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1.  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.
PMCID: PMC3084665  PMID: 21372178
2.  Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly 
Eukaryotic Cell  2013;12(9):1258-1270.
Like 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.
PMCID: PMC3811559  PMID: 23873865
3.  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.
PMCID: PMC3276157  PMID: 22384359
eyespot; photoreception; organelle biogenesis; MIN2; MLT2
4.  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.
PMCID: PMC3166873  PMID: 21555459
5.  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.
PMCID: PMC3201734  PMID: 21766471
Chlamydomonas; eyespot; microtubule rootlet; organelle positioning; pey1
6.  C2 Domain Protein MIN1 Promotes Eyespot Organization in Chlamydomonas reinhardtii▿ †  
Eukaryotic Cell  2008;7(12):2100-2112.
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.
PMCID: PMC2593190  PMID: 18849467
7.  The Ultrastructure of a Chlamydomonas reinhardtii Mutant Strain Lacking Phytoene Synthase Resembles that of a Colorless Alga 
Molecular Plant  2008;1(6):925-937.
Chlamydomonas reinhardtii strains lacking phytoene synthase, the first enzyme of carotenoid biosynthesis, are white. They lack carotenoid pigments, have very low levels of chlorophyll, and can grow only heterotrophically in the dark. Our electron and fluorescence microscopic studies showed that such a mutant strain (lts1-204) had a proliferated plastid envelope membrane but no stacks of thylakoid membranes within the plastid. It accumulated cytoplasmic compartments that appeared to be autophagous vacuoles filled with membranous material. The lts1 mutants apparently lacked pyrenoid bodies, which normally house ribulose bisphosphate carboxylase–oxygenase (Rubisco), and accumulated many starch granules. Although these mutant strains cannot synthesize the carotenoid and carotenoid-derived pigments present in the phototactic organelle (eyespot), the mutant we examined made a vestigial eyespot that was disorganized and often mislocalized to the posterior end of the cell. The absence of a pyrenoid body, the accumulation of starch, and the disorganization of the eyespot may all result from the absence of thylakoids. The ultrastructure of lts1 mutant strains is similar to but distinct from that of previously described white and yellow mutant strains of C. reinhardtii and is similar to that of naturally colorless algae of the Polytoma group.
PMCID: PMC2902904  PMID: 19825593
8.  Expression Dynamics and Protein Localization of Rhabdomeric Opsins in Platynereis Larvae 
The larval stages of polychaete annelids are often responsive to light and can possess one to six eyes. The early trochophore larvae of the errant annelid Platynereis dumerilii have a single pair of ventral eyespots, whereas older nectochaete larvae have an additional two pairs of dorsal eyes that will develop into the adult eyes. Early Platynereis trochophores show robust positive phototaxis starting on the first day of development. Even though the mechanism of phototaxis in Platynereis early trochophore larvae is well understood, no photopigment (opsin) expression has yet been described in this stage. In late trochophore larvae, a rhabdomeric-type opsin, r-opsin1, expressed in both the eyespots and the adult eyes has already been reported. Here, we identify another Platynereis rhabdomeric opsin, r-opsin3, that is expressed in a single photoreceptor in the eyespots in early trochophores, suggesting that it mediates early larval phototaxis. We also show that r-opsin1 and r-opsin3 are expressed in adjacent photoreceptor cells in the eyespots in later stages, indicating that a second eyespot-photoreceptor differentiates in late trochophore larvae. Using serial transmission electron microscopy (TEM), we identified and reconstructed both photoreceptors and a pigment cell in the late larval eyespot. We also characterized opsin expression in the adult eyes and found that the two opsins co-express there in several photoreceptor cells. Using antibodies recognizing r-opsin1 and r-opsin3 proteins, we demonstrate that both opsins localize to the rhabdomere in all six eyes. In addition, we found that r-opsin1 mRNA is localized to, and translated in, the projections of the adult eyes. The specific changes we describe in opsin transcription and translation and in the cellular complement suggest that the six larval eyes undergo spectral and functional maturation during the early planktonic phase of the Platynereis life cycle.
PMCID: PMC3687135  PMID: 23667045
The cytoplasmic organization of a normal green strain of the alga Chlamydomonas reinhardi has been investigated with the electron microscope using thin sections of OsO4 fixed material. The detailed organization of the chloroplast has been of special interest. The chloroplast, a cup-shaped organelle, surrounded by a double membrane, consists of: (1) discs about 1 micron in diameter, considered to represent the basic structural unit of the chloroplast, and each composed of a pair of membranes joined at their ends to form a flat closed vesicle; the discs are grouped into stacks resembling the grana of higher plants; (2) matrix material of low density in which the discs are embedded; (3) starch grains; (4) the pyrenoid, a non-lamellar region associated with starch synthesis, and containing tubules which connect with the lamellae; (5) the eyespot, a differentiated region containing two or three plates of hexagonally packed, carotenoid-containing granules, located between discs, and associated with phototaxis. In addition to the chloroplast, the cytoplasm contains various membranous and granular components, including mitochondria, endoplasmic reticulum, and dictyosomes, identified on the basis of morphological comparability with structures seen in animal cells. The nucleus, not investigated in detail in this study, contains a large, granular nucleolus and is surrounded by a nuclear envelope which is provided with pores and exhibits instances of continuity with the endoplasmic reticulum of the cytoplasm.
PMCID: PMC2224040  PMID: 13438931
10.  The UNI3 Gene Is Required for Assembly of Basal Bodies of Chlamydomonas and Encodes δ-Tubulin, a New Member of the Tubulin Superfamily 
Molecular Biology of the Cell  1998;9(6):1293-1308.
We have cloned the UNI3 gene in Chlamydomonas and find that it encodes a new member of the tubulin superfamily. Although Uni3p shares significant sequence identity with α-, β-, and γ-tubulins, there is a region of Uni3p that has no similarity to tubulins or other known proteins. Mutant uni3–1 cells assemble zero, one, or two flagella. Pedigree analysis suggests that flagellar number in uni3–1 cells is a function of the age of the cell. The uniflagellate uni3–1 cells show a positional phenotype; the basal body opposite the eyespot templates the single flagellum. A percentage of uni3–1 cells also fail to orient the cleavage furrow properly, and basal bodies have been implicated in the placement of cleavage furrows in Chlamydomonas. Finally when uni3–1 cells are observed by electron microscopy, doublet rather than triplet microtubules are observed at the proximal end of the basal bodies. We propose that the Uni3 tubulin is involved in both the function and cell cycle-dependent maturation of basal bodies/centrioles.
PMCID: PMC25351  PMID: 9614175
The Journal of Cell Biology  1967;35(3):521-552.
This paper describes the morphology and photosynthetic activity of a mutant of Chlamydomonas reinhardi (y-1) which is unable to synthesize chlorophyll in the dark. When grown heterotrophically in the light, the mutant is indistinguishable from the wild type Chlamydomonas. When grown in the dark, chlorophyll is diluted through cell division and the photosynthetic activity (oxygen evolution, Hill reaction, and photoreduction of NADP) decays at a rate equal to or faster than that of chlorophyll dilution. However, soluble enzymes associated with the photosynthetic process (alkaline FDPase, NADP-linked G-3-P dehydrogenase, RuDP carboxylase), as well as cytochrome f and ferredoxin, continue to be present in relatively high concentrations. The enzymes involved in the synthesis of the characteristic lipids of the chloroplast (including mono- and digalactoside glycerides, phosphatidyl glycerol, and sulfolipid) are still detectable in dark-grown cells. Such cells accumulate large amounts of starch granules in their plastids. On onset of illumination, dark-grown cells synthesize chlorophyll rapidly, utilizing their starch reserve in the process. At the morphological level, it was observed that during growth in the dark the chloroplast lamellar system is gradually disorganized and drastically decreased in extent, while other subchloroplast components are either unaffected (pyrenoid and its tubular system, matrix) or much less affected (eyespot, ribosomes). It is concluded that the dark-grown mutant possesses a partially differentiated plastid and the enzymic apparatus necessary for the synthesis of the chloroplast membranes (discs). The advantage provided by such a system for the study of the biogenesis of the chloroplast photosynthetic membranes is discussed.
PMCID: PMC2107153  PMID: 6064364
12.  The power of functional proteomics 
Plant Signaling & Behavior  2008;3(7):433-435.
One of the key modifications of proteins that can affect protein functions, activities, stabilities, localizations and interactions, represents phosphorylation. For functional phosphoproteomics, phosphopeptides are enriched from isolated sub-cellular fractions of interest and analyzed by liquid chromatography-electrospray ionization-mass spectrometry. Such an approach was recently applied to the eyespot apparatus of the green flagellate alga Chlamydomonas reinhardtii, which represents a primordial visual system. Thereby, 32 phosphoproteins of known eyespot proteins along with 52 precise in vivo phosphorylation sites were identified. They include enzymes of carotenoid and fatty acid metabolism, (putative) light signaling components and proteins with unknown function. Strikingly, the two unique green algal photoreceptors, channelrhodopsin-1 and -2 were found to be phosphorylated in the cytoplasmic loop next to their seven transmembrane regions in a similar distance as observed in vertebrate rhodopsins.
PMCID: PMC2634420  PMID: 19513232
Chlamydomonas reinhardtii; eyespot; phosphoproteins; proteomics; signaling; rhodopsin
13.  A Rhodopsin-Guanylyl Cyclase Gene Fusion Functions in Visual Perception in a Fungus 
Current Biology  2014;24(11):1234-1240.
Sensing light is the fundamental property of visual systems, with vision in animals being based almost exclusively on opsin photopigments [1]. Rhodopsin also acts as a photoreceptor linked to phototaxis in green algae [2, 3] and has been implicated by chemical means as a light sensor in the flagellated swimming zoospores of the fungus Allomyces reticulatus [4]; however, the signaling mechanism in these fungi remains unknown. Here we use a combination of genome sequencing and molecular inhibition experiments with light-sensing phenotype studies to examine the signaling pathway involved in visual perception in the closely related fungus Blastocladiella emersonii. Our data show that in these fungi, light perception is accomplished by the function of a novel gene fusion (BeGC1) of a type I (microbial) rhodopsin domain and guanylyl cyclase catalytic domain. Photobleaching of rhodopsin function prevents accumulation of cGMP levels and phototaxis of fungal zoospores exposed to green light, whereas inhibition of guanylyl cyclase activity negatively affects fungal phototaxis. Immunofluorescence microscopy localizes the BeGC1 protein to the external surface of the zoospore eyespot positioned close to the base of the swimming flagellum [4, 5], demonstrating this is a photoreceptive organelle composed of lipid droplets. Taken together, these data indicate that Blastocladiomycota fungi have a cGMP signaling pathway involved in phototaxis similar to the vertebrate vision-signaling cascade but composed of protein domain components arranged as a novel gene fusion architecture and of distant evolutionary ancestry to type II rhodopsins of animals.
•A rhodopsin-guanylate cyclase gene fusion is involved in B. emersonii phototaxis•The rhodopsin fusion protein BeGC1 is localized to the zoospore eyespot apparatus•Endogenous retinal substitution by retinalA1 reconstitutes green light phototaxis•Zoospore phototaxis uses cGMP as a second messenger similar to vertebrate vision
Avelar et al. use genome sequencing, molecular inhibition, and light-sensing phenotype experiments, combined with immunolocalization data, to show that a type I rhodopsin-guanylyl cyclase fusion protein localizes to the “eyespot” and is involved in green light phototaxis in zoospores of the Blastocladiomycete fungus Blastocladiella emersonii.
PMCID: PMC4046227  PMID: 24835457
14.  Cell Division in Apicomplexan Parasites Is Organized by a Homolog of the Striated Rootlet Fiber of Algal Flagella 
PLoS Biology  2012;10(12):e1001444.
Apicomplexan parasites undergo cell division using an evolutionarily conserved mechanism first described in the positioning and assembly of flagella in algae.
Apicomplexa are intracellular parasites that cause important human diseases including malaria and toxoplasmosis. During host cell infection new parasites are formed through a budding process that parcels out nuclei and organelles into multiple daughters. Budding is remarkably flexible in output and can produce two to thousands of progeny cells. How genomes and daughters are counted and coordinated is unknown. Apicomplexa evolved from single celled flagellated algae, but with the exception of the gametes, lack flagella. Here we demonstrate that a structure that in the algal ancestor served as the rootlet of the flagellar basal bodies is required for parasite cell division. Parasite striated fiber assemblins (SFA) polymerize into a dynamic fiber that emerges from the centrosomes immediately after their duplication. The fiber grows in a polarized fashion and daughter cells form at its distal tip. As the daughter cell is further elaborated it remains physically tethered at its apical end, the conoid and polar ring. Genetic experiments in Toxoplasma gondii demonstrate two essential components of the fiber, TgSFA2 and 3. In the absence of either of these proteins cytokinesis is blocked at its earliest point, the initiation of the daughter microtubule organizing center (MTOC). Mitosis remains unimpeded and mutant cells accumulate numerous nuclei but fail to form daughter cells. The SFA fiber provides a robust spatial and temporal organizer of parasite cell division, a process that appears hard-wired to the centrosome by multiple tethers. Our findings have broader evolutionary implications. We propose that Apicomplexa abandoned flagella for most stages yet retained the organizing principle of the flagellar MTOC. Instead of ensuring appropriate numbers of flagella, the system now positions the apical invasion complexes. This suggests that elements of the invasion apparatus may be derived from flagella or flagellum associated structures.
Author Summary
Malaria, toxoplasmosis, and related diseases are caused by infection with unicellular parasites called Apicomplexa. Their name refers to the elaborate invasion machinery that occupies the apical end of the parasite cell. This apparatus allows the parasite to force its way into the cells of its host, and to deliver factors that will manipulate host cell structure, gene expression, and metabolism. Once in the host cell the parasite will begin to grow. The parasite replicates its genome and organelles numerous times and then loads these various elements into numerous daughter cells that will further spread the infection. Here we report a fiber that coordinates the daughter cell budding process. The fiber links the centrosome, which controls the mitotic spindle, and the genome with the microtubule organizing center of the budding daughter. Parasite mutants lacking the proteins that build the fiber fail to form daughter cells at the earliest step. The fiber and its components are remarkably similar to fibers that coordinate flagella in algae. While Apicomplexa are not flagellated (with the exception of certain gamete stages) they evolved from flagellated algae. We propose that elements of the invasion apparatus evolved from the flagellum or flagellum associated structures.
PMCID: PMC3519896  PMID: 23239939
15.  Comparative insights into questions of lepidopteran wing pattern homology 
Butterfly and moth eyespots can share a similar appearance, involving multiple concentric rings of colored scales, but usually occuring in non-homologous positions on the wing. Within the butterflies, on the other hand, spots that share the same homologous position may not share the concentric ring structure; and, in butterfly species that have eyespots with concentric rings, ectopic eyespots with a similar ring structure can be induced by means of a simple epidermal wound. The extent to which all these eyespots, natural or induced, share similar genes and developmental mechanisms is investigated here by means of protein in-situ localizations in selected butterfly and moth species. In addition to looking at some of the transcription factors previously identified as being involved in eyespot formation, we also tested the involvement of candidate genes from the Wingless and TGF-β signaling pathways as putative morphogens for eyespot development.
Saturniid moth and nymphalid butterfly eyespots with concentric rings of color express at least two transcription factors, Distal-less and Engrailed, in the center of the future pattern. Nymphalid eyespots centers also express the ligand Wingless and an activated signal transducer, a phosphorylated Smad protein, but neither these proteins nor the previous two proteins are found in pierid spot centers, which consist of a single patch of color. Both butterfly wing patterns, however, express a third transcription factor, Spalt, a portion of whose expression domain maps to the black scales on the adult wing. Wounding a nymphalid wing, on the other hand, leads to upregulation of Distal-less, engrailed and spalt in subsets of cells around the wounding site, mimicking concentric eyespot development.
Wingless and TGF-β ligands are both candidate morphogens involved in nymphalid butterfly eyespot formation. These eyespots, as well as saturniid moth eyespots with concentric circles, share two genes that are associated with the differentiation of the signaling cells in nymphalid eyespots. This commonality suggests that they may be produced via the same developmental mechanism despite their non-homologous location. By contrast, pierid butterfly spots of a single color share some of the same genes but appear to be produced by a different mechanism. Eyespots with concentric rings may have co-opted a wound healing genetic network during their evolution.
PMCID: PMC1654149  PMID: 17090321
16.  Nymphalid eyespots are co-opted to novel wing locations following a similar pattern in independent lineages 
Variation in the number of repeated traits, or serial homologs, has contributed greatly to animal body plan diversity. Eyespot color patterns of nymphalid butterflies, like arthropod and vertebrate limbs, are an example of serial homologs. These eyespot color patterns originated in a small number of wing sectors on the ventral hindwing surface and later appeared in novel wing sectors, novel wings, and novel wing surfaces. However, the details of how eyespots were co-opted to these novel wing locations are currently unknown.
We used a large data matrix of eyespot/presence absence data, previously assembled from photographs of contemporary species, to perform a phylogenetic investigation of eyespot origins in nine independent nymphalid lineages. To determine how the eyespot gene regulatory network acquired novel positional information, we used phylogenetic correlation analyses to test for non-independence in the origination of eyespots. We found consistent patterns of eyespot gene network redeployment in the nine lineages, where eyespots first redeployed from the ventral hindwing to the ventral forewing, then to new sectors within the ventral wing surface, and finally to the dorsal wing surface. Eyespots that appeared in novel wing sectors modified the positional information of their serial homolog ancestors in one of two ways: by changing the wing or surface identity while retaining sector identity, or by changing the sector identity while retaining wing and surface identity.
Eyespot redeployment to novel sectors, wings, and surfaces happened multiple times in different nymphalid subfamilies following a similar pattern. This indicates that parallel mutations altering expression of the eyespot gene regulatory network led to its co-option to novel wing locations over time.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-015-0300-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4335541
Serial homology; Correlation analysis; Ancestral states; Phylogeny; Wing patterns
17.  Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography 
eLife  null;4:e04889.
Chloroplast function is orchestrated by the organelle's intricate architecture. By combining cryo-focused ion beam milling of vitreous Chlamydomonas cells with cryo-electron tomography, we acquired three-dimensional structures of the chloroplast in its native state within the cell. Chloroplast envelope inner membrane invaginations were frequently found in close association with thylakoid tips, and the tips of multiple thylakoid stacks converged at dynamic sites on the chloroplast envelope, implicating lipid transport in thylakoid biogenesis. Subtomogram averaging and nearest neighbor analysis revealed that RuBisCO complexes were hexagonally packed within the pyrenoid, with ∼15 nm between their centers. Thylakoid stacks and the pyrenoid were connected by cylindrical pyrenoid tubules, physically bridging the sites of light-dependent photosynthesis and light-independent carbon fixation. Multiple parallel minitubules were bundled within each pyrenoid tubule, possibly serving as conduits for the targeted one-dimensional diffusion of small molecules such as ATP and sugars between the chloroplast stroma and the pyrenoid matrix.
eLife digest
Many organisms can harvest light to produce their own energy through a process called photosynthesis. In plant and algal cells, photosynthesis takes place within the chloroplasts, which are compartments that contain stacks of structures called thylakoids.
Inside the thylakoids, proteins absorb energy from light and convert it into biochemical energy that can be used by the cell. This energy then powers a series of reactions that result in carbon dioxide being incorporated into energy-rich sugars. The enzyme RuBisCO is essential for this process, and is believed to be the most abundant protein on Earth. In land plants, RuBisCO is found throughout the chloroplast, but in algae it is limited to a specialized area called the pyrenoid.
Much of our current knowledge of chloroplast structure comes from transmission electron microscopy (TEM) images. However, the traditional methods used to prepare cells for TEM can damage their internal structures. Also, previous studies have focused primarily on the chloroplasts of land plants, even though aquatic organisms—including the alga Chlamydomonas—account for over 50% of photosynthesis on the planet.
Here, Engel et al. provide the first three-dimensional structures of Chlamydomonas chloroplasts in their natural state. They used several recently-developed techniques to study cells that were preserved in a close-to-living condition. The cells were rapidly frozen, thinned with a technique called cryo-focused ion beam milling, and then imaged by a type of TEM called cryo-electron tomography.
The three-dimensional images provide many insights into the Chlamydomonas chloroplast, including evidence that lipids and proteins move between the membrane that surrounds the chloroplast—called the chloroplast envelope—and the tips of the thylakoids. These images show how thylakoids may be built by the transport of molecules from the chloroplast envelope. In addition, the images reveal the detailed structures of the tubes that connect the thylakoids to the pyrenoid, which could explain how the two stages of photosynthesis (light harvesting and the conversion of carbon dioxide) can be coordinated even though they occur at different places within the chloroplast.
Engel et al. also observed that RuBisCO enzymes are arranged in a hexagonal pattern inside the pyrenoid, but are spaced too far apart to make direct contact with each other. To understand how the pyrenoid is assembled, a future goal will be to determine what causes RuBisCO to be arranged in this way.
PMCID: PMC4292175  PMID: 25584625
Chlamydomonas; focused ion beam; cryo-electron tomography; chloroplast; thylakoid; RuBisCO; other
18.  Green Autofluorescence in Dinoflagellates, Diatoms, and Other Microalgae and Its Implications for Vital Staining and Morphological Studies▿ †  
Green autofluorescence (GAF) has been described in the short flagellum of golden and brown algae, the stigma of Euglenophyceae, and cytoplasm of different life stages of dinoflagellates and is considered by some researchers a valuable taxonomic feature for dinoflagellates. In addition, green fluorescence staining has been widely proposed or adopted to measure cell viability (or physiological state) in areas such as apoptosis of phytoplankton, pollutant stresses on algae, metabolic activity of algae, and testing treatment technologies for ships' ballast water. This paper reports our epifluorescence microscopic observations and quantitative spectrometric measurements of GAF in a broad phylogenetic range of microalgae. Our results demonstrate GAF is a common feature of dinoflagellates, diatoms, green algae, cyanobacteria, and raphidophytes, occurs in the cytoplasm and particularly in eyespots, accumulation bodies, spines, and aerotopes, and is caused by molecules other than chlorophyll. GAF intensity increased with time after cell death or fixation and with excitation by blue or UV light and was affected by pH. GAF of microalgae may be only of limited value in taxonomy. It can be strong enough to interfere with the results of green fluorescence staining, particularly when stained samples are observed microscopically. GAF is useful, however, for microscopic study of algal morphology, especially to visualize cellular components such as eyespots, nucleus, aerotopes, spines, and chloroplasts. Furthermore, GAF can be used to visualize and enumerate dinoflagellate cysts in marine and estuarine sediments in the context of anticipating and monitoring harmful algal blooms and in tracking potentially harmful dinoflagellates transported in ships' ballast tanks.
PMCID: PMC1855647  PMID: 17277199
19.  How 5000 independent rowers coordinate their strokes in order to row into the sunlight: Phototaxis in the multicellular green alga Volvox 
BMC Biology  2010;8:103.
The evolution of multicellular motile organisms from unicellular ancestors required the utilization of previously evolved tactic behavior in a multicellular context. Volvocine green algae are uniquely suited for studying tactic responses during the transition to multicellularity because they range in complexity from unicellular to multicellular genera. Phototactic responses are essential for these flagellates because they need to orientate themselves to receive sufficient light for photosynthesis, but how does a multicellular organism accomplish phototaxis without any known direct communication among cells? Several aspects of the photoresponse have previously been analyzed in volvocine algae, particularly in the unicellular alga Chlamydomonas.
In this study, the phototactic behavior in the spheroidal, multicellular volvocine green alga Volvox rousseletii (Volvocales, Chlorophyta) was analyzed. In response to light stimuli, not only did the flagella waveform and beat frequency change, but the effective stroke was reversed. Moreover, there was a photoresponse gradient from the anterior to the posterior pole of the spheroid, and only cells of the anterior hemisphere showed an effective response. The latter caused a reverse of the fluid flow that was confined to the anterior hemisphere. The responsiveness to light is consistent with an anterior-to-posterior size gradient of eyespots. At the posterior pole, the eyespots are tiny or absent, making the corresponding cells appear to be blind. Pulsed light stimulation of an immobilized spheroid was used to simulate the light fluctuation experienced by a rotating spheroid during phototaxis. The results demonstrated that in free-swimming spheroids, only those cells of the anterior hemisphere that face toward the light source reverse the beating direction in the presence of illumination; this behavior results in phototactic turning. Moreover, positive phototaxis is facilitated by gravitational forces. Under our conditions, V. rousseletii spheroids showed no negative phototaxis.
On the basis of our results, we developed a mechanistic model that predicts the phototactic behavior in V. rousseletii. The model involves photoresponses, periodically changing light conditions, morphological polarity, rotation of the spheroid, two modes of flagellar beating, and the impact of gravity. Our results also indicate how recently evolved multicellular organisms adapted the phototactic capabilities of their unicellular ancestors to multicellular life.
PMCID: PMC2920248  PMID: 20663212
20.  Cellular Differentiation in Moss Protonemata: A Morphological and Experimental Study 
Annals of Botany  2008;102(2):227-245.
Background and Aims
Previous studies of protonemal morphogenesis in mosses have focused on the cytoskeletal basis of tip growth and the production of asexual propagules. This study provides the first comprehensive description of the differentiation of caulonemata and rhizoids, which share the same cytology, and the roles of the cytoskeleton in organelle shaping and spatial arrangement.
Light and electron microscope observations were carried out on in vitro cultured and wild protonemata from over 200 moss species. Oryzalin and cytochalasin D were used to investigate the role of the cytoskeleton in the cytological organization of fully differentiated protonemal cells; time-lapse photography was employed to monitor organelle positions.
Key Results
The onset of differentiation in initially highly vacuolate subapical cells is marked by the appearance of tubular endoplasmic reticulum (ER) profiles with crystalline inclusions, closely followed by an increase in rough endoplasmic reticulum (RER). The tonoplast disintegrates and the original vacuole is replaced by a population of vesicles and small vacuoles originating de novo from RER. The cytoplasm then becomes distributed throughout the cell lumen, an event closely followed by the appearance of endoplasmic microtubules (MTs) in association with sheets of ER, stacks of vesicles that subsequently disperse, elongate mitochondria and chloroplasts and long tubular extensions at both poles of the nucleus. The production of large vesicles by previously inactive dictysomes coincides with the deposition of additional cell wall layers. At maturity, the numbers of endoplasmic microtubules decline, dictyosomes become inactive and the ER is predominantly smooth. Fully developed cells remain largely unaffected by cytochalasin; oryzalin elicits profound cytological changes. Both inhibitors elicit the formation of giant plastids. The plastids and other organelles in fully developed cells are largely stationary.
Differentiation of caulonemata and rhizoids involves a remarkable series of cytological changes, some of which closely recall major events in sieve element ontogeny in tracheophytes. The cytology of fully differentiated cells is remarkably similar to that of moss food-conducting cells and, in both, is dependent on an intact microtubule cytoskeleton. The disappearance of the major vacuolar apparatus is probably related to the function of caulonema and rhizoids in solute transport. Failure of fully differentiated caulonema and rhizoid cells to regenerate is attributed to a combination of endo-reduplication and irreversible tonoplast fragmentation. The formation of giant plastids, most likely by fusion, following both oryzalin and cytochalasin treatments, suggests key roles for both microtubules and microfilaments in the spatial arrangement and replication of plastids.
PMCID: PMC2712367  PMID: 18508779
Bryophyta; caulonemata; cytoskeletal inhibitors; food-conducting cells; protonemal differentiation; rhizoids; sieve elements
21.  Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella 
The Journal of Cell Biology  1981;91(2):352-360.
The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.
PMCID: PMC2111981  PMID: 7309786
22.  Stress induces the assembly of RNA granules in the chloroplast of Chlamydomonas reinhardtii 
The Journal of Cell Biology  2008;182(4):641-646.
Eukaryotic cells under stress repress translation and localize these messenger RNAs (mRNAs) to cytoplasmic RNA granules. We show that specific stress stimuli induce the assembly of RNA granules in an organelle with bacterial ancestry, the chloroplast of Chlamydomonas reinhardtii. These chloroplast stress granules (cpSGs) form during oxidative stress and disassemble during recovery from stress. Like mammalian stress granules, cpSGs contain poly(A)-binding protein and the small, but not the large, ribosomal subunit. In addition, mRNAs are in continuous flux between polysomes and cpSGs during stress. Localization of cpSGs within the pyrenoid reveals that this chloroplast compartment functions in this stress response. The large subunit of ribulosebisphosphate carboxylase/oxygenase also assembles into cpSGs and is known to bind mRNAs during oxidative stress, raising the possibility that it plays a role in cpSG assembly. This discovery within such an organelle suggests that mRNA localization to granules during stress is a more general phenomenon than currently realized.
PMCID: PMC2518703  PMID: 18710928
23.  Three-dimensional Organization of Basal Bodies from Wild-Type and δ-Tubulin Deletion Strains of Chlamydomonas reinhardtiiV⃞ 
Molecular Biology of the Cell  2003;14(7):2999-3012.
Improved methods of specimen preparation and dual-axis electron tomography have been used to study the structure and organization of basal bodies in the unicellular alga Chlamydomonas reinhardtii. Novel structures have been found in both wild type and strains with mutations that affect specific tubulin isoforms. Previous studies have shown that strains lacking δ-tubulin fail to assemble the C-tubule of the basal body. Tomographic reconstructions of basal bodies from the δ-tubulin deletion mutant uni3-1 have confirmed that basal bodies contain mostly doublet microtubules. Our methods now show that the stellate fibers, which are present only in the transition zone of wild-type cells, repeat within the core of uni3-1 basal bodies. The distal striated fiber is incomplete in this mutant, rootlet microtubules can be misplaced, and multiflagellate cells have been observed. A suppressor of uni3-1, designated tua2-6, contains a mutation in α-tubulin. tua2-6; uni3-1 cells build both flagella, yet they retain defects in basal body structure and in rootlet microtubule positioning. These data suggest that the presence of specific tubulin isoforms in Chlamydomonas directly affects the assembly and function of both basal bodies and basal body-associated structures.
PMCID: PMC165693  PMID: 12857881
24.  Chloroplast to chromoplast transition in tomato fruit: spectral confocal microscopy analyses of carotenoids and chlorophylls in isolated plastids and time-lapse recording on intact live tissue 
Annals of Botany  2011;108(2):291-297.
Background and Aims
There are several studies suggesting that tomato (Solanum lycopersicum) chromoplasts arise from chloroplasts, but there is still no report showing the fluorescence of both chlorophylls and carotenoids in an intermediate plastid, and no video showing this transition phase.
Pigment fluorescence within individual plastids, isolated from tomato fruit using sucrose gradients, was observed at different ripening stages, and an in situ real-time recording of pigment fluorescence was performed on live tomato fruit slices.
Key results
At the mature green and red stages, homogenous fractions of chloroplasts and chromoplasts were obtained, respectively. At the breaker stage, spectral confocal microscopy showed that intermediate plastids contained both chlorophylls and carotenoids. Furthermore, an in situ real-time recording (a) showed that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell; and (b) confirmed that all chromoplasts derived from pre-existing chloroplasts.
These results give details of the early steps of tomato chromoplast biogenesis from chloroplasts, with the formation of intermediate plastids containing both carotenoids and chlorophylls. They provide information at the sub-cellular level on the synchronism of plastid transition and pigment changes.
PMCID: PMC3143050  PMID: 21788376
Chloroplast; chromoplast; confocal; pigment fluorescence; Solanum lycopersicum; tomato
25.  Phytochrome-dependent coordinate control of distinct aspects of nuclear and plastid gene expression during anterograde signaling and photomorphogenesis 
Light perception by photoreceptors impacts plastid transcription, development, and differentiation. This photoreceptor-dependent activity suggests a mechanism for photoregulation of gene expression in the nucleus and plastid that serves to coordinate expression of critical genes of these two organelles. This coordinate expression is required for proper stoichiometric accumulation of components needed for assembly of plastids, photosynthetic light-harvesting complexes and components such as phytochromes. Chloroplast-targeted sigma factors, which function together with the plastid-encoded RNA polymerase to regulate expression of plastid-encoded genes, and nuclear-encoded plastid development factors, such as GLK1 and GLK2, are targets of phytochrome regulation. Such phytochrome-dependent functions are hypothesized to allow light-dependent regulation, and feasibly tuning, of plastid components and function in response to changes in the external environment, which directly affects photosynthesis and the potential for light-induced damage. When the size and protein composition of the light-harvesting complexes are not tuned to the external environment, imbalances in electron transport can impact the cellular redox state and cause cellular damage. We show that phytochromes specifically regulate the expression of multiple factors that function to modulate plastid transcription and, thus, provide a paradigm for coordinate expression of the nuclear and plastid genomes in response to changes in external light conditions. As phytochromes respond to changes in the prevalent wavelengths of light and light intensity, we propose that specific phytochrome-dependent molecular mechanisms are used during light-dependent signaling between the nucleus and chloroplast during photomorphogenesis to coordinate chloroplast development with plant developmental stage and the external environment.
PMCID: PMC4012200  PMID: 24817873
anterograde signaling; light signaling; nuclear gene expression; plastid gene expression; phytochrome; sigma factor

Results 1-25 (614088)