FLAlO Locus: Suppressors and Synthetic Phenotypes That Affect the Cell Cycle and Flagellar Function in Chlamydomonas reinhardtii

Through the isolation of suppressors of temperature-sensitive flagellar assembly mutations at the FLAlO locus of Chlamydomonas reinhardtii, we have identified six other genes involved in flagellar assembly. Mutations at these suppressor loci, termed SUFI-SUF6, display allele specificity with respect to which jZalOmutant alleles they suppress. An additional mutation, apml-122, which confers resistance to the plant herbicides amiprophos-methyl and oryzalin, was also found to interact with mutations at the FLAlO locus. The apmlI22 mutation in combination with threeJal0mutant alleles results in synthetic cold-sensitive cell division defects, and in combination with an additional pseudo-wild-type jlalOallele yields a synthetic temperature-sensitive flagellar motility phenotype. Based upon the genetic interactions of these loci, we propose that the FLAlO gene product interacts with multiple components of the flagellar apparatus and plays a role both in flagellar assembly and in the cell cycle. T HE flagellar apparatus of Chlamydomonas consists of a pair of basal bodies and their associated flagella (TUCKER 1984; LeDizet and PIPERNO 1986; HOLMES and DUTCHER 1989). In addition to the microtubules of the flagella, the majority of the cytoplasmic microtubules and the rootlet microtubules originate in the vicinity of the basal bodies at the anterior of the cell (RINGO 1967). Prior to mitosis, the flagella disassemble and the components are resorbed, basal body replication/elongation is completed, and the two pairs of basal bodies separate and migrate to opposite sides of the nucleus where they take up residence in the region of each mitotic spindle pole (JOHNSON and PORTER 1968; Coss 1974; TRIEMER and BROWN 1974; HOLMES and DUTCHER 1989). The cytoplasmic microtubules also disassemble but the rootlet microtubules remain intact. After cell division is completed, new flagella, cytoplasmic microtubules, and two new rootlet microtubules are assembled. Most evidence suggests that the basal bodies are required only for the assembly of the flagella and not for the assembly of the other microtubule systems (PICKETTHEAPS 1971). In other words, basal bodies and centrioles are not required for mitosis (MCINTOSH 1983; BRINKLEY 1985). Basal bodies are tubular structures composed of nine blades of triplet microtubules that are approximately 400 nm in length. Basal bodies are morphologically similar to centrioles, which are found in many but not all eukaryotic cells (KUBAI 1975; PETERSON and BERNS 1980). Basal bodies are complex organelles Genetics 128: 549-561 uuly, 1991) by morphological and biochemical criteria. Isolated basal bodies from Chlamydomonas reinhardtii contain more than 200 polypeptide components (DUTCHER 1986) and isolated centrosomes from mammalian cells contain a similar number of components (KLOTZ et al. 1990). At least 15 different polypeptides have been identified by antisera that recognize components of this organelle (CALARCO-GILLAM et al. 1983; BARON and SALISBURY 1988; SNYDER and DAVIS 1988; KELLOGG, FIELD and ALBERTS 1989; KURIYAMA 1989; RAO et al. 1989). One approach to the study of a complex system is the application of a genetic analysis. To this end, we have initiated a genetic and phenotypic analysis of mutations that appear to affect the basal body of Chlamydomonas (DUTCHER, GIBBONS and INWOOD 1988; DUTCHER 1989). In this paper we report the study of mutations in the FLA10 gene of Chlamydomonas. This gene was originally identified by temperature-sensitive mutations that are unable to assemble flagella at 32" (HUANG, RIFKIN and LUCK 1977; ADAMS, HUANG and LUCK 1982). We have shown that mutations at this locus have an effect on cell division and flagellar motility as well as on flagellar assembly. In addition to their requirement for flagellar assembly, basal bodies may also play a role in cell division. MATERIALS AND METHODS Mutant strains: The mutant strains dd-a-224, 519, and 544 were isolated in screens for temperature-sensitive flagellar assembly-defective strains of Chlamydomonas reinhardtii (HUANG, RIFKIN and LUCK 1977; ADAMS, HUANC and 550 F. G. Lux and S. K. Dutcher LUCK 1982). Each of the mutant JalOstrains was backcrossed to the isogenic wild-type strain, 137c, a minimum of three times to assure that the flagellar-defective phenotype segregated 2+:2and was due to a single gene mutation. The act2 (CC-1590), arg2 (CC-48 and CC-49), and arg7 (CC-50 and CC-51) strains were obtained from the Chlamydomonas Genetics Center (HARRIS, BOYNTON and GILLHAM 1987). The original argstrains yielded less than 10% viable spores in crosses. Consequently, argspores were backcrossed to wild type (137c) a minimum of six times to generate strains that yielded meiotic spore viability of 9099%. All haploid strains used in the construction of diploid strains were derived from these argstrains and yielded similar spore viability. Stable diploid strains were constructed by mating strains that carried the complementary arginine markers, arg2 and arg7, and selecting for prototrophic progeny (EBERSOLD 1967). These haploid parents also carried various recessive drug-resistance mutations. The apmlmutations were isolated in this laboratory following ultraviolet irradiation on rich medium containing 15 p~ oryzalin. They were designated as alleles at the APMl locus based on map distance from the UNZI locus, failure of the apml-122 allele to recombine with the apml-7 allele from P. LEFEBVRE (JAMES et al. 1988) in 48 random spores, failure of the 14 apmlalleles used in this study to recombine with each other in a total of 320 tetrads, and a failure to observe complementation in diploid strains in any of the 33 pairwise combinations of the 14 apmlalleles isolated in this laboratory. The pdr3-1 allele, isolated in the same selection, shows pleiotropic drug resistance phenotypes as do the original pdrmutations (JAMES and LEFEBVRE 1989), and is unlinked to them. Resistance to oryzalin is semidominant in heterozygous pdr3-1IPDR3" diploid strains. The apm3-1 allele, also isolated in the same selection, has not been mapped but is semidominant in heterozygous apm3-l/ APM3+ diploid strains. The pdrl-1 allele was obtained as strain CC-399 from the Chlamydomonas Genetics Center. Media and culture conditions: Rich growth medium is a modified version of medium I of SAGER and GRANICK (1953) in which the concentration of K2HP04 has been raised from 0.57 mM to 1.0 mM and the trace metal solution of HUTNER et al. (1950) is substituted. All water used in media preparation is derived from a Milli-Q water filtration system equipped with an ultrafilter cartridge that has an effective pore size o€ 0.1 pm (Millipore Corporation, Bedford, Massachusetts). Strains that are auxotrophic for arginine are grown on rich medium with one-tenth the normal concentration of ammonium nitrate, supplemented with 200 pg/ ml of arginine-HCI. Decreased concentrations of ammonium nitrate are reported to stimulate the uptake of exogenous amino acids in some algae (NORTH and STEPHENS 197 1, 1972). In Volvox, nitrogen starvation elevates arginine uptake, but arginine is insufficient to support growth when supplied as the sole nitrogen source (KIRK and KIRK 1977). Oryzalin-containing plates are rich medium supplemented with 15 pM oryzalin (courtesy of GLENN EVANS, Eli Lilly Greenfield Laboratories). M-N/5 is a low nitrogen, minimal liquid medium that lacks acetate and has one-fifth the normal concentration of ammonium nitrate of rich medium. Low sulfate medium is modified rich medium with onetenth the normal concentration of magnesium sulfate, supplemented with magnesium chloride at a concentration of 0.28 mg/ml. Low sulfate plates contain 1.5% Difco Bactoagar washed a minimum of five times in Milli-Q water to remove endogenous sulfates and other contaminants and air dried. Unless otherwise noted, all cells were grown under constant light at an intensity of 20 pE/m2/sec. Genetic analysis: Standard methods were employed for mating and for tetrad analysis (LEVINE and EBERSOLD 1960; for detailed description, see HARRIS 1989). Centromere distances were determined by the method of WHITEHOUSE (1 950) and linkage analysis was performed using the method of PERKINS (1 952). Mutagenesis:JalOmutant cells were exposed to ultraviolet (UV) irradiation or were treated with the alkylating agents; nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), or methyl methanesulfonate (MMS). W irradiation: The basic procedure for mutagenesis was that of LUCK et al. (1 977). Lawns of approximately 10" cells on 100-mm diameter Petri plates containing either rich or low sulfate medium, or suspensions of 10' cells in 10 ml of M-N/5 medium in 100-mm diameter Petri plates, were irradiated under a General Electric 30 W germicidal UV lamp (G3T08) at a distance of 15 cm for 45 or 60 sec. Chemical mutagenesis: Suspensions of 10' cells in M-N/5 medium were incubated in appropriate concentrations of MNNG (2-50 pg/ml), EMS (10-30 pl/ml), or MMS (0.2 or 1.0 pl/ml) for 15-120 min (LUX 1990). Cells were then pelleted, washed three times in M-N/5 medium, and diluted into 20 ml of rich liquid medium and incubated in the light at 32" for the isolation of suppressed strains (see below). EMS-treated cells were washed three times in 1% sodium thiosulfate in rich medium or in M-N/5 medium to inactivate the EMS. Isolation of suppressors: Following mutagenesis, suppressors of theflulostrains were isolated through the use of one of two schemes. The first regimen involved the enrichment for mutagenizedJul0cells that had acquired the ability to swim at the nonpermissive temperature of 32" (swimming screen). The second regimen selected for ja l0cells that had acquired the ability to mate at 32" (mating selection). This selection only required that cells be competent to assemble flagella, whether or not the flagella were functional and motile. Cells must only assemble flagella and express the appropriate surface molecules for the recognition of cells of the opposite mating type (BERGMAN et al. 1975; GOODENOUGH and WEISS 1975). Zygotes can be selectively recovered because their thick protective cell wall is impermeable to chloroform vapors, while the cell wall of vegetative haploid and diploid cells is not. Swimming screen: Approximately 1 O5 to 1 O6 mutagenized cells were placed in 20 ml rich liquid medium in 25 mm X 175 mm tubes. After 12-24 hr at 32", the upper 2 ml of medium were transferred to 20 ml of fresh rich medium in the same size tubes at 32 O until visible numbers of swimming cells became apparent, which was usually 3-4 days. At that point, the upper 2 ml were again transferred to 20 ml of fresh rich medium in order to further enrich for suppressed ja lOstrains that were able to swim. Cells were then plated on solid medium to isolate single colonies. Eighteen to 24 single colonies from each plate were inoculated individually into rich liquid medium and the motility phenotype was scored at 21 O and 32" to confirm the presence of a suppressor mutation. In general, only one revertant isolate from each original selection tube was retained in order to assure the independence of each suppressor allele. However, in cases where two single colonies from a particular tube yielded cells with significantly different phenotypes, both isolates were saved for further study and labeled "isolate #" and "isolate #A." Mating selection: Mutagenized populations were made competent for mating at the restrictive temperature of 32 O . Mutagenized cells were mated after nitrogen starvation either to jZal0-1 or to wild-type tester cells at 32" for approximately 12 hr to maximize the mating of suppressed Interactions at the FLAlO Locus 551 JalOcells in the population. Mixtures of mating cells were then inoculated onto rich medium plates, incubated for another 12 hr in the light at 32', and allowed to undergo the remainder of zygotic maturation under standard conditions (HARRIS 1989). Following zygotic maturation, plates were treated with chloroform and then placed in the light at 21 ' to allow for germination of zygotes and growth of the resultant meiotic progeny cells. After approximately 1 week, these zygote clones were transferred into rich liquid medium and grown for 1 or 2 days at 2 1 '. Cells were then diluted and plated on rich medium for the isolation of single colonies. These single colonies were picked into rich liquid medium and their flagellar assembly phenotypes were scored at 21' and 32". Identification of intragenic us. extragenic suppressors: Each suppressedf2alOisolate was crossed to the isogenic wild-type strain 137c. Aflagellate cells are nonmotile; when grown in liquid culture they fall under the influence of gravity and form a pellet. By contrast, wild-type cells either accumulate at the air-liquid interface or distribute themselves throughout the culture medium. Based upon this phenotypic difference at 32", mutant JalOstrains are easily distinguished from wild type or from suppressed strains. If only PD tetrads were recovered, then the suppressor was considered to be intragenic or linked [less than 7 centiMorgans (cM)] to the originalJal0mutation. Those strains that yielded tetratype (TT) and nonparental ditype (NPD) tetrads were considered to contain an extragenic suppressor mutation. At least seven tetrads were examined in these crosses and an average of 33 tetrads were analyzed. To confirm that suppression resulted from a single mutational event, strains that contained an extragenic suppressor mutation were backcrossed to the unmutagenized parentalJal0allele. At least nine tetrads were examined in the backcrosses and an average of 19 tetrads were analyzed. Recovery of only PD tetrads indicated that suppression resulted from a single extragenic mutation. Recovery of TT or NPD tetrads indicated that suppression resulted from more than one extragenic mutation.