Thioredoxin-family protein EYE2 and Ser/Thr kinase EYE3 play interdependent roles in eyespot assembly

Boyd, J. Morton; Mittelmeier, Telsa M.; Lamb, Mary Rose; Dieckmann, Carol L.

doi:10.1091/mbc.e10-11-0918

Cited by 26 publications

(59 citation statements)

References 39 publications

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“…The fixed cells were rehydrated in PBS plus 1% bovine serum albumin (BSA; block) for 30 min at RT and incubated in block containing 0.1% Triton X-100 for 30 min at RT, followed by incubation overnight at 4°C in block containing 1:20 anti-acetylated ␣-tubulin clone 6-11B-1 (␣-Ac-tub) (Sigma-Aldrich, St. Louis, MO) (54) plus one of the following antibodies: 1:20 anti-␣-tubulin clone DM1a (␣-tub; Sigma-Aldrich, St. Louis, MO), 1:50 polyclonal anti-ChR1 (37), 1:50 polyclonal anti-ChR2 (37) (see Fig. S2B in the supplemental material), 1:50 polyclonal anti-EYE2 (41). Cells were washed with block containing 0.05% Tween 20, incubated for 2 h at room temperature in block containing a 1:1,000 dilution of the appropriate Alexa Fluor-conjugated secondary antibody (Invitrogen, Carlsbad, CA), washed thoroughly, and coverslipped with Mowiol mounting medium prepared as follows: 2.4 g Mowiol 4-88 (Calbiochem, EMD Biosciences, Inc., La Jolla, CA) and 6 g of glycerol were combined, 6 ml of water was added, and the solution was stirred at RT for 2 h before the addition of 12 ml of 0.2 M Tris (pH ϭ 8.5), being heated to 50°C for 10 min, and being clarified by centrifugation at 5,000 ϫ g for 15 min.…”

Section: Methodsmentioning

confidence: 99%

“…Channelrhodopsins ChR1 and ChR2, rhodopsin family light-gated cation channels that initiate the phototactic response, are localized in the plasma membrane at the eyespot (36)(37)(38)(39)(40). EYE2, an eyespotspecific, thioredoxin superfamily protein of unknown function, is thought to reside in one of the two chloroplast envelope membranes (39,41,42). Following cell division, one or more eyespot proteins must respond directly to a positioning cue, such as the D4 rootlet, to initiate de novo eyespot assembly.…”

mentioning

confidence: 99%

“…Alternatively, plasma membrane and plastid components might localize independently of one another in response to the same cue. Immunofluorescence microscopy (IF) of eye2 (eyeless) mutant cells, which lack the pigment granule layers and the EYE2 protein, showed that the ChR1 photoreceptor can localize independently of the missing plastid components, prompting the hypothesis that D4-directed localization of the photoreceptor(s) is the initial step in eyespot assembly (36,41,43). This hypothesis predicts that following cell division, assembly of the D4 rootlet occurs before localization of eyespot components to the position of the nascent eyespot and that photoreceptor localization occurs before that of EYE2 or the pigment granule layers.…”

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Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

et al. 2013

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bLike many algae, Chlamydomonas reinhardtii is phototactic, using two anterior flagella to swim toward light optimal for photosynthesis. The flagella are responsive to signals initiated at the photosensory eyespot, which comprises photoreceptors in the plasma membrane and layers of pigment granules in the chloroplast. Phototaxis depends on placement of the eyespot at a specific asymmetric location relative to the flagella, basal bodies, and bundles of two or four highly acetylated microtubules, termed rootlets, which extend from the basal bodies toward the posterior of the cell. Previous work has shown that the eyespot is disassembled prior to cell division, and new eyespots are assembled in daughter cells adjacent to the nascent four-membered rootlet associated with the daughter basal body (D4), but the chronology of these assembly events has not been determined. Here we use immunofluorescence microscopy to follow assembly and acetylation of the D4 rootlet, localization of individual eyespot components in the plasma membrane or chloroplast envelope, and flagellar emergence during and immediately following cell division. We find that the D4 rootlet is assembled before the initiation of eyespot assembly, which occurs within the same time frame as rootlet acetylation and flagellar outgrowth. Photoreceptors in the plasma membrane are correctly localized in eyespot mutant cells lacking pigment granule layers, and chloroplast components of the eyespot assemble in mutant cells in which photoreceptor localization is retarded. The data suggest that plasma membrane and chloroplast components of the eyespot are independently responsive to a cytoskeletal positioning cue.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

et al. 2013

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“…In addition, we identified several unique proteins in the D. bardawil bC-plastoglobuli: the major lipid-associated protein CGP (Katz et al, 1995;Davidi et al, 2014), the eyespot assembly protein EYE3 (Boyd et al, 2011), the vesicle-inducing plastid protein VIPP1, and plant synaptotagmin. The proteome includes several enzymes involved in the synthesis and/or degradation of lipids, carotenoids, terpenoids, quinones, enzymes involved in carbohydrate and energy metabolism, stress-related proteins, protein kinases and phosphatases, as well as signaling proteins, suggesting diverse metabolic and regulatory roles (Tables II and IV).…”

Section: Core Cld and Bc-plastoglobuli Proteomesmentioning

confidence: 99%

“…EYE3 is a Ser/Thr kinase belonging to the ABC1 superfamily. A recent study localized EYE3 in eyespot lipid droplets in C. reinhardtii and proposed that this protein is involved in pigment granule biogenesis (Boyd et al, 2011). The identification of two homologs of EYE3 in high abundance (21 and 35 peptides) and high enrichment in bC-plastoglobuli suggests that they are integral core components of bC-plastoglobuli in D. bardawil and may be involved in their biogenesis or structural stabilization.…”

Section: Abc1 Kinasesmentioning

confidence: 99%

Proteome Analysis of Cytoplasmatic and Plastidic β-Carotene Lipid Droplets in Dunaliella bardawil

et al. 2014

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The halotolerant green alga Dunaliella bardawil is unique in that it accumulates under stress two types of lipid droplets: cytoplasmatic lipid droplets (CLD) and b-carotene-rich (bC) plastoglobuli. Recently, we isolated and analyzed the lipid and pigment compositions of these lipid droplets. Here, we describe their proteome analysis. A contamination filter and an enrichment filter were utilized to define core proteins. A proteome database of Dunaliella salina/D. bardawil was constructed to aid the identification of lipid droplet proteins. A total of 124 and 42 core proteins were identified in bC-plastoglobuli and CLD, respectively, with only eight common proteins. Dunaliella spp. CLD resemble cytoplasmic droplets from Chlamydomonas reinhardtii and contain major lipid droplet-associated protein and enzymes involved in lipid and sterol metabolism. The bC-plastoglobuli proteome resembles the C. reinhardtii eyespot and Arabidopsis (Arabidopsis thaliana) plastoglobule proteomes and contains carotene-globule-associated protein, plastid-lipid-associated protein-fibrillins, SOUL hemebinding proteins, phytyl ester synthases, b-carotene biosynthesis enzymes, and proteins involved in membrane remodeling/lipid droplet biogenesis: VESICLE-INDUCING PLASTID PROTEIN1, synaptotagmin, and the eyespot assembly proteins EYE3 and SOUL3. Based on these and previous results, we propose models for the biogenesis of bC-plastoglobuli and the biosynthesis of b-carotene within bCplastoglobuli and hypothesize that bC-plastoglobuli evolved from eyespot lipid droplets.

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Algal Eyes

Hegemann

Dieckmann

2011

Encyclopedia of Life Sciences

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‘Vision’ is defined in a very general sense as recognition of the ambient light pattern by a motile organism and its use for orientation in a local environment. The ‘eye’ is the organ or organelle in which the light absorption and transformation into a transient intracellular signal occurs. The pigmented eyespot is functioning as the optical system that operates in most motile green algae as an interference reflecting device (quarter wave stack). In Chlorophyceae the light sensors are light‐gated ion channels (Channelrhodopsin (ChR)), whereas in Euglenoida photo‐activated adenylyl cyclases (PAC) fulfil this function. ChR is located within the eyespot overlaying part of the plasma membrane whereas PAC is found as a paraflagellar swelling at the basis of the long flagalla. Key Concepts: Motile green algae are able to detect the light direction by means of a pigmented eye. Algal eyes are designed for detection of defuse light. Chlorophyceae use light‐gated ion channels (Channelrhodopsins) for fast depolarisation of the plasma membrane. The depolarisation changes of the plasma membrane (PM) follow intensity changes recorded by the photoreceptor according to the orientation of the cells respective to the direction of the light source. Depolarisation of the PM is continued to the flagellar membrane. Flagallar membrane depolarisation causes activation of voltage gated Ca‐channel and influx of Ca 2+ . Intraflagellar Ca 2+ changes modulate the flagellar beating pattern resulting in directional changes of the swimming. As a sensor, Euglenoids use a photo‐activated adenylyl cyclases (PAC) that produces cAMP in the light. Since the PAC is attached to the long flagella, the produced cAMP is immediately sensed by the cAMP‐regulated ion channels at the flagaller base. The intraflagallar Ca 2+ changes alter the flagellar bending and the swimming direction accordingly.

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Thioredoxin-family protein EYE2 and Ser/Thr kinase EYE3 play interdependent roles in eyespot assembly

Cited by 26 publications

References 39 publications

Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

Proteome Analysis of Cytoplasmatic and Plastidic β-Carotene Lipid Droplets in Dunaliella bardawil

Algal Eyes

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