Role of the Golgi Apparatus and Extracellular Wall Assembly

J. Cell Set. S3, 35I-37I (1981) 351 Printed in Great Britain © Company of Biologists Limited 1981

DEVELOPMENT OF THE CELL WALL IN TETRASELMIS: ROLE OF THE GOLGI APPARATUS AND EXTRACELLULAR WALL ASSEMBLY

DAVID S. DOMOZYCH,* KENNETH D. STEWART AND KARL R. MATTOX Department of Botany, Miami University, Oxford, Ohio 45056, U.SJl.

SUMMARY The green algal flagellate, Tetrasetmis, is a key transition organism in the phylogeny of green algae. It has been proposed that the cell wall of Tetraselmis arose evolutionarily from the fusion of scales and that this event secondarily caused the alteration of some cytoplasmic processes such as mitotic and cytokinetic mechanisms. Ultrastructural and developmental studies of the cell wall were performed with several strains of Tetraselmis. Two major wall types are reported. The wall of type 1 cells consists of a thick inner region covered by a layer of regularly repeating subunits of 26 ran,comparabl e to the subunits found in the median W2-W6 layer of Chlamy- domonas. The more elaborate type 2 cell wall consists of a thick median wall layer, homologous to the type 1 inner wall, with additional inner and outer strata of hairs, grains and scales. Development of the cell wall begins in the endomembrane system, particularly the Golgi apparatus, where fibrillar tufts and electron-dense droplets are synthesized, modified and transported to the outside. Here, the tufts and droplets are displaced around the protoplast and assemble in several steps to yield the intact wall. Edge-growth assembly of the wall occurs here synchronously with cytoplasmic developments to yield the characteristic anterior flagellar pit. Models explaining various aspects of this development are discussed. When released from the cell, the wall subunits are not completely comparable to stellate scales, but appear to correspond to developmental stages of scales in green flagellates possessing body scales.

INTRODUCTION Tetraselmis (Chlorophyta) is an unusual green algal flagellate possessing intermediate characteristics between 2 groups often separated at the class level, the Prasino- phyceae and the Chlorophyceae. Like many members of the more primitive Prasino- phyceae such as Pyramimonas (Norris & Pearson, 1975; see also Norris, 1980), Tetraselmis possesses scaly and hair-covered flagella that emerge from a central cellular depression, called the pit. In common with members of the advanced Chlorophyceae (Mattox & Stewart, 1977; Stewart & Mattox, 1978), this organism possesses an early-collapsing telophase spindle during mitosis and phycoplast- mediated cytokinesis (Pickett-Heaps, 1975; Mattox & Stewart, 1977). Prasinophytes with body scales lack such phycoplasts and usually possess persistent interzonal spindles during telophase (Rogers, Mattox & Stewart, 1981; Mattox & Stewart, 1977;

• To whom all correspondence and reprint requests should be forwarded at: Section of Plant Biology, Plant Science Building, Cornell University, Ithaca, N.Y. 14853, U.S.A. 352 D. S. Domozych, K. D. Stewart and K. R. Mattox Norris & Pearson, 1975). Therefore, the intermediate position of Tetraselmis makes it crucial to our understanding of green algal phylogeny, in particular the evolution of the Chlorophyceae from the Prasinophyceae. It has been proposed that the early-collapsing telophase spindle and the phycoplast originated in conjunction with the unique evolution of walls from scales in flagellates very much like Tetraselmis (Mattox & Stewart, 1977). According to this hypothesis, the presence of a rigid wall prevents cell elongation during mitosis and spindle elongation (as normally occurs in naked and scale-covered prasinophytes). The phycoplast is interpreted as a microtubular system that ensures the accurate occurrence of cytokinesis between daughter nuclei that do not become widely separated in flagellated cells covered by a rigid wall. From such flagellates as Tetraselmis arise the typical volvocalean flagellates and eventually the higher Chlorophyceae (see Domozych, Stewart & Mattox, 1980, for groups included). Manton & Parke (1965) originally reported that the walls of Tetraselmis developed from Golgi apparatus-derived stellate scales, which fuse extracellularly to yield the final wall-like structure. Also, early chemical analyses (Lewin, 1958; Gooday, 1971) have revealed that the wall consists of neutral and acidic polysaccharides associated with certain protein amino acids, unlike the cellulose walls reported in some higher green algae, but similar to the glycoprotein walls recently reported in volvocalean flagellates (Roberts, 1974). From these considerations, we can suppose that the evolution of the cell wall in a Tetraselmis-like organism was of paramount significance in the evolution of the Chlorophyceae. Unfortunately, a complete, detailed structural and developmental study of this cell wall is lacking. Therefore, this study of the cell wall of Tetraselmis was undertaken for the following reasons: (a) to test further the hypothesis of homology and evolutionary affinity between the Tetraselmis wall, prasinophycean scales and the walls of chlorophycean flagellates; and (b) to analyse the developmental sequence of wall formation; i.e. the mechanisms specifically involved in Golgi apparatus-wall precursor development and extracellular wall assembly.

MATERIALS AND METHODS Cultures used in this study (CCAP 66/8, Tetraselmis striata; CCAP 161/5, T. impellucida; collection of Drs Luigi Provasoli and Irma Pintner, Pisa 6a, T. comolutae) were routinely maintained on LDM (Starr, 1978) liquid medium in 250 ml Erlenmeyer flasks, under a photoregime of 16 h light/8 h dark using 1500 lux (m~* cd sr) of cool white fluorescent light at 18 ±2 deg. C. Cultures were transferred to fresh medium every 14 days. Partial synchrony (50 %) of cell division was obtained in cultures after several transfers, usually occurring at the 3-h mark of the dark cycle. To obtain higher synchrony (80-90 %), o-i ml of 10-day-old liquid culture was aseptically spread over the solid surface of 1-3 % agar/LDM medium contained in plastic Petri dishes. After 12 days of growth (in conditions as above), single colonies were aseptically isolated with a Teflon needle and placed in flasks in fresh LDM liquid and maintained as above. Observation of synchrony was performed using a Zeiss Nomarski optics light microscope and growth stages were monitored at 670 nm and 750 run on a Perkin-Elmer 770 spectrophotometer (1 cm path). Cultures of Pyramimonas inconstans (gift from Dr Luigi Provasoli) and Haematococcus capensis (UTEX-1022) were maintained on enriched ES (Starr, 1978) and modified WHV-o:i (Domozych, Mattox & Stewart, 1981), respectively, under the above conditions. Specimens were prepared for transmission electron microscopy (TEM) examination in the Wall of Tetraselmis: structure and development 353 following manner: log-phase cultures were harvested after 3 h in the dark cycle and centrifuged at 2000 rev./min for 5 min on an International clinical centrifuge (model CL). The resultant supernatant was discarded and the loose pellet was resuspended in 1 ml of fresh LDM medium. To this was added 1 ml of 0-9 % glutaraldehyde (Taab) in o-oi M-cacodylate buffer (pH 7-8). The cells were left in the fixative for 40 min at room temperature, washed in cacodylate buffer (3 times-for 20 min each) and postfixed for 2 h at 15 °C in the dark in 1-2 % OsO4 in o-oi % cacodylate buffer (pH 7'8). The cells were then washed as above, dehydrated through a 10 %- increment acetone series and embedded in Spurr's epoxy resin. Sections were cut on a Sorvall Porter-Blum MT-i ultramicrotome, using a diamond knife. Sections were stained with 1 % uranyl acetate/o-s % lead citrate (5 min each), thoroughly washed with water and observed on an HS-9 Hitachi TEM. To highlight the acidic polysaccharide nature of the large cell wall of T. convolutae, cells were treated as above except for postfixation in 0-5 % ruthenium red/i % OsO4 in o-oi cacodylate buffer (pH 78). Fragment analysis of the T. ttriata wall, Pyramimonas scales and Haematococcus walls were performed as follows: actively growing (log-phase) cells were centrifuged as above and the resultant pellet was resuspended in 3 ml of fresh LDM medium (or cacodylate buffer for the Pyramimonas and Haematococcus). The suspension was sonicated to cavitation for 30 s on a Branson sonifier (60 W). The fragment-containing solution was then centrifuged at 2000 g as above, and the resultant milky-white supernatant was collected and recentrifuged at 10 000 g for 20 min on a Sorvall RC-5 superspeed centrifuge. The resultant pellet was washed and recentrifuged (3 times) in a 0-05 M-Tris-HCl buffer (pH 8-o), then resuspended in 1 ml of the Tris buffer. Drops of this suspension were placed on Formvar-coated, 150-mesh copper grids. After 5 min, the excess drop was blotted off with filter paper and the grids were stained for TEM as above. Drops of the suspension were also recentrifuged (10000 g as above) and fixed for TEM as above. For shadow-cast specimens, specifically of developing walls of T. striata, actively growing cells were collected at 3 h after the onset of the dark cycle and centrifuged at 2000 rev./min as above. The resultant pellet was then macerated gently with a glass stirring rod and the resultant suspension was placed dropwise on Formvar-coated, 150-mesh copper grids. The grids were then blotted dry and washed in a continuous stream of 0-08 M-Tris-HCl (pH 8-2) buffer. Shadow-cast specimens were observed on the TEM as described above.

RESULTS AND DISCUSSION General The genus Tetraselmis (= Platymonas and Prasinocladus; see Norris, Hori & Chihara, 1980) includes flattened, unicellular green algae, which possess 4 scaly and hair-covered flagella and a rigid extracellular wall that covers the protoplast surface, including the characteristic apical depression, the pit (Figs. 1, 2). Another feature of this genus is the possession of a pyrenoid traversed by nuclear or cytoplasmic channels (Fig. 6). Detailed ultrastructural characterization of selected cellular components and events such as the flagellarapparatu s or cell division are not presented here for they have been described adequately elsewhere (Manton & Parke, 1965; Parke & Manton, 1965, 1967; Stewart, Mattox & Chandler, 1974; Melkonian, 1979; Ricketts & Davey, 1980).

Cell wall types and characteristics The entire outer surface of Tetraselmis (flagellar and cellular surfaces) is covered by a variety of structures. The flagella are ensheathed by 2 lateral rows of thin rigid hairs (Fig. 3) and a regularly arranged series of small square scales (Figs. 2, 3). The presence of these flagellar coverings has long been employed as the key feature in 354 D. S. Domozych, K. D. Stewart and.K. R. Mattox

8 Wall of Tetraselmis: structure and development 355 distinguishing ' prasinophycean' algae (Norris, 1980; Manton, Rayns, Ettl & Parke, 1965). At the base of the central pit, where the flagella emerge from the cell, there is a row of stiff, thickened fibres (pit fibres), attached to the wall and protruding outwards towards the opening of the pit (Figs. 4, 5). Pit-fibre length varies among the different species, but all have circular cross-sections (Fig. 5). The function of these unusual fibres is unknown. The cell wall of Tetraselmis is sometimes called the theca (Manton & Parke, 1965). This covering loosely embraces the entire, outer cell body surface, lines the apical depression and terminates abruptly at the pit base in an outcurled formation (Figs. 1, 2). All 4 flagella emerge through a single slot in this curled region. During this survey of the cell walls of 25 strains of Tetraselmis, we noted the existence of 2 major types: simple bi- and trilayered walls exemplified by T. striata and T. impelhidda (type 1); and complex multilayered walls exhibited by T. convolutae (type 2). Different views of the type 1 wall can be seen in Figs. 7-10, 13, 14. Shadow-cast preparations of the T. striata wall reveal a smooth surface covered on the outside by many tiny hairs (Fig. 7). Thin sections of this wall type show an electron-dense, inner, thickened layer with an outer stratum of small subunits (Figs. 8, 9). Fragment analysis of the wall surface revealed the presence of the wall subunits (Fig. 10). Upon close examination, discontinuous linear arrays of regular repeating, circular to polygonal subunits, each approximately 26 run in size (measured from subunit centre to adjacent subunit centre), line the outer wall surface (Figs. 9, 10). The size and linear arrangement of the subunits closely resemble the regular repeating subunits of the 'central triplet' layer of the Chlamydomonas cell wall (Roberts, Gurney-Smith & Hills,

Fig. 1. Overall view of cell shape in a T. convolutae motile cell, just before release from the lumen of the old mother cell. Note the anterior depression, the pit (arrows), and the incurling of the cell wall in this region, x 7500. Fig. 2. Magnified view of the pit region in T. convolutae. Note the emergence of the flagella (/), covered by small square scales (arrow). The cell wall in this pit region in- curls. Also note the rhizanchora at the base of the pit (rh) and the abundance of vesicles in the adjacent cytoplasm, x 32000. Fig. 3. Shadow-cast preparation of a flagellum of T. striata, highlighting the % lateral rows of hairs (large arrows) and the regular pattern of ensheathing scales (small arrows) on the flagellar surface, x 20000. Figs. 4, 5. Various sections of the pit fibres of T. striata. In Fig. 4, note the thickened fibres (arrows) in the pit region. Fig. 5 shows a cross-section of these fibres (arrow) revealing their circular profile. Fig. 4, x 34000; Fig. 5, x 33000. Fig. 6. View of the nuclear projection, or channel (c), into the pyrenoid (p) of T. striata. Nuclear, cytoplasmic and chloroplastidic projections of the pyrenoid are key diagnostic features of the Tetraselmis generic complex, x 27000. Fig. 7. Shadow-cast preparation of the cell wall of T. striata. Note the fine hairs projecting outwards from this fragment (arrow), x 60000. Figs. 8, 9. Various sections of the type 1 wall exhibited in T. striata. Fig. 8 reveals the wall as an inner, electron-dense layer (large arrow) with an outer attached layer of subunits (small arrows) x 35000. Fig. 9. A glancing section of the type 1 wall reveals the regular repeating subunits (arrow). Note that in this section the centres of the subunits are dark surrounded by a light area and a darkened rim. x 39500. 356 D. S. Domozych, K. D. Stewart and K. R. Mattox * Wall of Tetraselmis: structure and development 357 1972; Roberts, 1974). A wall fragment of the central triplet in the Chlamydomonas-\\kt genus, Haematococcus, can be observed in Fig. 12 to offer visual comparisons of the 2 wall types (see also Roberts, 1974). These observations of ultrastnictural similarities strongly suggest that the cell wall of Tetraselmis is related (and probably ancestral) to the Chlamydomonas cell wall. It is also intriguing to note that the linear pattern of subunit distribution in Tetraselmis is similar to the design of the small, square, inner body scales of scaly prasinophycean genera, such as Pyramimonas (see Fig. 11; also see Moestrup & Walne, 1979). This structural similarity can now be added to the developmental evidence that suggests that the Tetraselmis cell wall arose evolutionarily from the fusion of small body scales (Manton & Parke, 1965; Mattox & Stewart, 1977)-

Fig. 10. Fragment analysis of the cell wall of T. striata. Note the regular, linear arrays of subunits (arrows). Each subunit is about 26 nm in diameter, x 24000. Fig. 11. Inner body scales and their arrangement in regular arrays (arrows) in P. inconstant, x 90000. Fig. 12. Fragment analysis of the cell wall of H. lacustris. Note the regular, linear arrays of subunits (seen here as parallel striations, marked by arrows), x 42000. Figs. 13, 14. Various views of the cell wall of T. hnpelludda. Note that this wall possesses a much thicker inner wall layer (arrow, Fig. 13), and the regular, repeating subunits of the outer surface are not as plainly visible (arrows, Fig. 14) as seen in T. striata. Fig. 13, x 44500; Fig. 14, x 40000. Fig. 15. Overall view of the complex, multilayered wall of T. convolutae (type 2). Note the median, electron-dense layer (large arrow), the inner and outer hairy layers (small arrows), the outermost layer of small grains (tg) and the inner layer of attached scales (sc). Note the presence of another older wall exterior to the wall of a young daughter cell, x 40000. Fig. 16. Magnified view of the cell wall of T. convolutae. Note the large, thickened outer hairs (oh) and the thinner, inner hairs (ih). The innermost, scale-like entities are intimately attached to the rest of the wall by adjacent hairs (arrows), x 79000. Fig. 17. Ruthenium red staining of the cell wall of T. convolutae. Note the intense up- take of the stain in the electron-dense layer (large arrow) and the outer layers of the wall (small arrow), x 60000. Figs. 18, 19. Various surface views of the 2 outer layers of the T. convolutae wall. Fig. 18 shows the outermost layer of fine grains (arrow) arranged in no specific pattern, x 62000. Fig. 19 shows the branched nature (see arrows for branches) of the hairs merging to- gether to yield the thickened outer projection, seen in other sections (Fig. 16). x 63 000. Fig. 20. Cross-section through the pit region of a T. convolutae cell revealing the orien- tation of the Golgi body to the pit base marked by the basal bodies of the nagella (bb). Note that the maturing face (Af) is directed towards this pit region. Various tubules and vesicles (large arrows) can be seen in stages of transport in this region. Note the presence of microtubules in this region (small arrows), x 16000. Fig. 21. Longitudinal section through a Golgi body of an interphase T. convolutae cell. Note the underlying rough/transitional endoplasmic reticulum (er) and the vesicles emerging from this reticulum and travelling to the forming-face cisternae (F). Two major vesicle types occur in association with the Golgi body, smooth vesicles (asterisk) and small coated vesicles (small arrows) located in the forming-face or peripheral regions of the cisternae. Note that in the maturing-face region (M) of the Golgi body large vesicles emerge (large arrow). Also note the inner compact regions of the cisternae and swollen peripheries, x 36000. 358 D. S. Domozych, K. D. Stewart and K. R. Mattox The similar cell wall of T. impellucida is shown in Figs. 13, 14. This wall appears slightly thicker than the T. striata wall, but seems to have a similar outer coating of subunits. The cell wall of T. convolutae (type 2) is much more elaborate than that observed in the type 1 wall. In addition to the coherent, thickened, median wall region (Figs. 15- 19, particularly Figs. 15, 16), which appears to be homologous to the major portion of the type 1 wall (e.g. the inner electron-dense layer covered by the subunits), there exist inner and outer layers of attached hairs. The outer series of these hairs is much longer and thicker than the inner series, and readily takes up the polyanion-complexing agent, ruthenium red (Fig. 17). This staining highlights the acidic polysaccharide contained in the cell wall (Gooday, 1971). A glancing section of these outer hairs (Fig. 19) shows that they consist of a series of thin, underlying branched fibrils that fuse with neighbouring branches as they extend outwards. External to the hairs is a series of fine grains that exhibits no set periodicity (Fig. 18). The inner attached series of hairs is distinctly shorter and thinner than the outer series (Figs. 15, 16). The hairs terminate inwards towards the plasma membrane in small, square to polygonal, scale- like structures (Fig. 16). Though at first believed to be flagellar-scale contaminants, closer examination of this layer suggests that they are real wall components for the following reasons: (a) the scales are located along the entire, inner circumference of the wall and not only in the region of flagellarpenetrance . (b) The scales are intimately attached to the inner wall layers in a precise way, by the scale rim to the tip of the hair (Fig. 16). (c) The scale layer was observed during 5 independent periods of fixation spanning different stages of the cell cycle and photoregime. The appearance of such a scale layer remains somewhat of a mystery and requires further research. The variety of extracellular structures in Tetraselmis links it to the scaly prasinophytes, particularly by its possession of flagellar decoration (e.g. scales, hairs). Primi- tive prasinophytes usually possess cell-covering layers of variously shaped scales (1-5 layers) and a flagellar covering of scales and hairs (2-3 layers) (Norris, 1980). The organisms studied in this paper do not have complex body scales, but possess a thick, rigid layered wall and a reduced number of flagellarhair s and scales. Advanced chlorophycean flagellates (Mattox & Stewart, 1977), such as Haematococcus and Chlamy- domonas, usually possess thin layered walls (Roberts, 1974) and either naked or fine- haired flagella. It appears from this comparison that the body and flagellar coverings became modified and reduced as evolution proceeded from primitive, scaly prasinophytes to walled chlorophycean flagellates.

Development of the cell wall: the interphase Golgi apparatus The development of the Tetraselmis cell wall from a complex extracellular assembly system of fibrillar and electron-dense droplet subunits begins within the endomembrane system of the cell (i.e. endoplasmic reticulum, Golgi bodies, vesicles, etc.). Interphase cells of the examined strains commonly possess 2 Golgi bodies situated on either side of the large central nucleus. The maturing face of each Golgi body faces the base of the flagellar pit (Fig. 20). During interphase, vesicles emerging from this face apparently migrate to the base of the pit and fuse with the plasma membrane. In Wall of Tetraselmis: structure and development 359 the region between the pit and the maturing faces of the Golgi bodies, there is a series of microtubules (Fig. 20, arrows). It is important to note that the Golgi vesicles are directed towards a specific cell area (the pit) that is traversed by cytoskeletal elements (e.g. microtubules) and that they fuse with the plasma membrane in this region. Recent ultrastructural evidence obtained from other eukaryotic systems has also shown directed Golgi vesicle transport as well as the related presence of microtubular and microfilamentous systems (Brown & Franke, 1971; Morre, 1977; Mollenhauer & Morre, 1978). It has been proposed that such systems may be involved in actively promoting vesicle transport and release (microfilaments) or providing directional tracks (microtubules) for vesicle movement. The role of the microtubules of the pit region in vesicle transport in Tetraselmis remains obscure and further research is required to determine their precise function. A longitudinally sectioned profile of a typical Golgi body (Fig. 21) reveals that it is composed of approximately 14 cisternae, flanked by several strata of vesicles at the faces and sides of the Golgi body. No marked membrane polarity (i.e. thickness, staining gradients) from forming to maturing face was observed, as has been reported in some other eukaryotes (Whaley, 1975; Whaley & Dauwalder, 1979; Morre, 1977). The Golgi body seems to be fed by vesicles from either the nuclear envelope or more often from short stretches of rough endoplasmic reticulum (Fig. 21). Transition vesicles can be seen forming in the region between the forming face and underlying endoplasmic reticulum. Coated vesicles with dense coverings are also observed in the region near the forming face, but are more often located at the periphery of the intact Golgi body, either between the nuclear envelope and the Golgi body or between 2 recently divided Golgi bodies (Figs. 21, 26, 27). These vesicles appear to have blebbed off from intact cisternae or are vesicles ready to fuse with the maturing cisternae (Figs. 26, 27). The location of these vesicles suggests that they may be part of a com- munication system between 'non-facial' cisternae and other elements of the endomembrane system (e.g. nuclear envelope, other Golgi bodies, plasma membrane). A series of thick cross-sections through a Golgi body stack can be seen in Figs. 22- 25. In the first 2 views of this series (Figs. 22, 23), intact cisternae from the middle of the Golgi stack can be examined. The central regions of these cisternae are smooth and uninterrupted while the peripheral regions contain irregular fenestrations. Small coated vesicles appear to bleb off from these peripheral regions and surround the cisternae. Cisternae of the maturing face are included in the latter 2 views of this series. In these membranes, vesicles and tubes occur, particularly in the peripheral regions. These cisternae also seem somewhat more irregular than middle-of-the-stack cisternae.

Golgi division Before the onset of cell division each Golgi body divides (Figs. 26, 27). During this process the Golgi body appears structurally similar to the interphase Golgi body except that the cisternae expand laterally to yield very elongate membrane profiles (Fig. 26). Division of the Golgi bodies begins at the region of the maturing face. This process continues until cleavage is complete throughout the forming face to yield 2 360 D. S. Domozych, K. D. Stewart and K. R. Mattox

r- Wall of Tetraselmis: structure and development 361 Golgi bodies (Fig. 27). The daughter Golgi bodies are much smaller than normal interphase Golgi bodies and remain this size until cell division, at which time they separate and migrate to their respective positions around the daughter nuclei. Separa- tion of the Golgi bodies occurs by means of the cell division furrow led by the phycoplast microtubules (Fig. 28). The presence of 4 Golgi bodies per cell in preprophase indicates that the Golgi division products are stable. In many instances there appear to be coated vesicles in the region between the Golgi bodies (Fig. 27). After the Golgi bodies migrate to their respective areas in the young daughter cells, they expand to their interphase size and become active in wall ontogenesis. It should be noted that the beginning of wall development in the endomembrane system does not always coin- cide with the division of the Golgi body and subsequent cell division. During periods of non-motility (Manton & Parke, 1965; Parke & Manton, 1965; Norris et al. 1980), the cell can activate the wall-producing machinery of the endomembrane system to yield multiple wall layers. The triggering device and precise physiological conditions causing multiple-walled conditions, especially in the stalked ' Prastnocladus'-Yike strains are unknown. De novo formation of Golgi bodies was not observed in this study.

Figs. 22-25. Thick serial, cross-sections through an interphase Golgi body of T. convolutae. x 30500. Fig. 22 represents a cisterna in the middle of the Golgi stack. Note the coherent central region of the cisterna with peripheral fenestrations (large arrows). Note also that the peripheral region is in the process of vesiculation (small arrows) and is surrounded by a series of small vesicles. Fig. 23 illustrates a cisterna approaching the maturing face. Note the increased tubu- lization and fenestration in the peripheral regions (arrows). Figs. 24, 25 show cisternae at the maturing face. Note the emergence of tubular vesicles (large arrows) and the small and medium-sized circular vesicles (small arrows, Fig. 25) in this region. Fig. 26. View of Golgi body division in T. striata. Note the lateral expansion of the cisternae at the forming face (F) and the cleavage of the Golgi body (arrow) to yield 2 daughter organelles. This cleavage begins at the maturing face (M). x 30000. Fig. 27. View of post-division Golgi bodies in a transient position, adjacent to each other, in a dividing cell. Note that these Golgi bodies are directed towards the centriole (c) of the dividing cell. On either side of this daughter Golgi body complex, one can discern microtubules (arrow), x 21000. Fig. 28. The separation of daughter Golgi bodies during early cytokinesis. Note the centriole (c) and the phycoplast microtubules (small arrow) between the daughter Golgi bodies. Also observe the approaching cleavage furrow (large arrow), x 24000. Figs. 29-31. Sequence of pit development during wall ontogenesis. Fig. 29 shows the anterior region of a recently divided daughter cell that is as yet pitless. In this region note the emergence of the rhizanchora from the basal body region (bb), making contact with the plasma membrane of the future pit region (arrow), x 27000. Fig. 30 shows that cytoplasmic lobes emerge in this pit region, mimicking the shape of the future pit region. Note the projecting flagellar roots (arrows), lining and perhaps maintaining these cytoplasmic lobes, x 28000. Fig. 31 shows the normal pit configuration, after final wall self-assembly in the anterior region. Note that the wall is in its final form (to) and the flagellar roots now only extend to the rhizanchora (arrow), and not up the cytoplasmic lobes, x 33000. 362 D. S. Domozych, K. D. Stewart andK. R. Mattox

Wall ontogenesis The Golgi bodies of all strains examined undergo major structural and developmental changes during cell-wall ontogenesis. The precise mechanics of the activation of the Golgi apparatus to yield wall precursors vary according to the type of wall produced, either type 1 or type 2. In all test strains activation of the Golgi apparatus occurs immediately after the recently divided Golgi bodies move to their respective positions around the daughter nuclei during phycoplast-mediated cytokinesis (Figs. 43-45). Longitudinally-sectioned views of a wall component producing Golgi body of a type 1 (T. striata) wall producer reveal the following: the overall shape, size and number of cisternae in the Golgi body appear similar to those observed during interphase. However, the cisternae and vesicles, located specifically at the maturing face, swell to yield greatly expanded sacs. Also at this time, the ensheathing endoplasmic reticulum becomes filled with fibrils (Figs. 32, 46) and apparently transfers vesicles to the forming face of the associated Golgi body. Approximately 4 cisternae removed from the forming face, fibrillar material aggregates into irregular tufts on the internal cisternal membrane (Figs. 32, 33). On approaching the maturing face, the fibrillar tufts dissociate from the inner membrane of the cisterna and fill the lumen of the maturing sacs and emerging vesicles (Figs. 33, 34). Magnified views of the swollen maturing-face vesicles reveal the fibrillar tufts aggregating into loose bundles closely resembling the geometry of the ensheathing cisterna or vesicle. These aggregates are

Fig. 32. Longitudinal section of a Golgi body of T. striata (type 1) producing wall precursors. Note in the forming-face region (/) the production of fibrillar tufts on the inner cisternal membranes (small arrows). At the maturing face (m) in cisternae and emerging vesicles, note that the fibrillar tufta disassociate from the membrane and enter the lumen (large arrows), x 35000. Figs. 33, 34. Magnified views of the cisternae and vesicles of wall-producing Golgi bodies. Note the fibrillar tufts in the swollen cisternal lumen (arrows). Fig. 33, x 50000; Fig. 34, x 60000. F'g- 35- View of the pit region of a cell of T. striata revealing transport of wall precursor-containing vesicles and their release to the outside. Note early fusion of a large vesicle with the plasma membrane (large arrow) and the release of the fibrillar tufts (small arrow) from a vesicle that fused earlier, x 54000. Figs. 36, 37. Views of subunit assembly of T. striata, after release around the plasma membrane. Note the fine fibrillar connections (arrows) between the main parts of the fibrillar connections (arrows) between the main parts of the fibrillar tufts. Fig. 36 represents an early stage of self-assembly, x 84000. Fig. 37 shows a later stage when the subunits begin to condense into the final wall, x 76000. Figs. 38-41. Shadow-cast preparations of various stages of wall self-assembly in T. striata. Fig. 38 shows the aggregation of the fibrillar tufts (arrow) into the intact wall in an edge-growth manner, x 44000. Figs. 39, 40 represent magnified views of the aggregation of these subunits (arrows). Note that the subunits aggregate in linear arrays (Fig. 40) before final assembly. Fig. 39, x 56000; Fig. 40, x 43000; Fig. 41 represents an intact type 1 wall after final assembly, x 28000. Wall of Tetraselmis: structure and development 363

41 364 D. S. Domozych, K. D. Stewart and K. R. Mattox Wall of Tetraselmis: structure and development 365 connected to each other by radiating fibrils (Figs. 33, 34). The fibril-filled vesicles travel to the plasma membrane near the basal body complex, fuse with the plasma membrane and release their constituents to the outside of the young daughter cells (Fig. 35). This fibrillar tuft-Golgi membrane association is very similar (and probably phylogenetically homologous) to the Golgi apparatus-associated development of the small, inner-body scales in scaly prasinophytes, such as Pyramimonas (Moestrup & Walne, 1979). In these more primitive scaly flagellates, the fibrillar tufts associated with the inner cisternal membrane are quickly modified into small scales but in Tetraselmis they are released to the outside before further development. After release, the irregularly shaped, fibrillar tufts quickly form a thin stratum around the young daughter cells within the lumen of the old parent cell wall. Precise categorization of the geometry of these wall precursors (i.e. stellate scales, Manton & Parke, 1965) is not warranted. The assembly of these wall subunits into the rigid wall occurs in 2 temporally spaced steps. First, the tufts form loose connections with neighbouring tufts by thin fibrillar contacts (Fig. 36); these loose aggregates appear somewhat stable. Second, the tufts quickly assemble into the finished wall-product by forming an ever-increasing series of fibrillar connections, resulting in a mass

Fig. 42. View of wall assembly in T. impellncida. Unlike other type 1 wall-producing organisms, self-assembly of fibrillaraggregate s rather than fibrillar tufts occurs (arrow), x 60000. Figs. 43-45. Overall views of cell division, cytokinesis and wall ontogenesis in T. convolutae (type 2 wall). Fig. 43 represents a late telophase spindle with 2 daughter nuclei (dn) reforming. Note the random appearance of microtubules in the region between the separated nuclei (arrows), x 30000. Fig. 44 reveals the collapsing spindle, as the 2 daughter nuclei closely approach each other. Note that the maturing face of the Golgi bodies is directed (arrows) towards the centrioles (c). x 18000. Fig. 45 shows a cell during cytokinesis. Note the enlarged, wall-producing Golgi bodies (large arrow) and the thin stratum of wall precursors in the furrow (small arrow), x 23000. Fig. 46. View of the endoplasmic reticulum in a wall-producing cell of T. convolutae. Note that fibrillar material can be seen in the lumen (large arrow) and the overlying Golgi body vesicles (small arrow), x 41000. Figs. 47, 48. Longitudinal sections of wall-producing Golgi bodies in T. convolutae. Fig. 47 shows that fibrillar tufts (small arrows) arise in the forming-face cistemae (/). At the maturing face, these tufts become masked with electron-dense droplets (large arrows). Note that the emergence of the wall precursor-containing vesicles is directed towards the basal bodies (bb). x 30000 Fig. 48 shows the early fibrillar tufts arising from the inner cisternal membranes (small arrows) and the latter electron-dense droplets (large arrows) masking the tufts, x 495°o- Figs. 49-50. Thick cross-sections through a wall-producing Golgi body of T. convolutae. Figs. 49, 50. Note the fibrils attached to the inner cisternal membrane (small arrows) and, in more mature cistemae, the presence of electron-dense droplets (large arrows). Abo note the peripheral fenestrations and vesiculation of the Golgi bodies. Fig. 49, x 32000; Fig. 50, X42500. 366 D. S. Domozych, K. D. Stewart and K. R. Mattox coagulation (Fig. 37). Shadow-cast analysis (Figs. 38-41) of these stages highlights the aggregation of these wall precursors and reveals that the final developmental stage occurs in an edge-growth manner (i.e. the final aggregation of the tufts occurs on a limited front with unfinished aggregates lying ahead of this edge and the finished wall lying behind). More importantly, this edge-growth wall assembly begins in the posterior region of the cell and travels up the plasma membrane towards the anterior or flagellar region (see below; Ricketts & Davey, 1980; Manton & Parke, 1965). It should be noted that the final extracellular, wall assembly in type 1 organisms begins only after the Golgi apparatus ceases producing fibrillar tufts. This extracellular assembly of a multilayered wall, composed of regular repeating subunits from edge-growth (posterior to anterior) assembly of fibrillar tufts, is important for 2 reasons: first, it indicates that elements of the released tufts must differentiate before final assembly to yield the multilayered covering. Second, this type of development is remarkably similar to extracellular assembly of wall precursors in walled, volvocalean flagellates (Fulton, 1978). This evidence, along with evidence previously described (see above), supports the pre-existing hypothesis that close affinities exist between chlorophycean flagellates (Mattox & Stewart, 1977) and the walled Tetraselmis. Recently divided daughter protoplasts of Tetraselmis are spherical and enclosed in the lumen of the old parent cell wall. Before final wall assembly occurs, the central pit must form and the flagella must emerge from the cell. The development of the pit was examined in this study and selected stages can be seen in Figs. 29-31. As final edge- growth assembly of the cell wall approaches the anterior region of the cell, several outstanding cytoplasmic events occur. First, the flagellar roots begin to reform around the basal bodies; the rhizoplasts develop downward and the rhizanchora extend upward attaching to the adjacent plasma membrane (Fig. 29). At this time, the flagellar axonemes emerge from the cell. Next, 2 cytoplasmic lobes, one on either side of the basal bodies, extend upward to yield a mould of the future pit (Fig. 30). These lobes are at least subtended, if not maintained, by the microtubular flagellar roots that extend the entire length of the lobes (Fig. 30). At this time, the edge-growth assembly of the wall continues around these lobes and into the pit region to produce finally a walled, pitted daughter cell. After completion of the wall, the flagellar roots sub- tending the cytoplasmic moulds retract to their normal interphase position just under the rhizanchora (Fig. 31; see also Melkonian, 1979). From this evidence we can conclude that the development of the unusual, pitted cell shape is brought about by the close temporal coordination of cytoplasmic mould extension, movement of the flagellar roots and the final stages of extracellular wall assembly. Once the rigid wall forms around the moulds, the root microtubules retract, suggesting that they function in some fashion to mould the pit. The development of the anterior cell shape in Tetraselmis is different from that observed in related flagellates.I n scaly prasinophytes, the pit is maintained by a persistent, underlying series of parallel, closely set microtubules (Norris & Pearson, 1975). In walled, papillate chlorophycean flagellates (i.e. Carteria), the formation of the papilla occurs by the appearance of similar cytoplasmic moulds underlain by a parallel series of cytoskeletal microtubules, until final ontogenesis of the rigid wall occurs extracellularly. After the wall forms, the cytoplasmic Wall of Tetraselmis: structure and development 367 moulds retract (unlike Tetraselmis) and the underlying, parallel microtubular series disappears (Domozych et al. 1981). Hence, green algal flagellatesposses s many ways of maintaining and forming diverse cell shape. Cell wall assembly in T. impellucida is similar to the process observed in T. striata (Fig. 42). The only major difference in T. impellucida is that the extracellular precursors appear less compact and appear to interweave rather than aggregate. The development of the type 2 cell wall (T. convolutae) shares many common features with wall ontogenesis of type 1 walled organisms. The fibril-filled endoplasmic reticulum (Fig. 46) underlies and apparently feeds the forming face of the Golgi body. In the cisternal stack, fibrillar tufts arise on the inner membrane surface and remain tightly connected through the maturing face, only to dissociate into the lumen during vesiculation (Figs. 47-51). A unique modification of the precursors takes place in maturing-face cisternae. Electron-dense droplets appear and aggregate over the fibrillar tufts, masking them out (Figs. 47-61). The tuft-droplet associations remain closely connected to the cisternal membrane and are transported in vesicles to the plasma membrane where they are released outside (Fig. 52). Once again, their point of release is marked at the flagellar-apparatus region of the cell. Once released, the precursors form a thin stratum around the daughter protoplasts in the lumen of the old parent cell wall (Figs. 53, 54). Edge-growth assembly of the subunits occurs in the following 2-step sequence: first, the underlying tufts of the tuft-droplet complex make contact with the adjacent tufts by means of small radiating fibrils (Figs. 55-57). The underlying tufts then assemble further into a coherent underlying wall (probably the median wall layer), with an external and internal coating of electron-dense droplets (Figs. 58, 59). Second, the electron-dense droplets expand or 'fibre' outwards or inwards to yield the hairs of the inner and outer wall layers (Figs. 58, 59). The development of the outer grainy layer and inner scaly layer corresponds to the development of the hairy layers but details are lacking because of the rapidity of these events. Therefore, in type 2 walled organisms, the development of the cell wall occurs not only by the aggregation of fibrillar tufts but also by associated expansion of electron-dense droplets into the other wall layers in a precisely timed sequence. A major difference between the production of the type 1 cell wall and type 2 cell wall involves the timing of the shut-off of the precursor-producing Golgi apparatus and final wall assembly. In type 1 walled organisms, final assembly does not take place until the entire Golgi-wall-producing system is shut off. In type 2 walled organisms, this shut-off may not come until sometime after final assembly of the released precursors. Fig. 60 illustrates strange wall-like inclusions trapped between the finished wall and the plasma membrane of the pit region. These inclusions, composed of large subunits, probably represent trapped wall materials that were produced and released after final wall assembly.

General conclusions The development of the cell wall in Tetraselmis consists of a complex series of intracellular and extracellular events. The events and their sequence are important in understanding the evolutionary relationships between cell-wall ontogenesis in 368 D. S. Domozych, K. D. Stewart and K. R. Mattox

1 *.» t

/J i FT* - -

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60 Wall of Tetraselmis: structure and development 369 chlorophycean flagellates, scale development in more primitive prasinophytes, and development of other cell coverings in green algae. The evolution of the diverse form, structures and developmental patterns in chlorophycean algae are often closely related to wall evolution and wall development. Tetraselmis appears to be intermediate between the scaly green flagellatesan d typical walled green flagellates.Th e similarities between wall development and structure in Tetraselmis and Chlamydomonas suggest that wall development in the latter genus also develops by the extracellular assembly of components homologous to early stages of scale development in green flagellates with body scales. Ease of culture and manipulation with Tetraselmis make it a potentially valuable subject for studies of the endomembrane system, wall development, and related cellular processes in a unicellular organism. Several problems become apparent and require further research, including: (a) the manner by which the fibrillar tufts or tuft aggregates become regularly dispersed around the daughter cells after being released at the pit region; and (b) the mechanism that effects posterior-to-anterior assembly of wall components and the coordination of that process with cytoskeletal development so as to yield an anterior flagellar pit. It would be of great interest to know whether directional wall assembly is controlled by sequential release of an enzyme or by an effector molecule (e.g. an ion or sugar).

Fig. s 1. A thinner cross-section; note the close association of the precursors (arrows) with the cisternal membranes, x 31000. Figs. 52, 53- Overall views of wall precursor-containing vesicles in their transport and release to the outside and the displacement of the wall precursors around the cell, Fig. 52 shows 2 adjacent Golgi bodies before separation and release of their vesicles, containing wall precursors, to the plasma membrane region near the pit (arrows), as marked by the basal bodies (bb). x 16000. Fig. 5 3 shows early displacement of released wall precursors in the lumen of an old parent cell wall around the young daughter cells (arrows). Note the close association between the precursors and the plasma membrane, x 8000. Fig- 54- View of wall precursor material around plasma membrane of 2 young daughter cells. Note the electron-dense droplets (large arrows) overlying the fibrillar tufts (small arrows), x 62500. Figs. 55-57. Views of cell wall assembly in early stages in T. convolutae. Figs- 55> 56 show the close aggregation of electron-dense droplets (large arrows) and underlying fibrillar tufts (small arrows) as they begin to condense into the final wall product. Fig. ss, x 55000; Fig. 56, x 58000. Fig. 57 shows the subunit complexes making contact with each other by a series of underlying fibrils (arrows), x 54500. Figs. 58,59. View of latter stages of wall self-assembly in T. convolutae. After the under fibrillar layer of material makes contact and condenses into the median electron-dense layer (see Figs. 15, 16) the electron-dense droplets fibre outwards and inwards (arrows) to yield the inner and outer hairs ofthe type 2 wall. Fig. 58, x 49000; Fig. 59, x 53000. Fig. 60. Overall view of the strange wall-like inclusion in T. convolutae. Note the regular repeating subunits of this entity (1) entrapped in the pit region of the cell, x 24000. 37° D. S. Domosych, K. D. Stewart

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{Received 10 April 1981 - Revised 5 June 1981)