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Defective temporal and spatial control of flagellar assembly in a mutant of Chlamydomonas reinhardtii with variable flagellar number.

Wild-type Chlamydomonas reinhardtii carry two flagella per cell that are used for both motility and mating. We describe a mutant, vfl-1, in which the biflagellate state is disrupted such that the number of flagella per cell ranges from 0 to as many as 10. vfl-1 cells possess the novel ability to assemble new flagella throughout the G1 portion of the cell cycle, resulting in an average increase of about 0.05 flagella per cell per hour. Such uncoupling of the flagellar assembly cycle from the cell cycle is not observed in other mutants with abnormal flagellar number. Rather than being located in an exclusively apical position characteristic of the wild type, vfl-1 flagella can be at virtually any location on the cell surface. vfl-1 cells display abnormally wide variations in cell size, probably owing to extremely unequal cell divisions. Various ultrastructural abnormalities in the flagellar apparatus are also present, including missing or defective striated fibers and reduced numbers of rootlet microtubules. The pleiotropic defects observed in vfl-1 result from a recessive Mendelian mutation mapped to Chromosome VIII.


Measurement of Flagellar Number, Length, and Location:
Cells were fixed by the addition of ~/2 vol of 1% aqueous glutaraldehyde and examined at x800 using phase-contrast optics for determination of flagellar number and location. Flagellar length was measured with an ocular micrometer using phase-contrast or Nomarski differential interference contrast optics. Distributions of flagellar number and location were based on samples of at least 200 cells and flagellar length averages were based on samples of at least 20 cells per flagellar number class. Cell volumes were determined by measuring cell length and width and applying the equation ~r/6 x (width) 2 x length (13).

Determination of Flagellar Number Changes in Living Cells
and Mitotic Pedigree Analysis: Cells in the first hour of the light segment of the cycle were suspended in 0.5% melted agar. A drop was placed within a ring of petroleum jelly on a slide and sealed with a coverslip. Approximately 1 h later, cell positions were determined using the microscope stage's vernier scales, and cell size, flagellar number, and flagellar lengths were recorded. The slides were maintained at standard illumination for an additional 5 h and the cells were observed again. They were then returned to standard illumination conditions overnight, during which one or more rounds of mitosis per cell division typically took place. The next morning the number and size of daughter cells were measured along with the number of flagella on each cell. Genetic Analysis: Standard techniques were used for crosses and tetrad dissections (6,9). Mapping stocks ware those listed in Adams et al. (1).
Electron Microscopy: Log-phase cells between the second and fourth hour of the light segment of the illumination cycle were harvested by centrifugation (5 min at 2,500 rpm). The pellet was resuspended in residual culture medium and 2 ml freshly prepared fixative (2.5% glutaraldehyde, 2% osmium tetroxide in 0.1 M sodium cacodylate buffer, pH 7.8) was added (7). After 5 min on ice the cells were repelleted and the initial fixative was replaced with an equal volume of fresh fixative. After 30 min the cells were washed at least five times with 0. l M sodium cacodylate buffer and stored overnight at 4°C. Pellets were postfixed for 2 h in 2% osmium tetroxide in 0.1 M cacodylate and then rinsed twice with water. Ifen bloc staining was to be performed, the cells were given an additional two washes with 50% aqueous ethanol. En bloc staining was accomplished with 0.25-0.5% aqueous uranyl acetate for 15 rain or, for cases in which enhanced microtubule contrast was desired, 4% tannic acid in 0. l M sodium cacodylate. Samples were rapidly dehydrated in a graded alcohol series, rinsed twice in propylene oxide, and infiltrated with resin (60 ml dodececenyl succinic anhydride, 20 ml Araldite 502, 20 ml Embed 812, 3 ml 2,4,6-tri(dimethylaminomethyl)phenol-30); Electron Microscopy Sciences, Fort Washington, PA). Infiltration was facilitated by use of a vacuum oven (50°C, l0 psi) and the resin was polymerized at the same setting for 12-24 h. Silver to gold sections were cut using a diamond knife on a Sorvall MT-1 microtome (DuPont Instrnments--SorvalI Biomedical Div., DuPont Co., Wilmington, DE). Sections were stained with 3% aqueous uranyl acetate and Reynold's lead citrate and examined with a Phillips 300 electron microscope at 60kV.

Flagellar Number, Location, and Motility in vfl-1
vfl-1 was isolated after nitrosoguanidine mutagenesis of wild-type C reinhardtii (1). Most mutant cells carry from zero to four flagella each, but rare cells can have as many as 10. Fig. 1 shows the distribution of flagellar number classes in a typical vfl-1 culture, and Fig. 2 shows differential interference contrast micrographs of a number of vfl-1 cells. In some cells the flagella are paired as in wild type (Fig. 2, B and G) but in others they clearly are not (Fig. 2, C, D, and H).
Flagella can be anywhere on the vfl-1 surface, in marked contrast to wild type in which the flagella are always at the anterior cell apex. However, on multi-flagellated cells the flagella are usually clustered in the same general area.
Motility in vfl-I is severely impaired, even for those cells carrying two flagella. Many cells spin in place; others tumble wildly through the medium. Fewer than 1% of cells show a motility pattern that could be confused with that of wild type.

Cell Morphology and Growth
vfl-1 cells show extreme variation in size and are often abnormal in shape. Measured cell volumes for 100 mid-Gt vfl-I ceils ranged from 24 to 1423 /~m 3, whereas wild-type controls ranged from 72 to 733 gm 3. The range in size seen in these controls is typical of rapidly growing light/darksynchronized Chlamydornonas cultures, in which many cells undergo two or more rounds of mitosis during the dark portion of the cycle (4). No obvious correlation was seen between cell size and flagellar number (Fig. 3A), but, as in wild type, cells with larger volumes tended to have somewhat longer flagella (Fig. 3 B).
Microscopic observation of microcolonies derived from    single agar-immobilized cells indicated frequent segregation of inviable progeny at cell division. Extremely unequal divisions were common, producing inviable daughter cells as small as 1 #m diam. Counts of cell number in day-old microcolonies gave values of from 2 to 23 cells per colony for vfl-1, whereas wild-type control colonies were much more uniform, containing between two and eight cells in almost every case (Fig. 4). Note that nearly all wild-type colonies contained an even number of cells, whereas a great many vfl-I colonies contained an odd number, consistent with the death of some mitotic progeny or the failure of some daughters to divide again. 17% of the mutant cells had not divided at all and of these 6% were clearly dead.

vfl-I Cells Acquire New Flagella throughout G1
A number of results demonstrate clearly that vfl-I cells acquire new flagella throughout the G, phase of the cell cycle. In one experiment, a synchronous culture growing on a 12-h light/dark regime was sampled at intervals and the mean number of flagella per cell was determined. As indicated in Fig. 5, the number was lowest immediately after cell division and showed a linear increase throughout G~ (i.e., throughout the light period of the cell cycle) at a rate of 0.056 per cell per hour.
In another experiment, single cells of synchronized vfl-1 culture were immobilized in agar and the number of flagella on individual cells was recorded 1 and 6 h later. As the results presented in Table I  closely fit a Poisson distribution. Furthermore, the addition of new flagella during the 5-h interval was random ( Table  IIb).

Flagellar number was also observed for individual agarimmobilized vfl-1 cells before and again after cell division.
The results, summarized in Table III, indicated a tendency for cells with few flagella to yield progeny with few, and for cells with many flagella to yield progeny with many. This is not surprising because basal bodies persist through cell division and presumably can nucleate new flagella in the next generation. Consistent with these results was the observation that small clones (approximately 2,000 cells each) varied widely in terms of average flagellar number per cell (Table  IV). Nonetheless, flagellar distributions within individual clones were random (i.e., closely fit Poisson distributions) irrespective of average flagellar number.
vfl-1 Ultrastmcture As Fig. 6 illustrates, the geometrical relationships among the major cell

Flagella per cell
Change in fla-Initial After 5 h gella number Occurrences organelles were frequently observed to be aberrant in vfl-1. In   Fig. 6A, the normal positioning of the flagellar apparatus, nucleus, chloroplast, and pyrenoid in a wild-type cell is evident. Note the abnormality of flagellar position on the vfl-1 cell in Fig. 6B: the flagellum appears on the side of the cell, with a lobe of the chloroplast passing between the basal body and the nucleus, a situation never observed in wild-type sections. The vfl-1 cell membrane was often thrown into deep folds, especially at the anterior end of the cell (Fig. 6C). Chloroplasts appeared somewhat irregular in shape relative to the cup-shaped chloroplasts of wild type, and eyespots often displayed abnormal morphology or were present in multiple     locations in the chloroplast. Binucleate and/or bipyrenoidal cells were fairly common (Fig. 6, D and E). Golgi apparatuses were prominent, with as many as five appearing in a single section through a cell, and cells were generally highly vacuolated. Aberrent morphology was especially apparent in sections through "palmelloid clusters" in which several daughter cells were retained within a mother wall. In such cases nuclei were often found very close to the plasma membrane rather than in the normal cytocentric location.

BASAL BODY, STRIATED FIBER~ AND C Y T O S K E L E T O N
DEFECTS: Most vfl-1 basal bodies appeared normal in ultrastructure, but their positions in the cell and their orientations with respect to each other and to other cellular organelles were highly variable and rarely normal (Fig. 7). Basal bodies were sometimes found in the cell interior, either oriented parallel to the cell surface (Fig. 7A) or inverted, with the transition region, if present, pointing inward (Fig. 7, B and C). In several instances a basal body bearing an axoneme was observed deep within the cell (Fig. 7 D). These axonemes were always membrane bounded, so that they resembled the primary cilia which have been observed in cultured animal cells (16). Many basal bodies at the cell surface carried no axonemes at all, terminating instead just above the transition region (Fig. 8 D). In some instances tunnels were present in the cell wall overlying the basal bodies, but in other instances no tunnels (Fig. 8 C) were apparent. Other basal bodies at the cell surface appeared to carry only very short axonemes. Electron-dense rings of approximate basal body diameter were frequently observed in the cytoplasm of vfl-1 cells (Fig.   8, A and B); such rings were not observed in wild type or in vfl-3, another variable flagella mutant whose ultrastructure was closely examined (17). Occasional basal bodies with doublet rather than triplet microtubules were also seen in vfl-1 (Fig.  8E) but not in vfl-3 or wild type controls. We suspect that the rings and doublet basal bodies represent intermediates in basal body assembly (see Discussion).
Basal body-associated striated fibers were absent, incomplete, or misoriented in vfl-1. The position and ultrastructure of striated fiber connections in wild-type cells are depicted in Fig. 9, A and B. The striated fiber abnormalities observed in vfl-1 include the apparent absence of the distal fiber (Fig. 9 C) and the presence of abnormal projections from basal bodies at a level where a distal fiber should normally appear (Fig. 9, D, E, and F). Both electron-dense and striated projections were observed. In an occasional case, an intact distal fiber connected two basal bodies, but even then the angle between the basal bodies was abnormally large or small.
Cytoplasmic microtubules were present in some vfl-1 sections, but they were not nearly as frequent as in wild type, and the normal cruciate convergence of rootlet microtubules at the basal body complex was never observed. In addition, microtubule ultrastructure was not well resolved. Because so few sections showed cytoplasmic microtubules, we do not know whether this observation is significant; perhaps the vfl-1 microtubules are structurally or chemically different from those in wild type, but perhaps we simply didn't observe enough examples to find good clean sections. In either case, we conclude that vfl-1 has an incomplete and misarranged microtubular cytoskeleton.

Genetic Analysis
Tetrad analysis (Table V)  that the vfl-1 mutation is recessive to wild type, in that vfl-1/ wild type vegetative diploid cells were biflagellate like wild type/wild type diploids.

Defective Basal Body Cycle in vfl-1
In wild-type Chlamydomonas, the assembly of new flagella is confined to a short interval in the cell cycle (2). As mitosis approaches, the cell resorbs its flagella and then remains flagella-less throughout mitosis and cytokinesis (2,12). After cell division is completed, each daughter cell assembles and x 35,900. (D) Three fibrous structures (arrows) are present on the cross-sectioned basal bodies. The wedge shape is typical of proximal fibers, but, in wild type, these would never be present at the same level as transitional fibers, x 34,900. (E) An apparently half-length distal fiber is associated with the basal body in this section, x 55,000 (F) The basal bodies here are in a normal position (see Fig. 9A) but no distal fiber spans the space between them. A short, distal fiber-like structure is present on one basal body, in the appropriate position, x 37,100. ax, axoneme; b, basal body; dr, distal fiber; pf, proximal fiber; tr, transition region between basal body and axoneme. maintains two flagella until the next mitosis, whereupon the cycle begins again. The cycle of flagellar assembly described above refects and is dependent upon an underlying cycle of basal body assembly, maturation, and segregation. Early in Gl two probasal bodies appear adjacent to the two mature axoneme-bearing basal bodies. By late Gl these probasal bodies have matured into ultrastructuraUy complete basal bodies (2,8). These newly matured basal bodies are not yet competent to assemble flagella, since the cells remain biflagellate and regenerate only two flagella following deflagella-962 THE JOURNAL OF CELL BtOLOGY -VOLUME 100, 1985 tion although four basal bodies are present in the cytoplasm. During mitosis, the basal bodies, now without axonemes, can be observed in pairs near the spindle poles (3). It is probable that basal body segregation at cytokinesis is semiconservative; if so, each basal body pair consists of one newly matured and one experienced (i.e., previously flagellated) basal body (11). Frequently, daughter cells immediately undergo one or two further rounds of mitosis before flagellar assembly is initiated (4); the pattern of basal body biogenesis and segregation in these cases is unknown. Results presented in this paper demonstrate that in vfl-1 flagellar assembly is not tightly coupled to the cell cycle as it is in wild type. Instead of assembling flagella only during a narrow, well-defined "window" at the beginning of G~, vfl-1 Electron microscopic results suggest that the defect in the flagellar assembly cycle reflects an underlying defect in the basal body cycle such that basal body synthesis occurs throughout G~. In particular, we have observed objects in vfl-I cells that appear to be intermediates in basal body assembly. These include basal bodies composed of doublet rather than triplet microtubules and electron-dense rings of approximate basal body diameter. The latter resemble the "amorphous discs" observed by Cavalier-Smith (2) in early-G1 cells at sites where probasal bodies would later appear. Neither doubletcontaining basal bodies nor electron-dense rings were seen in wild-type controls or in the other vfl mutants examined. Of course, it is possible that the objects that appear to be early intermediates in basal body assembly are something else entirely. Perhaps the increase in flagellar number observed during interphase reflects the completion of late stages in basal body maturation--that is, the conversion of basal bodies that appear ultrastructuraUy complete but are unable to nucleate axoneme formation into fully mature basal bodies that are capable of initiating axoneme assembly. Even if the production of additional flagella on vfl-1 cells is due to maturation of basal bodies already present in the cytoplasm, rather than to the initiation and completion of new basal assembly, vfl-1 can still be properly said to be defective in the temporal control of the basal body cycle, though in a different and rather more limited sense than implied previously.
Empty tunnels through the cell wall were not observed in vfl-1 sections, which suggests that flagella formation and tunnel formation may be concurrent. It seems reasonable that, as Cavalier-Smith suggested (2), the flagella themselves could serve as templates for tunnel formation or even actively participate in the process. In any case, the existence or formation of a tunnel is not a limiting factor for the addition of

Defective Cytokinesis and Organelle Placement in vfl-I
Organelles are often misplaced in vfl-1, and cytokinesis frequently fails to partition the cell evenly in two. Basal bodyassociated striated fibers are absent or incomplete, and rootlet microtubules are typically misplaced. Another vfl mutant, vfl-3, shows a similar phenotype (17). It is not known how organelle or cleavage furrow localization is achieved, and so it is not possible to interpret these defects in molecular terms. Two contrasting possibilities are worth mentioning, however. One is that the basal bodies themselves play important roles in organizing the cytoplasm so that other organelles are properly placed and so that the cleavage furrow passes cleanly between basal body pairs and daughter nuclei. If this were the case, then the presence of basal bodies at abnormal locations in vfl-1 and vfl-3 might lead directly to misplaced organelles and misplaced cleavage furrows. Such organizing functions might be mediated by the rootlet microtubules that radiate into the cytoplasm from the region beneath the distal striated fiber and/or by the numerous microtubules that radiate from the basal body region at cytokinesis and line the cleavage furrow (12). Alternatively, the abnormalities in basal body placement and number might derive from defects in an asyet-unidentified cytoplasmic organizing system that is also responsible for localizing other organelles and/or the cleavage furrow. If this were the case, then the relation between the basal body defects and the other defects would not be one of cause and effect.

Aberrant Motility in vfl-1 Is Due to Abnormal Placement or Rotational Orientation of Flagella
We attribute the aberrant motility of vfl-I to defective flagellar location or orientation and not to defective flagellar structure per se. While flagellar location in many vfl-1 cells is decidedly abnormal (Fig. 2), and this should be sufficient to produce aberrant motility, some cells do have closely paired anterior flagella, and it might seem that these should swim normally. However, normal motility requires that the two flagellar wave forms possess 180* rotational symmetry with respect to one another. The basal body-associated striated fibers are probably required to ensure that this symmetry exists (10,17), and since the fibers are defective in vfl-1, abnormal motility in almost every vfl-1 cell is expected. Because the defects ofvfl-1 at the basal body level are sufficient to produce the aberrant motility phenotype, there is no reason to invoke abnormalities in the flagella themselves. In fact, we have failed to note any defects in axoneme ultrastructure in vfl-1 nor were any protein-composition abnormalities revealed by two-dimensional gel electrophoresis/autoradiography of S-35 labeled vfl-1 flagella (data not shown).

Relation of vfl-1 to Other vfl Mutants
vfl-1 is genetically and phenotypically distinct from other C. reinhardtii variable flagella mutants that have been described in the literature. In phenotype it most closely resembles vfl-3 (see previous discussion), but vfl-3 cells do not accumulate flagella throughout Gt as vfl-1 cells clearly do. The vfl-3 and vfl-1 loci are unlinked genetically: vfl-3 is linked to the centromere of chromosome VI (17) and vfl-1 is on chromosome VIII, also centromere linked. Another vfl gene, vfl-2 (13), has not yet been mapped to a particular chromosome, but it has been demonstrated to be unlinked to either vfl-1 or vfl-3 (data not shown). Furthermore, the vfl-2 phenotype differs from that of vfl-1 or vfl-3 in that vfl-2 cell size is fully normal (13). A variable flagella number mutant designated cyt-1 was described some years ago (15). cyt-1 cells often fail to complete cytokinesis, which leads to the generation of many large multinucleate cells with multiple pairs of flagella.