Genetics of the Deflagellation Pathway in Chlamydomonas

Corresponding author: Lynne M. Quarmby, Department of Cell Biology, Emory University School of Medicine, 1648 Pierce Drive, Atlanta, GA 30322-3030, quarmby{at}cellbio.emory.edu (E-mail).

Communicating editor: P. J. PUKKILA

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Signal-induced deflagellation in Chlamydomonas involves Ca²⁺-activated breakage of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. In this study, we isolated 13 new deflagellation mutants that can be divided into two phenotypic classes, the Adf class and the Fa class. Cells with the Adf deflagellation phenotype are defective in acid-stimulated Ca²⁺ influx, but can be induced to deflagellate by treatment with nonionic detergent and Ca²⁺. Genetic analyses show that the five new Adf mutations, as well as the previously identified adf1 mutation, are alleles of the ADF1 gene. Mutants in the second phenotypic class, the Fa mutants, fail to deflagellate in response to any known chemical stimulus and are defective in Ca²⁺-activated microtubule severing. Genetic analysis of these eight new Fa strains demonstrated that they define two complementation groups, and one of these contains the previously identified fa1 mutation. Diploid analysis showed that five alleles map to the FA1 gene, whereas four alleles define a novel gene that we have named FA2. The isolation of multiple mutant alleles of each gene, generated by either ultraviolet irradiation or insertional mutagenesis, indicates that ADF1, FA1, and FA2 may be the only genes that can be identified in a loss-of-function screen. These alleles should provide a better understanding of the regulation of microtubule severing by Ca²⁺.

DEFLAGELLATION in Chlamydomonas reinhardtii is triggered by a number of stimuli, including treatment with weak organic acids (WITMAN 1986 ; QUARMBY et al. 1992 ; HARTZELL et al. 1993 ). The key event in deflagellation is the severing of the axonemal outer doublet microtubules at a precise site, distal to the transition region between the basal body and the beginning of the flagellar shaft (ROSENBAUM and CARLSON 1969 ; SATIR et al. 1976 ; LEWIN and LEE 1985 ; SANDERS and SALISBURY 1989 ; JARVIK and SUHAN 1991 ). Severing of axonemal microtubules during deflagellation may be homologous to the severing of cytoplasmic microtubules discovered in mitotic Xenopus and starfish oocytes and attributed to the ATPase katanin (VALE 1991 ; MCNALLY and VALE 1993 ; KARSENTI 1993 ; MCNALLY et al. 1996 ; LOHRET et al. 1998 ). Axonemal microtubule severing is accompanied by Ca²⁺-mediated contraction of a stellate array of centrin-containing fibers in the transition zone (SANDERS and SALISBURY 1994 ). Although a variety of stimuli can cause deflagellation in vivo (MINZ and LEWIN 1954 ; THOMPSON et al. 1974 ; LEWIN et al. 1980 ; WITMAN 1986 ; QUARMBY et al. 1992 ), axonemal severing (deflagellation) in vitro requires ~1 µM Ca²⁺ (SANDERS and SALISBURY, 1994 ). This suggests that all agents that cause deflagellation in living cells do so by elevating intracellular [Ca²⁺] (QUARMBY and HARTZELL 1994 ; QUARMBY 1996 ; L. M. QUARMBY, unpublished results).

In Chlamydomonas, Ca²⁺ also regulates other flagellar functions, such as mating (GOODENOUGH et al. 1993 ) and the motile responses to light (HARZ et al. 1992 ; WITMAN 1993 ; PAZOUR et al. 1995 ). Presumably, the Ca²⁺ influx pathway and the Ca²⁺-sensing protein involved in deflagellation must have particular properties that allow the cell to distinguish this Ca²⁺ signal from the Ca²⁺ signals that regulate other flagellar behaviors. Our aim is to identify the proteins involved in the regulation of Ca²⁺ influx and the severing of microtubules in response to Ca²⁺.

The first C. reinhardtii deflagellation-defective mutant is the fa1 (flagellar autotomy) strain, and this strain does not shed its flagella in response to any known stimulus (LEWIN and BURRASCANO 1983 ). Moreover, the addition of Ca²⁺ to permeabilized fa1 cells does not trigger the axonemal severing and deflagellation observed in wild-type cells (QUARMBY and HARTZELL 1994 ; LOHRET et al. 1998 ; J. JARVIK, personal communication). The second deflagellation mutant, adf1 (acid deflagellation), was discovered as a second unlinked mutation carried by the imp4 strain (T. SAITO and U. GOODENOUGH, Washington University, St. Louis). The Adf phenotype is distinct from the Fa phenotype because (Adf) cells do not shed their flagella when treated with a weak organic acid, but they deflagellate if Ca²⁺ is added to permeabilized cells (QUARMBY and HARTZELL 1994 ; L. M. QUARMBY, unpublished observations). Apparently, the proton-activated Ca²⁺ influx pathway is defective in adf1 cells, whereas Ca²⁺-activated microtubule severing is defective in fa1 cells (QUARMBY and HARTZELL 1994 ; QUARMBY 1996 ; LOHRET et al. 1998 ).

Microtubule severing is the breakage of microtubules along their length. It is distinct from dynamic instability where polymer shortening and elongation occurs by tubulin subunit addition or loss at the polymer ends (MITCHISON and KIRSCHNER 1984 ). A mitotically activated protein constituent of Xenopus egg extracts can sever microtubules in vitro (VALE 1991 ). MCNALLY and VALE 1993 subsequently purified a heterodimeric ATP-dependent, microtubule-severing protein, katanin, composed of 81- and 60-kD subunits from mitotic sea urchin oocytes. Katanin appears to cause microtubule severing by a localized, ATP hydrolysis–dependent depolymerization without proteolysis. Although it is present throughout the cytoplasm, MCNALLY et al. 1996 found an enrichment of katanin surrounding the pericentriolar -tubulin–containing region (STEARNS et al. 1991 ).

We have found that an antibody that specifically recognizes katanin from humans, sea urchins, and Xenopus also recognizes a single band at ~55 kD on Chlamydomonas protein blots (LOHRET et al. 1998 ). A bright signal in the basal body/flagellar transition zone region is observed in indirect immunofluorescence experiments with this antibody. Furthermore, this antibody blocks Ca²⁺-stimulated deflagellation in a preparation of Chlamydomonas flagella-basal body complexes (FBBC; LOHRET et al. 1998 ). We concluded that a katanin-like mechanism probably mediates axonemal microtubule severing during deflagellation. A genetic dissection of the deflagellation pathway could, therefore, provide new insights into katanin regulation.

From a screen of more than 26,000 mutants generated by either UV irradiation or nonhomologous insertion of exogenous DNA, we isolated 13 new deflagellation mutants. Five mutants have the Adf phenotype, and all are in the same complementation group as the previously identified adf1 mutation. Analysis of stable diploid strains showed that all the mutations are recessive. We were able, therefore, to determine from adf/adf diploids that the five adf mutations are allelic, and we have named the gene ADF1. Eight of the new deflagellation mutants have the Fa phenotype and define two complementation groups. One group, FA1, mapped to the same locus as the previously identified fa1 mutation. Diploid analysis showed that the five mutations in this complementation group are allelic. Similarly, we demonstrate the discovery of four mutant alleles in the newly defined FA2 gene. ADF1, FA1, and FA2 are likely to be the only genes that can be identified in a loss-of-function screen because we have isolated multiple alleles of each of the three genes using two independent methods of mutagenesis. In our model for deflagellation, we propose that Adf1p is an essential component of proton-stimulated Ca²⁺ influx. Fa1p and Fa2p are proposed to play essential roles in the regulation of Ca²⁺-activated microtubule severing.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cell strains and culture:
Table 1 provides a summary of the strains used. C. reinhardtii wild-type strains g1 (nit1, agg1, and MT+) and B214 (nit1, agg1, and MT-; obtained from G. PAZOUR, Worchester Foundation, Shrewsbury, MA; PAZOUR et al. 1995 ) were mutagenized by either UV irradiation or by nonhomologous insertion of exogenous DNA (see below). Strains g1 and B214 also served as parental strains for most backcrosses. Strains cc620 and cc621 (obtained from the Chlamydomonas Genetics Center, Durham, NC) served as (1) wild-type strains in some backcrosses and all dikaryon experiments, (2) tester strains used to assess mating type, and (3) sources of gametic lytic enzyme (HARRIS 1989 ). Strains ac17 and nit2 (provided by S. DUTCHER, University of Colorado, Boulder, CO) were used for stable diploid experiments. The nit2 strain cannot grow on nitrate as the sole nitrogen source, whereas the ac17 strain requires acetate to grow (HARRIS 1989 ). A paralyzed mutant, pf18, defective in assembly of the central pair microtubules, was obtained from the Chlamydomonas Genetics Center. Cells were maintained on TAP plates (1.5% agar; HARRIS 1989 ) at 22° with constant illumination.

Mutagenesis scheme:
For UV mutagenesis, wild-type cells on 1.5% agar TAP plates were suspended 6 cm above the surface of a TM-36 Transilluminator (UVP, San Gabriel, CA) powered at 7.0 mW/cm². We used an exposure time of 2.6 min, which was determined empirically to be the LD₅₀ for these cells. After UV light exposure, cells were placed in the dark for 24 hr to prevent photoreactivation and then placed in constant light for one cell division, as assessed by stereomicroscopy. Cells were resuspended in liquid medium, and 10³ cells were spread on 150-mm TAP agar plates.

Nuclear transformation by exogenous DNA was done essentially as described previously (KINDLE et al. 1989 ; TAM and LEFEBVRE 1993 ), using the nitrate reductase gene NIT1 (FERNANDEZ et al. 1989 ; NELSON et al. 1994 ). Cells defective for nitrate reductase were grown in liquid TAP medium bubbled with 5% CO₂ under bright light for several days. Cells were concentrated by centrifugation (4°, 4100 g, 3 min), and their walls were removed by treatment with gametic lytic enzyme. Cells were pelleted and resuspended in SGII(NO₃) (SAGER and GRANICK 1953 ; modified by replacement of NH₄NO₃ with 2 mM KNO₃ as described by FERNANDEZ et al. 1989 ) at a final concentation of 2 x 10⁸ cells/ml. In a 15-ml polypropylene conical tube, 0.3 ml cells, 1 µg of pMN56 plasmid (NIT1 in pUC119, linearized by digestion with EcoRI), 0.3-g glass beads (0.7–1.2 mm in diameter; Sigma Chemical Co., St. Louis, MO), and PEG-8000 (final concentration = 0.64%; Sigma) were combined and vortexed at maximum speed for 45 s on a Vortex Genie Mixer. Cells were diluted with 10 ml SGII(NO₃), transferred to a new tube (leaving the glass beads behind), concentrated by centrifugation, and plated on SGII(NO₃) agar plates to select for Nit1⁺ transformants.

Screen for deflagellation mutants:
Colonies from UV light–exposed cells and Nit1⁺ transformants were picked into TAP medium in 96-well plates and placed under bright light for a few hours. Cells were then treated with an equal volume of a solution containing 40 mM Na acetate, pH 4.5, with 1 mM CaCl₂ for 60 sec to induce deflagellation and neutralized with 0.75 volumes of 0.1 N NaOH. Motility was assessed by stereomicroscopy. To determine whether the mutation was in the Adf pathway or the Fa pathway, cells grown on TAP plates for 6–7 days were innoculated into liquid TAP medium and incubated for 6–7 hr. An aliquot of cells was treated with an equal volume of 40 mM Na acetate, pH 4.5, with 1 mM CaCl₂ for 30 sec, followed by fixation with 2% glutaraldehyde. Another aliquot of cells was treated with an equal volume of 0.05% Triton X-100 with 1 mM CaCl₂. Cells were scored by phase contrast microscopy for the number of attached flagella.

Genetic analyses:
Methods outlined by HARRIS 1989 were followed for mating and tetrad analysis. Meiotic progeny of backcrossed deflagellation-defective mutants were assayed for deflagellation phenotype and mating type. To assess mating type, we modified the pellicle assay (described in HARRIS 1989 ). Duplicate 50-µl aliquots of the strain of unknown mating type were placed in pairs of wells in a 96-well plate, one well contained 50 µl of MT⁺ (cc620) cells, and the other well contained 50 µl of MT^- (cc621) cells. Plates were incubated overnight under bright lights and examined by stereomicroscopy the next day for formation of a zygote pellicle.

Mutants generated by NIT1 transformation were analyzed for nitrate reductase (Nit1) expression by assessing growth on SGII(NO₃) agar plates. To determine the number of linkage groups, F₁ progeny of adf or fa mutants were crossed with each other, and meiotic progeny (determined by the occurance of both mating types in a tetrad) were assayed for deflagellation phenotype.

Stable diploids were generated for complementation and dominance tests (EBERSOLD 1967 ). To construct suitable strains for diploid analysis, deflagellation-defective mutants were crossed with either nit2 or ac17 strains. SGII medium lacking acetate was used to assay Ac phenotypes. SGII medium with NaNO₂ substituted for NH₄NO₃ was used to assay Nit2 phenotypes. Nit2 and ac17 were mated, plated on selective medium, and exposed to a 12-hr dark, 12-hr light cycle for several days. Mitotic division of heterozygous zygotes on selective medium generated colonies of stable diploid prototrophs that were assayed for mating type and deflagellation phenotype. MT^- is dominant in stable diploids (EBERSOLD 1967 ), and all our isolates mated as MT^-.

For dikaryon rescue experiments (STARLING and RANDALL 1971 ), wild-type cells and F₁ deflagellation-defective mutants, grown on TAP plates for 6–7 days, were incubated in nitrogen-free liquid medium under light for 6–7 hr to promote completion of gametogenesis. Equal numbers of cells were mated at a density of 10⁷ cells/ml. In some experiments, cells were incubated with cycloheximide (Sigma; stock solution of 2 mg/ml in water).

DNA isolation and Southern analysis:
The protocol for obtaining genomic DNA was modified from one provided by J. WOESSNER (Washington University, St. Louis, MO) and is described below. Cells (cultured for 4–6 days) were harvested from an agar plate, resuspended in 1 ml TAP in a 1.5 ml microfuge tube, and pelleted at 16,000 g for 20 sec. Cells were resuspended in 400 µl TEN, pH 8 (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl), followed by addition of 40 µl of 20% SDS, 40 µl of 20% Sarkosyl, and 2 mg Proteinase K (Promega, Madison, WI), and were incubated at 65° for 1.5 hr. Samples were phenol-chloroform extracted (SAMBROOK et al. 1989 ) and treated with RNase (Sigma). After ethanol precipitation, the DNA was resuspended in 100 µl TE, pH 8 (10 mM Tris-HCl, 1 mM EDTA).

For Southern analysis, genomic DNA was restriction digested, size fractionated by agarose gel electrophoresis, and vacuum blotted (VacuGene XL; Pharmacia, Uppsala, Sweden) to Zeta Probe GT membranes (Bio-Rad, Richmond, CA). DNA was UV cross-linked to membranes (Stratalinker 1800; Stratagene, La Jolla, CA), which were then hybridized with either pUC119 plasmid linearized with BamHI or a fragment of the NIT1 gene. A 1.2-kb fragment from the 3' end of NIT1 was obtained by restriction digestion of the pMN56 plasmid with BamHI and EcoRI. The DNA fragment was isolated after agarose gel electrophoresis using the QIAquick Gel Extraction kit (Qiagen, Chatsworth, CA). We used [-³²P]dCTP (Amersham Life Science, Arlington Heights, IL) and the Prime-It II kit (Stratagene) to generate ³²P-labeled probes from the templates described above. Rapid-hyb buffer (Amersham) was used for all hybridizations following the instructions of the manufacturer, except for the following modifications: (1) hybridizations were typically done for 4 hr and (2) several rinses with 0.1x SSC (SAMBROOK et al. 1989 ) followed the washes. PhosphorImager SI computer hardware and ImageQuaNT v4.2 software (Molecular Dynamics, Sunnyvale, CA) were used to visualize the blots.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Identification and characterization of deflagellation-defective mutants:
It has been previously shown that brief treatment with acetate induces wild-type Chlamydomonas cells to deflagellate (WITMAN 1986 ; QUARMBY et al. 1992 ). Isolates of mutagenized cells, swimming in 96-well plates, were treated briefly with an equal volume of 40 mM acetate, pH 4.5. Deflagellation-defective mutants briefly stopped swimming in response to the acid shock, but when their medium was neutralized, they rapidly regained motility because they retained their flagella. In contrast, wild-type cells took more than 15 min to regrow flagella of sufficient length to achieve motility. Mutant cells were treated with either acetate, detergent, or buffer (control) to determine whether the mutation was in the acid-activated Ca²⁺ influx (Adf) pathway or in the Ca²⁺-activated microtubule severing (Fa) pathway. Figure 1 shows that the new deflagellation mutants could be clearly identified as either adf or fa mutants. F₁ deflagellation mutants were used for the quantification of deflagellation shown in Figure 1. Approximately 6000 clones of UV-mutagenized g1 cells were screened for defects in deflagellation. One adf (PK14) and two fa mutants (PK46 and PK33) were identified. Insertional mutagenesis resulted in more than 20,000 Nit1⁺ clones that were screened for deflagellation defects. As shown in Figure 1, four Adf (VF95, VF107, LQ206, and LQ216) and five Fa mutants (RF44, RF46b, CH56, RF88, and VF99) were identified. An additional Fa mutant, LQ1, was identified from ~1000 ARG7 (arginosuccinate lyase) insertional mutants generated by V. AMBRUST (University of Washington, Seattle, WA; Figure 1).

View larger version (32K): In this window In a new window Download PPT slide   Figure 1. —The deflagellation phenotypes of the new mutants were determined by treating cells with either acid or detergent. Adf and fa strains both fail to deflagellate in response to treatment with a weak organic acid, but only the fa strains fail to deflagellate when permeabilized with detergent in the presence of Ca2+. An aliquot of cells was treated with an equal volume of 40 mM Na acetate, pH 4.5, with 1 mM CaCl2 for 30 sec, followed by fixation with 2% glutaraldehyde. Another aliquot of cells was treated with an equal volume of 0.05% Triton X-100 with 1 mM CaCl2. Treatment with medium served as a control for the proportion of cells that were flagellated before treatment (generally >95%). Cells were scored by phase contrast microscopy for the number of flagella. Mean ± SD for two experiments, 100 cells scored for each experiment.

Nitrate reductase expression did not cosegregate with the deflagellation defect in backcrosses of the Adf Nit1⁺ transformants VF95, VF107, LQ206, and LQ216. Nor did it cosegregate in Fa Nit1⁺ transformant RF88. Similarly, arginosuccinate lyase expression, assessed by growth on medium lacking arginine, did not cosegregate with the deflagellation mutation in backcrosses of LQ1. Each of these strains was analyzed by Southern blot to determine whether a fragment of the insertional DNA cosegregated with the deflagellation mutation. Probes were generated from either the original bacterial vector, pUC119, or from fragments of the NIT1 or ARG7 gene. We did not detect the cosegregation of any insertional DNA with the deflagellation mutation in any of these strains.

On the other hand, as assessed in at least six complete tetrads, nitrate reductase expression cosegregated with the deflagellation mutation in the four other Fa Nit1⁺ transformants RF44, RF46b, CH56, and VF99. Complete tetrads of progeny from these backcrosses were examined by Southern analysis to determine the number of insertion events in each of these strains. A single insertion was found in each of the four tagged fa alleles (for example, see Figure 2). In summary, the results presented thus far indicate that we have identified eight new fa mutants and five new adf mutants. Of these 13 strains, four carry mutations that are linked to the inserted mutational DNA.

View larger version (40K): In this window In a new window Download PPT slide   Figure 2. —Southern analysis demonstrated cosegregation of a single insertion with the mutant phenotype. Progeny from complete tetrads, derived from backcrosses of CH56, VF99, and RF46b were used to prepare genomic DNA. The DNA was digested with PstI. pUC119 was used as the probe. Deflagellation phenotypes were determined as either wild type (+) or mutant (-). Nitrate reductase (Nit1) expression was either functional (+) or defective (-). A single band cosegregated with both Nit1 expression and the deflagellation mutation. Southern analysis using restriction endonucleases SmaI, SacI, BamHI, XhoI, and HindIII was performed to confirm that a single insertion was responsible for the deflagellation defect (data not shown).

Genetic characterization of the adf mutants:
To determine whether the five adf strains were allelic to adf1, each mutant was independently crossed with adf1, and meiotic progeny were scored for deflagellation. As shown in Table 2, all the progeny from crosses of strains PK14, VF95, VF107, or LQ206 with adf1 showed the Adf phenotype. Of 135 meiotic progeny from a cross between adf1 and LQ216, a single progeny with wild-type deflagellation behavior was recovered (Table 2). These data suggested that the five mutant strains identified in our screen are allelic to adf1. To confirm this result, complementation tests using stable diploids were performed. At least seven independent diploid strains from each cross were characterized for deflagellation phenotype and mating type. All diploid strains were MT^-. Stable diploids of adf1 with PK14, VF95, LQ206, and LQ216, as well as PK14 with VF107, are all phenotypically mutant for deflagellation. ADF/adf diploids did not exhibit any deflagellation defects; therefore, all the adf alleles are recessive. Because all the mutant alleles are recessive, we can conclude from the mutant phenotype of the adf/adf diploids that the five adf mutants recovered in our screen are alleles of adf1. The single wild-type progeny isolated from the cross of adf1 with LQ216 may have arisen either from an intragenic recombination event or as a spontaneous revertant.

Genetic characterization of new fa genes:
Previous results indicate that the deflagellation defect of fa1 is closely linked to the mating type locus (FERRIS and GOODENOUGH 1994 ). As shown in Table 3, the deflagellation defect cosegregated with the mating type locus in backcrosses of LQ1, CH56, RF88, and PK46. These results indicate linkage between these fa mutations and the mating type locus, although we did observe a presumed recombination event in a cross involving PK46. In contrast, in strains PK33, RF44, RF46b, and VF99, the deflagellation defect was not linked to mating type, indicating that these strains are not alleles of fa1 (Table 3). As shown in Table 4, no wild-type progeny were identified from several crosses between strains in this group, suggesting that PK33, RF44, VF99, and RF46b are allelic.

The mating type locus is a 1.1-Mb region on the left arm of linkage group VI, where meiotic recombination is suppressed and comprises 12 known gene loci, some of which have no obvious role in mating type or the sexual cycle (GILLHAM 1969 ; FERRIS and GOODENOUGH 1994 ; FERRIS 1995 ). Although fa1, LQ1, PK46, CH56, and RF88 all showed close linkage to mating type, it was important to determine whether the same gene was mutated in these five strains. Similarly, we wanted to confirm that PK33, RF44, VF99, and RF46b are alleles. Therefore, stable diploids were used for dominance and complementation tests. Deflagellation of FA/fa diploids was wild type, indicating that the fa alleles are recessive. Deflagellation of fa/fa diploids from crosses of MT⁺ fa strains fa1, LQ1, PK46, and CH56 with the MT^- fa strain RF88 showed the Fa phenotype, confirming that these five strains are alleles. Similarly, deflagellation of fa/fa diploids generated from crosses of PK33 and either RF44 or VF99, or from a cross between RF46b and VF99, showed the Fa phenotype, confirming that these four strains are allelic. From the crosses and diploid analyses, we conclude that our fa mutants define two genes, which we will call FA1 and FA2 (Table 5).

We also determined the phenotype of diploids heterozygous for mutations in the two FA genes. On the basis of our observation that all fa alleles were recessive, we predicted that these heterozygotes would be rescued for deflagellation. As expected, fa1FA2/FA1fa2 diploids, from a cross of fa1 (fa1-1) with PK33 (fa2-2), and a cross of fa1 (fa1-1) with RF46b (fa2-4), exhibited wild-type deflagellation.

The results described thus far indicate that we have identified three genes required for acid-induced deflagellation, ADF1 (disrupted in strains adf1, PK14, VF95, VF107, LQ206, and LQ216), FA1 (fa1, LQ1, CH56, RF88, and PK46), and FA2 (PK33, RF44, VF99, and RF46b). The FA1 gene maps near the mating type locus on the left arm of linkage group VI. The FA2 and ADF1 genes will be mapped by RFLP, after we have cloned DNA from these loci.

Fa1/fa2 double mutants (in haploid cells):
It is often the case that double mutants for genes that are from the same or a related pathway exhibit a phenotype different from that of either of the two mutants. To test this possibility, we crossed fa1 with fa2. MT⁺ was used as a marker for fa1, and Nit1⁺ was used as a marker for tagged alleles of fa2. In a total of 36 meiotic progeny from crosses of fa1-5 to fa2-1 and fa2-4, 8 MT⁺/Nit1⁺ progeny were recovered. These presumptive fa1/fa2 double mutants exhibited the Fa deflagellation phenotype and appeared to have wild-type motility, growth rates, phototaxis, and mating (data not shown).

Dikaryon analysis of adf and fa mutants:
In Chlamydomonas, after the mating and fusion of opposite mating type gametes, temporary dikaryons form (STARLING and RANDALL 1971 ). These dikaryons persist for about 2 hr with four flagella, two nuclei, and a common cytoplasm. At ~2 hr after mating, in preparation for meiosis, flagella are resorbed and a zygotic cell wall is formed. Temporary dikaryons can be used to assess the ability of wild-type components to rescue mutations in the context of preformed complexes and structures. The basal bodies, flagellar transition zone, and proximal outer-doublet microtubules are multiprotein structures that appear stable during the 2-hr life span of the dikaryon. These structures are the location of microtubule-severing activity in Chlamydomonas flagella; therefore, we wanted to examine the ability of wild-type components to rescue the adf and fa mutations in such dikaryons.

Immediately after mating, ADF/adf dikaryons shed two of their four flagella upon acid treatment. However, after 30 min, the Adf phenotype is rescued by the wild-type gamete, and all four flagella are shed (QUARMBY and HARTZELL 1994 ). To determine whether protein synthesis was required for this rescue, we incubated gametes in nitrogen-free medium with 15 µg/ml cycloheximide for 15 min before and during mating. As a control for the effectiveness of this cycloheximide treatment, we deflagellated gametes with or without cycloheximide and observed flagellar regeneration. As previously reported, cells in cycloheximide grew short flagella, whereas control cells, incubated in parallel, grew full-length flagella (BAKER et al. 1989 ). In dikaryon experiments, rescue of the Adf phenotype by the wild-type gamete occurred in the presence of cycloheximide, demonstrating that protein synthesis is not required for rescue (data not shown). As shown in Figure 3, the five other adf strains were also rescued in dikaryons, demonstrating that a wild-type component was sufficient to restore deflagellation competence to the two adf flagella of the quadriflagellate. Conversely, FA/fa dikaryons shed only two of their four flagella when treated with acid as late as 90 min after mating. Alleles of both FA1 and FA2 behaved in this manner. To confirm that the flagella retained by the cell were those of the fa mutant, we mated the paralyzed flagella mutant pf18, which is not rescued in dikaryons with wild type (STARLING and RANDALL 1971 ), with fa1-4 and assayed motility of the dikaryon after deflagellation. This motility assay indicated that the flagella retained were that of fa and not the flagella contributed by pf18 (data not shown). These results confirm that the recessive deflagellation defect of fa mutants is not rescued by wild-type cytoplasm in dikaryons.

View larger version (58K): In this window In a new window Download PPT slide   Figure 3. —In temporary dikaryon experiments, adf/ADF dikaryons (A) shed all four flagella, whereas fa/FA dikaryons (B) shed only two of their four flagella. Unmated wild-type gametes lost both flagella when treated with acid. Unmated gametes of adf or fa mutants retained both flagella when exposed to acid. Dikaryons are easily distinguished from unmated gametes on the basis of cell body shape and size. These results are presented quantitatively in C. Equal numbers of cells of opposite mating type were mated at a density of 107/ml. After 90 min, cells were treated with an equal volume of 40 mM Na acetate, pH 4.5, with 1 mM CaCl2 for 30 sec, followed by fixation with 2% glutaraldehyde. The control treatment was identical, except that media was substituted for the Na acetate solution. Cells were scored by phase contrast microscopy for the number of flagella. Mean ± SE for two experiments, 100 cells counted in each experiment.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have identified three genetically mutable loci involved in deflagellation, ADF1, FA1, and FA2. The recovery of multiple alleles of each gene (six ADF1, five FA1, and four FA2 alleles) leads us to postulate that either only these three gene products are required for deflagellation, or that other proteins that are involved play essential roles in the cell and are thus covered by genetic redundancy or cause lethality when mutated. The proposal that we have saturated this pathway is further supported by the identification of the same three genes from each of two independent methods of mutagenesis. Any bias in susceptibility to mutation by ultraviolet irradiation is likely to be different from any bias intrinsic to insertional mutagenesis; therefore, recovery of the same three genes from both forms of mutagenized cells suggests that no other genes will be identified by loss-of-function screens of this pathway.

Our previous physiological studies indicate that a unique, proton-activated Ca²⁺ influx pathway regulates deflagellation (QUARMBY and HARTZELL 1994 ; QUARMBY 1996 ). As discussed below, the results presented here indicate that Adf1p is a component of this Ca²⁺ entry pathway. Fa1p and Fa2p appear to be components of an axonemal transition zone complex that regulates Ca²⁺-activated microtubule severing.

The ADF1 gene may encode a proton-activated Ca²⁺ channel:
In this paper, the Adf phenotype is defined as deflagellation that is defective in acid-treated intact cells, but occurs when Ca²⁺ is added to detergent permeabilized cells. This suggests that Adf mutants have a defect in Ca²⁺ entry or the Ca²⁺-dependent signal transduction pathway. If true, cells with the Adf phenotype could carry mutations in a Ca²⁺-permeant channel, a Ca²⁺-activated phospholipase C, the IP₃ receptor, or as yet unknown regulators of these components (see Figure 4). Ca²⁺ influx from extracellular sources is clearly required for acid-induced deflagellation (QUARMBY and HARTZELL 1994 ; QUARMBY 1996 ). It is unknown if the Ca²⁺ that enters the cell during this influx directly stimulates Ca²⁺-activated microtubule severing or if it causes a release of intracellular Ca²⁺, possibly via activation of phospholipase C and subsequent release of Ca²⁺ from I(1,4,5)P₃-sensitive stores (QUARMBY et al. 1992 ; YUEH and CRAIN 1993 ; EVANS et al. 1997 ).

View larger version (28K): In this window In a new window Download PPT slide   Figure 4. —Intracellular acidification, caused by treatment of cells with a weak organic acid such as acetate, activates Ca2+ influx. Entry of Ca2+ via this pathway is required for acid-activated deflagellation, but the source of Ca2+ that triggers deflagellation in vivo has not been firmly established. It has been demonstrated in vitro that 1 µM Ca2+ activates axonemal microtubule severing at the base of the flagella, distal to the flagellar transition zone. Adf mutants are defective in acid-activated Ca2+ influx, whereas fa mutants are defective in Ca2+-activated microtubule severing.

We have shown previously that adf1-1 is defective in acid-stimulated Ca²⁺ influx (QUARMBY and HARTZELL 1994 ). Further Ca²⁺ influx studies with strains adf1-2 and adf1-3 revealed that these mutants also lack acid-stimulated Ca²⁺ influx (L. M. QUARMBY, unpublished observations), suggesting that the ADF1 gene product, Adf1p, plays a critical role in acid-activated Ca²⁺ influx. Because all four flagella of the ADF/adf temporary dikaryons show wild-type deflagellation (Figure 3), the defective component(s) in adf flagella can be replaced by wild-type counterpart(s). Physiological studies suggest that the acid-activated Ca²⁺ influx pathway, like known Ca²⁺ channels, is specifically localized to a plasma membrane subdomain (QUARMBY 1996 ), consistent with the idea that Adf1p is a plasma membrane protein, such as an ion channel. Restoration of wild-type function to adf flagella occurs ~30 min after cell fusion and does not require protein synthesis (QUARMBY and HARTZELL 1994 , for adf1-1; data not shown for other adf alleles). This latency is consistent with limited lateral mobility of a membrane protein localized to the flagellar transition zone. If Adf1p were soluble or were expressed over the entire cell surface, the expectation would be that deflagellation would be rescued soon after cell fusion. Based on several lines of evidence, such as the Adf phenotype, Ca²⁺ influx studies, and the delayed rescue of dikaryons, we hypothesize that the ADF1 gene encodes a critical subunit of a transition zone–localized, proton-activated Ca²⁺ channel.

There is evidence that phospholipase C is activated during deflagellation (QUARMBY et al. 1992 ; YUEH and CRAIN 1993 ), and that release of intracellular Ca²⁺ might trigger microtubule severing (QUARMBY and HARTZELL 1994 ; EVANS et al. 1997 ). We do not think that ADF1 encodes either phospholipase C or the IP₃ receptor because adf mutants are defective in the Ca²⁺ entry (QUARMBY and HARTZELL 1994 ; QUARMBY 1996 ) that is required for release of Ca²⁺ from internal stores (QUARMBY et al. 1992 ; EVANS et al. 1997 ). We suggest that phospholipase C and the IP₃ receptor are either not important for acid-induced deflagellation, or that mutations in these genes are lethal.

The FA genes may encode components of the microtubule-organizing center:
Centrioles, which are morphologically similar to basal bodies (FAWCETT and PORTER 1954 ), are surrounded by pericentriolar material, forming a structurally and functionally complex region known as the microtubule-organizing center (STERNS and WINEY 1997). The flagellar transition zone, immediately distal to the basal bodies, is a region that may perform some functions analogous to those of the pericentriolar region, including microtubule nucleation (the central pair of flagellar microtubules are not extended directly from the basal bodies) and microtubule severing. For example, a 20-kD member of the EF hand superfamily of Ca²⁺-binding proteins, centrin (also known as caltractin), was originally isolated as a protein associated with flagellar basal bodies (HUANG et al. 1988A , HUANG et al. 1988B ; SALISBURY et al. 1988 ), but has been found associated with the pericentriolar region of many cell types (BARON et al. 1992 ; PAOLETTI et al. 1996 ; LEVY et al. 1996 ). Two other proteins that have been identified as pericentriolar proteins, -tubulin and katanin (STEARNS et al. 1991 ; MCNALLY et al. 1996 ), are also found at the base of the flagella (SANDERS and SALISBURY 1989 ; TAILLON et al. 1992 ; DIBBAYAWAN et al. 1995 ; LOHRET et al. 1998 ). None of our fa mutants appears to affect katanin expression (assessed by Western analysis) or disrupt the centrin gene (assessed by Southern analysis; LOHRET et al. 1998 ). However, because of the deflagellation phenotype, we postulate that the two FA gene products play a role in axonemal microtubule severing.

As indicated in our model (Figure 4), we suggest that Fa1p and Fa2p are localized to the flagellar transition zone. Temporary dikaryons of any of the fa strains with wild-type are not rescued for deflagellation (Figure 3). There are a number of possible explanations for the lack of rescue in the fa dikaryons, but the simplest include (1) restricted access of proteins to the transition zone, (2) requirement for sequential assembly of proteins in a complex, (3) slow, or cell cycle–coupled turnover of the Fa proteins, and (4) low abundance of the Fa proteins. All of these explanations are consistent with our suggestion that a stable complex of Fa proteins is a component of the flagellar transition zone. Furthermore, in preparations of FBBC from wild-type and fa mutant cells, Ca²⁺-induced deflagellation occurs in the wild-type but not fa mutant FBBCs (LOHRET et al. 1998 ), suggesting that the entire machinery for axonemal microtubule severing, presumably including Fa1p and Fa2p, is a component of the detergent-resistant FBBC.

Our current thinking is that Fa1p and Fa2p transduce a particular Ca²⁺ signal into the activation of Chlamydomonas katanin, resulting in axonemal microtubule severing. This model raises the possibility that homologues of Fa1p and Fa2p might regulate the activity of pericentriolar katanin.

	ACKNOWLEDGMENTS

This work was supported by the National Science Foundation, award number MCB-9603716 to L.M.Q. We thank ELIZABETH SEAL, CHRIS HAYNES, and VAN FONGNALY for their contributions to the mutagenesis and screening. We are grateful for productive interactions with members of the Hartzell, Sale, and Quarmby labs in the Department of Cell Biology at Emory. We thank SUSAN DUTCHER (Boulder, CO) for consultation and for providing strains and protocols for diploid analysis. KRISHNA BHAT, STEVE L'HERNAULT, BETH and DAVID MITCHELL, and WIN SALE provided useful comments on the manuscript.

Manuscript received December 4, 1997; Accepted for publication March 12, 1998.

	LITERATURE CITED

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