Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the~120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

Daughter four-membered rootlet microtubules direct eyespot positioning and assembly.

EYE2 is a key protein in connecting the positioning information of the microtubule rootlet cytoskeleton and channelrhodopsin 1 (ChR1) photoreceptor to the formation and positioning of the eyespot pigment granules in the chloroplast of Chlamydomonas. EYE3, a ser/thr kinase of the ABC1 family, is found in pigment granules and is required for their biogenesis.

Assembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green alga Chlamydomonas reinhardtii requires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast. min1 (mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. The MIN1 gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly in Chlamydomonas. In min1 null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth of min1 cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1.A variety of organelles and multicomponent structures are assembled and maintained within eukaryotic cells. These structures occupy defined, often asymmetric, intracellular locations and must be correctly partitioned or reassembled at every cell division. Elucidation of the mechanisms that govern the assembly and localization of complex structures is critical to our understanding of how basic cellular processes are regulated, how they may have evolved, and the factors driving their ongoing diversification.Assembly and asymmetric localization of the photosensory eyespot in cells of the biflagellate, unicellular, photosynthetic green alga Chlamydomonas reinhardtii provide useful models for genetic, molecular, and microscopic analyses of organelle biogenesis (11,14,23). Chlamydomonas is phototactic, using two anterior flagella to swim toward or away from a light source to locations where light intensity is optimal for photosynthesis but minimally damaging to the photosynthetic membranes (82). The eyespot (Fig. 1A, wild type) is a light-sensing structure positioned near the equator of the cell at an asymmetric location relative to the flagella (25). Stimulation of the rhodopsin family photoreceptors in the eyespot activates a Ca 2ϩ -dependent signal transduction pathway(s) that affects flagellar movement and the swimming behavior of the cell (24,58,69). Asymmetric localization of the eyespot is required for the transmission of information about the direction of the light source (82).The sensitivity of the eyespot to light is a result of the properties and organization of the...

The photosensory eyespot of the green alga Chlamydomonas reinhardtii is a model system for the study of organelle biogenesis and placement. Eyespot assembly and positioning are governed by several genetic loci that have been identified in forward genetic screens for phototaxis-defective mutants. These include the previously described miniature-eyespot mutant min1, the multiple-eyespot mutant mlt1, the eyeless mutants eye2 and eye3, and two previously uncharacterized eyespot mutants, min2 and mlt2. In this study, effects of miniature- and multiple-eyespot mutations and their combinations on the localization and expression levels of the rhodopsin photoreceptor channelrhodopsin-1 (ChR1) and the localization of the eyespot-assembly proteins EYE2 and EYE3 were examined. min2 mutants assemble a properly organized, albeit nonfunctional, eyespot that is slightly smaller than wild-type; however, combination of the min2 and mlt1 mutations resulted in drastic reduction of photoreceptor levels. Both stationary-phase mlt1 and mlt2 cells have supernumerary, mislocalized eyespots that exhibit partial or total dissociation of the eyespot layers. In these mutant strains, photoreceptor patches in the plasma membrane were never associated with pigment granule arrays in the chloroplast stroma unless EYE2 was present in the intervening envelope. The data suggest that MIN2 is required for the photoreceptive ability of the eyespot and that MLT2 plays a major role in regulating eyespot number, placement, and integrity.

Asymmetric placement of the photosensory eyespot organelle in Chlamydomonas is patterned by mother-daughter differences between the two basal bodies, which template the anterior flagella. Each basal body is associated with two bundled microtubule rootlets, one with two microtubules and one with four, forming a cruciate pattern. In wild type cells, the single eyespot is positioned at the equator in close proximity to the plus end of the daughter rootlet comprising four microtubules, the D4. Here we identify mutations in two linked loci, MLT1 and MLT2, which cause multiple eyespots. Antiserum raised against MLT1 localized the protein along the D4 rootlet microtubules, from the basal bodies to the eyespot. MLT1 associates immediately with the new D4 as it extends during cell division, before microtubule acetylation. MLT1 is a low-complexity protein of over 300,000 daltons. The expression or stability of MLT1 is dependent on MLT2, predicted to encode a second large, low-complexity protein. MLT1 was not restricted to the D4 rootlet in cells with the vfl2-220 mutation in the gene encoding the basal body-associated protein centrin. The cumulative data highlight the role of mother-daughter basal body differences in establishing asymmetry in associated rootlets, and suggest that eyespot components are directed to the correct location by MLT1 on the D4 microtubules.

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 Ca++-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.

No abstract