{'fraction_non_alphanumeric': 0.02809856317980395, 'fraction_numerical': 0.011078286558345642, 'mean_word_length': 4.996175523349437, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Organization of the flagellar apparatus and associate cytoplasmic microtubules in the quadriflagellate alga Polytomella agilis

The organization of microtubular systems in the quadriflagellate unicell Polytomella agilis has been reconstructed by electron microscopy of serial sections, and the overall arrangement confirmed by immunofluorescent staining using antiserum directed against chick brain tubulin. The basal bodies of the four flagella are shown to be linked in two pairs of short fibers. Light microscopy of swimming cells indicates that the flagella beat in two synchronous pairs, with each pair exhibiting a breast-stroke-like motion. Two structurally distinct flagellar rootlets, one consisting of four microtubules in a 3 over 1 pattern and the other of a striated fiber over two microtubules, terminate between adjacent basal bodies. These rootlets diverge from the basal body region and extend toward the cell posterior, passing just beneath the plasma membrane. Near the anterior part of the cell, all eight rootlets serve as attachment sites for large numbers of cytoplasmic microtubules which occur in a single row around the circumference of the cell and closely parallel the cell shape. It is suggested that the flagellar rootless may function in controlling the patterning and the direction of cytoplasmic microtubule assembly. The occurrence of similar rootlet structures in other flagellates is briefly reviewed.

The presence of flagellar rootlets, constructed of microtubules in a characteristic grouping and/or striated fibers, originating near flagellar basal bodies, is a common feature of motile algal cells. In some of these algae, for example, Chlamydomonas (34) and the zoospores of Microthamnion (43), the microtubules comprising the flagellar rootlets appear to constitute the entire cytoplasmic microtubule system. In others, Ochromonas (2) and the zoospores of Schizomeris (1), large numbers of additional cytoplasmic microtubules appear to attach to the flageUar rootlets. In an earlier series of papers (2,4,5) it was shown that in the formation of this attachment in Ochromonas the flagellar rootlets, the rhizoplast and kineto-beak fiber in this organism function as nucleating sites (or microtubule-organizing centers, 29) for the initial assembly of cytoplasmic microtubules. These observations were made on cells in which the cytoplasmic microtubule system was regenerating after an exposure to hydrostatic pressure or antimitotic chemicals. To further clarify this proposed function, we suggested that it was important to examine the role of such sites during the normal development of the cytoplasmic microtubule system (e.g. in synchronously dividing cultures), and ultimately to analyze the polymerization capabilities of these structures in an in vitro 106 THE JOURNAL OF CELL BIOLOGY 9 VOLUME 69, 1976 9 pages 106-125 system. The flagellate Polytomella agilis offers several advantages for these and other studies on the role of cytoplasmic microtubules and microtubule initiation sites in development.
Members of the genus Polytomella (Chlorophyceae, Volvocales) are colorless, quadriflagellate unicells (32). The nutritional requirements (46,47), growth characteristics (36), and life cycle and reproduction (20) of the genus have been examined. In exponentially growing cultures, P. agilis is a free-swimming flagellate lacking a cell wall. It grows rapidly in liquid culture (mean generation time of 4-5 h) and can be synchronized by a repetitive temperature cycle (7). The basic structure of the swimming cell has been examined and the presence of numerous cytoplasmic microtubules noted (28). In late exponential and stationary phases of growth, increasing numbers of cells become immotile thick-walled cysts, which, if resuspended in fresh medium, will excyst and release quadriflagellate swimming cells. During the excystment period the complete complement of basal bodies, flagella, and cytoplasmic microtubules is formed (3). The basic objective of this report is to provide a detailed description of the flagellar apparatus and associated microtubular structures of the freeswimming quadriflagellate stage of P. agilis. The 6tructure and organization of the basal body apparatus are correlated with the flagellar activity of the swimming cell, and comparisons are made with the flagellar apparatus and activity of other biflagellate and quadriflagellate algae. It is shown that flagellar rootlets terminating near the basal bodies serve as attachment sites for large numbers of cytoplasmic microtubules. Separate reports will describe the role of these rootlets in initiating the assembly of cytoplasmic microtubules in vivo (see ref. 3 for a preliminary report) and in the in vitro assembly of microtubules from brain microtubule protein subunits (40). Samples for scanning microscopy were prepared by freezedrying or critical point procedures. For freeze-drying, the cells were fixed for 30 rain in 0.5% glutaraldehyde in 0.05 M sodium phosphate at pH 7.4 and washed in distilled water. A thin layer of cells was then spread on a cover glass and rapidly frozen in liquid nitrogen-cooled Freon 12. The cover glass was then transferred to the cooled stage (-80~ of a Speedyvac Pearse Tissue Dryer Model l and dried for 12-16 h. Cells for critical point drying were fixed in glutaraldehyde as described above, washed, and postfixed for 30 min in 1% osmium tetroxide. The cells were then dehydrated in a graded ethanol series, gradually transferred to Freon 113 and finally to Freon 13 (8), and dried in a Bomar SPC 900 critical point apparatus (The Bomar Co., Tacoma, Wash.). Samples prepared by both drying methods were then coated with gold-palladium and examined in a Cambridge Mark 2A Stereoscan (Cambridge Thermionic Corp., Cambridge, Mass.). IMMUNOFLUORESCENCE: The immunofluorescent staining was carried out in the laboratory of Dr. V. Kalnins, Department of Anatomy, University of Toronto, Toronto, Canada, using antiserum directed against electrophoretically purified chick brain tubulin? Swimming cells were fixed for 30 min in 2% paraformal-Kalnins, V. I., and L. Subrahmanyan. 1976. Staining of microtubule-containing regions by immunofluorescence with antibodies to electrophoretically purified chick brain tubulin. Manuscript submitted for publication).

Cultures
BROWN ET AL. Flgellar Apparatus in Polytomella dehyde in 0.1 M phosphate buffer at pH 7.0. The cells were then washed several times in phosphate-buffered saline by gentle centrifugation and resuspension, and stained in suspension by the indirect fluorescein-labeled antibody technique (for details on preparation of antiserum and staining methods, see footnote I).
After the staining, some samples were subjected to vortex agitation. The cells are fragile after a brief paraformaldehyde fixation and some of them lyse, releasing the intact flagellar apparatus. Samples were photographed with a Zeiss microscope equipped with epifluorescence or phase-contrast optics, using llford FP4 film developed in Diafine.

General Cell Structure
The external morphology ofP. agilis is best seen in the scanning electron micrographs (Figs. I and 2). The cell shape is basically ovoid, averaging 16 um in length and 8 #m in width. Four anterior flagella of equal length (11 #m) emerge from depressions below a cruciform papilla (Figs. l and 2).
The distribution of organelles in the swimming cell has been described already (28) and will be considered only briefly here. The anterior papilla contains a fine fibrillar network (Figs. 3 and 6 8) and is devoid of larger organelles. The four basal bodies are situated directly below the papilla. A single row of cytoplasmic microtubules occurs just beneath the naked plasma membrane. Mitochondria (or the single mitochondrion, 6) are usually located in the anterior half of the cell near the periphery, and the Golgi bodies surround the central nucleus. The starch-containing plastids generally occupy the posterior half of the cell.

Flagellar Apparatus
TERMINOLOGY" Although the arrangement of basal bodies and their interconnecting fibers is more complex in Polytomella than in Chlamydomonas (34), there are several similarities; hence, whenever possible, the terminology of Ringo (34) will be used. The arrangement of the basal bodies is best explained by first referring to the diagrammatic reconstruction (Fig. 4). The four basal bodies are organized in two pairs, with each pair having distinctive interconnecting fibers. One of the pairs (termed the A pair) is situated slightly more anterior in the cell than the other pair of basal bodies (termed the B pair).
FLAGELLAR MOTILITY: The most frequently observed forward motion of P. agillis is a straight line path, with the anterior end of the cell facing in the direction of movement (Fig. 5 a-e).
The four flagella do not beat synchronously (Fig. 5 a-c), but there is good coordination between the opposite pairs of flagella. Both pairs show the breast stroke type of movement described for Chlamydomonas (34), with one pair sweeping farther back along the cell at the end of the power stroke ( Fig. 5 b), although occasionally all four flagella appear in almost the same position of power ( As far as we can tell, there are no fibers directly connecting A basal bodies to B basal bodies. However, the four basal bodies are held together by connections to rootlets which terminate between adjacent A and B basal bodies (see below).
The basal bodies of either the A or B pair are not in a directly opposite arrangement, and we refer to them as two nearly opposite pairs (Table I). By this we mean that if a line is drawn longitudinally through the center of one basal body of a pair (

ORGANIZATION OF ROOTLETS
SERIAL SECTIONS" The organization of the rootlet system has been reconstructed from serial sections cut at both right angles (transverse sections) and parallel (longitudinal sections) to the long axis of the cell. The overall arrangement is shown diagrammatically in Fig. 10 as it would appear in a view looking down on the anterior part of the cell. The flagellar rootlets are of two structural types: one consists of four microtubules in a 3 over l pattern (microtubule rootlet), and the other of a striated fiber over two microtubules (compound rootlet). One rootlet of each type terminates between adjacent basal bodies for a total of eight rootlets (numbered l to 8) in the cell. The exception is the compound rootlets numbered l and 5 in which the striated portion appears continuous between the closely spaced A basal bodies. To aid the reader in interpreting the electron micrographs, all the transverse sections through the basal body region are printed as if viewed from the anterior of the cell, and the basal bodies of the A and/or B pair are labeled. IMMUNOFLUORFSCENCE: The use of an antibody directed against chick brain tubulin has allowed us to observe the extent of the rootlet system by immunofluorescence. In the intact cell ( Fig. 12 a), the flagella and four basal bodies fluoresce brightly and a less intense background fluorescence is apparent in the cytoplasm. In samples lysed by agitation (Fig. 12 b), some of the cell contents are released from the cell, leaving a "ghost" which has retained the normal cell shape. Some of the flagellar rootlets clearly extend the length of the cell.
Since the rootlets consist of only a few microtubules (two to four), it has not been possible to detect them or demonstrate their length in intact or

110
THE JOURNAL OF CELL BIOLOGY -VOLUME 69, 1976 partially lysed cells by phase microscopy (Fig. 12  d). In the same samples, some cells have lysed to release the entire flagellar apparatus as a unit (Fig.  12 c). Both the compound rootlets (containing two microtubules) and the microtubule rootlets (consisting of three or four microtubules) are retained and can be visualized by immunofluorescence microscopy. The fluorescent material seen as aggregates or faint lines between, and sometimes on, the rootlets may represent remnants of the cytoplasmic microtubules and does not fluoresce in samples treated with normal preimmune serum (Fig. 12J).

DETAILED STRUCTURE OF THE ROOTLETS
The substructure of the rootlets and their association with the basal bodies are illustrated in transverse and longitudinal sections through the basal body region. Fig. 13 is a transverse section through the cruciform papilla just anterior to the basal bodies. Although the papilla contains only a network of fine filaments, numerous cytoplasmic microtubules are observed just beneath the plasma membrane in the ridges at the base of the papilla (Fig. 13). In sections slightly lower in the cell (Figs. 14 and 15), the distal striated fiber connecting the A basal bodies is seen in surface view, and, running beneath it, the striated portion of the compound rootlets I and 5. The striation pattern of the compound rootlet consists of repeating groups of three closely spaced dark lines with a center-tocenter spacing of 300 A. The two tubules subtending the striated part of this rootlet (Fig. 15) terminate near the proximal end of a basal body (Figs. [16][17][18] so that, in longitudinal sections through the center of the A basal bodies, only the striated part of the rootlet is seen (Figs. 6 and 7). In similar sections farther from the center, the two tubules are evident in cross section below the striated part of the rootlet (Figs. 24 and 25). The other two compound rootlets (numbered 3 and 7, Fig. 10) are of similar construction, but both the striated and tubular portions terminate near the proximal end of a basal body. In all sets of serial sections, it has been possible to detect the continuity of rootlets 1 and 5 but the continuity of rootlets 3 and 7 was never observed. One microtubule rootlet terminates near the proximal end of each basal body on the side opposite a compound rootlet. All four of these rootlets consist of four microtubules in a 3 over 1 pattern (Fig. 23). As a microtubule rootlet approaches the basal body, one of the tubules in the • GO Free complex similar to that in (c) but treated with normal preimmune serum and printed on high contrast paper. The film exposure time was identical for (c) and (/), and when these figures were printed, using the same grade of paper and exposure time, no structure could be seen in (/). • 3,000. row of three ends to leave a 2 over 1 arrangement (Fig. 26). The remaining three tubules end closer to the basal body at, or very near, the same point.
One compound rootlet and one microtubule rootlet terminate between each set of adjacent A and B basal bodies (Figs. 16, 18, 21, and 24) and diverge in passing to the cell surface. In transverse sections, therefore, if one of the rootlets is seen in cross section the other appears in oblique view (Fig. 24). The rootlets appear to be firmly attached tO the basal bodies, and by lateral association link the adjacent basal bodies (Fig. 16). Two observations support this suggestion. (a) No other connections are observed between A and B basal bodies, yet the entire basal apparatus can be isolated as a unit. (b) in cells exposed to hydrostatic pressure (Brown, unpublished), the four basal bodies become separated in the cytoplasm but each still has two rootlets attached to the proximal end.
As the rootlets approach the basal body region,

BROWN ET AL. Flagellar Apparatus in Polytomella
they are surrounded by a dense-staining amorphous material (Figs. [23][24][25][26] which can also be seen running parallel to the rootlets (Fig. 17). This material may function in interconnecting or attaching rootlets to basal bodies, but it could also simply obscure finer attaching fibers. However, it may have a very different function (see below).

CYTOPLASMIC MICROTUBULES
All eight basal body rootlets serve as attachment sites for large numbers of cytoplasmic microtubules (Figs. II c, 15, 16, and 18). These microtubules diverge at an angle from the rootlet toward the cell surface and occur in a single row around the circumference of the cell just beneath the plasma membrane. The site(s) of attachment of the cytoplasmic microtubules appears specific. Tubules have not been observed to terminate on the striated fiber of the compound rootlets. All microtubules attach to the side of the rootlets and all terminate very close to, but not directly on, a rootlet tubule (Figs. 15, 16 and 18), It appears that the cytoplasmic microtubules insert on the amorphous electron-dense material surrounding the rootlet tubules. As the rootlets approach the basal bodies, the number of attached microtubules increases and the dense material is most prominent. The juncture of the rootlets and basal bodies is often obscured by this material. At points in the posterior part of the cell, the dense material is not observed around the rootlet extensions (Fig. 27), nor are cytoplasmic microtubule attachments observed.

Cell Motility
P. agilis normally swims in straight, helical, or curved paths with its anterior, flagella-bearing end facing in the direction of movement. The most frequently observed swimming path is a straight line, and the average speed of swimming is up to 220 gm/s (14). The flagellar activity during forward movement has been analyzed by highspeed cinephotomicrography (13), and some correlations with our observations on the structure of the flagellar apparatus can be made. Gittleson and Jahn (13) point out that two flagella always appear to beat on one side of the cell and two on the other side. In all views in which all four flagella are seen, the two on the same side of the cell are adjacent flagella (in our terminology, one A flagellum and one B flagellum). One of the two flagella on each side of the cell beats farther back along the cell than the other. Since the B basal bodies are set deeper in the cytoplasm, it seems likely that these are the B flagella (Fig. 5 b). Gittleson and Jahn also examined the beat frequency of the individual flagella and found that a flagellum which beats farther back along the cell has a beat frequency of 7.1 times per second and that the other flagellum beats 33.3 times per second. We suggest that the slower flagellum is a B flagellum and the faster an A flagellum. This difference in beat frequency would account for our observation that there is good coordination between opposite flagella but usually a lack of synchrony between the A and B pairs of flagella.
This type of breast stroke-like coordination between two flagella (of either the A or B pairs) which beat in opposite directions at the same time is identical to that observed in the biflagellate Chlamydomonas. Ringo (34) (19). The recent demonstration (17) that the isolated flagellar apparatus (consisting of two flagella with basal bodies and accessory structures) of Chlamydornonas when reactivated in vitro shows coordinated movement of the two flagella lends some support to Ringo's suggestion. However, it is still not clear which accessory structures, the distal fibers, the proximal fibers, or the microtubule rootlets, are responsible for the coordination. The similarity in size between the filaments comprising distal and proximal fibers and actin filaments has been pointed out (19), and it is tempting to speculate that these fibers have some contractile function. Now that methods are available to isolate and purify the basal bodies with

Phylogenetic Considerations
The phyletic significance of the fine structure of the flagellar apparatus in the motile cells of algae and primitive land plants has been recognized for some time (c.f. the review of Manton,23). The structure of the mitotic and cytokinetic apparatus has proven most useful in analyzing evolutionary trends in the green algae (for a complete review, see 31). Pickett-Heaps (and others) has recognized a "class" of green algae (including Chara, Nitella, Coleochaete, and Klebsormidium) that he has termed the "bryophytan" type which clearly resembles the primitive land plants in the structure of the mitotic spindle and cytokinetic apparatus.
Chaetosphaeridium (26) and Trentepohlia (16) will also probably be added to this "bryophytan" class. The presence of a multilayered structure (a complex flagellar rootlet) associated with the basal bodies of the motile cells of organisms in this class and in the motile sperm of primitive plants adds further support to the suggestion that this group of organisms is very likely closely related to the green algal progenitor(s) of the land plants (I, 26, 3 I).
The use of the structure of the flagellar apparatus and that of the associated flagellar rootlets as characteristics in the phylogenetic analysis of other green algae has been less successful, and in many cases confusing, mainly because too few organisms have been studied, and in those that have been studied many of the descriptions are incomplete. Probably the most complete study to date is that of the biflagellate Chlamydomonas reported by Ringo (34). Ringo described the detailed structure of the striated fibers interconnecting the basal bodies, and showed that four flagellar rootlets, each consisting of four microtubules in a 3 over I pattern, were present in a cruciate arrangement. The cruciate arrangement of four rootlets is a common feature of chlorophycean algae (for a review, see 25), exclusive of the "bryophytan" type, and has been used as one characteristic in differentiating this group of Chlorophyceae from the green algae of the class Prasinophyceae. This interpretation appears to have been premature, as the prasinophyte Pyramimonas (27) has a cruciate arrangement of four rootlets, two consisting of two microtubules and two of four microtubules in a 3 over I pattern. Stewart et al. (41) and Moestrup (26) have recently questioned the validity of the class Prasinophyceae on the basis of other fine structural criteria (e.g. structure of spindle and cytokinetic apparatus, presence of flagellar scales).
With the limited amount of evidence available in 1967, Ringo (34) noted the basic similarity in the flagellar apparatus of biflagellate, quadriflagellate and some multiflagellate (e.g. Oedogonium) algal cells and suggested that the quadriflagellate and multiflagellate apparatus may be "polymers" of a basic biflagellate unit as is found in Chlamydomonas. However, in the quadriflagellate Carteria, which is usually considered most closely related to Chlamvdomonas, the structure of the flagellar apparatus is quite different than that of Chlamydomonas (19). In the quadriflagellate algae examined so far, the flagellar apparatus in Several of the quadriflagellate green algae examined (see Table I for a partial list) appear to have the four basal bodies arranged in two Lshaped pairs which are in a mirror image relationship (the "ulotrichalean" type of Mattox and Stewart,24). The fibers interconnecting the basal bodies are not detailed for most of these organisms, but in Schizorneris it appears that the adjacent basal bodies are linked by distal and proximal fibers. The four basal bodies in Polytomella also superficially appear to be two pairs in a mirror image relationship (Figs. 10, 18 and 19). However, serial sectioning clearly shows that opposite flagella are linked in pairs. The positioning of the flagella, with one pair situated more anterior in the cell than the other, has also not been noted for other quadriflagellates, although this may have been missed if adjacent serial sections were not examined.
The two types of flagellar rootlets found in   (Table I). in Polytomella, six of the eight rootlets are arranged in a bilateral symmetry on either side of the compound rootlets numbered I and 5 in Fig. 10. The four microtubule rootlets do appear in a cruciate arrangement in surface view (as shown in the diagram, Fig. 10), but the two attached to the A basal bodies are situated more anterior in the cell than the two attached to the B basal bodies.
Asymmetry of motile cells is generally considered to be a primitive characteristic in the green algae, and the radial symmetry as in Chlamydomonas or Schizomeris, both of which have four equal rootlets in a cruciate arrangement, is presumed to be a derived characteristic. In comparison to these organisms, Polytomella exhibits considerable asymmetry in the structure of the flagellar appratus, and also has the primitive characteristics (3 I) of a completely closed spindle and cytokinesis by furrowing (unpublished results). On the basis of the structure of the flagellar apparatus, we can make only some very tentative conclusions at this time. Polytomella seems to be one of the more primitive Volvocales, and appears to be closely related to Chlamydomonas. The appearance of the four basal bodies in surface view as two L-shaped pairs in a mirror image relation-ship does resemble the arrangement in other quadriflagellates, but the type of basal body interconnection in most of these is unknown. It seems likely, though, that the structure of the flagellar apparatus will prove to be an increasingly important characteristic in phylogenetic analysis as detailed information on more flagellates becomes available.
A phylogenetic consideration of a much broader nature is the conservation of microtubule protein (20). Several studies have shown that antisera directed against tubulin from sea urchin sperm flagell~ (44), bovine brain (12), and chick brain (footnote I) cross-react with microtubules in very diverse animal species. Our observation that antibodies produced against chick brain tubulin react with microtubule structures (flagellar axonemes, basal bodies, rootlets and spindle) in a primitive phytoflagellate extend the idea that tubulin is highly conserved. This method should be useful in visualizing microtubule systems in other flagellates.

Cytoplasmic Microtubule System
Cytoplasmic microtubules have been shown to have a cytoskeletal function in the development and maintenance of cell asymmetry in a variety of 122 THE JOURNAL OF CELL BIOLOGY . VOLUME 69,1976 organisms (see Roberts,35, for a recent review). In Chlamydomonas, in which relatively few cytoplasmic microtubules are observed, this type of cytoskeletal function is less obvious, and these tubules may play a greater role in anchoring or stabilizing the flagellar apparatus (34). However, cytoplasmic microtubule systems are well developed in many of the flagellated algal cells, particularly in those lacking cell walls. In the multiflagellate zoospores (30) and sperm (t0) of Oedogonium, there is a proliferation in the number of microtubule rootlets, which extend the length of the cell and presumably function as a cytoskeleton. In Polytornella, and in the naked zoospores of many green algae (e.g. Enteromorpha, Schizomeris, Urospora, Table 1), the number of rootlets remains relatively low and large numbers of additional microtubules are present, which appear to attach to the flagellar rootlets and underlie the plasma membrane.
The correlation between cytoplasmic microtubule distribution and the development of cell shape is especially evident in asymmetric flagellated cells, and many examples could be cited from all classes of algae that have motile cells.
Ochromonas is a particularly clear example (2,4,5). In this unicellular Chrysophycean alga, the characteristic asymmetry is mediated by two sets of cytoplasmic microtubules which are initiated at two distinct sites in the cell at different times during development of cell shape. Both of these sites are flagellar rootlets, the rhizoplast (a striated rootlet) and the kineto-beak fiber (a microtubule rootlet), and each site is related to the development and maintenance of specific portions of the asymmetric cell form.
The existence of a "structure" which could control the spatial and temporal distribution of microtubules by controlling the initiation of microtubule assembly has been postulated several times [for example, the microtubule organizing center of Pickett-Heaps (29); the nucleating site of Tilney and Goddard, (42)]. Basal bodies are highly structured assembly sites for axoneme microtubules of cilia and flagella. The pattern and orientation of microtubules in the axoneme appears to result, at least in part, from the distal assembly of tubulin subunits onto the existing (pattern of) tubules of the basal body, and this can be partially mimicked in vitro (37). What established that pattern of tubules in the basal body is still not clear, but this may also be a nucleated assembly (15). More commonly, however, the sites (i.e. organizing centers) that are presumed to be associated with microtubule assembly consist of an amorphous electron-dense material that has no obvious structural organization [e.g. the polar bodies of the mitotic spindle in Oedogonium (9); the granular aggregate in homogenates of surf clam eggs which will polymerize asters and spindles in vitro (45)].
A more complex type of organizing center appears to result from an ordering of the amorphous material (that described above) onto some other structure to provide a template which could control the patterning and direction of microtubule assembly. The rhizoplast and kineto-beak fiber of Ochromonas are examples of this type of organizing center (for a more complete discussion see Brown and Bouck,ref. 4). We suggest, on the basis of the structural information that cytoplasmic microtubules terminate on amorphous material associated with rootlets, that the flagellar rootlets in Polytomella are further examples of structured organizing centers. We have demonstrated that these organizing centers appear to initiate microtubule assembly in vivo and in vitro (3,40). This system should be useful in analyzing the mechanism of spatial and possibly temporal control of microtubule assembly.