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Coral reefs are the biodiversity hot spots of the oceans, but they have suffered from increasing environmental stresses caused principally by anthropogenic global warming. The keystone species of coral reefs are scleractinian corals, which maintain obligatory symbiotic relationships with photosynthetic dinoflagellates. Understanding cellular and molecular mechanisms of symbiosis is therefore essential for future preservation of coral reefs. To date, however, almost no in vitro experimental systems have been devised to illuminate such mechanisms. To this end, our previous study established stable in vitro cell culture lines, including IVB5, originating from planula larvae of the scleractinian coral, Acropora tenuis. Here, we show that soon after mixture with the dinoflagellate, Breviolum minutum, flattened amorphous coral cells with endodermal properties exhibited elevated locomotor activity using filopodia and lamellipodia and interacted with dinoflagellates. Several minutes thereafter, coral cells began to incorporate B. minutum, and in vitro symbiosis appeared to have been accomplished within 30 min. Nearly a half of the coral cells had incorporated algal cells within 24 h in a reproducible manner. Coral cells that harbored algal cells gradually became round and less mobile, and the algal cells sometimes settled in vacuole-like structures in coral cell cytoplasm. This symbiosis state was maintained for at least a month. The IVB5 line of A. tenuis therefore provides an experimental system to explore cellular and molecular mechanisms involved in coral-dinoflagellate symbiosis at the single-cell level, results of which may be useful for future preservation of coral reefs.

The cell wall of the tip‐growing cells of the giant‐cellular xanthophycean alga Vaucheria frigida is mainly composed of cellulose microfibrils (CMFs) arranged in random directions and the major matrix component into which the CMFs are embedded throughout the cell. The mechanical properties of a cell‐wall fragment isolated from the tip‐growing region, which was inflated by artificially applied pressure, were measured after enzymatic removal of the matrix component by using a protease; the results showed that the matrix component is involved in the maintenance of cell wall strength. Since glucose and uronic acid are present in the matrix component of Vaucheria cell walls, we measured the mechanical properties of the cell wall after treatment with endo‐1,3‐s‐glucanase and observed the fine structures of its surfaces by atomic force microscopy. The major matrix component was partially removed from the cell wall by glucanase, and the enzyme treatment significantly weakened the cell wall strength without affecting the pH dependence of cell wall extensibility. The enzymatic removal of the major matrix component by using a protease released polysaccharide containing glucose and glucuronic acid. This suggests that the major matrix component of the algal cell walls contains both proteins (or polypeptides) and polysaccharides consisting of glucose and glucuronic acid as the main constituents.

The cell walls of the giant-celled green alga Valonia exhibit the "crossed-fibril" arrangement of cellulose microfibrils (CMFs). The existence of fibrous matrix components coiling around the CMFs was recently reported using atomic force microscopy (AFM). To understand the biological role of this fibrous wall component, we attempted to reveal the detailed morphology of the fibrous structure of the cell walls isolated from V. utricularis specimens in artificial seawater (ASW), by using an improved method of AFM. We also investigated the effect of incubation in an acidic environment that had been known to make the cell walls liable to separate into layers, on the morphology of the fibrous structures. Thin, fibrous structures were found to entangle around a single CMF or multiple adjacent CMFs, the thickness of which was 0.85 nm on average. Incubation in acidic ASW (pH 4), greatly modified the morphology of the fibrous structures in quality and quantity, compared with those incubated in the original ASW (pH 8). Thickness of the fibrous structures was increased to 4.63 nm on average, remarkable deformations were observed, and the density of the structures was reduced to less than half by incubation in the acidic ASW. In addition, same fibrous structures extended over CMFs and forming aggregates were observed on the surface of the cell wall layers separated artificially. Fluorescence microscopy of cell walls treated with Vicia villosa lectin conjugated with fluorescein isothiocyanate showed specific labelling of cell wall regions where the fibrous structures were present abundantly as observed by AFM. From these observations, it can be assumed that the fibrous structures of V. utricularis are the matrix component of the cell wall containing N-acetylgalactosamine, involved in the maintenance of the cell wall integrity through bonding the neighboring cell wall layers, and their morphology and function of V. utricularis are sensitive to acidic pH.

The unicellular green alga Haematococcus pluvialis accumulates large amounts of the red ketocarotenoid astaxanthin to protect against environmental stresses. Haematococcus cells that accumulate astaxanthin in the central part (green-red cyst cells) respond rapidly to intense light by distributing astaxanthin diffusively to the peripheral part of the cell within 10 min after irradiation. This response is reversible: when astaxanthin-diffused cells were placed in the dark, astaxanthin was redistributed to the center of the cell. Although Haematococcus possesses several pigments other that astaxanthin, the subcellular distribution and content of each pigment remain unknown. Here, we analyzed the subcellular dynamics and localization of major pigments such as astaxanthin, β-carotene, lutein, and chlorophylls under light irradiation using time-lapse and label-free hyperspectral imaging analysis. Fluorescence microscopy and freeze-fracture transmission electron microscopy showed that, preceding/following exposure to light, astaxanthin colocalized with lipid droplets, which moved from the center to the periphery through pathways in a chloroplast. This study revealed that photoresponse dynamics differed between astaxanthin and other pigments (chlorophylls, lutein, and β-carotene), and that only astaxanthin freely migrates from the center to the periphery of the cell through a large, spherical, cytoplasm-encapsulating chloroplast as a lipid droplet. We consider this to be the Haematococcus light-protection mechanism.

The cells in the foliose thallus of trebouxiophycean alga Prasiola japonica apparently develop into 2 × 2 cell groups composed of two two-celled groups, each of which is a pair of derivative cells of the latest cell division. In the present study, the structural features of cell walls of the alga P. japonica concerning the formation of the cell groups were investigated using histochemical methods. Thin cell layers stained by Calcofluor White appeared to envelope the two-celled and four-celled groups separately and, hence, separated them from neighboring cell groups, and the Calcofluor White-negative gaps between neighboring four-celled groups were specifically stained by lectins, such as soybean agglutinin, jacalin, and Vicia villosa lectin conjugated with fluorescein. These results indicated that the Calcofluor White-positive cell wall layer of parent cell that existed during two successive cell divisions structurally distinguished two-celled and four-celled groups from others in this alga. Moreover, the results suggested that the cell wall components of the Calcofluor White-negative gaps would possibly contribute to the formation of the planar thallus through lateral union of the cell groups.

SUMMARY Monostroma angicava and Protomonostroma undulatum are monostromatic green benthic algae (Ulvophyceae), which grow together in the same intertidal habitat of Muroran, Hokkaido, Japan, during the spring season. Commonly, both species have a single chloroplast with one pyrenoid per cell. The parietal chloroplast is located on the periphery of the thallus in both species, although the location of the chloroplast differs in the two. In M. angicava, the chloroplast was observed to be arranged on one-side of the thallus surface, whereas, in P. undulatum, it was dispersed and randomly located on either side of the thallus or on the lateral face. The density of chlorophylls (Chls) assessed from the absorption spectra of the thallus and its solvent extract was higher in M. angicava, which appeared dark-green in color, than in the light-green colored P. undulatum. The maximum photosynthetic rate per thallus area (μmol O2 m−2 s−1) was higher in M. angicava, whereas, per total chlorophyll content (μmol O2 g Chl a + Chl b−1 s−1) was higher in P. undulatum. Both species showed similar efficiency of photosynthesis at light-limiting conditions. The efficiency of light absorption by photosystem II (PSII) in P. undulatum was higher than M. angicava, whereas the photoprotective response was higher in M. angicava. This indicates that more energy is utilized in M. angicava to protect its PSII due to the chloroplast position, which has more direct exposure to light and, therefore, lowers the efficiency of light absorption by PSII. The higher density of chlorophylls in M. angicava could explain higher photosynthesis per thallus area, whereas, higher efficiency of light absorption by PSII in P. undulatum could explain higher photosynthesis per total chlorophyll content. The differences in light absorption efficiency and quantum efficiency of PSII might be an important ecological strategy in these two species for their coexistence in the intertidal area.

SUMMARY The detailed segregative cell division (SCD) processes and changes in the arrangement of cortical microtubules and actin filaments were examined in two species of S truvea. SCD was initiated by the appearance of annular constrictions along the lateral side of a mother cell. The constrictions decreased in diameter, became thin, tubular in shape, and pinched the protoplasm of the mother cell into several protoplasmic sections. The protoplasmic sections expanded and developed into daughter cells, which appressed each other, and were arranged in a single row. Lateral branches protruded from the upper parts of the daughter cells. The protoplasm of the lateral branches was divided by secondary SCDs and was distributed amongst the new daughter cells. SCD and lateral branch formation were essential for morphogenesis in S truvea. Cortical microtubules were arranged parallel and longitudinally to the cell axis before SCD. When SCD was initiated, there was considerable undulation of the cortical microtubules and several transverse bundles appeared in the cytoplasmic zone where annular constrictions occurred. A microtubule-disrupting drug (amiprophos methyl) inhibited SCD. Actin filaments maintained reticulate patterns before and during SCD. These results demonstrated that SCD in Struvea species was quite distinct from that in D ictyosphaeria cavernosa reported previously.

Summary Because of their large sizes and simple shapes, giant-celled algae have been used to study how the structural and mechanical properties of cell walls influence cell growth. Here we review known relationships between cell wall and cell growth properties that are characteristic of three representative taxa of giant-celled algae, namely, Valonia ventricosa, internodal cells of characean algae, and Vaucheria frigida. Tip-growing cells of the genus Vaucheria differ from cells undergoing diffuse growth in V. ventricosa and characean algae in terms of their basic architectures (non-lamellate vs. multilamellate) and their dependence upon pH and Ca2+ for cell wall extensibility. To further understand the mechanisms controlling cell growth by cell walls, comparative analyses of cell wall structures and/or associated growth modes will be useful. The giant-celled algae potentially serve as good models for such investigations because of their wide variety of developmental processes and cell shapes exhibited.

Asexual bud development in the budding tunicate Polyandrocarpa misakiensis involves transdifferentiation of multipotent epithelial cells, which is triggered by retinoic acid (RA), and thrives under starvation after bud isolation from the parent. This study aimed to determine cell and molecular mechanisms of dedifferentiation that occur during the early stage of transdifferentiation. During dedifferentiation, the numbers of autophagosomes, lysosomes, and secondary lysosomes increased remarkably. Mitochondrial degradation and exosome discharge also occurred in the atrial epithelium. Autophagy-related gene 7 (Atg7) and lysosomal proton pump A gene (PumpA) were activated during the dedifferentiation stage. When target of rapamycin (TOR) inhibitor was administered to growing buds without isolating them from the parent, phagosomes and secondary lysosomes became prominent. TOR inhibitor induced Atg7 only in the presence of RA. In contrast, when growing buds were treated with RA, lysosomes, secondary lysosomes, and mitochondrial degradation were prematurely induced. RA significantly activated PumpA in a retinoid X receptor-dependent manner. Our results indicate that in P. misakiensis, TOR inhibition and RA signals act in synergy to accomplish cytoplasmic clearance for dedifferentiation.

We performed a molecular cytogenetic investigation of the scleractinian coral Acropora solitaryensis, which is dominant in the temperate region of Japan (30-35°N). Molecular cytogenetic analysis, using fluorescence in situ hybridization (FISH), was carried out for karyotyping and gene mapping. We propose the karyotype of this coral (2n = 30) based on C-banding and FISH analyses. FISH mapping of the rRNA gene was carried out with a probe generated by PCR amplification using rRNA gene primers. Furthermore, the telomeres and centromeres of all chromosomes were visualized using FISH. By comparative genomic hybridization using DNA from sperm and unfertilized eggs of this coral, we offer evidence suggesting the existence of sex chromosomes in this species. Collectively, these data advance our understanding of coral genetics.

Colonies of the Japanese red coral Corallium japonicum Kishinouye, 1903 collected off Cape Ashizuri, Japan were gonochoric and produced gonads in siphonozooids annually, mainly during the spring season. Polyp anatomy, gonadal morphology and gametogenesis in this species were revealed by light and electron microscopy. A siphonozooid had a pharynx with a prominent siphonoglyph and eight mesenteries: two sulcal, two asulcal, and four lateral. A rudimentary retractor was found on one side of each mesoglea of these mesenteries. The retractor arrangement in the siphonozooid was reverse of what was described in the autozooids of octocorals. Gonads initiated as small protrusions on the mesenteries, except in the asulcal ones, and even at an incipient stage they were covered with a sac-shaped thin layer of mesoglea, which was continuous with the mesoglea of mesenteries. Gastrodermis enveloped the complete outer surface of the thin layer of mesoglea throughout gametogenesis in both oocytes and sperm cysts. Oocytes produced many microvilli on their cortical surfaces beneath the thin layer of mesoglea concomitantly with the accumulation of lipid globules in the cells, whereas in sperm cysts spermatocytes and spermatids increased in number without microvilli production, followed by synchronous spermiogenesis involving remarkable changes in the shape and position of organelles. Based on the comparison of patterns in gonadal development between octocorals including C. japonicum, hexacorals and scyphozoans, octocoral and stauromedusa species may be characterized by the fact that gametogenesis never occurs in the matrix of mesoglea, but rather exclusively within the thin sac of mesoglea surrounded by gastrodermis.

The possible role of cortical microtubules in dinoflagellates was studied using high-pressure treatments applied to nonmotile cells (just after ecdysis) of Scrippsiella hexapraecingula T. Horig. et Chihara. Whereas considerable disorganization of cortical microtubules was observed when cells were exposed to high-pressure treatments of 98 MPa or more for 5-15 min, they were mostly intact in cells exposed to a pressure of98 MPa for 5 min. After nonmotile cells were exposed to high-pressure treatments sufficient to disorganize the cortical microtubules, they produced new motile cells with thecal plate patterns that differed considerably from the pattern known for this species. Increasing the intensity of high pressure applied to nonmotile cells resulted in an increase in the number of cells that exhibited disorganized cortical microtubules as well as a change in their thecal plate pattern, suggesting that high pressure disorganizes cortical microtubules leading to a change in the thecal plate pattern.

Elvira P.R., Sekida S. and Okuda K. 2012. Rhizoid formation in Valonia (Siphonocladales, Chlorophyceae). Phycologia 51: 391–402. DOI: 10.2216/11-31.1Rhizoids were artificially induced by the contac...

Cell differentiation and cell type commitment are an integral part of plant growth and development. Investigations on how environmental conditions affect the formation of shoots, roots, and rhizoids can help illustrate how plants determine cell fate and overall morphology. In this study, we evaluated the role of substratum and light on rhizoid differentiation in the coenocytic green alga, Valonia aegagropila. Elongating rhizoids displayed varying growth modes and cell shape upon exposure to different substrata and light conditions. It was found that soft substrata and dark incubation promoted rhizoid elongation via tip growth while subsequent exposure to light prevented tip growth and instead induced swelling in the apical region of rhizoids. Swelling was accompanied by the accumulation of protoplasm in the rhizoid tip through expansion of the cell wall and uninhibited cytoplasmic streaming. Subsequent diffuse growth led to the transformation from slender, rod-shaped rhizoids into spherical thallus-like structures that required photosynthesis. Further manipulation of light regimes caused vacillating cell growth redirections. An elongating V. aegagropila rhizoid cell thus appears capable of growth mode switching that is regulated by immediate environmental conditions thereby influencing ultimate cell shape and function. This is the first description of inducible, multiple growth mode shifts in a single intact plant cell that directly impact its differentiation.

The possible role of cortical microtubules in dinoflagellates was studied using high-pressure treatments applied to nonmotile cells (just after ecdysis) of Scrippsiella hexapraecingula T. Horig. et Chihara. Whereas considerable disorganization of cortical microtubules was observed when cells were exposed to high-pressure treatments of 98 MPa or more for 5-15 min, they were mostly intact in cells exposed to a pressure of

In uniseriate ceramiacean red algae, intercalary cells grow in a longitudinal direction by the formation of new cell surface in narrow, transverse bands of the cell. This localized growth mode is known as ‘band growth’. In this study, the distribution of vesicle exocytosis in the axial cells of the red alga Antithamnion nipponicum was investigated by transmission electron microscopy in specimens prepared by freeze-substitution. Two zones of band growth, which were detected as dark, horizontal bands after fluorescent cell wall staining followed by 24-h incubation in stain-free medium, were observed in the apical and basal regions of the cell. The width of the band was much greater in the basal region than in the apical region. Comparisons between the total elongation of the cells and the total width of the apical and basal bands during a 24-h incubation indicated that all of the cell elongation could be attributed to band growth. A short incubation in stain-free medium revealed that cell elongation in the ...

Mine I., Yoshimatsu K., Kobanba A., Sekida S. and Okuda K. 2011. Outflow movement during gamete discharge in Bryopsis plumosa (Caulerpales, Chlorophyta). Phycologia 50: 363–369. DOI: 10.2216/10-66.1 Light-induced gamete discharge in the caulerpalean alga Bryopsis plumosa was analyzed using video microscopy. Gametangia kept in darkness discharged gametes 7–8 min after they were exposed to light. The dimensions of gametangia continued to decrease until gamete discharge was initiated. When the apical papilla of a gametangium broke down to make a pore, gametes were explosively expelled from the gametangium through the pore. This was immediately followed by the moderate, continuous movement of the inner contents of the gametangium towards the pore, which we call outflow movement, resulting in the exhaustive evacuation of gametes from the gametangium. The velocity of outflow movement gradually decreased and continued for several tens of seconds. Outflow movement in the apical and middle portions of the...

The coenocyte of Bryopsis gametophytes is composed of a main axis and side branches lateral to the main axis, both of which grow by cell growth at a dome-like tip of cylindrical axis or branches. Regular development of side branches along the main axis formed pinnate thallus of Bryopsis gametophyte and, therefore, the formation of new growing tip is a key event in the thallus morphogenesis of the alga. Previous inhibitory experiments showed that the microtubule cytoskeleton is involved in cell growth and regular development of side branches. To clarify the role of microtubules in the formation of new growing tip further, we observed the microtubule arrangement during 2 different modes of growing tip formation—formation of side branches and regeneration of protoplasm aggregates in B. plumosa by immunofluorescence microscopy. A longitudinal, dense arrangement of cortical microtubules was observed in the cytoplasm of the main axes. These microtubules appeared to be interrupted in the region of side branch formation during its earliest stage. In the spherical cells regenerated from protoplast aggregates, a radial array of cortical microtubules was observed in the cell protrusion, which later became the growing tip of the regenerating cell. Thus, microtubules showed specific morphological changes in the region of growing tip formation in B. plumosa.

SUMMARY Brief treatment with hypertonic solutions induced gamete discharge from gametangia in the coenocytic green alga Bryopsis plumosa (Hudson) C. Agardh. It is known that gamete discharge in this alga is triggered by a change from darkness to light. However, in this study mature gametangia, incubated for 30–120 s in artificial seawater (ASW) with an additional 0.4–0.6 M NaCl and then transferred into pure ASW, discharged gametes in darkness. The treatment did not affect the motility of the gametes. Addition of sucrose (1.0–1.2 M) to ASW also induced gamete discharge in darkness. Similar results were obtained by adding KCl (0.4 M) or mannitol (1.2 M) to ASW. Continuous incubation of gametangia in such hypertonic solutions also induced gamete discharge but led to a delay and a reduction in the rate of gamete discharge, and a loss of gamete motility. In gametangia treated with the hypertonic solutions, as well as in those exposed to light, shortening of the gametangial length was observed before gamete discharge.

The coenocytic thallus of Bryopsis gametophytes is composed of a main axis and side branches, both of which have a dome-like growing tip with a clear zone of protoplasm and a subsequent cylindrical, basal cell region. Pinnate thallus is formed by regular development of side branches lateral to the main axis. It has been reported that both cytoplasmic streaming and tip growth in the alga are controlled by the microtubule cytoskeleton. We examined the physiology of the tip-growing region in B. plumosa by observing changes in cytoplasmic streaming during 2 different modes of growing tip formation—the germination of spherical cells regenerated from protoplasm aggregates and the formation of side branches. In the main axes, longitudinal cytoplasmic streaming, indicating chloroplast movement, was evaluated using time-lapse videomicroscopy. In the spherical cells regenerated from protoplast aggregates, chloroplasts showed active movement before and after their germination, but showed just small wriggling motion during germination. Chloroplast motility was unchanged during the early stages of side branch formation, during which a clear zone developed in cell surface protrusion. Thus cytoplasmic streaming appeared not to be involved in the growing tip formation in B. plumosa and hence microtubules may play a role in the growing-tip morphogenesis in this alga via mechanisms not directly related to the cytoplasmic streaming.

Study of the north Alaskan brown algal epiphyte Chukchia pedicellata sp. nov. suggests an apparently close relationship to Phaeostroma. Phaeostroma endophyticum S. Lund from east Greenland, Bylot Island, Nunavut, Canada, shows generic identity with Chukchia and specific differences from C. pedicellata. Comparison of C. pedicellata and P. pustulosum Kuck. (the type species of Phaeostroma) shows morphological, cytological, reproductive, and distributional differences. We recognize novelty of C. pedicellata and necessity to broaden the species description of P. endophyticum, which becomes Chukchia endophytica. C. endophytica is distinguished by its dimorphic vegetative habit, its large plurilocular sporangia, and the putative ability of the sporangia to digest host tissue to accommodate development within the host. Absence of pyrenoids and novel plurilocular sporangia in both species suggests that they are not members of the Ectocarpales, sensu lato.

In the tip-growing filamentous cell of the xanthophycean alga Vaucheria terrestris sensu Götz, a new growing tip develops in the non-growing, cylindrical region of the cell that was exposed by local illumination. The present study examined changes in the strength and extensibility of the cell wall of the new growing tip and in the matrix components of the inner surface of the cell wall. The internal pressure required to rupture the cell walls decreased remarkably during the early to middle stages of growing tip development, but the cell wall hardly extended before rupture. In contrast, during the middle and late stages of development, cell walls were extended by internal pressure. Atomic force microscopy revealed that protease-resistant, fine granular matrix components were present only at the apical portion of a normal growing tip, and were absent in the non-growing cylindrical region. In the early and middle stages of new growing tip development, these matrix components appeared in the cell walls in patches. These results suggest that first cell wall strength decreases and then cell wall extensibility increases in the development of new growing tips, and that protease-resistant, fine granular matrix components may be involved in rendering a cell wall extensible.

Karyotype analysis was performed on the scleractinian coral Coelastrea aspera Verrill, 1866, commonly found along temperate coasts in Japan (30–35°N) and in coastal waters in the Indian and Pacific oceans. G-banding of C. aspera was successfully performed, although the banding pattern was not as clear as that in mammals. The karyogram clearly revealed that this coral had a homogeneously staining region (hsr) in chromosome 11. This hsr consisted of ribosomal RNA (rRNA) related genes, which was demonstrated by fluorescence in situ hybridization (FISH) with probes generated using 28S ribosomal DNA (rDNA) primers and those generated through chromosome microdissection. In addition, we conducted silver-stained nucleolus organizer region (Ag-NOR) analysis and found Ag depositions in the interphase nuclei but not on rRNA gene loci and hsr(s) in the mitotic stage. The hsr of this coral was observed in approximately 50% of the metaphase spreads analyzed. This may explain the diversity of coral rDNA based on the molecular study of sequence analysis. Furthermore, it was discovered that human telomere and Alu repeated sequences were present in this C. aspera. Probes derived from human Alu sequences are expected to play an important role in the classification of corals. Overall, our data can be of great value in discriminating among scleractinian coral species and understanding their genetics, including chromosomal evolution.

Freeze fracture investigations have been carried out to investigate cellulose-synthesizing terminal complexes (TCs) in some chromophyte algae. The xanthophycean alga Botrydiopsis intercedens has TCs that consist of 3–8 diagonal rows of particles in the PF of the plasma membrane. A new type of TC was found in the phaeothamniophycean algae Phaeoschizochlamys mucosa, Phaeothamnion confervicola and Stichogloea doederleinii, where TC particles formed 2–3 linear rows. The TC of the eustigmatophycean alga Pseudocharaciopsis minuta was composed of a single linear row of particles and similar to that of phaeophycean algae so far examined. Each of the TCs found in the present study synthesized thin, ribbon-like cellulose microfibrils. A possible pathway of TC development in Botrydiopsis intercedens and TC evolution in the chromophytes are discussed.

The development of the pellicle and thecal plates of the dinoflagellate Scrippsiella hexapraecingula Horiguchi et Chihara was investigated with particular emphasis on cellulose synthesis. In young motile cells, incipient thecal plates appear as several groups of granular material within individual sites that are thought to be amphiesmal vesicles and develop into thin, sheet-like plates. These plates subsequently thicken with the deposition of amorphous material and cellulose microfibrils to form mature thecal plates. After ecdysis, non-motile cells form a pellicle consisting of three layers, L1-3. L1 is electron dense, while L2 and L3 are electron transparent. L1 and L2 are non-cellulosic, but L3 contains cellulose microfibrils. A new type of putative cellulose-synthesizing enzyme complex was found in non-motile cells. It consists of two rows of particles occurring on the plasmatic fracture face of the plasma membrane and begins to appear 0.5 – 1 h after ecdysis. The periodic acid-thiocarbohydrazide-silve...

A novel type of body scale has been found in two strains of unidentified species of the dinoflagellate genus Amphidinium. The body scales are distributed over the entire outer surface of the plasma membrane of the cells, and are embedded in an amorphous material. No scales are present on the flagellar membrane. The body scales are oval, uniform and simple, and lack a basal plate; they are similar in shape in the two species, but differ in size. Scale formation takes place within the Golgi cisternae. The mature scales are then transported to the vicinity of the flagellar canal in Golgi vesicles in the cytoplasm, where they are extruded out of the cell. These two species are morphologically indistinguishable from Amphidinium carterae except by the presence of scales, and this distinction is supported by base pair differences in the small subunit ribosomal RNA (SSU rRNA) gene (28–29 base alterations). We consider the two strains to be different species on the basis of differences in the size of the ...

We examined the dynamics of nuclear histone H3 trimethylation related to cell differentiation and aging in a budding tunicate, Polyandrocarpa misakiensis. Throughout zooidal life, multipotent epithelial and coelomic cell nuclei showed strong trimethylation signals at H3 lysine27 (H3K27me3), consistent with the results of western blotting. Epidermal H3K27me3 repeatedly appeared in protruding buds and disappeared in senescent adult zooids. The budding-specific cytostatic factor TC14-3 allowed aging epidermal cells to restore H3K27me3 signals and mitochondrial gene activities via mitochondrial transcription factor a, all of which were made ineffective by an H3K27me3 inhibitor. Chromatin immunoprecipitation showed that TC14-3 enhances H3K27me3 of transdifferentiation-related genes and consequently downregulates the expression of these genes. In contrast, trimethylation signals at H3 lysine4 (H3K4me3) appeared transiently in transdifferentiating bud cells and stably lasted in undifferentiated adult cells without affecting H3K27me3. A transdifferentiation-related gene external signal-regulated kinase heavily underwent H3K4me3 in developing buds, which could be reproduced by retinoic acid. These results indicate that in P. misakiensis, TC14-3-driven H3K27 trimethylation is a default state of bud and zooid cells, which serves as the histone code for cell longevity. H3K27me3 and H3K4me3 double-positive signals are involved in cell stemness, and absence of signals is the indication of senescence.

Zooids of the asexual strain of Polyandrocarpa misakiensis have a lifespan of 4-5 months; before dying, they produce many buds, enabling continuation of the strain. This study was designed to investigate the nature of gene inactivation and reactivation during this continuous process of senescence and budding. During senescence, the zooidal epidermis showed acid β-galactosidase activity, lost proliferating cell nuclear antigen immunoreactivity and became ultrastructurally worn, indicating that the epidermis is a major tissue affected by the ageing process. Semi-quantitative PCR analysis showed that the genes encoding mitochondrial respiratory chains (MRCs) engaged in decreased transcriptional activity in senescent adults compared with younger adults. The results of in situ hybridization showed that the epidermis dramatically attenuates MRC expression during ageing but restores gene activity when budding commences. During budding and ageing, the nuclear gene Eed (a polycomb group component) was activated and inactivated in a pattern similar to that observed in MRCs. In buds, RNA interference (RNAi) of Eed attenuated Eed transcripts but did not affect the gene expression of pre-activated MRCs. A tunicate humoral factor, TC14-3, could induce Eed, accompanying the reactivation of MRC in adult zooids. When RNAi of Eed and Eed induction were performed simultaneously, zooidal cells and tissues failed to engage in MRC reactivation, indicating the involvement of Eed in MRC activation. Results of this study provide evidence that the mitochondrial gene activities of Polyandrocarpa can be reversed during senescence and budding, suggesting that they are regulated by nuclear polycomb group genes.

A marine dinoflagellate Scrippsiella hexapraecingula Horiguchi et Chihara produces a cell covering called a pellicle in the non-motile phase. We found cellulose microfibrils present in the pellicle and new cellulose-synthesizing terminal complexes (TCs) that synthesize those microfibrils. Treatment of pellicle with acetic-nitric reagent yielded 2 to 14-nm microfibrils, which were shown to be cellulosic by electron diffraction. Negatively stained preparations revealed these microfibrils to be composed of 2-nm fibrils. The TCs were linear particle arrays that are associated with the tip of microfibril impressions in the PF of the plasma membrane. Five to forty particles, each with a diameter of 5-15 nm, were arranged in two rows, but the positions of the particles were almost random. The length and the width of the TCs ranged 62-290 nm and 15-31 nm, respectively. Several TCs up to 7 were often associated laterally with each other and consolidated as a cluster. Such a TC cluster synthesized bands of microfibrils. Thus, the TCs of Scrippsiella hexapraecingula are quite distinctive from those found so far both in the particle arrangement of TCs and in the structure of microfibrils synthesized.