Three-dimensional architecture and assembly mechanism of the egg-shaped shell in testate amoeba Paulinella micropora.

Unicellular euglyphid testate amoeba Paulinella micropora with filose pseudopodia secrete approximately 50 siliceous scales into the extracellular template-free space to construct a shell isomorphic to that of its mother cell. This shell-constructing behavior is analogous to building a house with bricks, and a complex mechanism is expected to be involved for a single-celled amoeba to achieve such a phenomenon; however, the three-dimensional (3D) structure of the shell and its assembly in P. micropora are still unknown. In this study, we aimed to clarify the positional relationship between the cytoplasmic and extracellular scales and the structure of the egg-shaped shell in P. micropora during shell construction using focused ion beam scanning electron microscopy (FIB-SEM). 3D reconstruction revealed an extensive invasion of the electron-dense cytoplasm between the long sides of the positioned and stacked scales, which was predicted to be mediated by actin filament extension. To investigate the architecture of the shell of P. micropora, each scale was individually segmented, and the position of its centroid was plotted. The scales were arranged in a left-handed, single-circular ellipse in a twisted arrangement. In addition, we 3D printed individual scales and assembled them, revealing new features of the shell assembly mechanism of P. micropora. Our results indicate that the shell of P. micropora forms an egg shape by the regular stacking of precisely designed scales, and that the cytoskeleton is involved in the construction process.

Rappemonads are haptophyte phytoplankton.

Rapidly accumulating genetic data from environmental sequencing approaches have revealed an extraordinary level of unsuspected diversity within marine phytoplankton,1-11 which is responsible for around 50% of global net primary production.12,13 However, the phenotypic identity of many of the organisms distinguished by environmental DNA sequences remains unclear. The rappemonads represent a plastid-bearing protistan lineage that to date has only been identified by environmental plastid 16S rRNA sequences.14-17 The phenotypic identity of this group, which does not confidently cluster in any known algal clades in 16S rRNA phylogenetic reconstructions,15 has remained unknown since the first report of environmental sequences over two decades ago. We show that rappemonads are closely related to a haptophyte microalga, Pavlomulina ranunculiformis gen. nov. et sp. nov., and belong to a new haptophyte class, the Rappephyceae. Organellar phylogenomic analyses provide strong evidence for the inclusion of this lineage within the Haptophyta as a sister group to the Prymnesiophyceae. Members of this new class have a cosmopolitan distribution in coastal and oceanic regions. The relative read abundance of Rappephyceae in a large environmental barcoding dataset was comparable to, or greater than, those of major haptophyte species, such as the bloom-forming Gephyrocapsa huxleyi and Prymnesium parvum, and this result indicates that they likely have a significant impact as primary producers. Detailed characterization of Pavlomulina allowed for reconstruction of the ancient evolutionary history of the Haptophyta, a group that is one of the most important components of extant marine phytoplankton communities.

A dynein-associated photoreceptor protein prevents ciliary acclimation to blue light.

Light-responsive regulation of ciliary motility is known to be conducted through modulation of dyneins, but the mechanism is not fully understood. Here, we report a novel subunit of the two-headed f/I1 inner arm dynein, named DYBLUP, in animal spermatozoa and a unicellular green alga. This subunit contains a BLUF (sensors of blue light using FAD) domain that appears to directly modulate dynein activity in response to light. DYBLUP (dynein-associated BLUF protein) mediates the connection between the f/I1 motor domain and the tether complex that links the motor to the doublet microtubule. Chlamydomonas lacking the DYBLUP ortholog shows both positive and negative phototaxis but becomes acclimated and attracted to high-intensity blue light. These results suggest a mechanism to avoid toxic strong light via direct photoregulation of dyneins.

Fine Structure Observation of Feeding Behavior, Nephroselmis spp.-derived Chloroplast Enlargement, and Mitotic Processes in the Katablepharid Hatena arenicola.

The difficult-to-cultivate katablepharid Hatena arenicola ingests green algae, Nephroselmis spp., and temporarily retains a Nephroselmis-derived cell compartment (kleptochloroplast), including a chloroplast within a phagocytotic vacuole. H. arenicola has a unique life history; during cell division, the Nephroselmis-derived cell compartment is only inherited by one of two daughter cells. However, the detailed morphological transition of the Nephroselmis cell to a kleptochloroplast and the mitotic process of the host cell remain unclear. Herein, we observed feeding behavior, enlargement of the Nephroselmis-derived chloroplast, and mitotic processes in H. arenicola using light and electron microscopy. During feeding behavior, H. arenicola peeled off the cell coverings and flagella of the Nephroselmis cell, which selectively accumulated in a vacuole separate to one containing a Nephroselmis cell body. An obvious nucleolus, but no heterochromatin was observed in the Nephroselmis-derived nucleus during the chloroplast-enlarging process, while compressed heterochromatin was explicitly observed in the nuclei of free-living Nephroselmis cells. The cell membrane of an ingested Nephroselmis cell disintegrated during enlargement of the Nephroselmis-derived chloroplast. The process of mitosis in H. arenicola was very similar to that of other katablepharids and cryptophytes.

Erratum: Publisher Correction: Calaxin is required for cilia-driven determination of vertebrate laterality.

[This corrects the article DOI: 10.1038/s42003-019-0462-y.].

Calaxin is required for cilia-driven determination of vertebrate laterality.

Calaxin is a Ca2+-binding dynein-associated protein that regulates flagellar and ciliary movement. In ascidians, calaxin plays essential roles in chemotaxis of sperm. However, nothing has been known for the function of calaxin in vertebrates. Here we show that the mice with a null mutation in Efcab1, which encodes calaxin, display typical phenotypes of primary ciliary dyskinesia, including hydrocephalus, situs inversus, and abnormal motility of trachea cilia and sperm flagella. Strikingly, both males and females are viable and fertile, indicating that calaxin is not essential for fertilization in mice. The 9 + 2 axonemal structures of epithelial multicilia and sperm flagella are normal, but the formation of 9 + 0 nodal cilia is significantly disrupted. Knockout of calaxin in zebrafish also causes situs inversus due to the irregular ciliary beating of Kupffer's vesicle cilia, although the 9 + 2 axonemal structure appears to remain normal.

Single-cell genomics unveiled a cryptic cyanobacterial lineage with a worldwide distribution hidden by a dinoflagellate host.

Cyanobacteria are one of the most important contributors to oceanic primary production and survive in a wide range of marine habitats. Much effort has been made to understand their ecological features, diversity, and evolution, based mainly on data from free-living cyanobacterial species. In addition, symbiosis has emerged as an important lifestyle of oceanic microbes and increasing knowledge of cyanobacteria in symbiotic relationships with unicellular eukaryotes suggests their significance in understanding the global oceanic ecosystem. However, detailed characteristics of these cyanobacteria remain poorly described. To gain better insight into marine cyanobacteria in symbiosis, we sequenced the genome of cyanobacteria collected from a cell of a pelagic dinoflagellate that is known to host cyanobacterial symbionts within a specialized chamber. Phylogenetic analyses using the genome sequence revealed that the cyanobacterium represents an underdescribed lineage within an extensively studied, ecologically important group of marine cyanobacteria. Metagenomic analyses demonstrated that this cyanobacterial lineage is globally distributed and strictly coexists with its host dinoflagellates, suggesting that the intimate symbiotic association allowed the cyanobacteria to escape from previous metagenomic studies. Furthermore, a comparative analysis of the protein repertoire with related species indicated that the lineage has independently undergone reductive genome evolution to a similar extent as Prochlorococcus, which has the most reduced genomes among free-living cyanobacteria. Discovery of this cyanobacterial lineage, hidden by its symbiotic lifestyle, provides crucial insights into the diversity, ecology, and evolution of marine cyanobacteria and suggests the existence of other undiscovered cryptic cyanobacterial lineages.

Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes.

The division of life into producers and consumers is blurred by evolution. For example, eukaryotic phototrophs can lose the capacity to photosynthesize, although they may retain vestigial plastids that perform other essential cellular functions. Chrysophyte algae have undergone a particularly large number of photosynthesis losses. Here, we present a plastid genome sequence from a nonphotosynthetic chrysophyte, "Spumella" sp. NIES-1846, and show that it has retained a nearly identical set of plastid-encoded functions as apicomplexan parasites. Our transcriptomic analysis of 12 different photosynthetic and nonphotosynthetic chrysophyte lineages reveals remarkable convergence in the functions of these nonphotosynthetic plastids, along with informative lineage-specific retentions and losses. At one extreme, Cornospumella fuschlensis retains many photosynthesis-associated proteins, although it appears to have lost the reductive pentose phosphate pathway and most plastid amino acid metabolism pathways. At the other extreme, Paraphysomonas lacks plastid-targeted proteins associated with gene expression and all metabolic pathways that require plastid-encoded partners, indicating a complete loss of plastid DNA in this genus. Intriguingly, some of the nucleus-encoded proteins that once functioned in the expression of the Paraphysomonas plastid genome have been retained. These proteins were likely to have been dual targeted to the plastid and mitochondria of the chrysophyte ancestor, and are uniquely targeted to the mitochondria in Paraphysomonas Our comparative analyses provide insights into the process of functional reduction in nonphotosynthetic plastids.

Microtubule stabilizer reveals requirement of Ca2+-dependent conformational changes of microtubules for rapid coiling of haptonema in haptophyte algae.

A haptonema is an elongated microtubule-based motile organelle uniquely present in haptophytes. The most notable and rapid movement of a haptonema is 'coiling', which occurs within a few milliseconds following mechanical stimulation in an unknown motor-independent mechanism. Here, we analyzed the coiling process in detail by high-speed filming and showed that haptonema coiling was initiated by left-handed twisting of the haptonema, followed by writhing to form a helix from the distal tip. On recovery from a mechanical stimulus, the helix slowly uncoiled from the proximal region. Electron microscopy showed that the seven microtubules in a haptonema were arranged mostly in parallel but that one of the microtubules often wound around the others in the extended state. A microtubule stabilizer, paclitaxel, inhibited coiling and induced right-handed twisting of the haptonema in the absence of Ca2+, suggesting changes in the mechanical properties of microtubules. Addition of Ca2+ resulted in the conversion of haptonematal twist into the planar bends near the proximal region. These results indicate that switching microtubule conformation, possibly with the aid of Ca2+-binding microtubule-associated proteins is responsible for rapid haptonematal coiling.

Taming chlorophylls by early eukaryotes underpinned algal interactions and the diversification of the eukaryotes on the oxygenated Earth.

Extant eukaryote ecology is primarily sustained by oxygenic photosynthesis, in which chlorophylls play essential roles. The exceptional photosensitivity of chlorophylls allows them to harvest solar energy for photosynthesis, but on the other hand, they also generate cytotoxic reactive oxygen species. A risk of such phototoxicity of the chlorophyll must become particularly prominent upon dynamic cellular interactions that potentially disrupt the mechanisms that are designed to quench photoexcited chlorophylls in the phototrophic cells. Extensive examination of a wide variety of phagotrophic, parasitic, and phototrophic microeukaryotes demonstrates that a catabolic process that converts chlorophylls into nonphotosensitive 132,173-cyclopheophorbide enols (CPEs) is phylogenetically ubiquitous among extant eukaryotes. The accumulation of CPEs is identified in phagotrophic algivores belonging to virtually all major eukaryotic assemblages with the exception of Archaeplastida, in which no algivorous species have been reported. In addition, accumulation of CPEs is revealed to be common among phototrophic microeukaryotes (i.e., microalgae) along with dismantling of their secondary chloroplasts. Thus, we infer that CPE-accumulating chlorophyll catabolism (CACC) primarily evolved among algivorous microeukaryotes to detoxify chlorophylls in an early stage of their evolution. Subsequently, it also underpinned photosynthetic endosymbiosis by securing close interactions with photosynthetic machinery containing abundant chlorophylls, which led to the acquisition of secondary chloroplasts. Our results strongly suggest that CACC, which allowed the consumption of oxygenic primary producers, ultimately permitted the successful radiation of the eukaryotes throughout and after the late Proterozoic global oxygenation.

Characterization of spliced leader trans-splicing in a photosynthetic rhizarian amoeba, Paulinella micropora, and its possible role in functional gene transfer.

Paulinella micropora is a rhizarian thecate amoeba, belonging to a photosynthetic Paulinella species group that has a unique organelle termed chromatophore, whose cyanobacterial origin is distinct from that of plant and algal chloroplasts. Because acquisition of the chromatophore was quite a recent event compared with that of the chloroplast ancestor, the Paulinella species are thought to be model organisms for studying the early process of primary endosymbiosis. To obtain insight into how endosymbiotically transferred genes acquire expression competence in the host nucleus, here we analyzed the 5' end sequences of the mRNAs of P. micropora MYN1 strain with the aid of a cap-trapper cDNA library. As a result, we found that mRNAs of 27 genes, including endosymbiotically transferred genes, possessed the common 5' end sequence of 28-33 bases that were posttranscriptionally added by spliced leader (SL) trans-splicing. We also found two subtypes of SL RNA genes encoded by the P. micropora MYN1 genome. Differing from the other SL trans-splicing organisms that usually possess poly(A)-less SL RNAs, this amoeba has polyadenylated SL RNAs. In this study, we characterize the SL trans-splicing of this unique organism and discuss the putative merits of SL trans-splicing in functional gene transfer and genome evolution.

Highly Dispersible and Bioavailable Curcumin but not Native Curcumin Induces Brown-Like Adipocyte Formation in Mice.

SCOPE: The induction of brown-like adipocytes in white adipose tissue (WAT) is a potential therapeutic target for the treatment of obesity and metabolic disorders via the ability of these cells to release excess energy as heat in association with uncoupling protein 1. Some experimental trials suggest that curcumin (a yellow pigment from turmeric) has a suppressive effect on the accumulation of body fat. However, there is little evidence to show that curcumin induces the formation of brown-like adipocytes and the molecular mechanisms involved remain elusive. In addition, in most experimental trials, high doses of curcumin are administered. METHODS AND RESULTS: Highly dispersible and bioavailable curcumin (HC, i.e., 4.5 mg native curcumin kg-1 ) but not the same dose of native curcumin induces the formation of brown-like adipocytes in mouse inguinal WAT. Moreover, the formation of brown-like adipocytes induced by HC in inguinal WAT may be mediated by the production of local norepinephrine from accumulated alternatively activated macrophages. CONCLUSION: These novel findings suggest that curcumin increases energy expenditure by inducing the formation of brown-like adipocytes via a unique molecular mechanism. Importantly, they show that HC has significant bioactive effects in vivo at lower doses of curcumin.

Fine-structural Observations on Siliceous Scale Production and Shell Assembly in the Testate Amoeba Paulinella chromatophora.

The fine structure of shell formation was observed in P. chromatophora. Scales were formed one by one in silica deposition vesicles (SDVs) that were supported by an array of microtubules, which are probably involved in determining the shape and size of scales. The timing of silicic acid transport into an SDV was shown to be at an early stage of scale production because silicon was detected within SDVs containing immature scales. During the shell construction process, vesicles containing two types of dense materials were observed. One type of vesicle contains lower-density material and is located at the front edge of the branched, thick pseudopodium, extending from the maternal shell to the newly formed shell. The other type of vesicle, which contains higher-density material, was also observed in the thick pseudopodium. It appears that microtubules are involved in the shell construction process.

Detailed process of shell construction in the photosynthetic testate amoeba Paulinella chromatophora (euglyphid, Rhizaria).

Most euglyphids, a group of testate amoebae, have a shell that is constructed from numerous siliceous scales. The euglyphid Paulinella chromatophora has photosynthetic organelles (termed cyanelles or chromatophores), allowing it to be cultivated more easily than other euglyphids. Like other euglyphids, P. chromatophora has a siliceous shell made of brick-like scales. These scales are varied in size and shape. How a P. chromatophora cell makes this shell is still a mystery. We examined shell construction process in P. chromatophora in detail using time-lapse video microscopy. The new shell was constructed by a specialized pseudopodium that laid out each scale into correct position, one scale at a time. The present study inferred that the sequence of scale production and secretion was well controlled.

Facilitated intracellular transport of TrkA by an interaction with nerve growth factor.

Intracellular transport of neurotrophin receptors together with neurotrophins is one of the key events of neurotrophin signaling for the growth and the survival of neurons. However, the involvement of neurotrophin signaling in the regulation of intracellular transport of neurotrophin receptors has been remained unclear. We visualized the behavior of TrkA, a receptor of nerve growth factor (NGF), by labeling with GFP in PC12 cells. We found remarkable changes of the behavior of TrkA-GFP upon the application of NGF. Before the application, only ~37% of the fluorescent dots of TrkA showed translocations along neurites of PC12 cells. After the application, number of the dots showing the directional movement increased to ~65%. The averaged velocities of the directional movement of TrkA-GFP dots became higher after the application of NGF. We tested the idea whether NGF binding accelerated the translocations of TrkA by simultaneously observing TrkA-GFP and fluorescently labeled NGF, Cy3.5-NGF. The velocity of TrkA-GFP dots associated with Cy3.5-NGF was remarkably higher than that of TrkA-GFP dots without Cy3.5-NGF. On the basis of these observations, we hypothesize that there is a signaling mechanism within a single vesicle that facilitates the intracellular transport of each vesicle containing the activated TrkA.

Possible involvement of aminopeptidase A in hypertension and renal damage in Dahl salt-sensitive rats.

BACKGROUND: Although aminopeptidase A (APA), which is abundant in the kidneys, is responsible for metabolizing angiotensin II (Ang II), its association with salt sensitivity remains uncertain. We aimed to clarify the involvement of APA in salt-induced hypertension and renal damage. METHODS: Male Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats were fed low-salt (0.3%) or high-salt diet (8%) from 6 weeks of age for 12 weeks. Tail-cuff-measured blood pressure (BP), renal APA activity, renal Ang II levels, histologic renal damage, and APA immunoreactivity were periodically examined. RESULTS: Systolic BP progressively increased only in DS rats given the high-salt diet (DS-8% rats). The DR-8% rats had approximately 3-fold higher renal APA activity than the rats given the low-salt diet (DR-0.3% rats) during the maintenance on the high-salt diet. However, although DS-8% rats also had 2.5-fold higher renal APA activity than DS-0.3% rats at 10 weeks, continuing the high-salt diet afterward suppressed the activity in DS-8% rats below the levels observed in DS-0.3% rats. High-salt diet reduced renal Ang II levels by 30% in DR rats, whereas it showed a small and nonsignificant decrease in DS rats. The number of injured glomeruli was markedly elevated in DS-8% rats after 10 weeks. The APA immunostaining in DS-8% rats was enhanced in glomeruli displaying mild damage, diminished in the severely injured glomeruli, and absent in lesions with hyalinization. CONCLUSIONS: High-salt diet in DS rats increased renal APA activity, although renal injury remained mild, but then reduced it along with the progression of glomerulosclerosis, suggesting that reduced APA activity may be involved in the deterioration of salt-induced hypertension and renal injury.