Ultrastructural organization and composition of carotenoids of the eyespot in Chlamydomonas reinhardtii mutants

Biogenesis of the ultrastructure of the eyespot in chloroplasts of unicellular green alga Chlamydomonas reinhardtii has been studied. It was established that development of the eyespot structure correlates with the accumulation of carotenoids. Due to their accumulation, the eyespot forms from one to four layers of lipid-carotenoid globules. It has been shown that, in eyespot globules, only carotenes are accumulated. It was found for the first time that, in mutants, the carotene composition in the eyespot may be changed due to changes of their composition in chloroplast membranes.     

Development of ultrastructure and experimental change in carotene composition of the eyespot in Chlamydomonas reinhardtii mutants

Biogenesis of the ultrastructure of the eyespot in chloroplasts of a unicellular green alga Chlamydomonas reinhardtii has been studied. It has been ascertained that the development of eyespot structure correlates with the accumulation of carotenoids. Owing to their accumulation, the eyespot may have one to four layers of lipid/carotenoid globuli. It has been shown that only carotenes are accumulated in the eyespot globuli. It has been found for the first time that in mutants, the composition of carotenes in the eyespot may change together with the changes of their composition in chloroplast membranes.     

Subfractionation of eyespot apparatuses from the green alga Spermatozopsis similis: isolation and characterization of eyespot globules

Despite the well-characterized function of the green-algal eyespot apparatus as a combined absorption/reflection screen for the photoreceptor for phototaxis, little is known about the proteins involved in the formation of this complex organelle. We therefore purified the carotenoid-rich lipid globules, which are the most conspicuous component of the eyespot sensu strictu from Spermatozopsis similis Preisig et Melkonian. Electron microscopy and an average carotenoid:chlorophyll ratio of 51, confirmed the high purity of the fraction. The diameter of isolated globules (approx. 112 nm) fell within their in vivo range (90–120 nm). Absorption spectra in aqueous media peaked at 535 nm. The predominant carotenoids were β,ψ-, β, β- and δ-carotene. Freeze-fracture studies with cells and whole-mount electron microscopy of isolated globules demonstrated regularly arranged particles at the globule surface. Sodium dodecyl sulfate–polyacrylamide gel electrophresis revealed specific enrichment of 10 tightly bound major proteins and several minor proteins with the globules. Proteases were used to analyze their topology and function. Upon treatment with thermolysin, globules were released from a fraction enriched in isolated eyespot apparatuses. Major proteins of these globules, and those treated with thermolysin after isolation, were identical. However, the purified proteins were sensitive to thermolysin, indicating that domains of them are normally hidden in the globule matrix. In contrast, pronase degraded all globule-associated proteins in situ. These globules were not stable and easily fused, whereas thermolysin-treated globules were relatively stable. Lipase did not affect globule stability. These results indicate that the five thermolysin-resistant proteins (apparent M _r values: 56, 52, 32, 29, 27 kDa) are close to the surface and might be crucial for globule stabilization, whereas the thermolysin-accessible proteins are probably involved in globule/globule interactions and/or globule/eyespot-membrane interactions. Received: 19 June 2000 / Accepted: 4 October 2000     

Lipid globules on the plastid surface in Iris tepal epidermis cells during tepal maturation and senescence

Epidermis cells in the outer tepals of Iris flowers (Iris × hollandica, cv. Blue Magic) start programmed cell death (PCD) prior to floral opening. The tepals show visible senescence symptoms three days after full opening. Visible senescence coincides with collapse (death) of the upper epidermis cells. In these cells, electron-dense particles (plastoglobuli), membranes, and oil bodies were observed in the plastid interior. Electron-dense globules similar to plastoglobuli, thus apparently mainly consisting of lipids, were found on the plastid surface, from before flower opening until cell death. Such electron-dense globules were also present in the cytosol. The size of some of the globules on the plastid surface increased with time. The globules are likely involved in transfer of lipidic/proteinaceous material from the plastid to the cytosol. As the plastids contained ample oil bodies, up to the time of cell death, cell death was likely not due to lack of reserves. Mitochondrial ultrastructure also remained the same until cell death. The role of mitochondria in PCD is discussed.     

Eyespot fine structure in Eudorina illinoiensis

Electron microscopy of the eyespot of the coenobic yolvocacean Eudorina illinoiensis (Kofoid) Pascher shows that besides the well-documented anterior-posterior decrease in size, congruity of structure is involved in eyespot differentiation.

In the anterior cells the eyespot lies adjacent to the plasmalemma with the thylakoid underlayers parallel to it, while in the posterior cells eyespot placement and orientation may vary considerably. Deep-sited eyespots of congruent organisation probably represent the mother-cell eyespot; discongruent ones are typical of posterior cells, but the smallest ones may be random associations of osmiophilic globules.

Eyespot structure is discussed in relation to photoreception and phototaxis.     

A simulation study of mutations in the genetic regulatory hierarchy for butterfly eyespot focus determination

The color patterns on the wings of butterflies have been an important model system in evolutionary developmental biology. A recent computational model tested genetic regulatory hierarchies hypothesized to underlie the formation of butterfly eyespot foci [Evans, T.M., Marcus, J.M., 2006. A simulation study of the genetic regulatory hierarchy for butterfly eyespot focus determination. Evol. Dev. 8, 273-283]. The computational model demonstrated that one proposed hierarchy was incapable of reproducing the known patterns of gene expression associated with eyespot focus determination in wild-type butterflies, but that two slightly modified alternative hierarchies were capable of reproducing all of the known gene expressions patterns. Here we extend the computational models previously implemented in Delphi 2.0 to two mutants derived from the squinting bush brown butterfly (Bicyclus anynana). These two mutants, comet and Cyclops, have aberrantly shaped eyespot foci that are produced by different mechanisms. The comet mutation appears to produce a modified interaction between the wing margin and the eyespot focus that results in a series of comet-shaped eyespot foci. The Cyclops mutation causes the failure of wing vein formation between two adjacent wing-cells and the fusion of two adjacent eyespot foci to form a single large elongated focus in their place. The computational approach to modeling pattern formation in these mutants allows us to make predictions about patterns of gene expression, which are largely unstudied in butterfly mutants. It also suggests a critical experiment that will allow us to distinguish between two hypothesized genetic regulatory hierarchies that may underlie all butterfly eyespot foci.     

Eyespot behavior during the fertilization of gametes in Ulva arasakii Chihara (Ulvophyceae, Chlorophyta)

Behavior of the eyespots during the fertilization of Ulva arasakii Chihara was studied using field emission scanning electron microscopy (FE‐SEM). FE‐SEM enabled the visualization of the eyespot of biflagellate male and female gametes. The smaller male gamete has one protruded smaller (1.3 ± 0.15 μm× 1.0 ± 0.29 μm) eyespot and the larger female gamete has a larger (1.6 ± 0.2 μm× 1.1 ± 0.13 μm) one on a posterior position of the cell. The cell membrane over the eyespot region is relatively smooth compared to other parts of the cell body and exhibits hexagonal arranged lipid globules. Because the size of the cell and the morphology of the eyespot are different between male and female gametes, we could follow the fate of the eyespots during the fertilization. The initial cytoplasmic contact and fusion of the gametes takes place at their anterior end, slightly posterior to the flagellar base. The morphology of the fusing gametes followed two clearly distinguishable patterns. About half the gamete pairs lie side‐by‐side with their longitudinal axes nearly parallel, while the rest are oriented anti‐parallel to each other. In all cases, the larger female gamete fused along the same side as the eyespot, while the smaller male gamete fused along the side away from its eyespot. As fusion proceeds, the gamete pair is transformed into the quadriflagellate planozygote, in which the eyespots are positioned side‐by‐side on the region of cell fusion. These observations indicated that the opposite positioning of the eyespot relative to the cell fusion site in male and female gametes is important for the proper arrangement of the eyespots in the planozygote. The significance of this feature in advanced green algae is briefly discussed.     

Hox genes are essential for the development of eyespots in Bicyclus anynana butterflies

The eyespot patterns found on the wings of nymphalid butterflies are novel traits that originated first in hindwings and subsequently in forewings, suggesting that eyespot development might be dependent on Hox genes. Hindwings differ from forewings in the expression of Ultrabithorax (Ubx), but the function of this Hox gene in eyespot development as well as that of another Hox gene Antennapedia (Antp), expressed specifically in eyespots centers on both wings, are still unclear. We used CRISPR-Cas9 to target both genes in Bicyclus anynana butterflies. We show that Antp is essential for eyespot development on the forewings and for the differentiation of white centers and larger eyespots on hindwings, whereas Ubx is essential not only for the development of at least some hindwing eyespots but also for repressing the size of other eyespots. Additionally, Antp is essential for the development of silver scales in male wings. In summary, Antp and Ubx, in addition to their conserved roles in modifying serially homologous segments along the anterior–posterior axis of insects, have acquired a novel role in promoting the development of a new set of serial homologs, the eyespot patterns, in both forewings (Antp) and hindwings (Antp and Ubx) of B. anynana butterflies. We propose that the peculiar pattern of eyespot origins on hindwings first, followed by forewings, could be due to an initial co-option of Ubx into eyespot development followed by a later, partially redundant, co-option of Antp into the same network.     

Carotenoids in the eyespot apparatus are required for triggering phototaxis in Euglena gracilis

Carotenoids are the most universal and most widespread pigments in nature. They have played pivotal roles in the evolution of photosensing mechanisms in microbes and of vision in animals. Several groups of phytoflagellates developed a photoreceptive organelle called the eyespot apparatus (EA) consisting of two separable components: the eyespot, a cluster of carotenoid‐rich globules that acts as a reflector device, and actual photoreceptors for photobehaviors. Unlike other algal eyespots, the eyespot of Euglenophyta lacks reflective properties and is generally considered to act as a shading device for the photoreceptor (paraflagellar body, PFB) for major photomovements. However, the function of the eyespot of Euglenophyta has not yet been fully proven. Here, we report that the blocking carotenoid biosynthesis in Euglena gracilis by suppressing the phytoene synthase gene (crtB) caused a defect in eyespot function resulting in a loss of phototaxis. Raman spectroscopy and transmission electron microscopy suggested that EgcrtB‐suppressed cells formed eyespot globules but had a defect in the accumulation of carotenoids in those packets. Motion analysis revealed the loss of phototaxis in EgcrtB‐suppressed cells: a defect in the initiation of turning movements immediately after a change in light direction, rather than a defect in the termination of cell turning at the appropriate position due to a loss of the shading effect on the PFB. This study revealed that carotenoids are essential for light perception by the EA for the initiation of phototactic movement by E. gracilis, suggesting one possible photosensory role of carotenoids in the EA for the phototaxis.     

Ultrastructure of the Harmful Unarmored Dinoflagellate Cochlodinium polykrikoides (Dinophyceae) with Reference to the Apical Groove and Flagellar Apparatus

ABSTRACT. The external and internal ultrastructure of the harmful unarmored dinoflagellate Cochlodinium polykrikoides Margalef has been examined with special reference to the apical groove and three‐dimensional structure of the flagellar apparatus. The apical groove is U‐shaped and connected to the anterior sulcal extension on the dorsal side of the epicone. The eyespot is located dorsally and composed of two layers of globules situated within the chloroplast. A narrow invagination of the plasma membrane is associated with the eyespot. The nuclear envelope has normal nuclear pores similar to other eukaryotes but different from the Gymnodinium group with diagnostic nuclear chambers. The longitudinal and transverse basal bodies are separated by approximately 0.5–1.0 μm and interconnected directly by a striated basal body connective and indirectly by microtubular and fibrous structures. Characteristic features of the flagellar apparatus are as follows: (1) a nuclear extension projects to the R1 (longitudinal microtubular root) and is connected to the root by thin fibrous material; (2) fibrillar structures are associated with the longitudinal and transverse flagellar canal; and (3) a striated ventral connective extends toward the posterior end of the cell along the longitudinal flagellar canal. We conclude, based on both morphological and molecular evidence, that Cochlodinium is only distantly related to Gymnodinium.     

BUTTERFLY EYESPOTS: THE GENETICS AND DEVELOPMENT OF THE COLOR RINGS

The butterfly Bicyclus anynana has a series of distal eyespots on its wings. Each eyespot is composed of a white pupil, a black disc, and a gold outer ring. We applied artificial selection to the large dorsal eyespot on the forewing to produce a line with the gold ring reduced or absent (BLACK) and another line with a reduced black disc and a broad gold ring (GOLD). High heritabilities, coupled with a rapid response to selection, produced two lines of butterflies with very different phenotypes. Other eyespots showed a correlated change in the proportion of their color rings. Surgical experiments were performed on pupal wings from the different lines at the time of eyespot pattern specification. They showed that the additive genetic variance for this trait was in the response of the wing epidermis to signaling from the organizing cells at the eyespot center (the focus). This response was found to vary across different regions of the wing and also between the sexes. The particular eyespot color composition found for each sex, as well as the maintenance of the high genetic variation, are discussed with reference to the ecology of the butterfly, sexual selection, and visual selection by predators.     

Ultrastructure of the gametes ofCoelomomyces dodgei Couch (Blastocladiales,Chytridiomycetes)

Summary As part of an investigation on the developmental biology ofCoelomomyces dodgei Couch (Blastocladiales, Chytridiomycetes), the ultrastructure of the male and female gametes was studied. The nucleus is central and conical in shape except for a basal spur that curves back towards the large plate-like mitochondrion. A nuclear cap of ribosomes sits on the flat anterior end of the nucleus. Approximately seven lipid globules are partially embedded in the mitochondrion and are interconnected by membrane cisternae. The lipid globules are covered by a single fenestrated microbody and a backing membrane lies between the microbody and the gamete plasma membrane. The kinetosome is at the base of the nucleus and is connected to a single, posterior, whiplash flagellum. A nonkinetosomal centriole is absent. In the peripheral cytoplasm of both mating types there is a paracrystalline body of unknown composition and function. No significant ultrastructural differences were found between the male and female gametes.     

不同发育时期哲罗鱼卵黄的超微结构

应用透射电镜观察了不同发育时期哲罗鱼(Hucho taimen)卵黄的超微结构.根据哲罗鱼卵黄物质在卵母细胞中的加工合成、积累以及卵母细胞中参与卵黄颗粒形成的细胞器的变化,可将该鱼卵黄发生分为4个特征时期,即卵黄发生前期、卵黄泡期、卵黄积累期和卵黄积累完成期.卵黄发生前期是指卵母细胞发育过程中的卵黄物质开始积累前的时期,此时期核仁不断分裂,出现线粒体云和早期的滤泡细胞层、基层和鞘细胞层;卵黄泡期特点主要是细胞器不断变化产生卵黄泡和皮层泡;卵黄积累期的滤泡膜由内向外依次为放射带、颗粒细胞层、基层和鞘细胞层,此时外源性卵黄前体物质不断经过血液汇集于鞘细胞层,后经微胞饮作用穿过胶原纤维组成的基层,经过多泡体作用转运至颗粒细胞内,在细胞内经过加工和修饰形成小的卵黄蛋白颗粒,卵黄蛋白颗粒经微胞饮穿过放射带进入卵母细胞边缘形成的空泡中,不断积累形成卵黄球;进入卵黄积累完成期,卵黄球体积变大,向细胞中心聚集,填满大部分卵母细胞,卵黄积累完毕.     

Phototactic responses of four marine dinoflagellates with different types of eyespot and chloroplast

Great structural variety is seen in the eyespot of dinoflagellates, a structure involved in phototaxis. Although there are several works on the phototactic responses in some species of dinoflagellates, none of the dinoflagellates used in these studies possessed an eyespot and, therefore, we have no knowledge of the relationship between eyespot type and phototactic response. In this study, we determined wavelength dependency curves for phototaxis in four marine dinoflagellates that possess a different type of either eyespot or chloroplast. These include: (i) a dinoflagellate possessing a peridinin-containing ohioroplast with an eyespot (Scrippsiella hexapraecingula Horiguchi et Chihara); (ii) a dinoflagellate containing a diatom endosymbiont and with the type B eyespot sensu Dodge (1984; (Peridinium foli-aceum (Stein) Biecheler); (iii) a dinoflagellate with peri-dinin-containing chloroplasts, but lacking an eyespot (Atexandrium hiranoi Kita et Fukuyo); and (iv) a dinoflagellate with fucoxanthin, 19′-hexanoyloxyfucoxanthin and 19′-butanoyloxyfucoxanthin, but lacking an eyespot (Gymnodinium mikimotoi Miyabe et Kominami ex Oda), Regardless of the eyespot or the chloroplast type, all four dinoflagellates showed similar wavelength dependency curves for phototaxis, with sensitivity between 380 and 520 nm, the highest peak at approximately 440 or 460 nm and smaller peaks or shoulders at 400–420 nm and 480–500 nm. Substantial peaks have also been noted in the ultraviolet range (260–280 nm). The ultrastructural study of the eye-spot of Scrippsiella hexapraecingula revealed that the eyespot consists of two layers of lipid globules and probably acts as a quarter-wave stack antenna.     

Ultrastructure,composition of neutral lipids and their fatty acids of Candida tropicalis strain D-2 mutants resistant to the polyene antibiotic nystatin

This report deals with data on the cell ultrastructure of Candida tropicals strain D-2 mutants resistant to the polyene antibiotic, nystatin, and with an analysis of the fractional composition of neutral lipids and their fatty acids. The ultrastructural organization of the mutant cells is characterized by thickening of the cell wall and formation of invaginations into the cytoplasm, the appearance of new formations, large vacuoles, and reduction of the system of mitochondrial cristae. Lipids of nys^r mutants differ from those of the nys^s variant in having a decreased content of steroids and some fractions of neutral lipids. Certain nys^r mutants manifest difference in the relative amounts of saturated and unsaturated fatty acids (C_16:0, C_16:1, C_18:0, C_18:1).     

Characterization of the Eyespot Regions of "Blind"Chlamydomonas Mutants after Restoration of Photophobic Responses

Chlamydomonas reinhardtii exhibits photophobic and positive and negative phototactic responses that can be defined for cell populations using computerized cell tracking and motion analysis. Mutants CC-2359 and FN68 are pigment deficient mutants that are blocked in carotenoid synthesis and lack these photo responses. In particular, neither mutant exhibits flash-induced photophobic responses to visible light stimuli to which wild-type gametic cells exhibit a strong response, with several behavioral stages. Upon addition of all-trans retinal to these mutants, the photophobic responses are restored with minor quantitative differences from wild-type populations. Using both light and electron microscopy, we have compared the ultrastructural characteristics of wild-type C. reinhardtii to those of both mutants. As previously described, wild-type cells contain an eyespot consisting of 2–4 layers of pigmented granules encased within thylakoid membranes, located between the distal extremities of the flagellar root. This structure is also visible as an orange-red spot in light microscopy. The photoreceptor is thought to be concentrated in the plasma membrane above the eyespot. The mutant, CC-2359, lacks this eyespot as seen by both light and electron microscopy, even when the photophobic response has been restored. FN68-like mutants studied earlier by Morel-Laurens and Feinlieb and others contain an eyespot which can be seen only by electron microscopy. In FN-68, the eyespot generally has the same dimensions as in wt cells, differing mainly in pigment granule appearance. Consistent with these findings, several laboratories have shown that the full range of phototactic responses can be reconstituted in FN68 and CC-2359, but that negative phototaxis requires a significantly stronger light stimulus in the latter strain. We confirm the suggestion that the eyespot is not necessary for the photophobic response, and is not critical for the appropriate assembly and function of the photophobic response receptor in the membrane. Furthermore, the locus of reconstitution of the functional receptor is not the eyespot. Because of the definitive demonstration of the absence of the eyespot in CC-2359, however, the eyespot may play a role in negative phototaxis.     

Eyespot-assembly mutants in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.     

The comparative ultrastructure and possible function of eyespots: Euglena granulata and Chlamydomonas eugametos

Summary The structure of Euglena granulata and Chlamydomonas eugametos has been studied using polarization and electron microscopy, cinematography, and chemical extraction procedures, with the main focus on the structure of the eyespot.The 50–60 granules which form the extrachloroplastic eyespot of E. granulata are large bodies, up to 1200 m in diameter. They are found in the cytoplasm near the base of the reservoir and are associated with the parabasal body which contains a large crystal. The eyespot granules are contained within membranes having a unit membrane structure; 2 or 3 are usually present in a single eyespot packet; microtubules are also contained within the packet. The eyespot granules have the structure of a positive spherite and clearly exhibit birefringence; this structure is modified by fixation.The granules of the chloroplastic eyespot of C. eugametos are about 75 m in diameter and are contained within the chloroplast in an ordered array. Occasionally, the eyespot contains elongate or helical bodies mixed with the granules. Extraction with organic solvents caused the removal of materials which formed the eyespot granules as well as that of the osmophilic globules in the chloroplasts.Several hypotheses which concern the function of the eyespots in these and other species are discussed in the light of our results. the possible origin and demise of the eyespot granules are also discussed.Supported in part by NSF Grant GB-313. We thank Dr. Harold C. Bold and Dr. W. Gordon Whaley for their support and encouragement, Dr. R.M. Brown, jr. and Dr. Tom Kantz for aid in cinephotography, Dr. Peter Sitte for his help with polarization microscopy, and Mrs. Virginia Stork for her excellent technical assistance.A preliminary report of this research was presented at the Annual Meeting, Phycological Society of America, American Institute of Biological Sciences, College Park, Maryland, August, 1966.Contribution No. 286 from the Department of Botany, The University of Tennessee, Knoxville.     

Dactylodinium pterobelotum gen. et sp. nov., a new marine woloszynskioid dinoflagellate positioned between the two families Borghiellaceae and Suessiaceae

A new marine woloszynskioid dinoflagellate Dactylodinium pterobelotum gen. et sp. nov., collected from a southern Vietnamese estuary, was described on the basis of LM, SEM, and TEM, and molecular phylogeny inferred from rDNA sequences. This species had the smallest number of amphiesmal vesicles (5 latitudinal series) in woloszynskioid dinoflagellates assigned to the Suessiaceae and Borghiellaceae. The eyespot was of type B, composed of osmiophilic globules and brick‐like material, located in‐ and outside of the chloroplast respectively. An apical structure comprised a pair of elongate anterior vesicles (PEV). A large peduncle was conspicuous, located in the sulcal extension in the epicone, and supported by a microtubular strand of ~140 microtubules. Ultrastructural features of trichocysts represent a novel type in the Dinophyceae, bearing lateral hairs besides anterior fibers. The molecular phylogeny based on partial LSU rDNA showed the species in a basal position in the family Suessiaceae; this indicates the eyespot type B and PEV of the Borghiellaceae are ancestral states of the eyespot comprising brick‐like material (type E) and an elongate apical vesicle of the Suessiaceae.     

Chemical Composition of Chlamydospores of Candida albicans

A variety of analytical techniques was employed to study the composition of the chlamydospore of Candida albicans. The outer, thin, electron-transparent layer was found to be composed of glucan, together with a small amount of chitin. The inner, thick, electron-dense layer is proteinaceous. The central structure is composed largely of ribonucleic acid and lipid globules. In addition to being acid-fast, the chlamydospore was found to contain glycolipids and to lose the property of acid-fastness on extraction with ethanol-ether.