The development of a cell-substrate attachment system in a euglenoid flagellate

JOURNAL OF ULTRASTRUCTURE RESEARCH 74, 165--174 (1981)

The Development of a Cell-Substrate Attachment System in a Euglenoid Flagellate K A T H L E E N A . W A R D 1 AND R U T H

L. WILLEY

Department of Biological Sciences, University of Illinois, Chicago, Illinois 60680 Received April 1, 1980, and in revised form December 9, 1980 The transition of the euglenoid Colacium libellae from the free-swimming flagellated to the nonflagellated stage attached by a stalk occurs in three distinct stages: (1) initial attachment, perhaps by the flagellum, (2) substrate contact by the anterior region of the cell concomitant with attachment disc formation, and (3) the extrusion of stalk material. The flagellum is resorbed within minutes of initial attachment. Polysaccharide and acid phosphatase derived from within intracellular cisternae initially accumulate in the reservoir, then transfer to the canal and finally become incorporated into the stalk, thus delineating the source and route of transport for most stalk material. Indeed, the inner "shaft" and central " c o r e " of the stalk contain a neutral polysaccharide that is continuous with similar material found in the reservoir and canal. The peripheral region of the stalk (the "cortex"), however, contains a polyanionic polysaccharide which may be extruded by a special secretory system composed of a ring of granules and canals located beneath the anterior pellicle of the organism.

The extracellular release of polysaccharide by algae is of interest in that these substances often maintain a distinctive morphology during a particular stage in the organism's life cycle (Hufford and Collins, 1972; Lee and Bold, 1974; Leedale, 1967, 1975; Willey et al., 1977). All members of the Euglenophyceae produce an extracellular polysaccharide, commonly referred to as mucilage (Leedale, 1967). There exist no definitive studies on the synthesis and release of this material. Previous investigations with various euglenoid genera have been concerned primarily with the pattern of extracellular carbohydrate deposition and qualitative estimates of the relative amounts of polysaccharide extruded (Rosowski and Glidden, 1977; Rosowski and Willey, 1977; Willey et al., 1977; Triemer, 1980). Little is known about the origin of this polysaccharide or its method of release. Members of the epibiotic genus Colacium may exist in any of three distinct stages during their life cycle: the free-swim1 Present address: Stritch School of Medicine, Loyola University of Chicago, Maywood, Ill. 60153.

ming flagellate, the nonflagellated cell attached by a stalk, or the immobile palmella stage. It is the transition from the flagellated to the sessile, stalked stage that is easily manipulated in culture and, as yet, has not been exploited for developmental investigations. Introduction of the cladoceran, Daphnia, into log or early stationary phase cultures of Colacium libellae yields immediate and maximal attachment to ceils onto the Daphnia cuticle, and is followed by stalk formation. Therefore, the genus Colacium is an ideal euglenoid system in which to study algal polysaccharide extrusion. Recently, a preliminary morphological and histochemical analysis of the mature stalk produced by Colacium mucronatum was described at the light microscope level (Willey et al., 1977). A detailed ultrastructural description of the Colacium stalk, as well as the more basic aspects of the process of stalk formation, however, needs to be explored. In particular, controversy still exists concerning the source of the extruded stalk polysaccharide. Some believe this to be due exclusively to a network of subpellicular muciferous bodies, which are

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considered ubiquitous among euglenoids (Rosowski, 1977). However, in Colacium and Euglena, the canal region recently has been implicated in polysaccharide extrusion for stalk and/or palmella sheath formation (Mignot, 1972; Willey et. al., 1977; Triemer, 1980). This is a question of taxonomic and morphogenetic importance. The present study of Colacium libellae, therefore, was undertaken in an effort to identify the immediate source of stalk polysaccharide extrusion, as a preliminary study of the intracellular events leading to the release of this material, to describe the extracellular process of stalk formation, and to determine the structure of the stalk material. As a result of this investigation, the feasibility of Colacium as a model system for future developmental studies can be better assessed. MATERIALS AND METHODS

Cultures Monoalgal cultures of C. libellae (Warren el. 15, available from the authors), were maintained in glass petri dishes containing soil-water-pea medium (Start, 1964). Axenic cultures were maintained in glass culture tubes containing a 3:1 mixture of Euglena medium and Alga-Gro (Carolina Biological Supply, Burlington, N.C.). All cultures received 1000 lx on a 12:12 hr, light:dark cycle at 25°C.

Staging Stalk formation in C. libellae can be induced and timed by the addition of Daphnia pulex to the Colacium cultures. At timed intervals, the Daphnia, or their exoskeletons, were removed for analysis. Stalk formation was examined at 15-rain intervals from 0 to 6 hr after flagellated cell attachment, and at 24-hr intervals through 96 hr.

Light Microscopy Whole mount preparations were fixed in Carnoy's fluid and stained according to the periodic acid-Schiff

(PAS), PAS-alcian blue (PAS-AB), or alcian blueCEC procedures. Prior to staining, aldehyde functional groups were blocked with the aniline-aldehyde condensation method, while other samples were extracted with trypsin (Pearse, 1968; Willey et al., 1977).

Scanning Electron Microscopy Specimens were fixed in glutaraldehyde, frozen on liquid nitrogen, and freeze-dried in a Speedivac Pearse Tissue-Dryer (Edward's High Vacuum Corp., Grand Island, N.Y.). Other specimens were dried by the critical-point method in a Bomar SPC-501 EX Apparatus (Bomar Inc., Tacoma, Wash.). Specimens were coated with 120 A of gold and examined with a Cambridge Stereoscan Mark IIA microscope operating at an accelerating voltage of 20 kV.

Transmission Electron Microscopy Specimens were fixed in glutaraldehyde, stained for specific catalase localization or acid phosphatase localization, and postfixed with buffered osmium tetroxide (Brody and White, 1973). Other specimens were fixed in a glutaraldehyde-ruthenium red mixture followed by osmium tetroxide-ruthenium red (Luft, 1971), or were fixed in glutaraldehyde and stained with Thi&y's procedure for carbohydrate localization (Mignot, 1972). RESULTS

The extracellular events involved in stalk formation may be divided into three main categories: (1) initial attachment, perhaps by the flagellum, (2) substrate contact by the anterior region of the cell and attachment disc formation, and (3) the extrusion of stalk material. Initial attachment appears to be directed by the flagellum, which is the first region observed to make contact with the substrate. The beating flagellum loops onto the substrate, and appears to be attached only at the flagellar base and extreme tip (Figs. 1 and 2). Settling of the cell body onto the substrate is the result of a quick series of downward thrusts, perhaps initiated by the flagellum. The flagellum will

Fro. 1. External cuticle of Daphnia pulex covered with erect C. libellae. Flagellum (arrow). x 1400. FIG. 2. Recently attached C. libellae with coiled flagellum (arrow). × 5900. FIG. 3. Anterior region of attached cell with a ring of thin strands (arrows) connecting cell to substrate. Flagellum (F) and its associated mastigonemes (M). × 15 000. Fro. 4. After attachment, anterior region of the organism extrudes material (G) from the canal and from between the pellicle ridges (arrow). × 23 000.

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then assume a characteristically coiled pattern which beats for a few minutes and then settles on the substrate, while the anterior end of the cell flattens out as it contacts the surface (Fig. 2). Deflagellated cells cannot or will not attach to the substrate until after a new flagellum is regenerated (approximately 10 min). Once the anterior region of the pellicle has contacted the substrate surface, the extrusion of the attachment disc material begins. Fine strands, 0.5-1 /zm in length in SEM material, appear connecting the anterior end of the cell to the substrate (Fig. 3). Within 15 rain of initial attachment, the canal opening appears to dilate, revealing the presence of large globules which are associated specifically with the canal ring region of the pellicle and seem to emerge from between the pellicle strips (Fig. 4). The globules emanating from the canal ring correspond with the relative position of the strands of material initially connecting the cell to the substrate (Figs. 3 and 4). The continual deposition of material from the canal and canal ring form a diffuse attachment disc beneath the canal, extending some 5 /zm on either side of the cell periphery (Fig. 5). Concomitant with attachment disc formation is the loss of the flagellum and the cell's immediate capacity for swimming (i.e., irreversible attachment). Observations with living organisms indicate that the flagellum is resorbed, sometimes within 15 min of initial attachment in newly isolated clones. A whole intact flagellum or axoneme, coiled or folded within the reservoir or the cytoplasm, was never visible with

either SEM or TEM. Flagellar loss, however, appears to be associated with a degradative process occurring over the entire external portion of the flagellum in SEM preparations. Degradation appears to be initiated at the proximal end of the flagellum and proceeds distally with time (Fig. 5). Stalk elongation is the result of continual extrusion and subsequent accumulation of stalk material beneath the canal (Figs. 6-8). All data indicate that the major portion of stalk polysaccharide is continuous with material accumulated in the canal during the stalked stage (Fig. 8). The reservoir, therefore, is considered to be the immediate source of this material. SEM and TEM studies reveal, however, that some attachment disc material may originate from between the pellicle strips of the anterior region of the stalk-forming cell (Figs. 3, 4, 79). Nascent material from the canal condenses into a stalk about 4 hr after initial attachment, and lifts the cell off the substrate (Fig. 6). The attachment disc remains as a distinct distal component of the mature stalk. A maximal stalk length of 75/zm and a maximal width of 5 tzm may be attained between 48 and 72 hr after stalk initiation on the Daphnia cuticle. Based on LM and TEM observations, there are three basic parts of stalk structure: (1) the outer "cortex," (2) an inner "shaft," and (3) a central " c o r e . " The cortex, in section, appears to be composed of an outer ornamentation or condensation, a homogeneous, fibrous region and up to 36 electron-dense fibrous segments which radiate laterally from the center axis of the

FIG. 5. Degradation of the flagellum (F) s e e m s to be initiated at the proximal end. Pellicle (P); a t t a c h m e n t disc (AD). x 7000. FIG. 6. Material extruded from the canal c o n d e n s e s into stalk (S) after initial attachment. A t t a c h m e n t disc (AD) remains distinct, x 6500. FIG. 7. Longitudinal section through the anterior region of the stalk (S) labeled with r u t h e n i u m red reveals ornamentation on the cortex (CX) and stalk material b e t w e e n pellicle strips (P) near the canal (CL). × 27 000. FI~. 8. Longitudinal section with evident stalk material within the canal (CL) and b e t w e e n the pellicle strips (arrows). x 27 000.

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Fro. 9. Strands of r u t h e n i u m red-labeled material (arrow) are evident b e t w e e n the pellicle strips (P) of the anterior region, x 15 000. FIG. 10. Cross section through the stalk j u s t below the cell reveals outer ornamentation of the cortex (CX) and electron-dense s e g m e n t s within the cortex (SG). x 20 200. FIG. 11. Acid p h o s p h a t a s e localized specifically in one-half of a biphasic granule (BG). × 29 500.

stalk (Fig. 10). The position of the cortex Acid phosphatase, which is a marker enseems to correlate directly with the pellic- zyme for lysosomes, was localized in the ular canal ring. The material comprising the Golgi complex, in vesicular membranes and major portion of the long axis of the stalk cisternae surrounding the reservoir, in contains the shaft and core, and originates amorphous masses in the reservoir, and in from the canal proper. The shaft material parts of the biphasic granules (Fig. 11). Catis continuous with the homogeneous fibers alase, which is generally diagnostic for miof the cortex and surrounds the low-density crobodies although it is not detectable in core. Euglena gracilis under normal culture conThe composition of the stalk of C. libel- ditions (Brody and White, 1973), could not lae, as examined by histochemical and cy- be localized at any stage in stalk formation. tochemical techniques, is summarized in Thi6ry-positive carbohydrate did not apTable I. The stalk is predominantly carbo- pear in either the Golgi complex nor in the hydrate with little detectable trypsin-labile biphasic granules. However, stainable maprotein. The PAS reaction indicates a gen- terial, visualized as amorphous masses or eral neutral polysaccharide content with an fine strands of material, appeared in the resadditional polyanionic component which is ervoir (increasing rapidly during the first 12 concentrated in the cortex as exhibited by hr of stalk formation) with the Thi6ry rethe reaction with AB and ruthenium red action, with ruthenium red, and with the (see also Figs. 7, 8, 10). Gomori test for acid phosphatase. Cell organelles which have been deDISCUSSION scribed in euglenoids and have been associated with carbohydrate metabolism inThe initiation of stalk formation in C. liclude dense bodies, lysosomes, micro- bellae seems to result from cell substrate bodies, and biphasic granules (Brody and contact, which may itself be the result of White, 1973; Buetow, 1968; Willey, 1980). either a chemotactic or phototactic behav-

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ATTACHMENT

ioral response. Initial cell attachment by the flagellum precedes stalk formation. However, the actual extrusion of stalk material does not occur until after the anterior region of the cell proper has contacted the substrate. The attachment of the flagellum to the substrate probably functions solely to bring the anterior region of C. libellae in contact with the substrate and may orient or modify that contact. This is consistent with the observations that attached C. libellae cells remain erect prior to stalk formation and that deflagellated cells seem inc a p a b l e of f o r m i ng stalks. It may be inferred, therefore, that the induction center of stalk formation probably resides with the anterior cell membrane. Models for cellular adhesion emphasizing the interaction of specific cell membrane components have been presented in the literature (Bosmann, 1977; Culp, 1978; Rothstein, 1978). Parts of the flagellar membrane and anterior cell membrane of C. libellae may be modified specifically for both initial cell attachment and stalk induction, and require further examination. Among the algae, evidence is accumulating for the specific role of the ftagellar surface membrane in the transduction of external stimuli (Snell, 1976; Crandall, 1977; H e s l o p - H a r r i s o n , 1978). B ouck et al., (1978) have found that the flagellar surface of Euglena gracilis is a structurally and biochemically distinct cell surface region with distinct glycoprotein surface structures (Rogalski and Bouck, 1980). In addition, the mastigonemes comprise a distinct subset of specific carbohydrate-containing surface antigens in this area. If the flagellum of C. libellae can be equated biochemically with that of the closely related E. gracilis, the adhesion of the flagellum of C. libellae to a substrate may conceivably involve a specific flagellar surface domain and/or the mastigonemes. The transduction of stimuli from such a flagellar domain to the anterior cell membrane may also be required for stalk extrusion.

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SYSTEM TABLE I

HISTOCHEMICAL REACTIONS OF THE STALK AND RESERVOIR CONTENTS a Stalk region~

Stain~ 1. P A S

2. T r y p s i n - P A S 3. Thi6ryprocess'z 4. AB 5. T r y p s i n - A B 6. Rutheniumreda

Correx

Shaft

Core

Reservoir contents

+ + + + + ++

+ + + +

+ + + _ _ +

+ + + + + +

( + ) P o s i t i v e ( r e a c t s w i t h the s t a i n or r e a g e n t ) ; (-) negative. PA, periodic acid; PAS, periodic acid-Schiff; AB, a l c i a n blue. c Stalks and reservoirs examined were from whole m o u n t s a n d s e c t i o n s b e t w e e n 12 a n d 24 h r a f t e r initial attachment. '~ T E M o b s e r v a t i o n s . All o t h e r s w e r e e x a m i n e d w i t h LM.

The permanent attachment of C. libellae to a substrate is achieved by the extrusion of a polysaccharide stalk. In most algae, the chemical nature of such an adhesive structure is unknown. However, the stalk of C. libellae consists of both neutral and acidic polysaccharide components, as does that of C. mucronatum (Willey et al., 1977). The stalk of C. mucronatum, however, contains a very apparent and stable polyanionic component in both the cortex and shaft. Since both C. mucronatum and C. libellae were prepared by identical procedures for LM, it is possible that a qualitative biochemical or subtle structural difference exists between the stalks of these two species. The extrusion of extracellular polysaccharides among euglenoids is often considered to be mediated through subpellicular muciferous bodies, which are membranous cisternae occurring in the cytoplasm in rows parallel to the pellicular strips. The generally distributed muciferous bodies are believed to be connected to the pellicle surface by a canal (Leedale, 1967). It is pos-

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sible that muciferous bodies may be involved in the extrusion of some of the polysaccharide during attachment disc formation in C. libellae, as indicated by the presence of presumptive stalk material in Fig. 4. The apparent restriction of secretory activity to the anterior region of the pellicle of C. libellae implies that a ring of highly specialized rnuciferous bodies might surround the canal. The source of the polysaccharide extruded by any anteriorly located system may be either the endoplasmic reticulum or specialized secretory vesicles: possibly the biphasic granules characteristic of the genus Colacium (Willey, 1980). Biphasic granules cytologically similar to those found in C. libellae have been located in the scopular region of the telotroch of Z o o t h a m n i u m and are also implicated with stalk formation (Suchard, 1978). The intense acid phosphatase activity localized in the osmiophilic portion of the biphasic granules of C. libellae, is indicative of a lysosomal function. Biphasic granules in other systems which contain a strongly Thi6ry-positive (carbohydrate) half as well as acid phosphatase have been established as secretory granules in mammalian parotid and gastric mucous neck cells (Garrett and Kidd, 1976; Simson et al., 1974; Spicer et al., 1978). While the biphasic granules of C. libellae do not exhibit a strong Thi6rypositive reaction under the conditions utilized in this study, they could contain carbohydrate that is masked by the osmiophilic (lipid) portion of the granule. The granule-associated acid phosphatase activity may be indicative of, or itself involved in, the modification of carbohydrate in the process of transport to an anterior ring of specialized muciferous bodies or to channels through the pellicle. Schiller (1924) described a ring of refractile granules located in the anterior region of Colacium, which he claimed were responsible for stalk secretion. The biphasic granules of C. libellae may, indeed, be equivalent to the granules seen by Schiller (Willey, 1980). After disc

formation is completed, further stages of stalk formation are largely the product of polysaccharide extrusion through the canal. Two major sources of Colacium stalk carbohydrate precursors are possible. De novo synthesis of the stalk precursors remains a tenable hypothesis, due to the fact that the closely related E. gracilis maintains an endogenous pool of sugar nucleotides (Barras and Stone, 1968). The cytoplasmic storage carbohydrate, paramylon, represents the alternative source of stalk carbohydrate precursors. Paramylon cannot be stained specifically at the light or ultrastructural level. Therefore, paramylon may be transported, and yet remain cytologically unreactive, until it is modified and extruded from the organism. The mechanism of utilization of paramylon by all euglenoids, however, remains speculative. No isolated enzyme has been demonstrated to degrade native paramylon (Barras and Stone, 1968; Huang and Larsen, 1974). There does exist indirect evidence that stalk precursors might somehow be modified in either the reservoir region or the anterior portion of the pellicle, as indicated by the appearance of stainable carbohydrate within these regions that is concomitant with or preceded immediately by the appearance of acid phosphatase in these same regions. Carbohydrate could not be localized cytologically beyond the immediate vicinity of the reservoir. Perhaps future autoradiographic studies could delineate the intracellular mobilization and recruitment of stalk precursors. The actual extrusion of stalk material through the canal occurs by a process facilitated by metaboly. Recent evidence indicates that the euglenoid reservoir is composed of a highly fluid membrane, capable of transmembrane functions including endocytotic and exocytotic activities (Kivic and Vesk, 1972). Other parts of the reservoir membrane are lined by microtubules (Buetow, 1968; Silverman and Hikida,

CELL-SUBSTRATE ATTACHMENT SYSTEM

1976). The canal represents a region of membrane transition, intermediate between the putative static pellicle region and the fluid reservoir membrane (Hoffman and Bouck, 1976; Miller and Miller, 1978). If the microtubules that line parts of the reservoir and the canal of C. libellae function as a cytoskeletal network, and the pellicular strips lining the canal restrict movement, a contractile or elastic system initiated by the reservoir could act as a stalk extrusion mechanism. Such a mechanism may be analogous to the hydrostatic system employed in the expulsion of material from the contractile vacuole (Leedale, 1967). Flagellar resorption, which accompanies stalk extrusion in C. libellae, may occur through a process of in situ disassembly and seems to exhibit intermediate stages of internalization (for review, see Bloodgood, 1974). TEM observations reveal that the emergent flagellum of the stalked Colacium is shortened, so as not to emerge from the canal (Willey et al., 1977). SEM micrographs also indicate that some form of ftagellar degradation occurs, perhaps concomitant with the resorption of the axoneme. This breakdown is initiated at the proximal end of the flagellum and could be restricted to the ftagellar membrane. Lost axonemal components would not be visible in material processed for this study. Clearly, the fact that flagellar resorption can be stimulated in C. libellae by the attachment of a cell to a substrate in situ, represents a novel system for determining the ultrastructural events of axonemal disassembly and the dissemination of associated structures such as mastigonemes. Mr. R. Wibel provided valuable assistance with the scanning electron microscope.

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