Journal of Cell Science 102, 611-627 (1992) 611 Printed in Great Britain © The Company of Biologists Limited 1992

Actin has multiple roles in the formation and architecture of zoospores of the , ferax and bisexualis

I. BRENT HEATH*

Biology Department, York University, 4700 Keele Street, North York, Ontario M3J 1P3, Canada and RUTH L. HAROLD

Department of Biochemistry, Colorado State University, Fort Collins, Colorado 80523, USA *Author for correspondence

Summary

Very similar changing patterns of actin are described encystment there is a dramatic increase in stained actin with rhodamine-phalloidin labelling during the zo- in the form of peripheral plaques associated with the osporic life cycle of the oomycetes, Saprolegnia ferax and newly synthesized cell wall. . By comparing the changes with When the cysts germinate, a fibrillar actin cap, previously described ultrastructural and functional comparable to that previously described in hyphal tips, changes, we show that actin functions in numerous forms in the germ tube apex, but only after cell wall previously unrecognized processes. softening to permit germ tube protrusion. This sequence Most spectacularly, the directed vesicle expansions of is consistent with the actin cap modulating turgor-driven the cytokinetic system involve newly formed actin which expansion of the tip as previously discussed for hyphae. outlines the developing zoospores. Disruption of this In addition to disrupting cleavage-associated actin, actin with cytochalasins leads to abnormal cleavage as cytochalasins show developmental stage, dose and drug witnessed by the formation of enlarged and irregular (CE>CD>CB) specific effects on zoosporulation-related cysts. Prior to cytokinesis, two new types of organelle are actin, which indicates that, contrary to previous sugges- synthesized and one, known as K bodies, clusters around tions, rhodamine-phalloidin staining is a useful indicator the nuclei. These organdies are actin-rich during of actin behaviour in response to cytochalasins. These development and clustering, consistent with actin func- responses include differential effects on adjoining actin tioning in their positioning. arrays, some of which are transient in the continued In the zoospores, actin is concentrated around the presence of the drugs, indicating a mechanism of drug water expulsion vacuoles, indicating that they are adaptation. contractile, and permeates the cytoplasm, probably with a skeletal role. This concept is supported by the first Key words: actin, rhodamine-phalloidin, cells, demonstration of actin specifically associated with a cytokinesis, organelle positioning, cytochalasins, fungi, microtubular root in the secondary zoospore. Upon contractile vacuoles, tip growth, oomycetes.

Introduction situation is seen during tip growth of pollen tubes, root hairs and fungal hyphae where the locally extensible, Differentiation during animal development involves newly synthesized apical cell wall yields to turgour many cellular shape changes mediated by both the pressure to generate the familiar tube shape. There is cytoskeleton and selective cell-substrate interactions. growing evidence that cytoskeletal elements, especially In contrast, in plants and fungi many aspects of actin, play some role in this morphogenesis (see reviews differentiation are mediated by selected cell wall by Heath, 1990a,b; Jackson and Heath, 1990a) but the extensibility with turgour pressure providing the force role is not easily understood because of both concomi- to produce cell expansion. Nevertheless, even in walled tant changes in the cell wall and the fact that tip growth cells the cytoskeleton clearly plays a role in aspects of involves at least six coordinated activities, each of morphogenesis but the role is not easily deduced which could utilize cytoskeletal components (Heath, because of the concomitant behaviour of the cell wall 1990b). Oomycetes, such as Saprolegnia and Achlya (see reviews by Lloyd, 1982). An example of this species, offer an unusual opportunity to clarify some of 612 /. B. Heath and R. L. Harold the uncertainties because the hyphal tip can be induced to Rhodamine B or Nile Red, all with the same protocol and stop growing and produce biflagellate zoospores which, in concentration (4 /AM) as RP. turn, can reinitiate tip growth. The changes are rapid and Zoospores were produced from colonies grown in liquid highly reproducible and we know the major organizatio- OM for 18 h followed by 5 X 1 h rinses in DS and a further 2.5 nal changes (e.g. see Heath et al., 1971; Heath and h (primary zoospores) or 6 h (secondary zoospores) in DS, all at approx. 24°C. Some secondary zoospores were examined Greenwood, 1970, 1971; Gay et al., 1971; Holloway and from colonies incubated in DS for a further 13 h at 4°C Heath, 1977a,b; Lehnen and Powell, 1991). These followed by 40 min at approx. 24°C prior to fixation. changes include new patterns of cell wall synthesis, Germinating cysts, mixed with zoospores and empty primary organelle rearrangements, new organelle synthesis, cyto- cysts, were produced via this latter protocol with a further 14 kinesis, development and maintenance of motile cell min at 24°C, followed by harvesting the free cells from the DS (zoospore) shape and the initiation of new polarized cell solution by centrifugation at 1,200 gmax for 2 min and growth. Such changes are common to many organisms. resuspension in OM for 1.7 h at 24°C. All zoospore and cyst We previously exploited the 'natural experiments' in populations were fixed by adding a volume of double strength these changes, together with anti-microtubule inhibitor fixative equal to the volume of DS or OM in the Petri dish. studies, to investigate the roles of microtubules (e.g. see Medium and fixative were mixed by gentle swirling and left for a few minutes prior to harvesting by pipette into centrifuge Holloway and Heath, 1977a; Heath et al., 1982). In this tubes, concentration by two spins at 1,200 gmax for approx. 2 report we extend our analysis by continuing our obser- min each, then resuspension in either buffer or RP for 5 min. vations on the behaviour of actin in Saprolegnia hyphae Some preparations were mounted on slides in a 1:1 mixture of (Heath, 1987,1988; Jackson and Heath, 1989,1990a, b; RP and Citifluor in phosphate-buffered saline (Marivac Ltd., McKerracher and Heath, 1987; Heath and Kaminskyj, Nova Scotia), others were rinsed (via centrifugation) and 1989) into the asexual reproductive phase of the life mounted in buffer and Citifluor. The total time in fixative cycle of both Saprolegnia and the related genus, varied between 5 and 23 min in different experiments but the Achlya. We show that actin is indeed intimately results were similar in all variations of fixative and staining involved in the processes mentioned above and also protocols, except that the longer fixative time failed to reveal eliminate some of the alternatives on the way it the intracellular staining patterns in the zoospores for which 5 functions during tip growth. Preliminary accounts of min total fixation seemed optimal. > ' •' • this work were presented at the International Society of Cytochalasin experiments were performed on. hyphae Evolutionary Protistology meeting in Maryland in June, grown on dialysis membrane overlying OM agar for 15 h, 1990 and the International Fungal meeting in fastened to the membrane by agar as described above, then incubated for 2 x 1 h in DS followed by between 2.2 h and Georgia in August, 1991. 22.2 h in DS containing cytochalasins and DMSO, all at approx. 24°C. Stock solutions contained 5 mg/ml (D) or 10 mg/ml (B and E) of cytochalasin in DMSO and were added to the DS to give the appropriate concentrations. For each Materials and methods cytochalasin treatment, 4-5 colonies were incubated in 10 ml of cytochalasin solution in the dark prior to fixation and RP Saprolegnia staining. Spore sizes were measured from unfixed 22.17 h Hyphae of Saprolegnia ferax (Gruith.) Thuret (ATCC no. colonies observed with DIC optics and a video camera giving 36051) were grown on dialysis membrane overlying a nutrient a screen magnification of X1750. Median optical sections of agar designated as OM by Heath and Greenwood (1968). in randomly picked fieldso f view were measured to the Hyphal tips were induced to form zoosporangia by two nearest mm with a rule from the screen. For aspherical spores different protocols, which gave identical results: the means of the maximum and minimum diameters were (1) Portions of the margins of colonies containing many recorded. Diameters of empty, full but ungerminated and hyphal tips were floated off the membrane in liquid OM, germinating cysts were combined because subjective obser- picked up on a glass coverslip, covered by a drop of OM and vations of the separate population measurements showed no incubated overnight, over water, in a Petri dish at approx. evident differences. The largest spore recorded from an 4°C. These hyphae were then floated in a dilute salts solution untreated population was 11.4 fim, thus the next largest size (DS; Sistrom and Machlis, 1955), picked up on a coverslip and recorded (=12 ,um) defined the lower limit of the 'large cyst' embedded in a thin layer of 0.5% water agar only at their most category. proximal ends. These colonies were further incubated for 2 h All fluorescent observations were made with a 1.32 NA, in fresh DS followed by a final incubation of 3 to 5 h X100 objective lens, a G2 filter cube for RP (Heath, 1988) and (depending on developmental stage required) in more DS, all a V2 cube (excitation 395-446 nm, barrier 470 nm, dichroic at 24°C. mirror 460 nm) for mithramycin. (2) The superior protocol involved dipping the proximal Developing sporangia were prepared for electron mi- edge of the hyphae growing on the dialysis membrane in cool croscopy as previously described (Heath and Greenwood, 2% water agar, which kept them attached throughout the DS 1971). transfers listed in (1) and subsequent staining. Zoosporangia were fixed in either 6% formaldehyde in Achlya PIPES buffer with protease inhibitors and selected ions or 4% Achlya bisexualis Coker, $ (ATCC no. 14524), was main- formaldehyde in PIPES alone for 5 min, as previously tained on a complete nutrient agar, PYG (Kropf et al., 1984), described (Heath, 1987). Both fixatives gave the same results. at 22.5-24°C. Subsequent rinses, rhodamine-phailoidin (RP) and mithra- Extending hyphae and the various stages of zoosporo- mycin staining, mounting and observation were as described genesis were prepared by growing Achlya on dialysis tubing previously (Heath, 1980, 1987, 1988). Some sporangia were (Heath, 1987). Dialysis tubing was autoclaved in medium stained with Bodipy-phallacidin (Molecular Probes, Oregon), DMA3.2 (Schreurs et al., 1989) containing 0.4% locust bean Actin in zoosporulation 613 gum (Sigma). Tubing was placed on DMA32 agar (2% w/v), (for hyphae) and PYG agar (for sporangiogenesis), and innoculated from the growing edge of a colony. After 20 h incubation, 5 x 10 mm rectangles of tubing were cut which included the edge of the hyphal mat as well as room for subsequent extension. For extending hyphae, rectangles were placed on fresh DMA3 2 and 1.5 h were allowed for recovery of growth to the normal extension rate of 4-5 fim/min. To prepare the various stages of zoosporogenesis, the cut hyphal mats on rectangles were placed on SS agar (1 mM potassium PIPES buffer, pH 6.5, 0.25 mM CaCI2, 0.25 mM KC1 and 0.5-0.8% Difco agar noble), and incubated in a humid box. The rectangles were transferred to fresh SS agar after 1 h to eliminate carry-over of nutrients. Hyphae on the edge of the cut rectangle extended into liquid surrounding the dialysis tubing and released spores 5-5.5 h later. Other hyphae remained on the tubing, were subjected to some dryness and released spores 5.5 to 6.5 h later. Development of some sporangia was abnormal with 2 discharge papillae but this did not affect actin distribution. The actin cytoskeleton was visualized using RP as for Fig. 1. Diagrammatic representation of the life cycle of Saprolegnia. Saprolegnia. A coenocytic hyphal tip (A) stops growing All fluorescent observations used a 1.30 NA, X100 when transferred to DS, swells (B) and becomes delimited Neofluar objective and a Zeiss No. 15 filter set (excitation 540- as a by a cross wall (C). Intercalary sporangia 552 nm, barrier 590 nm, dichroic mirror 580 nm). may also form sub-apically by insertion of two cross walls in an older region of a hypha. Within the sporangium the All figures are of Saprolegnia, except Figs 29-37, which nuclei (black circles) become arranged around the show Achlya, are reproduced at X1125 and show parfocal periphery (C, D), an apical papillum forms (D), and corresponding Nomarski differential interference contrast enlarging vacuoles cleave the cytoplasm (D) into (DIC) and fluorescence images, unless otherwise noted. uninucleate, biflagellate primary zoospores (E). These spores are released following rupture of the apical papillum Results (E), swim erratically and briefly as pyriform cells with apically inserted flagella (F) prior to withdrawing their flagella, becoming spherical and secreting a wall to become Overview of developmental stages a primary cyst (G). Primary cysts remaining in DS typically The asexual life cycles of Saprolegna and Achlya species produce a.short determinate exit tube through which the are very similar and are summarized in Fig. 1. Within secondary zoospore is released (H). The secondary this cycle there is synchrony but diverse stages do occur zoospore assumes a reniform shape with two, laterally together. However, the distinctive morphologies of the inserted, flagella (1) and swims rapidly and smoothly until stages of zoosporogenesis (Gay and Greenwood, 1966) it sheds its flagella, rounds up and becomes a secondary and primary and secondary zoospores permit their cyst (J). Repeated emergence of secondary type zoospores is known, but under our conditions the secondary cysts identification. We shall describe Saprolegnia first, typically remained as cysts until germination, via a germ followed by Achlya for comparison. tube, into a new hypha, was initiated by transfer to OM (K). This entire cycle, from A to J, occurs in about 7-10 h Hyphal actin after transfer to DS. The cycle of Achlya is similar except Because of the actin specificity of RP-staining (Heath, that the primary zoospores encyst in a cluster around the 1987), we shall refer to RP-positive material as actin. papillum immediately after release from the sporangium. Hyphae fixed during active tip growth contained an apical cap of fine fibrils which transformed sub-apically into coarser, more widely dispersed fibrils interspersed with peripheral plaques (Fig. 2). All of these structures The cytoplasmic spots were initially uniformly dis- were exclusively peripheral, as verified by optical persed throughout the cytoplasm (Fig. 5) but concomi- sectioning (Heath, 1987). The sub-apical pattern of tantly with cross wall formation some began to cluster fibrils and plaques were uniform throughout the hyphae (Figs 6-8). In a sample of 16 sporangia fixed at about the for many mm. time of cross wall insertion, 3 had no cross wall and no clusters, 2 had no cross wall and a few small clusters and Sporangial actin 11 had cross walls and larger and more abundant Early in sporangium development, prior to cross wall clusters. Clusters typically containing about 5 spots formation, the apical actin caps and most coarse sub- were associated with nuclei (Figs 6-8). In paired DIC apical actin fibrils dispersed (Figs 3, 4). Fibrils persisted and fluorescence micrographs of 18 sporangia, 73% of longer near the apex of the sporangium and were 143 clusters were associated with nucleus-like structures sparsely present during papillum formation (Figs 3, 4). in the DIC images (e.g. Figs 6, 7). The remaining 27% The dominant forms of actin in the developing may have been associated with nuclei above or below sporangia were residual peripheral plaques and a new the focal plane because through-focus series were not population of spots deeper in the cytoplasm (Fig. 5). taken. Most clusters lay between the nuclei and the 614 /. B. Heath and R. L. Harold

plasmalemma, rather than on the inner side of the ment until just prior to cytoplasmic cleavage. They were nuclei. The cytoplasmic spots, both clustered and also stained by Bodipy - phallacidin but not by either dispersed, persisted throughout sporangium develop- Nile Red or Rhodamine B, both of which showed rather Actin in oomycete zoosporulation 615

Fig. 2. DIC (A) and fluorescence (B) images of a typical incipient zoospores, their membranes associated with subapical (> 100 /m\ from tip) region of a RP-stained sheet-like arrays of actin which formed polygonal vegetative hypha. The optical plane of section is at the cell patterns in optical section (Figs 12-15). These arrays surface such that the peripheral actin plaques and initially co-existed with the peripheral plaques and prominent fibrils are clear (B). cytoplasmic spots (Figs 12, 13) but later the cleavage Figs 3 and 4. Paired images of two young sporangia with the DIC images (A) focussed at the centre of the sporangia actin dominated (Figs 14, 15). The nearly cleaved to show their early stages of development and the zoospores were permeated by a low concentration of fluorescent images (B) focussed at the cell surface to show diffuse actin which was heterogeneous as if excluded the abundant actin plaques and the almost complete from organelles (Fig. 15). This diffuse cytoplasmic actin absence of the fibrils (cf. Fig. 2). These sporangia appear persisted after cytokinesis (Fig. 16), but the actin to be at early stages of papillum formation (complete formerly associated with the cleavage vacuole mem- papillae are seen in Figs 12-14) as shown by the branes (which became part of the zoospore plasma- homogeneous cytoplasm in the slight apical protrusions. lemma) was lost (Fig. 16). The cleaved, pre-release Note the greater abundance of residual actin filaments in zoospores each contained a single hollow sphere of these regions (arrow). actin (Fig. 16). Fig. 5. An early stage in sporangium development. Both DIC (A) and RP (B) images are focussed at the centre of Throughout zoosporulation, the organization of actin the sporangium. Cytoplasmic spots dispersed throughout elsewhere in the colonies, including in the sporangium- the cytoplasm are evident in B. bearing hyphae, remained unchanged. Fig. 6. An unusually small sporangium at a later stage of development than that in Fig. 5. Dispersed cytoplasmic Primary zoospores spots permeate the cytoplasm but three prominent clusters Released zoospores retained their diffuse cytoplasmic in B can be correlated with DIC images of nuclei (arrows) actin (Figs 17,18) but the spheres of actin (Fig. 17) were in A. absent. However, similarly sized regions devoid of Fig. 7. A sporangium at a comparable stage of cytoplasmic actin coincided with water expulsion development to that shown in Fig. 6. Peripheral plaques vacuoles (wev) in DIC images (Fig. 17). The plasma- (brackets, B) and dispersed cytoplasmic spots are present lemmae of released zoospores bore uniformly distrib- with numerous clusters predominently located towards the cell periphery, adjacent to the DIC images of nuclei uted fine spots of much lower intensity and size than the (arrows in A and B). peripheral plaques found in walled cells. (Figs 17, 18). Fig. 8. A sporangium at a slightly earlier stage than those There was also a low level of stain in the flagella (Fig. shown in Figs 6 and 7 showing parfocal images stained with 18). mithramycin (A) for nuclei and RP (B). Whilst most cytoplasmic spots are still dispersed, some are clustered Primary cysts with nuclei (arrows in B). Primary cysts contained dramatically more actin than zoospores as shown by their relative fluorescence in a single field of view (cf. Figs 21, 25). This actin formed weak (Nile Red) or very bright (Rhodamine B) diffuse peripheral plaques, which were always present (Figs 19, cytoplasmic staining. 20), and an eccentric cluster of about 5 similarly sized In order to identify the cytoplasmic spots in pre- internal spots (Fig. 19), which were not always present cleavage sporangia, we compared the numbers and (Fig. 20). Younger cysts (with colonies in DS for shorter distribution of actin spots and organelles. There were times) more frequently lacked the clustered spots than 125 (mean = 124.5 ± 17.2, n = 4) spots per median cysts from older colonies. Serial optical sections optical section per sporangium. A median longitudinal through 15 cysts (e.g. Figs 19, 20) revealed 69.9 ± 8.7 section of a similar sporangium prepared for electron peripheral plaques per cyst. microscopy showed 290 dispersed dense bodies (Fig. During excystment of secondary zoospores, the 10) (Gay and Greenwood, 1966), 8 clustered K bodies cytoplasm contained a low level of diffuse actin with (Fig. 9) (Heath and Greenwood, 1971; Holloway and small punctate regions (Fig. 21). No actin remained in Heath, 1977a), 241 dispersed bars (Fig. 9) (Gay and the empty cysts (Figs 21, 25). Greenwood, 1966) and 70 unidentified and previously unreported dispersed "dark vesicles" (Fig. 10). The Secondary zoospores latter were characterized by a moderately stained The level of staining in secondary zoospores was lumen and apparently radially arranged material on consistently lower than in either primary or secondary their surfaces (Fig. 10). cysts (Figs 22, 25, 28). However, all secondary Septa judged to be undergoing synthesis, based on zoospores had diffuse cytoplasmic actin with inhomoge- the stage of development of the cytoplasm, were neities indicating exclusion from unidentified sub- associated with a high concentration of peripheral cellular compartments (e.g. Figs 23, 24) and about half plaques and diffuse cytoplasmic actin (Fig. 11). The had fine spots on the plasmalemma (Fig. 25). Spores plaques persisted until all plaques were lost late in fixed for more than 5 min showed no other staining, but sporogenesis but the diffuse actin dispersed rapidly. shorter fixations consistently showed strong staining One of the most conspicuous arrays of actin in around the wev and a linear structure running along the developing sporangia was associated with cytoplasmic groove towards the opposite end of the cell (Figs 22-25). cleavage. As the cleavage vacuoles began to outline The wev is located on one side in the anterior of the cell 616 /. B. Heath and R. L. Harold Actin in oomycete zoosporulation 617

Fig. 9. Portion of the cytoplasm from a developing Fig. 10. Another portion of cytoplasm from the sporangium, showing K bodies with fibrillar contents and sporangium shown in Fig. 9, showing dense bodies (db) undulating membrane (k), bars (b), a dense body (db), a and dark vesicles with amorphous contents and smooth nucleus (N), kinetosomes (K) and associated microtubular membranes (V) and apparently radially arranged material roots (r). Bar, 1 fjm. on their surfaces (arrows). Bar, 1 jxm.

(Holloway and Heath, 1977b). Thus the linear structure or 5 rather irregular dots interconnected with less was posterior and in 14 out of 18 spores it lay in the intensely stained material (Fig. 22), or as a simple linear opposite side of the cell. It appeared either as a line of 4 structure (Figs 23-25).

Fig. 11. Basal region of a developing sporangium judged by its cytoplasmic appearance (A) to be close to the time of septum synthesis. The RP image was exposed to highlight the high concentration of actin associated with the septum (arrow, B). x 562. Fig. 12. Sporangium at a later stage than those shown in Figs 5-8, as indicated by the mature apical papillum (large arrow, A). In the images near the surface (A, B) peripheral plaques (brackets, B) and cytoplasmic spots clustered with nuclei (small arrows, A and B) are evident but less intensely stained than comparable structures in Figs 5-7. Actin is beginning to be associated with the cleavage vacuoles (double arrows in A and B) and is more clearly seen as the somewhat diffuse polygonal arrays in the deeper focal plane in C (arrows). Fig. 13. Sporangium at a comparable stage of development to that in Fig. 12. Cleavage vacuoles, with associated actin, are clear between nucleate (N) masses of cytoplasm (arrows A and B). Peripheral plaques are still evident in B but cytoplasmic spots are less prominent. 618 /. B. Heath and R. L. Harold

Fig. 14. Sporangium at a stage inferred to be later than those in Figs 12 and 13. All cytoplasmic spots and peripheral plaques are absent (B) and the cleavage-associated polygonal arrays are sharper than in the earlier stages (B), although the cleavage vacuoles themselves are not well contrasted in the DlC image (A). Fig. 15. Sporangium at a comparable stage to Fig. 14 but with much more clearly detectable cleavage vacuoles in the DlC image (arrows, A). Actin is associated with the cleavage vacuoles (arrows, B). The difference in clarity of the cleavage vacuoles in the DlC images between Figs 14A and 15A may be related to unexplained fixation-induced cytoplasmic shrinkage, since such is evident in Fig. 15. Note the diffuse but inhomogeneous staining permeating the cytoplasm of the incipient zoospores in B. Note also the abundant peripheral plaques and fibrils in the hypha lying across the sporangium in B. Fig. 16. Post-cleavage sporangium. The primary zoospores have begun to assume their pyriform shape (e.g. arrows in A) and flagella profiles are detectable (circled in A). The image in B is from a slightly different focal plane and shows diffuse, but inhomogeneous, cytoplasmic staining and spherical concentrations of actin, some of which can be correlated with "depressions" in the DIC image, which most likely represent water expulsion vacuoles (arrows in B, correlate to A).

Secondary cysts similar presence and absence of the eccentrically Older cultures contained many unstained empty cysts located clusters of cytoplasmic spots (Fig. 25). The only (presumably primaries) and full ones (Fig. 25). We difference was that these clusters were typically larger assume that the latter were secondary cysts. Their than in primary cysts (compare Figs 19 and 25) with as staining pattern was similar to that of primary cysts with many as 20 spots detected per cluster. Actin in oomycete zoosporulation 619

Germinating cysts more abundant than in controls (compare Fig. 41 with Germinating cysts initially looked very similar to cysts, Fig. 2). Cleavage vacuole membranes did not stain, but showing peripheral plaques with and without a cluster pre-release zoospores showed normal diffuse cytoplas- of cytoplasmic spots. However, from the earliest sign of mic actin while lacking the peri-wev staining. Primary germ tube (or exit tube) formation, they developed a cysts showed normal staining but more appeared fine peripheral array of filaments enclosing the germ unstained. Many of the cysts were larger (mean tube apex (Figs 26, 27). In spite of an intensive search, diameter 12.67 ± 3.44 pm, n = 50; largest cyst = 20.5 this array, which is comparable to the apical cap of jum;% large cysts = 48.0) and more irregular (cf. Figs hyphae, was never found in cysts which lacked a 43, 44) compared with the remarkably uniform control protuberance of the wall, i.e., an incipient germ tube. populations (Fig. 42: mean diameter 9.83 ± 0.81 fim, n As the germ tube elongated, the peripheral plaques in = 56; largest cyst = 11.4 /im; % large = 0). In contrast, the cyst persisted and an apical cap and sub-apical germ tubes through which secondary zoospores were plaques and cables typical of hyphae developed in the released in the drug were of normal size and shape, germ tube (Fig. 28). even on enlarged cysts (Fig. 43). Increasing the concentration of cytochalasin D to 20 Achlya actin behaviour (igfm\ elicited similar but more dramatic effects than The behaviour of actin during active tip growth and observed with 10 /ig/ml. Again, enlarged cysts (mean zoosporogenesis in Achlya was similar to that in diameter 12.80 ± 3.34 fj.m, n = 72; largest cyst = 22.8 Saprolegnia. Growing hyphae contained an apical cap jum; % large cysts = 48.6), with normal germ tubes, and sub-apical peripheral plaques and cables (Fig. 29). were produced but many fewer sporangia and spores The cap and cables were lost early in sporangium formed (Fig. 45). The cytoplasm in most hyphae was formation and replaced by cytoplasmic spots, both abnormally clumped and deformed, suggesting general dispersed and clustered (Fig. 30). Actin concentrated toxicity. around the developing septa of the sporangia (Fig. 31). Cytochalasin E at 5 jug/ml produced comparable During cleavage, actin associated with the cleavage results to cytochalasin D at 10 fig/ml. Terminal vacuoles, forming polygonal boundaries; the cytoplas- sporangia were apparently shorter and wider, interca- mic spots were barely detectable (Fig. 32). After lary sporangia were abundant and hyphae showed a cleavage, the cleavage actin became less prominent high density of peripheral actin plaques but no fibrils (Fig. 33) and the incipient zoospores contained diffuse throughout the incubation period. Spore production cytoplasmic actin and, contrary to Saprolegnia, clus- and cyst sizes were normal, as were the actin patterns tered cytoplasmic spots (Fig. 34). This pattern persisted throughout zoosporulation and in the cysts. Only the during zoospore release (Fig. 35). Primary cysts peri-wev actin in the pre-release primary zoospores was contained much more stained actin than the zoospores, not observed. mostly in the form of peripheral plaques (Figs 36, 37) At 10 |Ug/ml, cytochalasin E totally suppressed with some cysts containing clustered cytoplasmic spots sporulation (Fig. 46). Hyphal tips swelled but probably (Fig. 37). only prior to addition of the drug. No cross walls, papillae or zoospores formed. After 2.7 h in the drug, Cytochalasin treatments the swollen hyphal tips (incipient sporangia) contained The effects of cytochalasins were determined for peripheral actin plaques and a few isolated cytoplasmic Saprolegnia only. Cytochalasins D and E (but not B at spots and the subtending hyphae had abundant plaques up to 25 f

Fig. 17. Two primary zoospores showing diffuse but inhomogeneous cytoplasmic staining (B) with fine peripheral spots and zones of unstained cytoplasm in regions likely to correlate to the water expulsion vacuoles (arrows in B). Fig. 18. A primary zoospore with the RP images exposed to show the diffuse cytoplasmic staining with fine peripheral spots in (B) and the much fainter staining in (C). Fig. 19. Through focus series of three primary cysts, all of which show the typical peripheral plaques and two of which (upper and lower in D and E, and E, F and G, respectively) show single clusters of spots within the cytoplasm. Note that these clusters are smaller than those in secondary cysts (Fig. 25). Fig. 20. Through focus series of six primary cysts none of which shows internal clusters in the cytoplasm. The DIC image (A) is parfocal with (D). Fig. 21. An excysting secondary spore showing a much lower level of diffuse but inhomogeneous cytoplasmic staining relative to adjacent cysts (c in B) with a few fine fibrils in the "tail" of cytoplasm (arrow in B) still in the nearly empty cyst (arrow in A). Fig. 22. A secondary zoospore showing actin concentrated around the water expulsion vacuole (open arrows) and along the opposite side of the groove (small arrow, A) in Figs 26-28. Three cysts at different stages of germ tube the posterior part of the spore (small arrow, B). Note also formation. The peripheral plaques, characteristic of the diffuse inhomogeneous staining permeating the ungerminated cysts, are evident in all cysts. At the earliest cytoplasm but at a low intensity relative to the cyst (c in stage of germ tube formation (Fig. 26) a few fine fibrils are B). present as a rudimentary cap in the germ tube apex (Fig. Figs 23 and 24. Secondary zoospores in different 26, B). This cap becomes more extensive as the germ tubes orientations, showing actin concentrated around the water become longer (Figs 27 B and 28 B). Note the low level of expulsion vacuoles (open arrows) and along the groove of staining, relative to the cysts, in the zoospore (Z) in Fig. the spores (small arrows). In each spore, the cytoplasm is 28 B. permeated by diffuse inhomogeneous stain. Fig. 25. A group of secondary cysts, one of which bears a germ tube (X), an empty cyst (arrow, A) and a secondary ever, it is directly applicable to the recently re-described zoospore (Z). (B) and (C) are exposed at different levels vesicle expansion-based cytokinetic apparatus in the to illustrate the dramatic differences in total stained actin zoosporangia of another oomycete, cin- between cysts and the zoospore, the actin pattern, namomi (Hyde et al., 1991), a genus in which zoospore including peripheral spots, in the zoospore (arrow, B) and cleavage sensitivity to cytochalasins has previously been the large clusters of cytoplasmic spots in the cysts (arrows, described (Oertel and Jelke, 1986). Actin involvement C). Note also the trace of fibrils in the germ tube apex in in oomycete cytokinesis fits the emerging generality C (open arrow). that cytokinesis in all systems apparently involves actin interacting with cleavage membranes (Hepler and Bonsignore, 1990; Wick, 1991). causal agent in cytokinesis. There has been no previous direct indication of actin involvement in any compar- Organelle arrangements during zoospore development able cytokinetic system. Punctate actin is widely reported in diverse fungi In Saprolegnia, cytokinesis is effected by directed (Heath, 1990b), but, like the peripheral plaques in vacuole expansion (Gay and Greenwood, 1966; Gay et Saprolegnia and Achlya hyphae, it is consistently al., 1971) guided, at least in part, by kinetosome- located exclusively in the peripheral cytoplasm, adjac- associated microtubules (Heath and Greenwood, 1971; ent to the plasmalemma. The cytoplasmic spots formed Heath et al., 1982). The morphology of the sheet-like in the sporangia are a novel arrangement for fungal arrays of actin associated with the cleavage vacuoles is actin. They look similar to the RP-positive vesicles most consistent with a role in directing their (presum- which Grolig et al., (1990) showed to be rhodamine, not ably) osmotically driven expansion by appropriately phalloidin, positive in Mougeotia. Such is not the case in localized differential expansions and/or contractions. the sporangia because neither Nile Red nor Rhodamine This role differs in detail from the well known B stained the cytoplasmic spots, yet both stained the contractile ring operating in animal cell cytokinesis (see vesicles of Mougeotia (Grolig et al., 1990). Further- Schroeder, 1975) but both must involve controlled more, Bodipy-phallacidin gave identical staining to RP, actin-based force generation and membrane linkages. It yet its fluorophore is anionic, in contrast to cationic differs more significantly from aiding vesicle translo- rhodamine, and thus unlikely to react in a similar way cations in plant phragmoplasts (Kakimoto and Shi- with any specific cytoplasmic organelle. We conclude baoka, 1987; Schmit and Lambert, 1987, 1990). How- that the cytoplasmic spots are indeed actin-rich. 622 /. B. Heath and R. L. Harold

Because neither conventional fixation nor freeze bars (Gay and Greenwood, 1966; Gay et al., 1971; substitution adequately preserves actin in electron Heath and Greenwood, 1970) are too numerous and microscopic preparations of Saprolegnia (Heath and incorrectly distributed. Only K bodies (Heath and Kaminskyj, 1989), it is not possible to identify directly Greenwood, 1971; Holloway and Heath, 1977a; Lehnen the ultrastructural equivalents of the cytoplasmic spots. and Powell, 1989,1991) coincide in both abundance and Consequently, we used a correlative approach. Organ- distribution with the cytoplasmic spots clustered with elles preexisting in the hyphal cytoplasm can be ruled the pre-cleavage nuclei and only the previously unre- out because their morphology, abundance and distri- ported "dark vesicles" (Fig. 10, cf. similar vesicles in bution do not match the cytoplasmic spots (Heath and pollen tubes (Kappler et al., 1986)) correlate with the Greenwood, 1971). The newly formed dense bodies and dispersed spots. The clustered spots in some of the Actin in oomycete zoosporulation 623

Figs 29-37. RP images of Achlya at various developmental Nevertheless some cells use actin filaments to anchor stages. and orientate flagella (Kleve and Clark, 1980; Uyeda Fig. 29. A hyphal tip focussed at its surface to reveal the and Furuya, 1987,1989), to produce specialized cellular finely fibrillarca p giving way to sub-apical peripheral processes (Pagh and Adelman, 1982), for vesicle plaques and fibrils. morphogenesis (Brugerolle and Bricheux, 1984) or for Fig. 30. A surface focal plane of an early sporangium showing the absence of fibrils, peripheral plaques and intracellular transport and cytokinesis (Cohen et al., dispersed and clustered cytoplasmic spots (arrows). 1984; Hirano et al., 1987; Metenier, 1984; Kersken et Fig. 31. A sporangium at an early stage of development. al., 1986a). Its expected role in the wev is controversial. This median optical section shows abundant unclustered Patterson (1980) suggested that the vacuole is contrac- cytoplasmic spots and extensive staining associated with the tile, implying a role for perivacuolar actin and myosin, developing septum (arrow). yet the data are conflicting (c.f. Allen and Fok, 1988) Fig. 32. Sporangium at a later stage of development than with evidence for (Cohen et al., 1984) and against that shown in Fig. 31. Peripheral plaques are no longer (Metenier, 1984; Kersken et al., 1986b; Cho and Fuller, evident and the clustered cytoplasmic spots in each 1989) the presence of actin and similarly contradictory incipient zoospore are very faint (arrows). However, the results from the effects of inhibitors (Cohen et al., 1984; polygonal arrays associated with the cleavage system are Kersken et al., 1986a). Our results, showing actin obvious against a diffuse and inhomogeneous background. Fig. 33. Base of a sporangium after cytokinesis but before around the vacuole of both primary and secondary the zoospores have developed detectable separate zoospores and the demonstration of myosin 1 around morphology. Actin permeates the cytoplasm and is slightly Acanthamoeba wevs (Baines and Korn, 1990), support more concentrated around the incipent zoospore a role for actin in wev function. Our actin labelling in peripheries. Note zones of reduced cytoplasmic staining the pre-released, but not released, primary zoospores coincident with the likely positions of nuclei (arrows) and suggests that it is easily dissociated during processing, rare faint peripheral plaques (brackets). possibly explaining negative results in previous studies. Fig. 34. Cleaved but pre-released zoospores showing The second novel concentration of zoospore actin is diffuse cytoplasmic stain, clumps of cytoplasmic spots that associated with one of the microtubular roots. The (large arrows) and spore outlines of very fine spots, presumably associated with the zoospore plasma linear actin in the end of the secondary zoospore membranes (small arrows). opposite the water expulsion vacuole coincides with the Fig. 35. Zoospores fixed in the process of release from the octet microtubular root (Holloway and Heath, 1977b; sporangium. Their cytoplasm is permeated by diffuse stain Barr and Allan, 1985; Barr and Desaulniers, 1989; which shows darker regions suggestive of exclusion from Hardham, 1987). Because the staining pattern varies organelles (small arrows) and they contain clustered from linear to interconnected dots, it may represent a cytoplasmic spots (large arrow). site of attachment of an actin population which Fig. 36. A cluster of primary cysts with abundant undergoes variable contraction. This actin is probably peripheral plaques around the tip of an empty sporangium connected to the third novel arrangement, the diffuse (approximate position indicated by white dashes). Note the array permeating the cytoplasm and outlining intra- absence of internal clusters of spots in the cysts. Fig. 37. A cluster of primary cysts, most of which show cellular structures in both zoospore types. We suggest only peripheral plaques, but a few contain faint clusters of that zoospore cytoplasm is permeated by actin connec- internal spots (arrows). ted to the octet and the plasmalemma (the spots on the zoospore surface). Such an array may help generate cell shape and organelle distributions (Holloway and primary and secondary cysts also correlate with the Heath, 1977a) as well as amoeboid excystment of the known behaviour of K bodies (Holloway and Heath, secondary zoospores (Holloway and Heath, 1977b). 1977a; Lehnen and Powell, 1989, 1991). We speculate In addition to the above actin arrays, zoospores must that the cytoplasmic spots are, in fact, actin filaments also contain high levels of G actin because there is a attached to the surface of the K bodies and "dark dramatic difference in RP-stainable (presumably F) vesicles" and that these filaments may be artefactually actin (mostly peripheral plaques) between the zoos- concentrated during fixation (possibly yielding the poric versus cyst stages. Since encystment takes ap- radial material on the vesicle surfaces) by collapsing to proximately 1 min (Holloway and Heath, 1974), de their attachment points from a more diffuse actin novo synthesis is unlikely. Because the cyst, unlike the network. Such a diffuse network may be a precursor to zoospores, is supported by a cell wall, such polymeriz- that seen later in the zoospores. Organelle-associated ation is unlikely to relate to structural integrity. This is actin with possible roles in organelle positioning and simply another example of the consistency of associ- morphogenesis is becoming a widely reported phenom- ation between plaques and walled cells. enon (Abe et al., 1991; Henderson and Locke, 1992a and b; Bourett and Howard, 1992) which may also Hyphal actin relate to previously described fungal cytoplasmic Growing hyphae of Saprolegnia contain an apical matrices (McKerracher and Heath, 1986, 1987). filamentous actin cap and sub-apical peripheral spots and cables (Heath, 1987). Similar arrays in Achlya Actin in the motile cell hyphae extend the generality of this pattern among the Studies of flagellate cells have concentrated on organ- oomycetes. elles or microtubules. Actin has been largely ignored. The actin cap may function in morphogenesis by 624 /. B. Heath and R. L. Harold Actin in oomycete zoosporulation 625

Fig. 38. Surface focal planes of sub-sporangial hyphae from a colony incubated for 2.7 h in DS containing 5 ^g/ml cytochalasin D. Peripheral plaques are present but the normal fibrils are absent (cf. Figs 2 and 39). Fig. 39. Surface focal plane of a sub-sporangial hypha from a colony incubated in DS containing 5 ^g/ml cytochalasin D for 5.2 h. Plaques and fibrils are present in normal abundance. Fig. 40. Typical sporangium from the same colony as Fig. 38. The RP image (B) is exposed such that the actin in the hypha below the cross wall and an adjacent hypha is grossly overexposed (cf. Figs 38, 39) yet virtually no staining is detectable in the sporangium. Fig. 41. Surface focal plane of a hypha incubated in 10 iUg/ml cytochalasin D for 2.7 h, showing increased abundance of plaques and absence of fibrils. Fig. 42. Empty cysts from a normal colony. Note uniform diameter. A full cyst (c) bears a germ tube. Other germ tubes are detectable but out of focus on the empty cysts (arrows). Figs 43 and 44. Enlarged and irregular cysts bearing normal sized germ tubes from a colony incubated in 20 ;Ug/ml cytochalasin D for 22.2 h. Fig. 45. Margin of a colony incubated in 20 |Ug/ml cytochalasin D for 22.2 h. Rather few spores or empty sporangia are present but hyphal tips are swollen and bear undischarged sporangia. Bar, 100 fim. Fig. 46. Margin of a colony incubated in 10 ^g/ml cytochalasin E for 22.2 h. No spores or empty sporangia are seen and the hyphal tips are distorted. Bar, 100 fim. controlling apical expansion (Jackson and Heath, 1990a) but could also generate or maintain factors essential to establishing polarity (Heath, 1990b). Dur- ing cyst germination, a non-polarized cell becomes polarized and produces a germ tube or exit tube. If actin caps establish polarity they should form before germ tube emergence. They did not. They only formed when the cyst wall had been softened to permit localized expansion, consistent with the morphogenic role (Jack- son and Heath, 1990a). However, the formation of normal exit tubes on enlarged cysts in cytochalasins is contrary to this suggestion. Jackson and Heath (1990a) showed that 20-50 ^g/ml of cytochalasin E was needed to disrupt hyphal actin caps, compared with the 10 jug/ml which totally suppressed zoosporulation. It seems likely that the actin caps in both hyphae and exit tubes are more resistant to cytochalasins than the cytokinesis- associated actin, possibly due to differing actin binding proteins (Cooper, 1987). The sub-apical actin cables in hyphae parallel the Fig. 47. Sporangium from a colony incubated in 10 long axes of both the hyphae and the elongated cytochalasin E for 2.7 h. The sporangial shape has formed mitochondria and nuclei. These organelles lose their but the cytoplasm is abnormal and highly vacuolate (A). Only abundant peripheral plaques are seen in the surface elongated shape and orientation along the hyphae focal plane (B). (Heath and Greenwood, 1971) at the same time as Fig. 48. Hyphae incubated in 10 ^g/ml cytochalasin E for cables are lost in the developing sporangia. This 5.2 h. Most peripheral actin plaques and fibrils are lost and correlation is consistent with a role for the cables in replaced by needle-like rods of actin (arrows). generating organelle shape and orientation and is the first indication of their possible function. Our results also help explain the role of the function in wall synthesis. However, their abundance in peripheral actin plaques. Because they form so abun- sub-apical regions of hyphae, where wall synthesis is dantly when new cell walls develop at encystment (cf. miminal (Fevre and Rougier, 1982), and absence in also Temperli et al., 1990), it may be inferred that they growing tips (Heath, 1987; Fig. 29), where synthesis is 626 /. B. Heath and R. L. Harold maximal, counters this inference. They may represent myosin II in Acanthamoeba castellanii by indirect adhesion plaque equivalents (Adams and Pringle, 1984) immunofluorescence and immunogold electron microscopy. J. Cell Biol. Ill, 1895-1904. with cytoskeletal linkages via the plasmalemma to the Barr, D. J. S. and Allan, P. M. E. (1985). A comparison of the cell wall comparable to animal cell adhesion plaque flagellar apparatus in Phytophthora, Saprolegnia, interactions with extracellular matrices (Burridge et al., Thraustochytrium and Rhizidiomyces. Can. J. Bot. 63, 138-154. 1987). This hypothesis is supported by our observation Barr, D. J. S. and Desaulniers, N. L. (1989). The flagellar apparatus in that the only walled stage lacking plaques is just prior to zoospores of Phytophthora sojae f. sp. glycine and P. megasperma Can. J. Bot. 67, 1916-1926. cytokinesis when the plasmalemma is likely to be Bourett, T. M. and Howard, R. J. (1992). Ultrastructural detaching from the sporangium wall prior to zoospore immunolocalization of actin in a . Protoplasma 163, 199-202. discharge. The alternative suggestion, that the plaques Brugerolle, G. and Bricheux, G. (1984). Actin microfilaments are are filasomes (Hoch and Staples, 1983), is unlikely involved in scale formation of the chrysomonad cell Synura. because filasomes are unknown in oomycetes (Heath et Protoplasma 123, 203-212. Burridge, K., Molony, L. and Kelly, T. (1987). Adhesion plaques: al., 1985; Heath and Kaminskyj, 1989; Hoch, 1986). sites of transmembrane interaction between the extracellular matrix and the actin cytoskeleton. J. Cell Sci. Suppl. 8, 211-229. Actin-cytochalasln-phalloidin interactions Cho, C. W. and Fuller, M. S. (1989). Observations of the water Tang et al. (1989) questioned the value of RP staining in expulsion vacuole of Phytophthora palmivora. Protoplasma. 149, cytochalasin-treated cells because RP failed to stain 47-55. Cohen, J., Garreau de Loubresse, N. and Beisson, J. (1984). Actin actin bundles specifically after cytochalasin treatment. microfilaments in Paramecium: localization and role in intracellular However, our observations of cytochalasin-induced movements. Cell Motil. 4, 443-468. rearrangements of RP staining patterns, show the Cooper, J. A. (1987). Effect of cytochalasin and phalloidin on actin. J. technique is indeed informative. For example, the Cell Biol. 105, 1473-1478. transient loss of actin cables, but not plaques in the Fevre, M. and Rougier, M. (1982). Autoradiographic study of hyphal cell wall synthesis of Saprolegnia. Arch. Microbiol. 131, hyphae, in the continued presence of the drug (5 ^g/ml 212-215. cytochalasin D) shows that cells can accommodate to Gay, J. L. and Greenwood, A. D. (1966). Structural aspects of cytochalasins. This, and the correlated cable loss and zoospore production in Saprolegnia ferax with particular reference plaque increase (cytochalasin D and 5 jUg/ml cytochal- to the cell and vacuolar membranes. In The Fungus Spore, Colston asin E) (cf. Schliwa, 1982) and crystal-like aggregate Papers, vol. 18 (ed. M. F. Madelin), pp. 95-110. London: Butterworths. formation (10 jUg/ml cytochalasin E), show that cyto- Gay, J. L., Greenwood, A. D. and Heath, I. B. (1971). The formation chalasin effects cannot be explained by a simple F-actin and behaviour of vacuoles (vesicles) during oosphere development capping model. Conversely, the transient lack of all RP and zoospore germination in Saprolegnia. J. Gen. Microbiol. 65, staining in developing sporangia (5 /ig/ml cytochalasin 233-241. D) suggests a transient cytochalasin-induced change in Grolig, F., Weigang-Kohler, K. and Wagner, G. (1990). Different extent of F-actin bundling in walled cells and in protoplasts of fixation permeabilization of the cell membrane or wall. Mougeotia scalaris. Protoplasma 157, 225-230. The present study has suggested that actin plays Hardham, A. R. (1987). Ultrastructure and serial section previously unrecognized roles in the asexual zoosporic reconstruction of zoospores of the fungus Phytophthora life cycle of the aquatic organisms, Saprolegnia ferax cinnamomi. Exp. My col. 11, 297-306. and Achlya bisexualis. Many of these roles are relevant Heath, I. B. (1980). Apparent absence of chromatin condensation in metaphase mitotic nuclei of Saprolegnia as revealed by to our understanding of general aspects of cell biology mithramycin staining. Exp. Mycol. 4, 105-115. and open up new avenues of investigation in terms of Heath, I. B. (1987). Preservation of a labile cortical array of actin cytoplasmic mechanics. They also reinforce the idea filaments in growing hyphal tips of the fungus Saprolegnia ferax. that these organisms are well suited to further studies of Eur. J. Cell Biol. 44, 10-16. cell development and function. Heath, I. B. (1988). Evidence against a direct role for cortical actin arrays in saltatory organelle motility in hyphae of the fungus Saprolegnia ferax. J. Cell Sci. 91, 41-47. It is a pleasure for one of us (R.L.H.) to acknowledge many Heath, I. B. (1990a). Tip Growth in Plant and Fungal Cells, pp. 351. stimulating discussions with Franklin M. Harold. This work San Diego: Academic Press. was supported by grants from the Natural Sciences and Heath, I. B. (1990b). The roles of actin in tip growth of fungi. Int. Rev. Engineering Research Council of Canada and the Atkinson Cytol. 123, 95-127. Charitable Foundation to I.B.H. and NSF Grant DGB 89 Heath, I. B., Gay, J. L. and Greenwood, A. D. (1971). Cell wall 96200 to F.M. Harold. The skilled assistance of Use Fulton formation in the : cytoplasmic vesicles underlying and Dan Moe is gratefully acknowledged. developing walls../. Gen. Microbiol. 65, 225-232. Heath, I. B. and Greenwood, A. D. (1968). Electron microscopic observations of dividing somatic nuclei in Saprolegnia. J. Gen. Microbiol. 53, 287-289. References Heath, I. B. and Greenwood, A. D. (1970). Wall formation in the Saprolegniales. II. Formation of cysts by the zoospores of Abe, S., You, W. and Davies, E. (1991). Protein bodies in corn Saprolegnia and Dictyuchus. Arch. Microbiol. 75, 67-79. endosperm are enclosed by and emeshed in F-actin. Protoplasma Heath, I. B. and Greenwood, A. D. (1971). Ultrastructural 165, 139-149. observations on the kinetosomes and Golgi bodies during the Adams, A. E. M. and Pringle, J. R. (1984). Relationship of actin and asexual life cycle of Saprolegnia. Z. Zellforsch. Mikrosk. Anat. 112, tubulin distribution to bud growth in wild-type and morphogenetic 371-389. mutant Saccharomyces cerevisiae. J. Cell Biol. 98, 934-945. Heath, I. B., Heath, M. C. and Herr, F. (1982). Motile systems in Allen, R. D. and Fok, A. K. (1988). Membrane dynamics of the fungi. In Prokaryotic and Eukaryotic Flagellae, pp. 563-588. contractile vacuole complex of Paramecium. J. Prolozool. 35, 63- Cambridge: Cambridge University Press. 71. Heath, I. B. and Kaminskyj, S. G. W. (1989). The organization of tip- Baines, I. C. and Korn, E. D. (1990). Localization of myosin 1C and growth related organelles and microtubulcs revealed by Actin in oomycete zoosporulation 627

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(1990). Microinjected fluorescent Jackson, S. L. and Heath, I. B. (1989). Effects of exogenous calcium phalloidin in vivo reveals the F-actin dynamics and assembly in ions on tip growth, intracellular Ca2+ concentration, and actin higher plant mitotic cells. Plant Cell. 2, 129-138. arrays in hyphae of the fungus Saprolegnia ferax. Exp. Mycol. 13, Schreurs, W. J. A., Harold, R. L. and Harold, F. M. (1989). 1-12. Chemotropism and branching as alternative responses of Achlya Jackson, S. L. and Heath, I. B. (1990a). Evidence that actin reinforces bisexualis to amino acids. J. Gen. Microbiol. 135, 2519-2528. the extensible hyphal apex of the oomycete Saprolegnia ferax. Schroeder, T. E. (1975). Dynamics of the contractile ring. In Protoplasma 157, 144-153. Molecules and Cell Movement (ed. S. Inoue and R. E. Stephens), Jackson, S. L. and Heath, I. B. (1990b). Visualization of actin arrays pp. 305-334. New York: Raven Press. in growing hyphae of the fungus Saprolegnia ferax. Protoplasma Sistrom, D. E. and Machlis, L. (1955). The effect of D-glucose on the 154, 66-70. utilization of D-mannose and D-fructose by a filamentous fungus. Kakimoto, T. and Shibaoka, H. (1987). Actin filaments and J. Bact. 70, 50-55. microtubules in the preprophase band and phragmoplast of tobacco Tang, X., Lancelle, S. A. and Hepler, P. K. (1989). Fluorescence cells. Protoplasma 140, 151-156. microscopic localization of actin in pollen tubes: comparison of Kappler, R., Kristen, U. and Morre, D. J. (1986). Membrane flow in actin antibody and phalloidin staining. Cell Motil. Cytoskel. 12, plants: fractionation of growing pollen tubes of tobacco by 216-224. preparative free-flow elcctrophoresis and kinetics of labeling of Temperli, E., Roos, U. P. and Hohl, H. R. (1990). Actin and tubulin endoplasmic reticulum and Golgi apparatus with [3H]leucine. Protoplasma 132, 38-50. cytoskeletons in germlings of the oomycete fungus Phytophthora Kersken, H., Momaye/.i, M., Braun, C. and Plattner, H. (1986a). infestans.. Eur. J. Cell Biol. 53, 75-88. Filamentous actin in Paramecium cells: functional and structural Uyeda, T. Q. P. and Furuya, M. (1987). ATP-induced relative changes correlated with phalloidin affinity labelling in vivo. J. movement between microfilaments and microtubules in Histochem. Cytochem. 34, 455-465. myxomycete . Protoplasma 140, 190-192. Kersken, H., Vilmart-seuwen, J., Momayez, M. and Plattner, H. Uyeda, T. Q. P. and Furuya, M. (1989). Evidence for active (1986b). Filamentous actin in Paramecium cells: mapping by interactions between microfilaments and microtubules in phalloidin affinity labeling in vivo and in vitro. J. Histochem. myxomycete flagellates. J. Cell Biol. 108, 1727-1735. Cytochem. 34, 443-454. Wick, S. M. (1991). Spatial aspects of cytokinesis in plant cells. Curr Kleve, M. G. and Clark, M. W. (1980). Association of actin with Opin. Cell Biol. 3, 253-260. sperm centrioles: isolation of ccntriolar complexes and immunofluorescent localization of actin. J. Cell Biol. 86, 87-95. Kropf, D. L., Caldwell, J. H., Gow, N. A. R. and Harold, F. M. {Received 2 December 1991 - Accepted 13 March 1992)