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 oomycetes, Saprolegnia ferax and Achlya 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. Achlya bisexualis. 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, flagellate 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 Spore 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). spores in randomly picked fields of 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).