Eyespot-Assembly Mutants in Chlamydomonas Reinhardtii

Eyespot-Assembly Mutants in Chlamydomonas reinhardtii

Mary Rose Lamb,* Susan K. Dutcher,† Cathy K. Worley‡ and Carol L. Dieckmann‡ *Department of Biology, University of Puget Sound, Tacoma, Washington 98416-0320, †Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309 and ‡Department of Biochemistry, University of Arizona, Tucson, Arizona 85721 Manuscript received April 28, 1999 Accepted for publication June 25, 1999

ABSTRACT Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light- sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Caϩϩ-channel signal transduction components in the plasma membrane of the cell and reﬂective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identiﬁed. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.

HLAMYDOMONAS reinhardtii is a phototactic, sin- roplast, the eyespot has a precise location in the cell C gle-celled green alga. The light-sensing eyespot is relative to the cytoskeleton. The eyespot is equatorial a complex sandwich composed of plasma membrane, with respect to the polar location of the basal bodies outer and inner chloroplast membranes, and multiple and flagella (see Figure 1B). A cruciate microtubule layers of granules in the stroma of the chloroplast inter- “rootlet” structure is oriented at a 45Њ angle to the plane leaved with thylakoid membranes (see Figure 1A). The of the flagella. One four-membered microtubule rootlet portion of the eyespot visible as a round orange patch and one two-membered rootlet are associated with each by light microscopy is located in the stroma of the of the basal bodies (mother and daughter) and extend chloroplast. It is formed by a hexagonal array of carot- from the basal body region to the equator of the cell. enoid-filled pigment granules (Nakamura et al. 1973). The eyespot is found just clockwise of the daughter four- In wild-type Chlamydomonas cells, two to four layers membered microtubule rootlet (see Figure 1C; Holmes of carotenoid lipid granules are observed, each layer and Dutcher 1989). subtended by a thylakoid membrane. In the region of The eyespot is oriented to detect light striking the cell the eyespot, both of the chloroplast membranes and surface orthogonal to the eyespot. Chlamydomonas cells the plasma membrane are closely apposed. The photo- rotate twice per second as they breaststroke forward, receptor, Chlamydomonas rhodopsin-like “chlamyop- scanning the environment perpendicular to the swim- sin,” localizes to the plasma membrane in this region ming path much as a radar antenna scans the sky. The (Deininger et al. 1995). As shown by suction pipette carotenoid granule layers were hypothesized to have recording, the light-triggered calcium channel is also two functions: they reflect photons that pass unabsorbed in the plasma membrane overlying the region of the through the plasma membrane back onto the photore- pigmented eyespot (Harz and Hegemann 1991). Ac- ceptors in the plasma membrane, and they block light cordingly, an increased density of membrane proteins coming through the cell body from other directions. in the plasma membrane in the region of the pigmented The spacing and periodicity of the carotenoid granule eyespot has been observed by freeze-fracture electron and thylakoid membrane layers were hypothesized to microscopy (Melkonian and Robenek 1980). act thus as a “one-way mirror” or what in the physics of In addition to the complex organization of the eyespot light is called a quarter-wave plate (Foster and Smyth components across the membranes of the cell and chlo- 1980). Confocal microscopy studies have confirmed that the pigmented layers act as a reflector of light (Kreimer and Melkonian 1990; Schaller and Uhl 1997). Analy- Corresponding author: Carol L. Dieckmann, Department of Biochem- istry, University of Arizona, P.O. Box 210106, Tucson, AZ 85721- sis of a mutant strain with defective eyespots supports 0106. E-mail: [email protected] the idea that the eyespot also enhances the shielding

Genetics 153: 721–729 ( October 1999) 722 M. R. Lamb et al.

Figure 1.—Chlamydomonas eyespot. (A) Diagram of the eyespot as seen by electron microscopy. The plasma membrane (PM) and two chloroplast envelope membranes (OM, outer membrane; IM, inner membrane) are tightly apposed in the region of the eyespot. Carotenoid granules subtended by thylakoid membranes (TM) are arranged in stacked rows. The signal transduction machinery including chlamyopsin and a light-gated calcium channel lie in the plasma membrane. The carotenoid granule and thylakoid layers act as a quarter-wave plate (mirror) to reflect light (h␷) back onto the plasma membrane. (B) The eyespot (E) is positioned at the equator of the cell if the basal bodies are positioned at the flagellar pole (F, flagella; C, chloroplast). (C) Looking down on the flagellar pole, the eyespot (E) is positioned just clockwise of the daughter four-membered microtubule rootlet (4, four-membered microtubule rootlets; 2, two-membered microtubule rootlets; F, flagella; Holmes and Dutcher 1989). The flagellum and rootlets in the left half of the cell (trans with respect to the eyespot) were retained from the mother cell. The right half structures (cis with respect to the eyespot) were assembled during the division that produced this daughter cell. The flagella are offset relative to each other by approximately half a flagellar diameter (Hoops and Witman 1983). properties of the chloroplast to block light coming the signal transduction components in the plasma mem- through the cell body (Kreimer et al. 1992; reviewed in brane are assembled with the chloroplast envelope, re- Kreimer 1999). Together, the reflective property of the flective carotenoid pigment granule layers, and thyla- carotenoid pigment layer and the absorptive property koid membranes to form a functional light-sensing of the eyespot and cell body give up to an eightfold organelle. To identify genes important in the assembly enhancement of the light striking the plasma mem- pathway, we have identified nonphototactic mutant brane over the eyespot relative to the light coming strains of Chlamydomonas reinhardtii and screened for through the cell from the other side (Harz et al. 1992). those with aberrant carotenoid pigment patterns. Here Light absorption by the rhodopsin-like photoreceptor we report the identification of four genes governing the leads to the transient opening of calcium channels in assembly of the pigment layer. Mutations in two genes the plasma membrane over the eyespot. The influx of result in the absence of the reflective pigment layer, calcium triggers the transient opening of voltage-gated mutations in the third gene result in small pigment calcium channels in the flagella (Harz and Hegemann patches, and mutations in a fourth gene result in multi- 1991). The subsequent change in intracellular calcium ple, pigmented eyespots. concentration is thought to affect the activity of the two flagella differentially, as has been demonstrated in vitro (Kamiya and Witman 1984). The cis (near the eyespot) MATERIALS AND METHODS flagellum beats weakly and the trans (away from the eyespot) beats strongly to cause the cell to turn toward Media, culture conditions, and strains: M medium is Sager and Granick medium I with Hutner’s trace elements (Harris the light as it swims forward. If the light intensity is too 1989). R medium is M medium with 0.1% sodium acetate strong, the response of the two flagella is reversed and added. In RNA and DNA medium the NH4Cl concentration the cell turns away from the light (Ru¨ffer and Nultsch is reduced 10-fold and 200 ␮g/ml of arginine is added. Solid 1997). In-depth coverage of eyespot structure, localiza- media contain 1.5% agar (R, RNA), 2% agar (D, DNA), or tion, and function in green algae can be found in two 4% agar (Z). Both liquid cultures and plates were incubated at 25Њ in excellent recent reviews (Hegemann and Harz 1998; continuous white fluorescent light. All of the mutant strains Kreimer 1999). described in this article were isolated in strain 137c mtϩ except Our interest in the Chlamydomonas eyespot is in how strain 6-9, which was isolated in mtϪ. Strains bearing intragenic Eyespot Mutants in Chlamydomonas 723 complementing arg7 alleles were used to recover diploids for pH 7.4, 5 mm MgSO4,1mm EGTA, 0.1 mm EDTA, 25 mm complementation analyses of eyespot-assembly mutations KCl, 1 mm DTT). Cells were incubated with microwaving in (Matagne 1978; Harris 1989). 0.5% OsO4 0.8% K3Fe(CN)6 in HMEEK (25 sec, 15Њ, three Mutagenesis, enrichment, and screening for phototaxis-neg- times), rinsed with HMEEK three times, and incubated in ative mutants: Mutagenesis: Strain 137c was grown on R plates 0.5% tannic acid in HMEEK for 1 min at room temperature for two days. The cells were scraped from the surface and (RT). Cells were rinsed with HMEEK three times and deion- resuspended in liquid M medium. Between 2 and 7 ϫ 107 ized water four times, and then incubated in 2% uranyl acetate cells in 1 ml were pipetted into 3-cm-diameter petri dishes (2 hr, RT, in the dark). Dehydration was carried out in ace- cm for 45 tone, and cells were embedded in Epon-Araldite resin using 10ف and exposed to UV radiation at a distance of survival for this exposure time. microwave treatment to accelerate the process. Sections were %80ف sec. A kill curve showed The cultures were kept in the dark for 8 hr and then trans- cut by D. Bentley (Imaging Facility, University of Arizona), ferred to 30 ml of liquid M medium in large test tubes (25 ϫ mounted on copper grids, and stained with a saturated solu- 150 mm) and grown at 25Њ in light. tion of uranyl acetate (10 min, RT) and Reynold’s lead citrate Enrichment: At 2-day intervals the tubes were masked with (1.5 min, RT). In Figure 4A and Figure 5A the tannic acid black paper except for the bottom 1 cm of the tube and placed incubation was omitted and cells were imbedded in LR White 40 cm from a fluorescent bar lamp for 20 min. Most of the instead of Epon-Araldite. In Figure 4D (provided by K. E. cells phototaxed to the lighted bottom portion of the tube. Huey), microwaving was omitted. Imaging was performed on One milliliter of culture was removed from the top surface a Phillips 420 transmission electron microscope at 80 kV. and transferred to 30 ml of fresh M medium (see Stavis and Light microscopy: Cultures of each strain were grown for Hirschberg 1973). In all cases, cultures were tested at an 6 days in M medium or for 2 days in R medium. Glutaraldehyde approximate density of 1 ϫ 106 cells/ml. (1 ␮l) was added to 1 ml of each culture, and cells were Screening: After four or five enrichments, individual cultures harvested at 5000 rpm. A yellow-green filter (Zeiss 467806) were diluted and spread on R plates. Ten single colonies from was used at the light source to enhance the contrast of the each original mutagenized culture were transferred into 2.5 eyespots. Photographs were taken with Kodak Technical Pan ml of M medium in 13 ϫ 100-mm tubes and grown for 2 days film. at 25Њ in 80 ␮E/m2/sec light. On the second day, the tubes were placed in a box with a 0.5-cm-wide slit that illuminated the tubes at the bottom. After 20 min of illumination at 40 RESULTS cm from a fluorescent bar lamp, the tubes were removed from the box and examined. Cultures of cells that did not form a Efficient method yields 168 independent nonpho- band at the bottom were scored as phototaxis negative. Only totactic strains: C. reinhardtii wild-type strain 137c was one mutant strain was kept from each original mutagenized mutagenized with ultraviolet light and enriched for non- culture. Screening for mutants with an aberrant eyespot morphol- phototactic cells as described in materials and meth- ogy: Each of the original mutant strains was grown on liquid ods (Stavis and Hirschberg 1973). From 200 separate medium, fixed with 0.1% glutaraldehyde in phosphate-buf- mutagenized cultures, 168 independent mutant strains fered saline (PBS), and observed at ϫ1000 magnification by were collected. Since the strains were grown on minimal oil immersion light microscopy. The strains with no eyespot, medium without an added carbon source throughout multiple eyespots, or smaller than normal eyespot were charac- the mutagenesis and enrichment procedure, all of the terized genetically. Linkage and complementation analyses: Each of the mu- mutants retain photosynthetic capability. Because carot- tants with aberrant eyespot morphology was crossed to each enoid biosynthetic pathway mutants are defective in pho- of two strains bearing complementing arg7 alleles, arg7-8 and tosynthesis (Sager and Zalokar 1958; Spreitzer and arg7-2 (Matagne 1978; Harris 1989). Haploid phototaxis- Mets 1981), the collection should not include strains Ϫ negative Arg progeny of opposite mating types were mated defective in steps leading up to carotene biosynthesis. and diploids were recovered on medium lacking arginine. Ten diploid colonies from each cross were tested for phototaxis as Since the enrichment and screening procedures re- described in the screening section above. If the diploid was quired that the cells be able to swim, mutant strains able to phototax, the two mutants were placed in different with gross defects in swimming should not be included complementation groups; if the diploid was unable to pho- in the collection. We expect that this collection should totax, the mutants were placed in the same group. To test include mutations that affect the signal transduction linkage, haploid, phototaxis-negative strains of opposite mating type were crossed and allowed to go through meiosis. pathway, those that affect the response of the flagella C. reinhardtii produces haploid progeny in tetrads, which can to the light-activated calcium influx, and those govern- be dissected and analyzed. Approximately 20 tetrads were ana- ing the assembly and localization of the eyespot. lyzed for recombination between two different phototaxis- Mutants with aberrant eyespot morphology: The 168 negative mutations. Methods for mating, recovery of diploids, mutant strains were screened visually with an oil immer- and tetrad dissection were as described (Harris 1989). sion objective (ϫ1000 magnification) for eyespot pig- Electron microscopy: Cultures of each strain were grown for 8 days in M medium or for 3 days in R medium. Cells were ment patches that were other than wild type in size and/ harvested at 5000 rpm and mixed 1:1 with 2% formaldehyde or location. In this screen, three aberrant phenotypes (100% paraformaldehyde stock diluted in heat-treated water were identified: (1) the “eyeless” phenotype, or cells and neutralized with sodium hydroxide), 0.25% glutaralde- with no pigment patches observable by light microscopy; hyde (50% stock) in 50 mm cacodylate, and microwaved three (2) the “miniature” phenotype, or cells with a smaller times for 30 sec to accelerate fixation (Giberson et al. 1997). Paraformaldehyde and glutaraldehyde were purchased from than normal eyespot; (3) the “multiple” phenotype, or Electron Microscopy Sciences (Fort Washington, PA). Cells cells with multiple eyespots. In all, 27 mutant strains were rinsed three times with HMEEK buffer (30 mm Hepes, had one of these three different phenotypes. Seventeen 724 M. R. Lamb et al.

TABLE 1 Complementation/recombination groups of mutants with aberrant eyespots

eye1- eye2- eye3- min1- mlt1-ND 1, ey-1,C 1, 10-18, C 1, 6-9, C 1, 12-6, RC 1, 10-13, RC 8-11 2, ey-550, C 2, 8-18, C 2, 12-12, RC 2, 11-15, RC 8-15 3, ey-627, C 3, 10-6, RC 3, 15-2, RC 3, 12-10, RC 4, 10-11, C 4, 15-12, RC 4, 13-14, RC 5, 10-15, C 5, 14-14, RC 6, 11-5, C 7, 11-8, C 8, 12-5, RC 9, 12-7, C 10, 12-18, RC 11, 12-19, RC 12, 13-6, R 13, 14-7, RC 14, 14-13, RC 15, 14-18, R ND, not determined; 8-11 has a multi-eyed phenotype; 8-15 has an eyeless phenotype. R, determined by recombination. C, determined by complementation. are eyeless, 6 have multiple eyespots, and 4 have minia- noncomplementing mutations and thus contain ey-1 ture eyespots. alleles on linkage group X (Smyth et al. 1975). We Eyespot morphology mutants define four loci: Both propose that this locus be renamed EYE1 to reflect linkage and complementation analyses were used to the recommendation for three-letter Chlamydomonas characterize the 27 morphologically defective mutant gene names. The alleles thus should be renamed eye1-1 strains. All of the mutations were shown to be recessive (ey-1), eye1-2 (ey-550), and eye1-3 (ey-627). Our two eye- in diploids obtained by crossing the mutant strains to less loci are unlinked to, and the mutations comple- wild-type 137c. As shown in Table 1, the 27 strains fall ment, eye1 alleles (see Table 1). Therefore, we have into four complementation/linkage groups. Fifteen eye- named the new loci EYE2 and EYE3. EYE2 is defined less mutants comprise one group. One additional eye- by the mutation in the 10-18 strain and an insertion less mutant strain is in a complementation group by mutation, H9-8, obtained in a later transformation/mu- itself. Five multiple eyespot mutants are all in the same tagenesis scheme (D. G. W. Roberts and C. L. Dieck- group and the fourth group is composed of the four mann, unpublished data). eye2 mutants never acquire miniature eyespot mutants. It should be noted that mu- eyespots or the ability to phototax during extended cul- tant 10-13 was not identified originally as a multiple turing. eye3 mutant strains do acquire phototactic ability eye mutant. The addition of this mutant to the group after extended time in stationary phase; however, we occurred during analysis of a linkage matrix of ten mu- have never observed them to acquire pigmented eye- tants in the large nonphototactic collection chosen at spots. random. In logarithmically growing cultures, 10-13 cells Multi-eyed, mlt1, and mini-eyed, min1, mutants: Ac- had mostly single eyespots. Stationary phase cultures tively growing cultures of the multi-eyed mutant cells, contained cells with multiple eyespots. Therefore, it may named mlt1, show some phenotypic variability within the be possible that additional members of the multiple eye population. Scoring over 600 cells, 72% of a logarithmic group and/or the other three groups have not been culture of the mlt1-3 multi-eyed mutant had two eye- identified in the collection as a whole. spots. Of these two-eyespot cells, the majority (63% of Eyeless mutations define two new loci, EYE2 and the total cell number) had one eyespot in an equatorial EYE3: As described by Hartshorne (1953), one of the position and an additional eyespot in a region of the first mutant strains of Chlamydomonas to be character- chloroplast near the base of the flagella. The additional ized was an eyeless strain. Named ey-1, this mutant strain eyespot was located either on the same side and in line is still available from the stock center, as are strains with the equatorial eyespot (36%) or 180Њ from the equa- named ey-550 and ey-627 (Morel-Laurens and Bird torial eyespot (27%). In addition to the two-eyespot cells, 1984; Kreimer et al. 1992). All three of these strains are 14% had one eyespot, and 13% had more than two eyeless and nonphototactic in logarithmic culture, but eyespots. Cells in stationary phase cultures acquire addi- acquire phototactic ability and eyespots in stationary tional eyespots with time in culture. The miniature eye phase cultures. By complementation testing, we deter- mutants, named min1, have small eyespots located most mined that the ey-1 strain, ey-550, and ey-627 harbor often in the equatorial position. Eyespot Mutants in Chlamydomonas 725

TABLE 2 Linkage data for the min1-mlt1-eye2 cluster

Cross PD:NPD:TT Map units min-1 (12-6, 12-12) ϫ mlt-1 (12-10) 115:0:9 3.6 min-1 (12-6, 12-12) ϫ ey-2 (10-18) 115:0:11 4.7 mlt-1 (12-10) ϫ ey-2 (10-18) 132:0:2 0.75

Eyespot assembly gene cluster: Surprisingly, the mlt1 and min1 mutations are linked to the eye2 mutations. To determine the map distances between these loci, two-point crosses of strains mutant at the three loci were analyzed (see Table 2). The three loci are tightly linked and most likely in the order min1-mlt1-eye2. We are un- certain of the order with respect to the centromere. Recently, a probe prepared from sequence adjacent to the EYE2 locus hybridized to two BAC clones that were also identified with a PTX4 probe (D. G. W. Roberts, G. J. Pazour, G. B. Witman and C. L. Dieckmann, unpublished results). ptx4 mutations result in multiple eyespots and are allelic with mlt1 mutations (Pazour et al. 1995; C. L. Dieckmann, unpublished results). This physically places the MIN1-MLT1(PTX4)-EYE2 gene cluster on the left arm of linkage group XII/XIII (Pushpa Kathir, map position of PTX4 with a probe provided by G. Pazour, reported on gene maps provided on the Chlamydomonas Stock Center webpage http://genome. cornell.edu/cgi-bin/WebAce/webace?dbϭchlamydb). The eye2 and eye3 phenotype is epistatic to that of min1 and mlt1: To investigate whether the four gene products function in the same or different eyespot assembly or positioning pathways, double-mutant strains were constructed and the eyespot phenotypes were examined. Both eye2 and eye3 were found to be epistatic to min1 and mlt1; i.e., any combination of an eyeless mutation and a mini- or multi-eye mutation resulted in the eyeless phenotype. min1 mlt1 double mutants have a synthetic phenotype different from either parent. In -of the popula %43ف ,a logarithmically growing culture tion of min1 mlt1 cells had no eyespot visible under oil immersion at ϫ1500 magnification (100 cells analyzed). Figure 2.—Light microscopy of mutant strains. (A) Wild type; (B) min1; (C and D) two configurations of mlt1; (E) min1 An equivalent fraction had very tiny pigment spots that mlt1; (F) eye1; (G) eye2; (H) eye3. Arrows indicate positions of were displaced in the cell toward the region of the eyespots. Bar, 5 ␮m. chloroplast closest to the base of the flagella. In a min1 mlt1 culture that remained at stationary phase for 2 days, the percentage of cells with eyespots decreased to 26% bly mutant strains, late-log or early stationary phase cells (100 cells analyzed). As might be expected, eye2 eye3 were fixed and prepared for electron microscopy. As double mutants are eyeless and do not acquire the ability shown in Figure 4A, wild-type eyespots show a close to phototax in stationary phase, which is similar to eye2 apposition of the plasma membrane with the chloro- single mutants. Light micrographs of the single-mutant plast envelope membranes. In cross-section, the carot- strains and the min1 mlt1 double-mutant strain are enoid-filled lipid granules (arrowheads) are in a closely shown in Figure 2. packed, linear array directly beneath the inner envelope Eyeless mutants have pigment granules: To examine membrane. As shown here, multiple pigment granule the ultrastructure of the eyespots in the eyespot-assem- layers are separated by thylakoid membranes. Surpris- 726 M. R. Lamb et al.

R as shown by light microscopy (Figure 3). The number of pigment granules is significantly less than that of wild type as shown by electron microscopy (Figures 4B and 5, A and B). Eyespots in cells grown on acetate medium have nearly wild-type morphology with apposed membranes (Figure 5B). However, eyespots of cells grown in acetate-free medium have an aberrant morphology in which cytoplasm is often apparent between the plasma membrane and the underlying chloroplast envelope (Figure 4B), and thylakoid membranes and stroma are seen between the chloroplast envelope and the pigment granules, which are often jumbled rather than in neat layers (Figures 4B and 5A). The loss of contact between the plasma membrane and the chloroplast envelope membrane is suggestive of a defect in the organization of the eyespot components across the three usually apposed membranes. The min1 phenotype is exacerbated when the mutation is in combination with a mlt1 allele. As shown in Figure 4D, the pigment granule arrays in the double mutant 12-12,12-10 are small, disordered, and not asso- Figure 3.—Light microscopy of min1 cells grown on mini- ciated with the chloroplast envelope, similar to the min1 mal (M) and minimal plus acetate (R) medium. (A) min1 single mutant (Figure 4B). In general, the double- grown in M medium. (B) min1 grown in R medium. (C) Wild mutant eyespots are smaller and more disordered than type grown in M medium. (D) Wild type grown in R medium. Arrows indicate positions of eyespots. Bar, 5 ␮m. those of min1 single mutants. The pigment granule patches often appear to be randomly located within the stroma of the chloroplast. ingly we found that both eye2 and eye3 mutants appear to have carotenoid pigment granules (Figure 4, F and DISCUSSION G). However, they are most often found singly and unor- One of the obstacles on the road from endosymbiont ganized in the chloroplast compartment. Since eyespots to becoming an organelle is to ensure segregation into are observed by light microscopy in eye1 mutants that new cells at mitosis and meiosis. The mechanism for have reached stationary phase, it was not surprising to the segregation of mitochondria and chloroplasts and observe organized eyespots by electron microscopy (Fig- other organelles during division is still a mystery. Organ- ure 4E). The eyeless phenotype is suggestive of a defect elles such as flagella and light-sensing eyespots must in organizing the granules into regular arrays, rather also be correctly positioned in the new cells for proper than in formation of the granules. function. In C. reinhardtii, the eyespot is located at the Multi-eyed mutant has normal eyespot ultrastructure: equator of the cell if the flagella are centered at one The mlt1 mutant examined, 12-10, has eyespots with an pole (see Figure 1B). The positioning of this structure ultrastructure similar to that of wild-type cells. The three is complex, in both a spatial and a temporal sense. The apposed membranes (inner and outer chloroplast enve- eyespot is positioned just clockwise of the daughter four- lope and plasma membranes) show no distortion and membered microtubule rootlet as viewed from the fla- the eyespots often show the regular, multilayer pheno- gellar pole (see Figure 1C). During mitosis, the cleavage type of wild-type eyespots (see Figure 4C). furrow forms along the four-membered microtubule The plasma membrane and chloroplast envelope are plane and the eyespot is resorbed. In the two daughter not apposed in min1 mutants grown on medium lacking cells, the new eyespots appear again just clockwise of acetate: All of the strains in this study can grow photosyn- the new daughter four-membered rootlet. While the thetically on a standard laboratory culture medium lack- daughter cells are still attached to one another, these ing a carbon source (M medium; see materials and new eyespots are 180Њ from each other and 90Њ from methods). Often strains are grown on the same me- the cleavage plane and the location of the old eyespot dium containing acetate (R medium), because they (Holmes and Dutcher 1989). The eyespot is organized grow more quickly. Interestingly, we found that the ul- across the plasma membrane and both chloroplast enve- trastructural phenotype of min1 mutants depends on lope membranes to include pigment granules in the whether the medium is supplemented with acetate. In stroma. These pigment granules are organized in hexag- the min1 mutant 12-12, the eyespots are smaller than onal arrays and are subtended by thylakoid membranes those of wild type whether the cells are grown on M or (Nakamura et al. 1973). Eyespot Mutants in Chlamydomonas 727

Figure 4.—Electron microscopy of eyespots in mutant strains. (A) Wild type; (B) min1; (C) mlt1; (D) min1 mlt1; (E) eye1; (F) eye2; (G) eye3. Ar- rowheads indicate positions of representative carotenoid par- ticles. Bars, 100 nm. Image in C is one-half the magniﬁcation of the rest to show both eyespots.

Our interest is in understanding both the structural eyespots. These mutations could eliminate a component basis of the organization of the eyespot across the cell/ of the eyespot structure or disrupt a regulatory pathway organelle boundary and the timing and localization of necessary for proper assembly of the structure. the organelle in the cell during growth and cell division. The eye2 and eye3 mutants do not assemble a pigment To this end, we have isolated and characterized several granule apparatus observable by light microscopy. Quite mutant strains with aberrant eyespot assembly. From different from the appearance of eyespots in stationary 168 nonphototactic mutants, 27 eyespot mutants were cultures of eye1 mutants, we have never observed pig- found. Three different phenotypes were observed: no ment spots in the eye2 or eye3 strains in our collection. eyespots, multiple eyespots, or smaller than wild-type However, eye3 cells starved for nitrogen or kept in sta- 728 M. R. Lamb et al.

Figure 5.—Electron microscopy of min1 eyespots in cells grown on minimal and acetate medium. (A) min1 grown in M medium. (B) min1 grown in R medium. (C) Wild type grown in M medium. (D) Wild type grown in R medium. Arrow- heads indicate positions of representative carotenoid parti- cles. Bars, 100 nm.

tionary phase for 2 days recover the ability to phototax. eyespot number. Occasionally in a wild-type population It is clear that the signal transduction machinery, at of cells a cell with two eyespots is observed following least in starved cells, is sufficient for phototaxis. In addi- cytokinesis (Holmes and Dutcher 1989). The addition, we have found both eye2 and eye3 strains will pho- tional eyespot is the result of retention of the parental toshock at high levels of light (D. G. W. Roberts and eyespot in the daughter cell along with the newly formed C. L. Dieckmann, unpublished data), also suggestive eyespot. In logarithmically growing cultures of the mlt1 that signal transduction is intact in these mutants. Thus, strains, most cells have two eyespots, 36% with both in the eye2 and eye3 mutants are primarily defective in pig- line on the same side of the cell, one in the normal ment granule organization in the chloroplast. These equatorial location and the other closer to the flagella. EYE genes could code for essential structural compo- We have observed that, like wild-type cells, mlt1 cells nents of the eyespot or positively regulate the pathway absorb the eyespots during division. Appearance of new of assembly. We expected that we might identify several eyespots seems delayed relative to wild-type cells. There- more loci of this type. Identifying only three loci may fore, supernumerary eyespots are not due to being re- suggest the following: (1) that the structure is quite tained from the parental cell. Future investigation of simple, (2) that other structural or regulatory elements the mlt1 mutants during mitosis should clarify the timing of the system are essential gene products and would not of the appearance and placement of eyespot relative to have been identified in this analysis, or (3) that the the cleavage furrow and four-membered microtubule genome has not been saturated for mutations that would rootlets. That 62% of the cells examined have eyespots result in the eyeless phenotype. that are aligned or are 180Њ from each other and are The min1 mutants have smaller than normal eyespots, all in the flagellar hemisphere is suggestive that some which are highly disordered when the cells are grown positional cues have been retained. Perhaps the signal in medium lacking acetate. The product of the MIN1 that designates the position of the four-membered root- gene could be a structural component that is involved lets or the four-membered rootlets themselves are cues in the apposition of the plasma membrane with the for eyespot localization. Recently we discovered that chloroplast envelope. This hypothesis would explain mlt1 mutations and ptx4 mutations are allelic (Pazour why we observe cytoplasm between the plasma mem- et al. 1995; C. L. Dieckmann, unpublished data), which brane and the chloroplast in min1 mutant strains. How- places the MLT1/(PTX4) locus on the left arm of linkage ever, min1 cells grown on acetate assemble small but group XII/XIII (P. Kathir, Chlamydomonas Stock Cen- more normal-looking eyespots with the membranes ap- ter webpage). posed. Future investigations will focus on discovering We find it intriguing that the MIN1, MLT1(PTX4), the basis of the phenotypic variation. and EYE2 loci are closely linked to each other; this The mlt1 mutants are defective in the regulation of clustering may allow cell-cycle or other coordinated con- Eyespot Mutants in Chlamydomonas 729 trol of expression. Alternatively, this group could have Holmes, J. A., and S. K. Dutcher, 1989 Cellular asymmetry in Chlamydomonas reinhardtii. J. Cell Sci. 94: 273–285. a common genetic origin, perhaps from the genome of Hoops, H. J., and G. B. Witman, 1983 Outer doublet heterogeneity the endosymbiont/organelle. reveals structural polarity related to beat direction in Chlamydomo- This study is a beginning for the understanding of nas flagella. J. Cell Biol. 97: 902–908. Kamiya, R., and G. B. Witman, 1984 Submicromolar levels of cal- eyespot structure and function in C. reinhardtii and other cium control the balance of beating between the two flagella related algae. The continued analysis of genes described in demembranated models of Chlamydomonas. J. Cell Biol. 98: here will increase our general knowledge of the cell- 97–107. Kreimer, G., 1999 Light reception and signal modulation during organelle interactions required for light reception, sig- photoorientation of flagellate green algae, in Comprehensive Series nal transduction, and eyespot assembly and positioning in Photosciences, edited by M. Lebert and D.-P. Ha¨der. Elsevier/ in the cell. North Holland, Amsterdam (in press). Kreimer, G., and M. Melkonian, 1990 Reflection confocal laser Many people have contributed to this study, including K. E. Huey, scanning microscopy of eyespots in flagellated green algae. Eur. L. Kenaga, D. L. Bentley, J. Ehle, M. Essary, J. J. Anderson, T. Little, J. Cell Biol. 53: 101–111. F. Sibayan, K. Campbell, and S. J. King. We thank them for their help Kreimer, G., C. Overla¨nder, O. A. Sineshchekov, H. Stolzis, W. with genetics, phenotypic characterization, and microscopy. Thanks Nultsch et al., 1992 Functional analysis of the eyespot in Chla- Ϫ to Elizabeth Harris for sending strains from the Chlamydomonas mydomonas reinhardtii mutant ey 627 mt . Planta 188: 513–521. Stock Center. Thanks to G. Pazour and G. Witman for the PTX4 probe Matagne, R. F., 1978 Fine structure of the arg-7 cistron in Chlamydo- and ptx4 mutant strains, to P. Kathir for mapping information, and monas reinhardi: complementation between arg-7 mutants defective in argininosuccinate lyase. Mol. Gen. Genet. 160: 95–99. to G. Pazour for critical reading of the manuscript. This work was Melkonian, M., and H. Robenek, 1980 Eyespot membranes of Chla- funded by a Biomedical Research Small Grant to C.L.D., a National mydomonas reinhardtii: a freeze-fracture study. J. Ultrastruct. Res. 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