RSU-1 regulates the integrity of dense bodies in muscle cells of aging Caenorhabditis elegans.

Muscle contraction is vital for animal survival, and the sarcomere is the fundamental unit for this process. However, the functions of many conserved sarcomere proteins remain unknown, as their mutants do not exhibit obvious defects. To address this, Caenorhabditis elegans was utilized as a model organism to investigate RSU-1 function in the body wall muscle. RSU-1 is found to colocalize with UNC-97 at the dense body and M-line, and it is particularly crucial for regulating locomotion in aging worms, rather than in young worms. This suggests that RSU-1 has a specific function in maintaining muscle function during aging. Furthermore, the interaction between RSU-1 and UNC-97/PINCH is essential for RSU-1 to modulate locomotion, preserve filament structure, and sustain the M-line and dense body throughout aging. Overall, these findings highlight the significant contribution of RSU-1, through its interaction with UNC-97, in maintaining proper muscle cell function in aging worms.

Autophagy and its mediated mitochondrial quality control maintain pollen tube growth and male fertility in Arabidopsis.

Macroautophagy/autophagy, a major catabolic pathway in eukaryotes, participates in plant sexual reproduction including the processes of male gametogenesis and the self-incompatibility response. Rapid pollen tube growth is another essential reproductive process that is metabolically highly demanding to drive the vigorous cell growth for delivery of male gametes for fertilization in angiosperms. Whether and how autophagy operates to maintain the homeostasis of pollen tubes remains unknown. Here, we provide evidence that autophagy is elevated in growing pollen tubes and critically required during pollen tube growth and male fertility in Arabidopsis. We demonstrate that SH3P2, a critical non-ATG regulator of plant autophagy, colocalizes with representative ATG proteins during autophagosome biogenesis in growing pollen tubes. Downregulation of SH3P2 expression significantly disrupts Arabidopsis pollen germination and pollen tube growth. Further analysis of organelle dynamics reveals crosstalk between autophagosomes and prevacuolar compartments following the inhibition of phosphatidylinositol 3-kinase. In addition, time-lapse imaging and tracking of ATG8e-labeled autophagosomes and depolarized mitochondria demonstrate that they interact specifically via the ATG8-family interacting motif (AIM)-docking site to mediate mitophagy. Ultrastructural identification of mitophagosomes and two additional forms of autophagosomes imply that multiple types of autophagy are likely to function simultaneously within pollen tubes. Altogether, our results suggest that autophagy is functionally crucial for mediating mitochondrial quality control and canonical cytoplasm recycling during pollen tube growth.Abbreviations: AIM: ATG8-family interacting motif; ATG8: autophagy related 8; ATG5: autophagy related 5; ATG7: autophagy related 7; BTH: acibenzolar-S-methyl; DEX: dexamethasone; DNP: 2,4-dinitrophenol; GFP: green fluorescent protein; YFP: yellow fluorescent protein; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PVC: prevacuolar compartment; SH3P2: SH3 domain-containing protein 2.

Interaction between DLC-1 and SAO-1 facilitates CED-4 translocation during apoptosis in the Caenorhabditis elegans germline.

Apoptosis is one of the major forms of programmed cell death, and it serves vital biological functions in multicellular animal and plant cells. The core mechanism of apoptosis is highly conserved in metazoans, where the translocation of CED-4/Apaf-1 from mitochondria to the nuclear membrane is required to initiate and execute apoptosis. However, the underlying molecular mechanisms of this translocation are poorly understood. In this study, we showed that SAO-1 binds DLC-1 and prevents its degradation to promote apoptosis in C. elegans germ cells. We demonstrated that SAO-1 and DLC-1 regulate CED-4/Apaf-1 nuclear membrane accumulation during apoptosis. Isothermal titration calorimetry-based assay and high-resolution crystal structure analysis further revealed that SAO-1 interacted with DLC-1 to form a 2:4 complex: each of the two ß-sheets in the SAO-1 peptide interacted with two DLC-1 dimers. Point mutations at the SAO-1-DLC-1 binding interface significantly inhibited apoptotic corpse formation and CED-4 nuclear membrane accumulation within C. elegans germ cells. In conclusion, our study provides a new perspective on the regulation of CED-4-mediated apoptosis.

From primordial germ cells to spermatids in Caenorhabditis elegans.

Development of a syncytial germline for gamete formation requires complex regulation of cytokinesis and cytoplasmic remodeling. Recently, several uncovered cellular events have been investigated in the Caenorhabditis elegans (C. elegans) germline. In these cellular processes, the factors involved in contractility are highly conserved with those of mitosis and meiosis. However, the underlying regulatory mechanisms are far more complicated than previously thought, likely due to the single syncytial germline structure. In this review, we highlight how the proteins involved in contractility ensure faithful cell division in different cellular contexts and how they contribute to maintaining intercellular bridge stability. In addition, we discuss the current understanding of the cellular events of cytokinesis and cytoplasmic remodeling during the development of the C. elegans germline, including progenitor germ cells, germ cells, and spermatocytes. Comparisons are made with relevant systems in Drosophila melanogaster (D. melanogaster) and other animal models.

A collection of toolkit strains reveals distinct localization and dynamics of membrane-associated transcripts in epithelia.

Visualizing mRNA in real time in vivo at high resolution is critical for a full understanding of the spatiotemporal dynamics of gene regulation and function. Here, using a PP7/PCP-based mRNA-tagging approach, we construct a collection of tissue-specific and differentially expressed toolkit strains for visualizing mRNAs encoding apical, basolateral, and junctional proteins in Caenorhabditis elegans epithelia. We precisely delineate the spatiotemporal organization and dynamics of these transcripts across multiple subcellular compartments and tissues. Remarkably, all the transcripts exhibit an asymmetric, membrane-associated localization during epithelial polarization and maturation, which suggests that mRNA localization is a prerequisite for epithelial polarization and function. Single-particle tracking reveals striking features of the transport dynamics of the mRNAs in a gene-specific, compartment-linked, and time-resolved manner. The toolkit can be used to identify the cis-regulatory elements and trans-acting factors for mRNA localization. This study provides a valuable resource to investigate complex RNA dynamics in epithelial polarity and morphogenesis.

Cortical recruitment of centralspindlin and RhoA effectors during meiosis I of Caenorhabditiselegans primary spermatocytes.

Haploid male gametes are produced through meiosis during gametogenesis. Whereas the cell biology of mitosis and meiosis is well studied in the nematode Caenorhabditis elegans, comparatively little is known regarding the physical division of primary spermatocytes during meiosis I. Here, we investigated this process using high-resolution time-lapse confocal microscopy and examined the spatiotemporal regulation of contractile ring assembly in C. elegans primary spermatocytes. We found that centralspindlin and RhoA effectors were recruited to the equatorial cortex of dividing primary spermatocytes for contractile ring assembly before segregation of homologous chromosomes. We also observed that perturbations shown to promote centralspindlin oligomerization regulated the cortical recruitment of NMY-2 and impacted the order in which primary spermatocytes along the proximal-distal axis of the gonad enter meiosis I. These results expand our understanding of the cellular division of primary spermatocytes into secondary spermatocytes during meiosis I.This article has an associated First Person interview with the first author of the paper.

Forchlorfenuron (CPPU) causes disorganization of the cytoskeleton and dysfunction of human umbilical vein endothelial cells, and abnormal vascular development in zebrafish embryos.

Forchlorfenuron (CPPU) has been used worldwide, to boost size and improve quality of various agricultural products. CPPU and its metabolites are persistent and have been detected frequently in fruits, water, sediments, and organisms in aquatic systems. Although the public became aware of CPPU through the exploding watermelon scandal of 2011 in Zhenjiang, China, little was known of its potential effects on the environment and wildlife. In this study, adverse effects of CPPU on developmental angiogenesis and vasculature, which is vulnerable to insults of persistent toxicants, were studied in vivo in zebrafish embryos (Danio rerio). Exposure to 10 mg CPPU/L impaired survival and hatching, while development was hindered by exposure to 2.5 mg CPPU/L. Developing vascular structure, including common cardinal veins (CCVs), intersegmental vessels (ISVs) and sub-intestinal vessels (SIVs), were significantly restrained by exposure to CPPU, in a dose-dependent manner. Also, CPPU caused disorganization of the cytoskeleton. In human umbilical vein endothelial cells (HUVECs), CPPU inhibited proliferation, migration and formation of tubular-like structures in vitro. Results of Western blot analyses revealed that exposure to CPPU increased phosphorylation of FLT-1, but inhibited phosphorylation of FAK and its downstream MAPK pathway in HUVECs. In summary, CPPU elicited developmental toxicity to the developing endothelial system of zebrafish and HUVECs. This was do, at least in part due to inhibition of the FAK/MAPK signaling pathway rather than direct interaction with the VEGF receptor (VEGFR).

In Vivo Screening of Xanthones from Garcinia oligantha Identified Oliganthin H as a Novel Natural Inhibitor of Convulsions.

Epilepsy is a chronic neurological disorder, characterized by recurrent, spontaneous, and transient seizures, and affects more than 70 million people worldwide. Although two dozen antiepileptic drugs (AEDs) are approved and available in the market, seizures remain poorly controlled in one-third of epileptic patients who are suffering from drug resistance or various adverse effects. Recently, the xanthone skeleton has been regarded as an attractive scaffold for the discovery and development of emerging anticonvulsants. We had isolated several dihydroxanthone derivatives previously, including oliganthin H, oliganthin I, and oliganthin N, whose structures were similar and delicately elucidated by spectrum analysis or X-ray crystallographic data, from extracts of leaves of Garcinia oligantha. These xanthone analogues were evaluated for anticonvulsant activity, and a novel xanthone, oliganthin H, has been identified as a sound and effective natural inhibitor of convulsions in zebrafish in vivo. A preliminary structure-activity relationship analysis on the relationship between structures of the xanthone analogues and their activities was also conducted. Oliganthin H significantly suppressed convulsant behavior and reduced to about 25% and 50% of PTZ-induced activity, in 12.5 and 25 µM treatment groups (P < 0.01 and 0.001), respectively. Meanwhile, it reduced seizure activity, velocity, seizure duration, and number of bursts in zebrafish larvae (P < 0.05). Pretreatment of oliganthin H significantly restored aberrant induction of gene expressions including npas4a, c-fos, pyya, and bdnf, as well as gabra1, gad1, glsa, and glula, upon PTZ treatment. In addition, in silico analysis revealed the stability of the oliganthin H-GABAA receptor complex and their detailed binding pattern. Therefore, direct interactions with the GABAA receptor and involvement of downstream GABA-glutamate pathways were possible mechanisms of the anticonvulsant action of oliganthin H. Our findings present the anticonvulsant activity of oliganthin H, provide a novel scaffold for further modifications, and highlight the xanthone skeleton as an attractive and reliable resource for the development of emerging AEDs.

A Deep-Red to Near Infrared (NIR) Fluorescent Probe Based on a Sulfur-Modified Rhodamine Derivative with Two Spirolactone Rings.

We report the synthesis, characterization, and photophysical and ion-binding properties of deep-red to near-infrared (NIR) fluorescent rhodamine derivatives, bearing two spirolactone rings and substitution of the oxygen atoms in the xanthene ring with sulfur atoms (1-S). The diastereoisomeric cis- and trans-forms of the rhodamine derivative were separated and the cis-form (cis-1-S) was structurally characterized by X-ray crystallography. Upon treatment with Hg2+ ion, cis-1-S was converted into the dual spirolactone ring-opened species, resulting in significant color change and fluorescence enhancement. Substitution of the oxygen atoms with sulfur and extended π-conjugation across the fused six-membered rings upon the two rings-opening processes in the presence of Hg2+ ion led to a significant red-shift of absorption (623 nm) and fluorescence (706 nm) peaks, compared to the ordinary rhodamine. Furthermore, the intracellular Hg2+ -sensing properties of the cis-1-S have been studied by confocal microscopy.

RSU-1 Maintains Integrity of Caenorhabditis elegans Vulval Muscles by Regulating α-Actinin.

Egg-laying behavior in Caenorhabditis elegans is a well-known model for investigating fundamental cellular processes. In egg-laying, muscle contraction is the relaxation of the vulval muscle to extrude eggs from the vulva. Unlike skeletal muscle, vulval muscle lacks visible striations of the sarcomere. Therefore, vulval muscle must counteract the mechanical stress, caused by egg extrusion and body movement, from inducing cell-shape distortion by maintaining its cytoskeletal integrity. However, the underlying mechanisms that regulate the cellular integrity in vulval muscles remain unclear. Here, we demonstrate that C. elegans egg-laying requires proper vulval muscle 1 (vm1), in which the actin bundle organization of vm1 muscles is regulated by Ras suppressor protein 1 (RSU-1). In the loss of RSU-1, as well as RasLET-60 overactivation, blister-like membrane protrusions and disorganized actin bundles were observed in the vm1 muscles. Moreover, RasLET-60 depletion diminished the defected actin-bundles in rsu-1 mutant. These results reveal the genetic interaction of RSU-1 and RasLET-60in vivo In addition, our results further demonstrated that the fifth to seventh leucine-rich region of RSU-1 is required to promote actin-bundling protein, α-actinin, for actin bundle stabilization in the vm1 muscles. This expands our understanding of the molecular mechanisms of actin bundle organization in a specialized smooth muscle.

A BODIPY-based fluorescent sensor for the detection of Pt2+ and Pt drugs.

A BODIPY-based fluorescent sensor PS with an NO4S2 podand ligand was studied for the selective detection of Pt2+ over 21 cations as well as selected platinum drugs in aqueous medium. The platinum sensor PS shows 28-fold, 22-fold and 14-fold fluorescence turn-on enhancements to Pt2+, cisplatin and nedaplatin, and was thereby employed to detect platinum drugs in A-549 human lung cancer cells.

Boosting autophagy in sexual reproduction: a plant perspective.

The key process of sexual reproduction is the successful fusion of the sperm and egg cell. Distinct from dynamic and flagellated animal sperm cells, higher flowering plant sperm cells are immotile. Therefore, plants have evolved a novel reproductive system to achieve fertilization and generate progenies. Plant sexual reproduction consists of multiple steps, mainly including gametophyte development, pollen-pistil recognition, pollen germination, double fertilization and postfertilization. During reproduction, active production, consumption and recycling of cellular components and energy are critically required to achieve fertilization. However, the underlying machinery of cellular degradation and turnover remains largely unexplored. Autophagy, the major catabolic pathway in eukaryotic cells, participates in regulating multiple aspects of plant activities, including abiotic and biotic stress resistance, pathogen response, senescence, nutrient remobilization and plant development. Nevertheless, a key unanswered question is how autophagy regulates plant fertilization and reproduction. Here, we focus on comparing and contrasting autophagy in several key reproductive processes of plant and animal systems to feature important distinctions and highlight future research directions of autophagy in angiosperm reproduction. We further discuss the potential crosstalk between autophagy and programmed cell death, which are often considered as two disconnected events in plant sexual reproduction.

Amplification-free library preparation with SAFE Hi-C uses ligation products for deep sequencing to improve traditional Hi-C analysis.

PCR amplification of Hi-C libraries introduces unusable duplicates and results in a biased representation of chromatin interactions. We present a simplified, fast, and economically efficient Hi-C library preparation procedure, SAFE Hi-C, which generates sufficient non-amplified ligation products for deep sequencing from 30 million Drosophila cells. Comprehensive analysis of the resulting data shows that amplification-free Hi-C preserves higher complexity of chromatin interaction and lowers sequencing depth for the same number of unique paired reads. For human cells which have a large genome, SAFE Hi-C recovers enough ligated fragments for direct high-throughput sequencing without amplification from as few as 250,000 cells. Comparison with published in situ Hi-C data from millions of human cells demonstrates that amplification introduces distance-dependent amplification bias, which results in an increased background noise level against genomic distance. With amplification bias avoided, SAFE Hi-C may produce a chromatin interaction network more faithfully reflecting the real three-dimensional genomic architecture.

An extracellular matrix protein promotes anillin-dependent processes in the Caenorhabditis elegans germline.

Cell division requires constriction of an actomyosin ring to segregate the genetic material equally into two daughter cells. The spatial and temporal regulation of the contractile ring at the division plane primarily depends on intracellular signals mediated by the centralspindlin complex and astral microtubules. Although much investigative work has elucidated intracellular factors and mechanisms controlling this process, the extracellular regulation of cytokinesis remains unclear. Thus far, the extracellular matrix protein Hemicentin (HIM-4) has been proposed to be required for cleavage furrow stabilization. The underlying molecular mechanism, however, has remained largely unknown. Here, we show that HIM-4 and anillin (ANI-1) genetically act in the same pathway to maintain the rachis bridge stability in the germline. Our FRAP experiments further reveal that HIM-4 restricts the motility of ANI-1. In addition, we demonstrate that HIM-4 is recruited to the cleavage site in dividing germ cells and promotes the proper ingression of the cleavage membrane. Collectively, we propose that HIM-4 is an extracellular factor that regulates ANI-1 for germ cell membrane stabilization and contractile ring formation in Caenorhabditis elegans germline cells.

Synthesis and study of Au(iii)-indolizine derivatives: turn-on luminescence by photo-induced controlled release.

The photo- and structural properties of a series of Au(iii) indolizine complexes were determined. Controlled release of halogenated indolizine derivatives from the corresponding Au(iii) complexes was achieved by photoinduced C-X bond formation, which provided turn-on luminescence with an increase in emission intensity of up to 67 times.

A Novel ShK-Like Toxic Peptide from the Transcriptome of the Cnidarian Palythoa caribaeorum Displays Neuroprotection and Cardioprotection in Zebrafish.

Palythoa caribaeorum (class Anthozoa) is a zoantharian which, together with other cnidarians, like jellyfishes, hydra, and sea anemones, possesses specialized structures in its tissues, the cnidocytes, which deliver an array of toxins in order to capture prey and deter predators. The whole transcriptome of P. caribaeroum was deep sequenced, and a diversity of toxin-related peptide sequences were identified, and some retrieved for functional analysis. In this work, a peptide precursor containing a ShK domain, named PcShK3, was analyzed by means of computational processing, comprising structural phylogenetic analysis, model prediction, and dynamics simulation of peptide-receptor interaction. The combined data indicated that PcShK3 is a distinct peptide which is homologous to a cluster of peptides belonging to the ShK toxin family. In vivo, PcShK3 distributed across the vitelline membrane and accumulated in the yolk sac stripe of zebrafish larvae. Notably, it displayed a significant cardio-protective effect in zebrafish in concentrations inferior to the IC50 (<43.53 ± 6.45 µM), while in high concentrations (>IC50), it accumulated in the blood and caused pericardial edema, being cardiotoxic to zebrafish larvae. Remarkably, PcShK3 suppressed the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish. The present results indicated that PcShK3 is a novel member of ShK toxin family, and has the intrinsic ability to induce neuro- and cardio-protective effects or cause cardiac toxicity, according to its effective concentration.

Rhodol Derivatives as Selective Fluorescent Probes for the Detection of HgII Ions and the Bioimaging of Hypochlorous Acid.

Invited for this month's cover picture is the group of Professor Keith Man-Chung Wong from the Southern University of Science and Technology (P.R. China). The cover picture illustrates a novel rhodol-based fluorescence probe from the structural combination of rhodamine and fluorescein motifs. Read the full text of their Full Paper at 10.1002/open.201700154.

Rhodol Derivatives as Selective Fluorescent Probes for the Detection of HgII Ions and the Bioimaging of Hypochlorous Acid.

Two sensors, 1 with a spirolactone group and 2 with a spirolactam group containing a phenyl isothiocyanate moiety, based on rhodol, were designed and synthesized in order to obtain materials with excellent optical properties for the detection of environmentally and biologically important Hg2+ and hypochlorous acid (HClO) ions. The crystal structure of 1 revealed two moieties, a rhodamine-like portion with a spirolactone and a fluorescein-like portion without a spirolactone. In the absence of analyte, 1 produced an optical output with a maximum absorption and emission at 475 and 570 nm, respectively, which was attributed to the fluorescein-like moiety without a spirolactone. In contrast, the rhodamine-like moiety containing a spirolactone was activated by the addition of H+ or Hg2+ ions, and 1 yielded new absorption and emission peaks at 530 and 612 nm, respectively. Further functionalization with a phenyl isothiocyanate group afforded 2, a fluorescent probe for HClO. High selectivity and sensitivity towards the hypochlorite ion were anticipated, owing to the stoichiometric and irreversible formation of a thiosemicarbazide group, which led to dramatic fluorescence responses. With good functionality at physiological pH, probe 2 was successfully used to image HClO in HeLa cells.

A Molecular Chameleon with Fluorescein and Rhodamine Spectroscopic Behaviors.

A new class of fluorescein/rhodamine hybrids with two spirolactone rings was reported to exhibit dual-output fluorescent behaviors independently. Isolation and characterization for two diastereomers, trans-RhOH and cis-RhOH, have been made and their X-ray crystal structures determined. In a basic environment, the spirolactone ring on the hydroxyl side will be opened to give a fluorescein-like optical output with the lowest absorptions at 485 and 530 nm emission. On the other hand, a rhodamine-like optical output, i.e., 528 nm absorption and 575 nm emission, will be switched on by a H(+) or a Hg(2+) ion, attributed to the spirolactone ring opening on the amino side. In a methanol-buffer system with different pH values, the corresponding pKa values for the hydroxyl and amino groups were determined as 5.7 and 2.3, respectively. Selective Hg(2+)-sensing properties have also been discussed, and log Ks values of about 3.60 and 3.73 were determined. Confocal microscopic images of Caenorhabditis elegans incubated with RhOH were found to show enhanced fluorescent intensity with a Hg(2+) ion, demonstrating the potential application of RhOH for in vivo biological imaging.