Cellular asymmetry in Chlamydomonas reinhardtii JEFFREY A. HOLMES and SUSAN K. DUTCHER* Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA •Author for correspondence Summary Although largely bilaterally symmetric, the two asymmetric. As a result of asymmetric, but differ- sides of the unicellular alga Chlamydomonas rein- ent, locations of the plus and minus mating struc- hardtii can be distinguished by the location of the tures, mating preferentially results in quadriflagel- single eyespot. When viewed from the anterior end, late dikaryons with parallel flagellar pairs and both the eyespot is always closer to one flagellum than eyespots on the same side of the cell. This asymmet- the other, and located at an angle of approximately ric fusion, as well as all the other asymmetries 45° clockwise of the flagellar plane. This location described, may be necessary for the proper photo- correlates with the position of one of four acetylated tactic behavior of these cells. The invariant handed- microtubule bundles connected to the flagellar ap- ness of the spindle pole, eyespot position, and paratus. Each basal body is attached to two of these mating structure position appears to be based on microtubule rootlets. The rootlet that positions the the inherent asymmetry of the basal body pair, eyespot is always attached to the same basal body, providing an example of how an intracellular pat- which is the daugher of the parental/daughter basal tern can be determined and maintained. body pair. At mitosis, the replicated basal body pairs segregate in a precise orientation that main- tains the asymmetry of the cell and results in mitotic poles that have an invariant handedness. Key words: Chlamydomonas, asymmetry, basal bodies, The fusion of gametic cells during mating is also acetylated tubulin, eyespot, uni mutants. Introduction Using the eyespot as a marker, it has been shown that the flagellar apparatus has a physiological cis/trans The number and position of organelles within a cell of asymmetry. Kamiya and Witman (1984) have shown that Chlamydomonas is highly stereotyped. Most of the or- reactivated cis and trans axonemes have different and ganelles are symmetrically positioned around the long opposite responses to calcium concentration, and Huang axis of the cell (Fig. 1). The most striking exception to et al. (1982) have described a number of mutations (the this rotational symmetry is the position of the eyespot, a uni mutants) whose defect appears to be at the level of the red-pigmented structure that acts as an antenna for the basal body and whose distinguishing feature is that they photoreceptor in the plasma membrane above it (Foster fail to assemble the cis flagellum, but remain able to and Smyth, 1980). Each cell normally contains a single assemble the trans flagellum. Clearly the cis and trans eyespot, which is always located at the periphery of the basal bodies and axonemes are not equivalent. cell, in a location closer to one flagellum than the other. The basal bodies of Chlamydomovas replicate and The asymmetric position of the eyespot can be used to segregate in a manner similar to that of mammalian differentiate the cell into two halves. By convention, the centrioles. During mitotic prophase in Chlamydomonas half of the cell containing the eyespot is denoted cis, and the two probasal bodies extend to full length and are the other half trans (Huang et al. 1982). An imaginary segregated in a semiconservative manner, so that one cis/trans plane that passes between the two basal bodies parental and one daughter basal body go to each mitotic can be used to differentiate these two halves. To aid in pole (Gaffel, 1988). The parental/daughter difference in description, we have added the terms syn and and to the two basal bodies is postulated to be the source of the describe the two halves of the cell differentiated by a physiological difference between the two basal bodies and second imaginary plane in which the two flagella beat. the basis of the Uni~ phenotype (Huang et al. 1982). This is commonly referred to as the flagellar plane. The Chlamydomonas is not unique in having two different locations of these two planes of reference are shown flagella, and in fact no example of truly identical flagella diagrammatically in Fig. 2. on a biflagellate algal cell has yet been reported. In several Journal of Cell Science 94, 273-285 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 273 algal species it has now been shown that the differences between the flagella correlate with the parental/daughter history of the two basal bodies (Beech et al. 1980; Melkonian et al. 1987; Wetherbeee^a/. 1988). Because of this we believe the correct positioning of the eyespot occurs with respect to an inherently asymmetric parental/ daughter basal body pair. The position of the eyespot is believed to be deter- mined by a unique bundle of microtubules attached to the flagellar apparatus (Melkonian, 1984a; Moestrup, 1978). Each of the two basal bodies has two cytoplasmic microtubule roots or rootlets attached to it (Ringo, 1967). These rootlets differ from one another both in the number of microtubules they contain and in their angular position with respect to the flagellar plane. The four- membered microtubule rootlets extend at an angle of approximately 45° clockwise of the flagellar plane (all clockface distinctions are made from a point of reference outside the cell, facing the cell's anterior end) and the two-membered rootlets extend at an approximately 45° counterclockwise angle. The hypothesis that the eyespot is positioned by a rootlet is supported by the coincidence of the eyespot position and the angular position of the four-membered rootlet, as well as by the observation of a Fig. 1. Schematic diagram of the flagellar apparatus of band of microtubules seen in association with the eyespot Chlamydomonas reinhardtii. The view is from the anterior in electron micrographs (Gruber and Rosario, 1974). In end of the cell and demonstrates the 180° rotational other green algae, the eyespot is always found associated symmetry of the flagellar apparatus, bb. The two mature with the microtubule rootlet that corresponds, both in basal bodies, pbb. The probasal bodies that assemble the microtubule number and position, to the four-membered daughter basal bodies prior to mitosis. Their connections to rootlet of Chlamydomonas (Moestrup, 1978). the flagellar apparatus are omitted, df. The distal striated fiber that connects the basal bodies. Two smaller proximal Presumably the angular position of the rootlets is striated fibers are mostly obscured in this diagram. determined by a specific site of attachment for each cv. Contractile vacuoles whose location is represented by the rootlet type to each basal body. Chlamydomonas basal broken circles. Two types of microtubule rootlets emanate bodies are not rotationally symmetric and each micro- from the flagellar apparatus, composed of two and four tubule triplet can be numbered around the basal body microtubules, respectively, sf. Striated fibers associated with cylinder (Hoops and Witman, 1983). Examination of a both rootlet types. All four microtubule rootlets terminate in Chlamydomonas mutant (vfi-3) in which the fibers con- the region beneath the distal fiber, although the striated necting basal bodies are incomplete (Hoops et al. 1984; component of the two-membered rootlets is continuous. Wright et al. 1983) as well as observations of other green algae, which undergo a rotation of their basal bodies during development (Greuel and Floyd, 1985; Hoops, cis trans 1984; Taylor et al. 1985), support the idea that the rootlets and other components of the flagellar apparatus are physically connected to the basal bodies at unique sites. syn The position of the eyespot not only makes cells of Chlamydomonas asymmetric, but enantiomeric as well. That is, they exist in only one mirror-image configur- ation. When viewed from the anterior end, the eyespot is always located at an angle of approximately 45° from the plane determined by the two flagella (Fig. 2). Only the enantiomorph in which the eyespot is clockwise of the I flagellar plane exists in nature (Ruffer and Nultch, 1985); the counterclockwise enantiomorph has not been ob- Fig. 2. Diagram of the eyespot position in an anterior view. served. This handedness of the cell is distinct from Two imaginary planes bisecting the cell are used for reference. The as/trans plane divides the cell between the cis/trans asymmetry, since it is an asymmetry within the two basal bodies. The syn/anti plane, or flagellar plane, cis half of the cell itself. contains the two basal bodies and the two flagella. The Like the cells of Chlamydomonas, basal bodies exist in eyespot is always located at an angular position of 45 ° only one enantiomeric configuration. When viewed from clockwise of the syn/anti plane, coincident with the meridian the anterior end, the microtubule triplets overlap in a containing the four-membered microtubule rootlets. clockwise direction (Fulton, 1971; Melkonian, 1984a). 274 J. A. Holmes and S. K. Dutcher Basal bodies or centrioles of the opposite handedness, 1:100; 6-11B-1 was diluted 1:10. Chlamydomonas cells were 6 7 1 where the overlap is in a counterclockwise direction, have grown in liquid to a density of approximately 10 -10 cells ml" not been observed in any organism to our knowledge. and pelleted in a clinical centrifuge. The cells were resuspended The enantiomeric position of the eyespot in the cell is in an equivalent volume of a buffer containing 30 mM-Pipes, 25mM-KCl, SmM-MgSO , 5mM-EGTA, and 12% hexylene thus believed to be a reflection of the fact that each basal 4 glycol (pH 6.8) for about 1 min and then repelleted. They were body has only one attachment site for the four-membered then lysed in 0.5 % saponin (Sigma) in the same buffer for rootlet and basal bodies exist in only one enantiomeric 1-2 min, pelleted, and fixed by the addition of 100—200/il of a form.