Defective Temporal and Spatial Control of Flagellar Assembly in a Mutant of Chlamydomonas reinhardtii with Variable Flagellar Number G. MIKE W. ADAMS, ROBIN L. WRIGHT,* and JONATHAN W. JARVIK* Department of Botan, Louisiana State University, Baton Rouge, Louisiana 70803; and *Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213 ABSTRACT 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-I 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 pleio- tropic defects observed in vfl-1 result from a recessive Mendelian mutation mapped to Chromosome VIII. The basal body developmental cycle of the unicellular bifla- screening for motility-defective mutants following mutagenesis with N-methyl- gellate green alga, Chlamydomonas reinhardtii, represents a N'-nitro-N-nitrosoguanidine (1). Cells were grown at 25°C in medium I of Sager and Granick (14). Synchronization was achieved by a 14-h light/10-h clear case in which organelle replication is coupled to cell dark illumination schedule unless otherwise specified. replication. In the basal body cycle, new basal bodies form at Measurement of Flagellar Number, Length, and Loca- determined times and places during interphase, and they tion: Cells were fixed by the addition of ~/2 vol of 1% aqueous glutaraldehyde segregate equally at cell division to yield biflagellate daughter and examined at x800 using phase-contrast optics for determination of flagellar cells (2, 8). Through the analysis of mutants with defects in number and location. Flagellar length was measured with an ocular micrometer the basal body cycle, we hope to gain insight into the mech- using phase-contrast or Nomarski differential interference contrast optics. Distributions of flagellar number and location were based on samples of at anisms of basal body biogenesis and segregation, and also into least 200 cells and flagellar length averages were based on samples of at least the mechanisms by which cell and organelle cycles are coor- 20 cells per flagellar number class. Cell volumes were determined by measuring dinated. In this communication we describe a mutant, vfl-1, cell length and width and applying the equation ~r/6 x (width)2 x length (13). in which the temporal and spatial regulation of the basal body Determination of Flagellar Number Changes in Living Cells cycle is defective. As a result, vfl-1 cells have the novel property and Mitotic Pedigree Analysis: Cells in the first hour of the light of assembling new flagella continuously throughout much of segment of the cycle were suspended in 0.5% melted agar. A drop was placed the cell cycle. 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 MATERIALS AND METHODS 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 Strains and Culture Conditions: vfl-1was derived from the strain per cell division typically took place. The next morning the number and size CC-70 mt- (Chlamydomonas Culture Collection, Duke University) in a general of daughter cells were measured along with the number of flagella on each cell. THE JOURNAL OF CELL BIOLOGY - VOLUME 100 MARCH 1985 955-964 © The Rockefeller University Press • 0021-9525]85103/0955110$1.00 955 Genetic Analysis: Standard techniques were used for crosses and Cell Morphology and Growth tetrad dissections (6, 9). Mapping stocks ware those listed in Adams et al. (1). Dominance was assessed in arg-2/arg-7, vfl-l/vfl-I ÷ diploids (5). vfl-1 cells show extreme variation in size and are often Electron Microscopy: Log-phase cells between the second and fourth abnormal in shape. Measured cell volumes for 100 mid-Gt hour of the light segment of the illumination cycle were harvested by centrif- vfl-I ceils ranged from 24 to 1423 /~m3, whereas wild-type ugation (5 min at 2,500 rpm). The pellet was resuspended in residual culture controls ranged from 72 to 733 gm 3. The range in size seen 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 in these controls is typical of rapidly growing light/dark- min on ice the cells were repelleted and the initial fixative was replaced with synchronized Chlamydornonas cultures, in which many cells an equal volume of fresh fixative. After 30 min the cells were washed at least undergo two or more rounds of mitosis during the dark five times with 0. l M sodium cacodylate buffer and stored overnight at 4°C. portion of the cycle (4). No obvious correlation was seen Pellets were postfixed for 2 h in 2% osmium tetroxide in 0.1 M cacodylate and between cell size and flagellar number (Fig. 3A), but, as in 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 wild type, cells with larger volumes tended to have somewhat staining was accomplished with 0.25-0.5% aqueous uranyl acetate for 15 rain longer flagella (Fig. 3 B). or, for cases in which enhanced microtubule contrast was desired, 4% tannic Microscopic observation of microcolonies derived from 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 anhy- dride, 20 ml Araldite 502, 20 ml Embed 812, 3 ml 2,4,6-tri(dimethylamino- methyl)phenol-30); Electron Microscopy Sciences, Fort Washington, PA). In- filtration 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 Instrn- ments--SorvalI Biomedical Div., DuPont Co., Wilmington, DE). Sections were stained with 3% aqueous uranyl acetate and Reynold's lead citrate and exam- ined with a Phillips 300 electron microscope at 60kV. RESULTS 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 inter- ference 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. Since -20% of vfl-1 cells are typically biflagellate, this means that even biflagellate cells swim aberrantly. 50 20 o 0 II ], i FIGURE 2 Nomarski differential interference contrast micrographs o I 2 ~ 4 5 of glutaraldehyde-fixed vfl-1 vegetative cells (x 3,400). Note wide Number of F'logello per Cell variations in cell size and in the number and location of flagella. FIGURE 1 Distribution of flagella on synchronous early G1 vfl-I The cell in B c)osely resembles wild type in size and flagellar vegetative cells. 440 cells were scored. placement. 956 THE JOURNAL OF CELL BIOLOGY . VOLUME 100, 1985 light/dark regime was sampled at intervals and the mean • • vfl - 1 number of flagella per cell was determined. As indicated in o Wild- type 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. _a3 qJ • O0 OO • In another experiment, single cells of synchronized vfl-1 ,'7 culture were immobilized in agar and the number of flagella o ~<~g~ o on individual cells was recorded 1 and 6 h later. As the results • ellqlIQie Itl • 4e • • presented in Table I indicate, cells of all flagellar number e Z classes could add new flagella in G~. When the data are eOOllOee ••8 ~allqp • O • • • considered together, they give an average flagellar number per cell of 1.26 at the time of the initial observation, and a value of 1.51 5 h later.