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1.  The Daughter Four-Membered Microtubule Rootlet Determines Anterior-Posterior Positioning of the Eyespot in Chlamydomonas reinhardtii 
Cytoskeleton (Hoboken, N.J.)  2011;68(8):459-469.
The characteristic geometry of the unicellular chlorophyte Chlamydomonas reinhardtii has contributed to its adoption as a model system for cellular asymmetry and organelle positioning. The eyespot, a photosensitive organelle, is localized asymmetrically in the cell at a precisely-defined position relative to the flagella and cytoskeletal microtubule rootlets. We have isolated a mutant, named pey1 for posterior eyespot, with variable microtubule rootlet lengths. The length of the acetylated daughter four-membered microtubule rootlet correlates with the position of the eyespot, which appears in a posterior position in the majority of cells. The correlation of rootlet length with eyespot positioning was also observed in the cmu1 mutant, which has longer acetylated microtubules, and the mlt1 mutant, in which the rootlet microtubules are shorter. Observation of eyespot positioning after depolymerization of rootlet microtubules indicated that eyespot position is fixed early in eyespot development and becomes independent of the rootlet. Our data demonstrate that the length of the daughter four-membered rootlet is the major determinant of eyespot positioning on the anterior-posterior axis and are suggestive that the gene product of the PEY1 locus is a novel regulator of acetylated microtubule length.
doi:10.1002/cm.20524
PMCID: PMC3201734  PMID: 21766471
Chlamydomonas; eyespot; microtubule rootlet; organelle positioning; pey1
2.  New insights into eyespot placement and assembly in Chlamydomonas 
Bioarchitecture  2011;1(4):196-199.
Aspects of cellular architecture, such as cytoskeletal asymmetry cues, play critical roles in directing the placement of organelles and establishing the sites of their formation. In the model green alga Chlamydomonas, the photosensory eyespot occupies a defined position in relation to the flagella and microtubule cytoskeleton. Investigations into the cellular mechanisms of eyespot placement and assembly have aided our understanding of the interplay between cytoskeletal and plastid components of the cell. The eyespot, which must be assembled anew after each cell division, is a multi-layered organelle consisting of stacks of carotenoid-filled pigment granules in the chloroplast and rhodopsin photoreceptors in the plasma membrane. Placement of the eyespot is determined on both the latitudinal and longitudinal axes of the cell by the daughter four-membered (D4) microtubule rootlet. Recent findings have contributed to the hypothesis that the eyespot photoreceptor molecules are directed from the Golgi to the daughter hemisphere of the cell and trafficked along the D4 microtubule rootlet. EYE2, a chloroplast-envelope protein, forms an elliptical patch together with the photoreceptors and establishes the site for assembly of the pigment granule arrays in the chloroplast, connecting the positioning information of the cytoskeleton to assembly of the pigment granule arrays in the chloroplast.
doi:10.4161/bioa.1.4.17697
PMCID: PMC3210518  PMID: 22069514
Chlamydomonas; eyespot; organelle placement; organelle assembly; microtubule rootlet; asymmetry; photoreception
3.  Asymmetric properties of the Chlamydomonas reinhardtii cytoskeleton direct rhodopsin photoreceptor localization 
The Journal of Cell Biology  2011;193(4):741-753.
Daughter four-membered rootlet microtubules direct eyespot positioning and assembly.
The eyespot of the unicellular green alga Chlamydomonas reinhardtii is a photoreceptive organelle required for phototaxis. Relative to the anterior flagella, the eyespot is asymmetrically positioned adjacent to the daughter four-membered rootlet (D4), a unique bundle of acetylated microtubules extending from the daughter basal body toward the posterior of the cell. Here, we detail the relationship between the rhodopsin eyespot photoreceptor Channelrhodopsin 1 (ChR1) and acetylated microtubules. In wild-type cells, ChR1 was observed in an equatorial patch adjacent to D4 near the end of the acetylated microtubules and along the D4 rootlet. In cells with cytoskeletal protein mutations, supernumerary ChR1 patches remained adjacent to acetylated microtubules. In mlt1 (multieyed) mutant cells, supernumerary photoreceptor patches were not restricted to the D4 rootlet, and more anterior eyespots correlated with shorter acetylated microtubule rootlets. The data suggest a model in which photoreceptor localization is dependent on microtubule-based trafficking selective for the D4 rootlet, which is perturbed in mlt1 mutant cells.
doi:10.1083/jcb.201009131
PMCID: PMC3166873  PMID: 21555459
4.  Miniature- and Multiple-Eyespot Loci in Chlamydomonas reinhardtii Define New Modulators of Eyespot Photoreception and Assembly 
G3: Genes|Genomes|Genetics  2011;1(6):489-498.
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.
doi:10.1534/g3.111.000679
PMCID: PMC3276157  PMID: 22384359
eyespot; photoreception; organelle biogenesis; MIN2; MLT2
5.  Thioredoxin-family protein EYE2 and Ser/Thr kinase EYE3 play interdependent roles in eyespot assembly 
Molecular Biology of the Cell  2011;22(9):1421-1429.
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.
The eyespot of the biflagellate unicellular green alga Chlamydomonas reinhardtii is a complex organelle that facilitates directional responses of the cell to environmental light stimuli. The eyespot, which assembles de novo after every cell division and is associated with the daughter four-membered (D4) microtubule rootlet, comprises an elliptical patch of rhodopsin photoreceptors on the plasma membrane and stacks of carotenoid-rich pigment granule arrays in the chloroplast. Two loci, EYE2 and EYE3, define factors involved in the formation and organization of the eyespot pigment granule arrays. Whereas EYE3, a serine/threonine kinase of the ABC1 family, localizes to pigment granules, EYE2 localization corresponds to an area of the chloroplast envelope in the eyespot. EYE2 is positioned along, and adjacent to, the D4 rootlet in the absence of pigment granules. The eyespot pigment granule array is required for maintenance of the elliptical shape of both the overlying EYE2 and channelrhodopsin-1 photoreceptor patches. We propose a model of eyespot assembly wherein rootlet and photoreceptor direct EYE2 to an area of the chloroplast envelope, where it acts to facilitate assembly of pigment granule arrays, and EYE3 plays a role in the biogenesis of the pigment granules.
doi:10.1091/mbc.E10-11-0918
PMCID: PMC3084665  PMID: 21372178

Results 1-5 (5)