williamson et al. 1984) that actin bundle assembly in Chara is unlikely to proceedthrough a single mechanism. The minimum requirement seems to be for twomechanisms. First, a mechanism to extend the upstream bundles from the windowedge that is highly cytochalasin-sensitive, most probably as a result of an obligatedependence on the extension of barbed filament ends. Second, one or more mech-anisms operating in the window and in growing cells, that assemble thickened butfunctional bundles in the presence of cytochalasin. An assembly mechanism wouldbe cytochalasin-resistant if it lacked an obligate requirement for barbed-end ex-tension of F-actin. These results suggest that low concentrations of cytochalasinaffect actin polymerization in characean cells, while earlier studies suggest thathigher concentrations rapidly inhibit streaming by abolishing force generation byassembled actin bundles.

We thank Jean Perkin and Peter Jablonski for their helpful comments.

REFERENCES

BLATT, M. R., WESSELS, N. K. & BRIGGS, W. R. (1980). Actin and cortical fiber reticulation in thesiphonaceous alga Vaucheria sessilis. Planta 147, 363-375.

BOSTROM, T. E. & WALKER, N. A. (1976). Intercellular transport in plants. II. Cyclosis and therate of intercellular transport of chloride in Chara. J. exp. Bot. 27, 347-357.

BRADLEY, M. O. (1973). Microfilaments and cytoplasmic streaming: inhibition of streaming bycytochalasin. jf. Cell Sci. 12, 327-343.


GREEN, P. B. (1964). Cinematic observations on the growth and division of chloroplasts inNitella.Am.J.Bot. 51,334-342.

HARTWIG, J. H. & STOSSEL, T. P. (1979). Cytochalasin B and the structure of actin gels.J. molec.Biol. 134, 539-553.

HOCH, H. C. & STAPLES, R. C. (1983). Visualization of actin in situ by rhodamine-conjugatedphalloin in the fungus Uromyces phaseoli. Eur.J. Cell Biol. 32, 52-58.

KAMITSUBO, E. (1972). A 'window technique' for detailed observation of cytoplasmic streaming.Expl Cell Res. 74,613-616.

KAMIYA, N. (1962). Protoplasmic streaming. In Handbuch derPflanzenphysiologie, vol. 17, part 2(ed. W. Ruhland), pp. 979-1035. Berlin, Gottingen, Heidelberg: Springer-Verlag.

KERSEY, Y. M., HEPLER, P. K., PALEVITZ, B. A. & WESSELLS, N. K. (1976). Polarity of actinfilaments in characean algae. Praoc. natn. Acad. Sci. U.SA. 73, 165-167.

KURODA, K. & KAMIYA, N. (1981). Behaviour of cytoplasmic streaming in Nitella duringcentrifugation as revealed by the television centrifuge-microscope. Biorheology 18, 633-641.

MACLEAN-FLETCHER, S. & POLLARD, T. (1980). Mechanism of action of cytochalasin B on actin.Cell 20, 329-341.

NAGAI, R. & KAMIYA, N. (1977). Differential treatment of Chara cells with cytochalasin B withspecial reference to its effect on cytoplasmic streaming. Expl Cell Res. 108, 231-237.

NOTHNAGEL, E. A., BARAK, L., SANGER, J. W. & WEBB, W. W. (1981). Fluorescence studies onmodes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara. J. Cell Biol.88, 364-372.

PALEVITZ, B. A. & HEPLER, P. K. (1975). Identification of actin in situ at the ectoplasm-endoplasm interface of Nitella. Microfilament-chloroplast association.^. Cell Biol. 65, 29-38.

PARTHASARATHY, M. V. (1985). F-actin architecture in coleoptile epidermal cells. Eur.J. Cell Biol.39, 1-12.

PERDUE, T. D. & PARTHASARATHY, M. V. (1985). In situ localisation of F-actin in pollen tubes.Eur.J. Cell Biol. 39, 13-20.

POLLARD, T. D. & MOOSEKER, M. S. (1981). Direct measurement of actin polymerisation rateconstants by electron microscopy of actin. Filaments nucleated by isolated microvillus cores.J. Cell Biol. 88, 654-659.

SHIMMEN, T. & TAZAWA, M. (1983). Control of cytoplasmic streaming by ATP, Mg2+ andcytochalasin B in permeabilised Characeae cell. Protoplasma 115, 18-24.

SUZAKI, T. & WILLIAMSON, R. E. (1985). Euglenoid movement in Euglena fusca: evidence forsliding between pellicular strips. Protoplasma 124, 137-146.

TAIZ, L., METRAUX, J.-P. & RICHMOND, P. A. (1981). Control of cell expansion in the Nitellainternode. In Cytomorphogenesis in Plants (ed. O. Kiermayer), pp. 231-264. Wien, New York:Springer-Verlag.

TELLAM, R. & FRIEDEN, C. (1982). Cytochalasin D and platelet gelsolin accelerate actin polymerformation. A model for regulation of the extent of actin polymer formation in vivo. Biochemistry21, 3207-3214.

WESSELS, N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. O., LUDUENA, M. A., TAYLOR, E. L.,

WRENN, J. T. & YAMADA, K. M. (1971). Microfilaments in cellular and developmentalprocesses. Contractile microfilament machinery of many cell types is reversibly inhibited bycytochalasin B. Science 171, 135-143.

WILLIAMSON, R. E. (1972). A light microscope study of the action of cytochalasin B on the cells andisolated cytoplasm of the Characeae. J. Cell Sci. 10, 811-819.

WILLIAMSON, R. E. (1975). Cytoplasmic streaming in Chara: a cell model activated by ATP andinhibited by cytochalasin B. J. Cell Sci. 17, 655-668.

WILLIAMSON, R. E. (1978). Cytochalasin B stabilises the sub-cortical actin bundles of Charaagainst a solution of low ionic strength. Cytobiologie 18, 107-113.

WILLIAMSON, R. E. (1985). Immobilisation of organelles and actin bundles in the corticalcytoplasm of the alga Chara corallina Klein ex. Wild. Planta 163, 1-8.

WILLIAMSON, R. E., HURLEY, U. A. & PERKIN, L. (1984). Regeneration of actin bundles in Chara:polarised growth and orientation by endoplasmic flow. Eur.J. Cell Biol. 34, 221-228.

WILLIAMSON, R. E., PERKIN, J. L. & HURLEY, U. A. (1985). Selective extraction of Chara actinbundles: identification of actin and two coextracting proteins. Cell Biol. Int. Rep. 9, 547-554.


WILLIAMSON, R. E., PERKIN, J. L., MCCURDY, D. W., CRAIG, S. & HURLEY, U. A. (1986).

Production and use of monoclonal antibodies to study the cytoskeleton and other components ofthe cortical cytoplasm of Cham. Eur.J. Cell Biol. (in press).

WITZTUM, A. & PARTHASARATHY, M. V. (1985). Role of actin in chloroplast clustering and bandingin leaves of Egeria, Elodea and Hydrilla. Eur.J. Cell Biol. 39, 21-26.

YAHARA, I., HARADA, F., SEKITA, S., YOSHIHIRA, K. & NATORI, S. (1982). Correlation betweeneffects of 24 different cytochalasins on cellular structures and cellular events and those on actinin vitw.J. Cell Biol. 92, 69-78.

{Received 7April 1986 -Accepted 1 May 1986)

Documents

GROWTH AND REGROWTH OF ACTIN BUNDLES IN CHARA: … · 2005-08-25 · maintained normal bundle orientation and rapid cytoplasmic streaming, ... albeit in modified form. It is suggested