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1.  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
2.  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
3.  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
4.  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
5.  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
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 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
8.  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
9.  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 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
11.  Lag, lock, sync, slip: the many ‘phases’ of coupled flagella 
In a multitude of life's processes, cilia and flagella are found indispensable. Recently, the biflagellated chlorophyte alga Chlamydomonas has become a model organism for the study of ciliary motility and synchronization. Here, we use high-speed, high-resolution imaging of single pipette-held cells to quantify the rich dynamics exhibited by their flagella. Underlying this variability in behaviour are biological dissimilarities between the two flagella—termed cis and trans, with respect to a unique eyespot. With emphasis on the wild-type, we derive limit cycles and phase parametrizations for self-sustained flagellar oscillations from digitally tracked flagellar waveforms. Characterizing interflagellar phase synchrony via a simple model of coupled oscillators with noise, we find that during the canonical swimming breaststroke the cis flagellum is consistently phase-lagged relative to, while remaining robustly phase-locked with, the trans flagellum. Transient loss of synchrony, or phase slippage, may be triggered stochastically, in which the trans flagellum transitions to a second mode of beating with attenuated beat envelope and increased frequency. Further, exploiting this alga's ability for flagellar regeneration, we mechanically induced removal of one or the other flagellum of the same cell to reveal a striking disparity between the beatings of the cis and trans flagella, in isolation. These results are evaluated in the context of the dynamic coordination of Chlamydomonas flagella.
PMCID: PMC3973360  PMID: 24573332
flagella; synchronization; Chlamydomonas
12.  Ultrastructure of Chlamydomonas eugametos palmelloids induced by chloroplatinic acid treatment. 
Journal of Bacteriology  1975;121(1):338-343.
Palmelloids induced in the unicellular green alga Chlamydomonas eugametos by chloroplatinic acid treatment have been studied electron microscopically. Thin-sectioned specimens revealed the multilayer nature of the cell walls after second division within the palmelloid. Although synchrony in cell division is lost, to a certain degree, within the palmelloid, the cells themselves appeared normal, and the presence of normal flagellar structure was confirmed. The presence of the eyespot was observed at the optical level as well as in the freeze-etched specimens. The above results support the hypothesis that the palmelloid condition of Chlamydomonas eugametos induced by chloroplatinic acid is due to an abnormality in cell wall formation rather than flagellar malfunction or loss.
PMCID: PMC285648  PMID: 1116991
13.  Female choice depends on size but not symmetry of dorsal eyespots in the butterfly Bicyclus anynana. 
The eyespots on the ventral wings of Bicyclus anynana butterflies are exposed when at rest and interact with predators. Those on the dorsal surface are not exposed in this way, and may be involved in courtship and mate choice. In this study, we examined whether the size and fluctuating asymmetry (FA) of dorsal eyespots are reliable signals of male quality. High developmental stability is considered to result in low FA, and to be associated with high quality. Individuals of high quality are predicted to produce sexually selected traits that are large and symmetrical, at a relatively low cost. In this study, we manipulated eyespot development to uncouple eyespot size and FA in order to examine their independent roles in signalling to the female. Individual females in cages were given the choice between two or three males differing in eyespot traits. The results indicate that although size per se of the eyespots is used as a signal, FA and wing size are not. We discuss the use of FA in studies of sexual selection and aspects of sexual selection on dorsal eyespot size.
PMCID: PMC1691026  PMID: 12065039
14.  Deflective and intimidating eyespots: a comparative study of eyespot size and position in Junonia butterflies 
Ecology and Evolution  2013;3(13):4518-4524.
Eyespots are conspicuous circular features found on the wings of several lepidopteran insects. Two prominent hypotheses have been put forth explaining their function in an antipredatory role. The deflection hypothesis posits that eyespots enhance survival in direct physical encounters with predators by deflecting attacks away from vital parts of the body, whereas the intimidation hypothesis posits that eyespots are advantageous by scaring away a potential predator before an attack. In the light of these two hypotheses, we investigated the evolution of eyespot size and its interaction with position and number within a phylogenetic context in a group of butterflies belonging to the genus Junonia. We found that larger eyespots tend to be found individually, rather than in serial dispositions. Larger size and conspicuousness make intimidating eyespots more effective, and thus, we suggest that our results support an intimidation function in some species of Junonia with solitary eyespots. Our results also show that smaller eyespots in Junonia are located closer to the wing margin, thus supporting predictions of the deflection hypothesis. The interplay between size, position, and arrangement of eyespots in relation to antipredation and possibly sexual selection, promises to be an interesting field of research in the future. Similarly, further comparative work on the evolution of absolute eyespot size in natural populations of other butterfly groups is needed.
PMCID: PMC3856750  PMID: 24340191
Butterflies; deflection; eyespots; intimidation; Junonia; Junonia almana
15.  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
16.  Predator-induced changes in the growth of eyes and false eyespots 
Scientific Reports  2013;3:2259.
The animal world is full of brilliant colours and striking patterns that serve to hide individuals or attract the attention of others. False eyespots are pervasive across a variety of animal taxa and are among nature's most conspicuous markings. Understanding the adaptive significance of eyespots has long fascinated evolutionary ecologists. Here we show for the first time that the size of eyespots is plastic and increases upon exposure to predators. Associated with the growth of eyespots there is a corresponding reduction in growth of eyes in juvenile Ambon damselfish, Pomacentrus amboinensis. These morphological changes likely direct attacks away from the head region. Exposure to predators also induced changes in prey behaviour and morphology. Such changes could prevent or deter attacks and increase burst speed, aiding in escape. Damselfish exposed to predators had drastically higher survival suffering only 10% mortality while controls suffered 60% mortality 72 h after release.
PMCID: PMC3722912  PMID: 23887772
17.  Kinesin-II Is Required for Flagellar Sensory Transduction during Fertilization in Chlamydomonas 
Molecular Biology of the Cell  2002;13(4):1417-1426.
The assembly and maintenance of eucaryotic flagella and cilia depend on the microtubule motor, kinesin-II. This plus end-directed motor carries intraflagellar transport particles from the base to the tip of the organelle, where structural components of the axoneme are assembled. Here we test the idea that kinesin-II also is essential for signal transduction. When mating-type plus (mt+) and mating-type minus (mt−) gametes of the unicellular green alga Chlamydomonas are mixed together, binding interactions between mt+ and mt− flagellar adhesion molecules, the agglutinins, initiate a signaling pathway that leads to increases in intracellular cAMP, gamete activation, and zygote formation. A critical question in Chlamydomonas fertilization has been how agglutinin interactions are coupled to increases in intracellular cAMP. Recently, fla10 gametes with a temperature-sensitive defect in FLA10 kinesin-II were found to not form zygotes at the restrictive temperature (32°C). We found that, although the rates and extents of flagellar adhesion in fla10 gametes at 32°C are indistinguishable from wild-type gametes, the cells do not undergo gamete activation. On the other hand, fla10 gametes at 32°C regulated agglutinin location and underwent gamete fusion when the cells were incubated in dibutyryl cAMP, indicating that their capacity to respond to the cAMP signal was intact. We show that the cellular defect in the fla10 gametes at 32°C is a failure to undergo increases in cAMP during flagella adhesion. Thus, in addition to being essential for assembly and maintenance of the structural components of flagella, kinesin-II/intraflagellar transport plays a role in sensory transduction in these organelles.
PMCID: PMC102279  PMID: 11950949
18.  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
19.  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
20.  Differences in the selection response of serially repeated color pattern characters: Standing variation, development, and evolution 
There is spectacular morphological diversity in nature but lineages typically display a limited range of phenotypes. Because developmental processes generate the phenotypic variation that fuels natural selection, they are a likely source of evolutionary biases, facilitating some changes and limiting others. Although shifts in developmental regulation are associated with morphological differences between taxa, it is unclear how underlying mechanisms affect the rate and direction of evolutionary change within populations under selection.
Here we focus on two ecologically relevant features of butterfly wing color patterns, eyespot size and color composition, which are similarly and strongly correlated across the serially repeated eyespots. Though these two characters show similar patterns of standing variation and covariation within a population, they differ in key features of their underlying development. We targeted pairs of eyespots with artificial selection for coordinated (concerted selection) versus independent (antagonistic selection) change in their color composition and size and compared evolutionary responses of the two color pattern characters.
The two characters respond to selection in strikingly different ways despite initially similar patterns of variation in all directions present in the starting population. Size (determined by local properties of a diffusing inductive signal) evolves flexibly in all selected directions. However, color composition (determined by a tissue-level response to the signal concentration gradient) evolves only in the direction of coordinated change. There was no independent evolutionary change in the color composition of two eyespots in response to antagonistic selection. Moreover, these differences in the directions of short-term evolutionary change in eyespot size and color composition within a single species are consistent with the observed wing pattern diversity in the genus.
Both characters respond rapidly to selection for coordinated change, but there are striking differences in their response to selection for antagonistic, independent change across eyespots. While many additional factors may contribute to both short- and long-term evolutionary response, we argue that the compartmentalization of developmental processes can influence the diversification of serial repeats such as butterfly eyespots, even under strong selection.
PMCID: PMC2322975  PMID: 18366752
21.  Marginal Eyespots on Butterfly Wings Deflect Bird Attacks Under Low Light Intensities with UV Wavelengths 
PLoS ONE  2010;5(5):e10798.
Predators preferentially attack vital body parts to avoid prey escape. Consequently, prey adaptations that make predators attack less crucial body parts are expected to evolve. Marginal eyespots on butterfly wings have long been thought to have this deflective, but hitherto undemonstrated function.
Methodology/Principal Findings
Here we report that a butterfly, Lopinga achine, with broad-spectrum reflective white scales in its marginal eyespot pupils deceives a generalist avian predator, the blue tit, to attack the marginal eyespots, but only under particular conditions—in our experiments, low light intensities with a prominent UV component. Under high light intensity conditions with a similar UV component, and at low light intensities without UV, blue tits directed attacks towards the butterfly head.
In nature, birds typically forage intensively at early dawn, when the light environment shifts to shorter wavelengths, and the contrast between the eyespot pupils and the background increases. Among butterflies, deflecting attacks is likely to be particularly important at dawn when low ambient temperatures make escape by flight impossible, and when insectivorous birds typically initiate another day's search for food. Our finding that the deflective function of eyespots is highly dependent on the ambient light environment helps explain why previous attempts have provided little support for the deflective role of marginal eyespots, and we hypothesize that the mechanism that we have discovered in our experiments in a laboratory setting may function also in nature when birds forage on resting butterflies under low light intensities.
PMCID: PMC2875403  PMID: 20520736
22.  Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas 
The Journal of Cell Biology  1984;98(1):97-107.
When detergent-extracted, demembranated cell models of Chlamydomonas were resuspended in reactivation solutions containing less than 10(-8) M Ca++, many models initially swam in helical paths similar to those of intact cells; others swam in circles against the surface of the slide or coverslip. With increasing time after reactivation, fewer models swam in helices and more swam in circles. This transition from helical to circular swimming was the result of a progressive inactivation of one of the axonemes; in the extreme case, one axoneme was completely inactive whereas the other beat with a normal waveform. At these low Ca++ concentrations, the inactivated axoneme was the trans-axoneme (the one farthest from the eyespot) in 70-100% of the models. At 10(-7) or 10(-6) M Ca++, cell models also proceeded from helical to circular swimming as a result of inactivation of one of the axonemes; however, under these conditions the cis-axoneme was usually the one that was inactivated. At 10(-8) M Ca++, most cells continued helical swimming, indicating that both axonemes were remaining relatively active. The progressive, Ca++-dependent inactivation of the trans- or cis-axoneme was reversed by switching the cell models to higher or lower Ca++ concentrations, respectively. A similar reversible, selective inactivation of the trans-flagellum occurred in intact cells swimming in medium containing 0.5 mM EGTA and no added Ca++. The results show that there are functional differences between the two axonemes of Chlamydomonas. The differential responses of the axonemes to submicromolar concentrations of Ca++ may form the basis for phototactic turning.
PMCID: PMC2112995  PMID: 6707098
23.  The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions 
Merchant, Sabeeha S. | Prochnik, Simon E. | Vallon, Olivier | Harris, Elizabeth H. | Karpowicz, Steven J. | Witman, George B. | Terry, Astrid | Salamov, Asaf | Fritz-Laylin, Lillian K. | Maréchal-Drouard, Laurence | Marshall, Wallace F. | Qu, Liang-Hu | Nelson, David R. | Sanderfoot, Anton A. | Spalding, Martin H. | Kapitonov, Vladimir V. | Ren, Qinghu | Ferris, Patrick | Lindquist, Erika | Shapiro, Harris | Lucas, Susan M. | Grimwood, Jane | Schmutz, Jeremy | Cardol, Pierre | Cerutti, Heriberto | Chanfreau, Guillaume | Chen, Chun-Long | Cognat, Valérie | Croft, Martin T. | Dent, Rachel | Dutcher, Susan | Fernández, Emilio | Ferris, Patrick | Fukuzawa, Hideya | González-Ballester, David | González-Halphen, Diego | Hallmann, Armin | Hanikenne, Marc | Hippler, Michael | Inwood, William | Jabbari, Kamel | Kalanon, Ming | Kuras, Richard | Lefebvre, Paul A. | Lemaire, Stéphane D. | Lobanov, Alexey V. | Lohr, Martin | Manuell, Andrea | Meier, Iris | Mets, Laurens | Mittag, Maria | Mittelmeier, Telsa | Moroney, James V. | Moseley, Jeffrey | Napoli, Carolyn | Nedelcu, Aurora M. | Niyogi, Krishna | Novoselov, Sergey V. | Paulsen, Ian T. | Pazour, Greg | Purton, Saul | Ral, Jean-Philippe | Riaño-Pachón, Diego Mauricio | Riekhof, Wayne | Rymarquis, Linda | Schroda, Michael | Stern, David | Umen, James | Willows, Robert | Wilson, Nedra | Zimmer, Sara Lana | Allmer, Jens | Balk, Janneke | Bisova, Katerina | Chen, Chong-Jian | Elias, Marek | Gendler, Karla | Hauser, Charles | Lamb, Mary Rose | Ledford, Heidi | Long, Joanne C. | Minagawa, Jun | Page, M. Dudley | Pan, Junmin | Pootakham, Wirulda | Roje, Sanja | Rose, Annkatrin | Stahlberg, Eric | Terauchi, Aimee M. | Yang, Pinfen | Ball, Steven | Bowler, Chris | Dieckmann, Carol L. | Gladyshev, Vadim N. | Green, Pamela | Jorgensen, Richard | Mayfield, Stephen | Mueller-Roeber, Bernd | Rajamani, Sathish | Sayre, Richard T. | Brokstein, Peter | Dubchak, Inna | Goodstein, David | Hornick, Leila | Huang, Y. Wayne | Jhaveri, Jinal | Luo, Yigong | Martínez, Diego | Ngau, Wing Chi Abby | Otillar, Bobby | Poliakov, Alexander | Porter, Aaron | Szajkowski, Lukasz | Werner, Gregory | Zhou, Kemin | Grigoriev, Igor V. | Rokhsar, Daniel S. | Grossman, Arthur R.
Science (New York, N.Y.)  2007;318(5848):245-250.
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.
PMCID: PMC2875087  PMID: 17932292
24.  DNA elements regulating alpha1-tubulin gene induction during regeneration of eukaryotic flagella. 
Molecular and Cellular Biology  1997;17(7):3858-3866.
Eukaryotic flagella are complex organelles composed of more than 200 polypeptides. Little is known about the regulatory mechanisms governing synthesis of the flagellar protein subunits and their assembly into this complex organelle. The unicellular green alga Chlamydomonas reinhardtii is the premier experimental model system for studying such cellular processes. When acid shocked, C. reinhardtii excises its flagella, rapidly and coordinately activates transcription of a set of flagellar genes, and ultimately regenerates a new flagellar pair. To define functionally the regulatory sequences that govern induction of the set of genes after acid shock, we analyzed the alpha1-tubulin gene promoter. To simplify transcriptional analysis in vivo, we inserted the selectable marker gene ARG7 on the same plasmid with a tagged alpha1-tubulin gene and stably introduced it into C. reinhardtii cells. By deletion of various sequences, two promoter regions (-176 to -122 and -85 to -16) were identified as important for induction of the tagged alpha1-tubulin gene. Deleting the region between -176 and -122 from the transcription start site resulted in an induction level which was only 45 to 70% of that of the resident gene. Deleting the region upstream of -56 resulted in a complete loss of inducibility without affecting basal expression. The alpha1-tubulin promoter region from -85 to -16 conferred partial acid shock inducibility to an arylsulfatase (ARS) reporter gene. These results show that induction of the alpha1-tubulin gene after acid shock is a complex response that requires diverse sequence elements.
PMCID: PMC232238  PMID: 9199320
25.  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

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