Spillover in the direct-type PSI-PSII megacomplex isolated from Arabidopsis thaliana is regulated by pH.

Various megacomplexes in which Photosystem I and Photosystem II are physically bound (PSI-PSII m.c.) have been found in many organisms. In terms of function, these can be divided into two groups: those in which PSII and PSI are closely coupled (direct-type, photoprotection), and those in which a large light-harvesting antenna is placed between PSII and PSI (bridged-type, energy sharing). Arabidopsis thaliana has been reported to use the direct-type, where fast energy transfer occurs from PSII to PSI (~20 ps, fast spillover). In this paper, we show that the fast spillover is reversibly regulated depending on pH.

Formation of a Stable PSI-PSII Megacomplex in Rice That Conducts Energy Spillover.

In green plants, photosystem I (PSI) and photosystem II (PSII) bind to their respective light-harvesting complexes (LHCI and LHCII) to form the PSI-LHCI supercomplex and the PSII-LHCII supercomplex, respectively. These supercomplexes further form megacomplexes, like PSI-PSII and PSII-PSII in Arabidopsis (Arabidopsis thaliana) and spinach to modulate their light-harvesting properties, but not in the green alga Chlamydomonas reinhardtii. Here, we fractionated and characterized the stable rice PSI-PSII megacomplex. The delayed fluorescence from PSI (lifetime ∼25 ns) indicated energy transfer capabilities between the two photosystems (energy spillover) in the rice PSI-PSII megacomplex. Fluorescence lifetime analysis revealed that the slow PSII to PSI energy transfer component was more dominant in the rice PSI-PSII supercomplexes than in Arabidopsis ones, suggesting that PSI and PSII in rice form a megacomplex not directly but through LHCII molecule(s), which was further confirmed by the negatively stained electron microscopy analysis. Our results suggest species diversity in the formation and stability of photosystem megacomplexes, and the stable PSI-PSII supercomplex in rice may reflect its structural adaptation.

The photosystem I supercomplex from a primordial green alga Ostreococcus tauri harbors three light-harvesting complex trimers.

As a ubiquitous picophytoplankton in the ocean and an early-branching green alga, Ostreococcus tauri is a model prasinophyte species for studying the functional evolution of the light-harvesting systems in photosynthesis. Here, we report the structure and function of the O. tauri photosystem I (PSI) supercomplex in low light conditions, where it expands its photon-absorbing capacity by assembling with the light-harvesting complexes I (LHCI) and a prasinophyte-specific light-harvesting complex (Lhcp). The architecture of the supercomplex exhibits hybrid features of the plant-type and the green algal-type PSI supercomplexes, consisting of a PSI core, an Lhca1-Lhca4-Lhca2-Lhca3 belt attached on one side and an Lhca5-Lhca6 heterodimer associated on the other side between PsaG and PsaH. Interestingly, nine Lhcp subunits, including one Lhcp1 monomer with a phosphorylated amino-terminal threonine and eight Lhcp2 monomers, oligomerize into three trimers and associate with PSI on the third side between Lhca6 and PsaK. The Lhcp1 phosphorylation and the light-harvesting capacity of PSI were subjected to reversible photoacclimation, suggesting that the formation of OtPSI-LHCI-Lhcp supercomplex is likely due to a phosphorylation-dependent mechanism induced by changes in light intensity. Notably, this supercomplex did not exhibit far-red peaks in the 77 K fluorescence spectra, which is possibly due to the weak coupling of the chlorophyll a603-a609 pair in OtLhca1-4.

Exaggeration and cooption of innate immunity for social defense.

Social insects often exhibit striking altruistic behaviors, of which the most spectacular ones may be self-destructive defensive behaviors called autothysis, "self-explosion," or "suicidal bombing." In the social aphid Nipponaphis monzeni, when enemies damage their plant-made nest called the gall, soldier nymphs erupt to discharge a large amount of body fluid, mix the secretion with their legs, and skillfully plaster it over the plant injury. Dozens of soldiers come out, erupt, mix, and plaster, and the gall breach is promptly sealed with the coagulated body fluid. What molecular and cellular mechanisms underlie the self-sacrificing nest repair with body fluid for the insect society? Here we demonstrate that the body cavity of soldier nymphs is full of highly differentiated large hemocytes that contain huge amounts of lipid droplets and phenoloxidase (PO), whereas their hemolymph accumulates huge amounts of tyrosine and a unique repeat-containing protein (RCP). Upon breakage of the gall, soldiers gather around the breach and massively discharge the body fluid. The large hemocytes rupture and release lipid droplets, which promptly form a lipidic clot, and, concurrently, activated PO converts tyrosine to reactive quinones, which cross-link RCP and other macromolecules to physically reinforce the clot to seal the gall breach. Here, soldiers' humoral and cellular immune mechanisms for wound sealing are extremely up-regulated and utilized for colony defense, which provides a striking case of direct evolutionary connection between individual immunity and social immunity and highlights the importance of exaggeration and cooption of preexisting traits to create evolutionary novelties.

Competition for Mitogens Regulates Spermatogenic Stem Cell Homeostasis in an Open Niche.

In many tissues, homeostasis is maintained by physical contact between stem cells and an anatomically defined niche. However, how stem cell homeostasis is achieved in environments where cells are motile and dispersed among their progeny remains unknown. Using murine spermatogenesis as a model, we find that spermatogenic stem cell density is tightly regulated by the supply of fibroblast growth factors (FGFs) from lymphatic endothelial cells. We propose that stem cell homeostasis is achieved through competition for a limited supply of FGFs. We show that the quantitative dependence of stem cell density on FGF dosage, the biased localization of stem cells toward FGF sources, and stem cell dynamics during regeneration following injury can all be predicted and explained within the framework of a minimal theoretical model based on "mitogen competition." We propose that this model provides a generic and robust mechanism to support stem cell homeostasis in open, or facultative, niche environments.

SHISA6 Confers Resistance to Differentiation-Promoting Wnt/ß-Catenin Signaling in Mouse Spermatogenic Stem Cells.

In the seminiferous tubules of mouse testes, a population of glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1)-positive spermatogonia harbors the stem cell functionality and supports continual spermatogenesis, likely independent of asymmetric division or definitive niche control. Here, we show that activation of Wnt/ß-catenin signaling promotes spermatogonial differentiation and reduces the GFRα1+ cell pool. We further discovered that SHISA6 is a cell-autonomous Wnt inhibitor that is expressed in a restricted subset of GFRα1+ cells and confers resistance to the Wnt/ß-catenin signaling. Shisa6+ cells appear to show stem cell-related characteristics, conjectured from the morphology and long-term fates of T (Brachyury)+ cells that are found largely overlapped with Shisa6+ cells. This study proposes a generic mechanism of stem cell regulation in a facultative (or open) niche environment, with which different levels of a cell-autonomous inhibitor (SHISA6, in this case) generates heterogeneous resistance to widely distributed differentiation-promoting extracellular signaling, such as WNTs.

Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells.

Accumulating evidence suggests that exosomes are heterogeneous in molecular composition and physical properties. Here we examined whether epithelial cells secrete a heterogeneous population of exosomes, and if that is the case, whether epithelial cell polarity affects release of different populations of exosomes, especially that of those carrying Wnt. Sucrose-density ultracentrifugation and molecular marker analysis revealed that different populations of exosomes or exosome-like vesicles were released from MDCK cells depending on the cell polarity. Wnt3a associated with these vesicles were detectable in culture media collected from both apical and basolateral sides of the cells. Basolaterally secreted Wnt3a were co-fractionated with a typical exosomal protein TSG101 in fractions having typical exosome densities. In contrast, most of apically secreted Wnt3a, as well as Wnt11, were co-fractionated with CD63 and Hsp70, which are also common to the most exosomes, but recovered in higher density fractions. Wnt3a exhibiting similar floatation behavior to the apically secreted ones were also detectable in the culture media of Wnt3a-expressing L and HEK293 cells. The lipidation of Wnt3a was required for its basolateral secretion in exosomes but was dispensable for the apical one. Thus, epithelial cells release Wnt via distinct populations of vesicles differing in secretion polarity and lipidation dependency.

Hierarchical differentiation competence in response to retinoic acid ensures stem cell maintenance during mouse spermatogenesis.

Stem cells ensure tissue homeostasis through the production of differentiating and self-renewing progeny. In some tissues, this is achieved by the function of a definitive stem cell niche. However, the mechanisms that operate in mouse spermatogenesis are unknown because undifferentiated spermatogonia (Aundiff) are motile and intermingle with differentiating cells in an 'open' niche environment of seminiferous tubules. Aundiff include glial cell line-derived neurotrophic factor receptor α1 (GFRα1)(+) and neurogenin 3 (NGN3)(+) subpopulations, both of which retain the ability to self-renew. However, whereas GFRα1(+) cells comprise the homeostatic stem cell pool, NGN3(+) cells show a higher probability to differentiate into KIT(+) spermatogonia by as yet unknown mechanisms. In the present study, by combining fate analysis of pulse-labeled cells and a model of vitamin A deficiency, we demonstrate that retinoic acid (RA), which may periodically increase in concentration in the tubules during the seminiferous epithelial cycle, induced only NGN3(+) cells to differentiate. Comparison of gene expression revealed that retinoic acid receptor γ (Rarg) was predominantly expressed in NGN3(+) cells, but not in GFRα1(+) cells, whereas the expression levels of many other RA response-related genes were similar in the two populations. Ectopic expression of RARγ was sufficient to induce GFRα1(+) cells to directly differentiate to KIT(+) cells without transiting the NGN3(+) state. Therefore, RARγ plays key roles in the differentiation competence of NGN3(+) cells. We propose a novel mechanism of stem cell fate selection in an open niche environment whereby undifferentiated cells show heterogeneous competence to differentiate in response to ubiquitously distributed differentiation-inducing signals.

Molecular characterization of embryonic gonads by gene expression profiling in Drosophila melanogaster.

In many animal species, germ-line progenitors associate with gonadal somatic cells to form the embryonic gonads (EGs) that later develop into functional organ producing gametes. To explore the genetic regulation of the germ-line development, we initiated a comprehensive identification and functional analysis of the genes expressed within the EGs. First, we generated a cDNA library from gonads purified from Drosophila embryos by FACS. Using this library, we catalogued the genes expressed in the gonad by EST analysis. A total of 17,218 high-quality ESTs representing 3,051 genes were obtained, corresponding to 20% of the predicted genes in the genome. The EG transcriptome is unexpectedly distinct from that of adult gonads and includes an extremely high proportion of retrotransposon-derived transcripts. We verified 101 genes preferentially expressed in the EGs by whole-mount in situ hybridization. Within this subset, 39 and 58 genes were expressed predominantly in germ-line and somatic cells, respectively, whereas four genes were expressed in the both cell lineages. The gonad-enriched genes encompassed a variety of predicted functions. However, genes implicated in SUMOylation and protein translation, including germ-line-specific ribosomal proteins, are preferentially expressed in the germ line, whereas the expression of various retrotransposons and RNAi-related genes are more prominent in the gonadal soma. These transcriptome data are a resource for understanding the mechanism of various cellular events during germ-line development.

Isolation of germline cells from Drosophila embryos by flow cytometry.

Primordial germ cells (PGC) are the earliest identifiable germ cells in the embryo. To understand the molecular basis of germline development, isolation of pure PGC is required. We report here the use of fluorescence-activated cell sorting (FACS) to isolate pure populations of Drosophila pole cells, which are the presumptive primordial germ cells in flies. In order to fluorescently mark pole cells, we used an EGFP-vasa transgenic line that expresses green fluorescent protein (GFP) specifically and continuously in the germ line throughout the life cycle. The purity of FACS-sorted pole cells from embryos was confirmed by microscopic inspection and quantitative polymerase chain reaction. Moreover, by optimizing the sample preparation and the sorting protocol, embryonic gonads could also be isolated. This technique opens the way for genome-wide transcriptome analysis of germline cells. In a pilot experiment, we generated a cDNA library from purified embryonic gonad and identified a novel germline-specific gene, RpL22-like.