The entry of unclosed autophagosomes into vacuoles and its physiological relevance.

It is widely stated in the literature that closed mature autophagosomes (APs) fuse with lysosomes/vacuoles during macroautophagy/autophagy. Previously, we showed that unclosed APs accumulated as clusters outside vacuoles in Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. However, the fate of such unclosed APs remains unclear. In this study, we used a combination of cellular and biochemical approaches to show that unclosed double-membrane APs entered vacuoles and formed unclosed single-membrane autophagic bodies after prolonged nitrogen starvation or rapamycin treatment. Vacuolar hydrolases, vacuolar transport chaperon (VTC) proteins, Ypt7, and Vam3 were all involved in the entry of unclosed double-membrane APs into vacuoles in Vps21-mutant cells. Overexpression of the vacuolar hydrolases, Pep4 or Prb1, or depletion of most VTC proteins promoted the entry of unclosed APs into vacuoles in Vps21-mutant cells, whereas depletion of Pep4 and/or Prb1 delayed the entry into vacuoles. In contrast to the complete infertility of diploid cells of typical autophagy mutants, diploid cells of Vps21 mutant progressed through meiosis to sporulation, benefiting from the entry of unclosed APs into vacuoles after prolonged nitrogen starvation. Overall, these data represent a new observation that unclosed double-membrane APs can enter vacuoles after prolonged autophagy induction, most likely as a survival strategy.

VAMP4 regulates insulin levels by targeting secretory granules to lysosomes.

Insulin levels are essential for the maintenance of glucose homeostasis, and deviations lead to pathoglycemia or diabetes. However, the metabolic mechanism controlling insulin quantity and quality is poorly understood. In pancreatic ß cells, insulin homeostasis and release are tightly governed by insulin secretory granule (ISG) trafficking, but the required regulators and mechanisms are largely unknown. Here, we identified that VAMP4 controlled the insulin levels in response to glucose challenge. VAMP4 deficiency led to increased blood insulin levels and hyperresponsiveness to glucose. In ß cells, VAMP4 is packaged into immature ISGs (iISGs) at trans-Golgi networks and subsequently resorted to clathrin-coated vesicles during granule maturation. VAMP4-positive iISGs and resorted vesicles then fuse with lysosomes facilitated by a SNARE complex consisting of VAMP4, STX7, STX8, and VTI1B, which ensures the breakdown of excess (pro)insulin and obsolete materials and thus maintenance of intracellular insulin homeostasis. Thus, VAMP4 is a key factor regulating the insulin levels and a potential target for the treatment of diabetes.

Rapid isolation and cryo-EM characterization of synaptic vesicles from mammalian brain.

Synaptic vesicles (SVs) store and release neurotransmitters at chemical synapses. Precise regulation of SV trafficking, exocytosis and endocytosis is crucial for neural transmission. Biochemical characterization of SVs, which is essential for research into neurotransmitter uptake and release, requires effective in vitro isolation methods. Here, we describe an improved and simple purification protocol for isolating SVs from mouse brain within 6 h, achieving a yield of approximately 0.4 mg of SVs per single brain. The use of track-etch membrane filtration and iodixanol cushion ensured the uniform morphology of SVs and low contaminants in the sample. Cryo-electron microscopy was used to show that the in vitro isolated SVs retained intact membrane-associated proteins, and observation of SVs in hippocampal neurons using cryo-electron tomography confirmed the abundance of protein coating. Thus, our protocol allows effective isolation of SVs from small volumes of mammalian brain tissue, and the properties of the isolated SVs are close to those in vivo, making them suitable for biochemical analysis.

Host cytoskeletal vimentin serves as a structural organizer and an RNA-binding protein regulator to facilitate Zika viral replication.

Emerging microbe infections, such as Zika virus (ZIKV), pose an increasing threat to human health. Investigations on ZIKV replication have revealed the construction of replication complexes (RCs), but the role of cytoskeleton in this process is largely unknown. Here, we investigated the function of cytoskeletal intermediate filament protein vimentin in the life cycle of ZIKV infection. Using advanced imaging techniques, we uncovered that vimentin filaments undergo drastic reorganization upon viral protein synthesis to form a perinuclear cage-like structure that embraces and concentrates RCs. Genetic removal of vimentin markedly disrupted the integrity of RCs and resulted in fragmented subcellular dispersion of viral proteins. This led to reduced viral genome replication, viral protein production, and release of infectious virions, without interrupting viral binding and entry. Furthermore, mass spectrometry and RNA-sequencing screens identified interactions and interplay between vimentin and hundreds of endoplasmic reticulum (ER)-resident RNA-binding proteins. Among them, the cytoplasmic-region of ribosome receptor binding protein 1, an ER transmembrane protein that directly binds viral RNA, interacted with and was regulated by vimentin, resulting in modulation of ZIKV replication. Together, the data in our work reveal a dual role for vimentin as a structural element for RC integrity and as an RNA-binding-regulating hub during ZIKV infection, thus unveiling a layer of interplay between Zika virus and host cell.

Vam6 reduces iNKT cell function in tumor via modulating AMPK/mTOR pathways.

Activation of mTORC1 is essential for anti-tumor function of iNKT cells. The mechanisms underlying impaired mTORC1 activation in intratumoral iNKT cells remain unclear. Via generating Vam6+/- mice and using flow cytometry, image approach, and RNA sequencing, we studied the role of Vam6 in controlling mTORC1 activation and intratumoral iNKT cell functions. Here, we find that increased Vam6 expression in intratumoral iNKT cells leads to impaired mTORC1 activation and IFN-γ production. Mechanistically, Vam6 in iNKT cells is essential for Rab7a-Vam6-AMPK complex formation and thus for recruitment of AMPK to lysosome to activate AMPK, a negative regulator of mTORC1. Additionally, Vam6 relieves inhibitory effect of VDAC1 on Rab7a-Vam6-AMPK complex formation at mitochondria-lysosome contact site. Moreover, we report that lactic acid produced by tumor cells increases Vam6 expression in iNKT cells. Given the key roles of increased Vam6 in promoting AMPK activation in intratumoral iNKT cells, reducing Vam6 expression signifificantly enhances the mTORC1 activation in intratumoral iNKT cells as well as their anti-tumor effificacy. Together, we propose Vam6 as a target for iNKT cell-based immunotherapy.

Sanxiapeptin, a linear pentapeptide from Penicillium oxalicum, inhibited the growth of citrus green mold.

Penicillium oxalicum has been used as a biocontrol fungus in agriculture for many years, but the antimicrobial substances are still uncertain. Herein, we isolated a linear peptide named Sanxiapeptin in the culture broth of Penicillium oxalicum SG-4 collecting from the Three Gorges riparian zone. Sanxiapeptin exhibited potent inhibitory effect on citrus green mold Penicillium digitatum, the main fungi responsible for postharvest decay. Sanxiapeptin was elucidated as composing of five amino acids, which were ß-amino-α-methoxybutyric acid (Amoba), N-Me-l-Thr, d-Thr, N-Me-l-Val and l-Ser. By analyzing three chemically synthesized oligopeptides with similar structures, we found that the first amino acid of Amoba was crucial to the antifungal activity, as was the methylation of peptide bond. Sanxiapeptin may act as an antimicrobial agent by affecting the function of cell membranes or walls. The antimicrobial spectrum, safety and stability analysis supported that Sanxiapeptin was a promising antifungal agent for citrus preservation.

Metabolite profiling reveals comprehensive effects of Chaetomium globosum on citrus preservation.

The huge economic loss of citrus fruit after harvest called for safe and efficient preservatives, as chemically synthesized agents threatened the environment and human health. Herein a biocontrol fungus Chaetomium globosum QY-1 near the orchard in riparian area was identified to have antimicrobial, antioxidant and tyrosinase inhibition activity, which meets the requirements of an ideal preservative. Metabolite profiling based on bioassay-guided fractionation was carried out, and eight polyketones were determined by MS and NMR. The most abundant CheA exhibited strong inhibition to Penicillium digitatum, the main pathogen caused citrus fruit rot. Among these metabolites, Epicoccone and Epicoccolide B showed higher antioxidant activity, while Epicoccone and CheA had higher tyrosinase inhibitory activity. All the activities were close to or even better than the positive control (Vc; glutathione; Vc and arbutin; Bellkute), implying that the metabolites of C. globosum had comprehensive effects as natural preservatives.

Cryogenic superresolution correlative light and electron microscopy of vitreous sections.

Fluorescence microscopy and electron microscopy complement each other as the former provides labelling and localisation of specific molecules and target structures while the latter possesses excellent revolving power of fine structure in context. These two techniques can combine as correlative light and electron microscopy (CLEM) to reveal the organisation of materials within the cell. Frozen hydrated sections allow microscopic observations of cellular components in situ in a near-native state and are compatible with superresolution fluorescence microscopy and electron tomography if sufficient hardware and software support is available and a well-designed protocol is followed. The development of superresolution fluorescence microscopy greatly increases the precision of fluorescence annotation of electron tomograms. Here, we provide detailed instructions on how to perform cryogenic superresolution CLEM on vitreous sections. From fluorescence-labelled cells to high pressure freezing, cryo-ultramicrotomy, cryogenic single-molecule localisation microscopy, cryogenic electron tomography and image registration, electron tomograms with features of interest highlighted by superresolution fluorescence signals are expected to be obtained.

ASC deglutathionylation is a checkpoint for NLRP3 inflammasome activation.

Activation of NLRP3 inflammasome is precisely controlled to avoid excessive activation. Although multiple molecules regulating NLRP3 inflammasome activation have been revealed, the checkpoints governing NLRP3 inflammasome activation remain elusive. Here, we show that activation of NLRP3 inflammasome is governed by GSTO1-promoted ASC deglutathionylation in macrophages. Glutathionylation of ASC inhibits ASC oligomerization and thus represses activation of NLRP3 inflammasome in macrophages, unless GSTO1 binds ASC and deglutathionylates ASC at ER, under control of mitochondrial ROS and triacylglyceride synthesis. In macrophages expressing ASCC171A, a mutant ASC without glutathionylation site, activation of NLRP3 inflammasome is GSTO1 independent, ROS independent, and signal 2 less dependent. Moreover, AscC171A mice exhibit NLRP3-dependent hyperinflammation in vivo. Our results demonstrate that glutathionylation of ASC represses NLRP3 inflammasome activation, and GSTO1-promoted ASC deglutathionylation at ER, under metabolic control, is a checkpoint for activating NLRP3 inflammasome.

Molecular-scale axial localization by repetitive optical selective exposure.

We introduce an axial localization with repetitive optical selective exposure (ROSE-Z) method for super-resolution imaging. By using an asymmetric optical scheme to generate interference fringes, a <2 nm axial localization precision was achieved with only ~3,000 photons, which is an approximately sixfold improvement compared to previous astigmatism methods. Nanoscale three-dimensional and two-color imaging was demonstrated, illustrating how this method achieves superior performance and facilitates the investigation of cellular nanostructures.

Cryogenic superresolution correlative light and electron microscopy on the frontier of subcellular imaging.

Electron microscopy (EM) reveals cellular ultrastructure at high definition but faces the challenges of identification of specific subcellular structures and localization of specific macromolecules, whereas fluorescence microscopy (FM) can label and localize specific molecules in cells. Correlative light and electron microscopy (CLEM) combines the advantages of both microscopic techniques. Imaging vitreous hydrated samples at cryogenic temperatures using CLEM enables observations of cellular components of interest and their cellular context in a near-native state. This cryo-CLEM approach is further strengthened by incorporation of superresolution fluorescence microscopy, which can precisely pinpoint targets on electron micrographs. Cryogenic superresolution correlative light and electron microscopy (csCLEM) is an emerging and promising imaging technique that is expected to unveil its full power in ultrastructural studies. The present review describes the logic and principles behind this technique, how the method is implemented, the prospects, and the challenges.

Chiral Os(II) Polypyridyl Complexes as Enantioselective Nuclear DNA Imaging Agents Especially Suitable for Correlative High-Resolution Light and Electron Microscopy Studies.

The high-resolution technique transmission electron microscopy (TEM), with OsO4 as the traditional fixative, is an essential tool for cell biology and medicine. Although OsO4 has been extensively used, it is far from perfect because of its high volatility and toxicity. Os(II) polypyridyl complexes like [Os(phen)2(dppz)]2+ (phen = 1,10-phenanthroline; dppz = dipyridophenazine) are not only the well-known molecular DNA "light-switches" but also the potential ideal candidates for TEM studies. Here, we report that the cell-impermeable cationic [Os(phen)2(dppz)]2+ can be preferentially delivered into the live-cell nucleus through ion-pairing with chlorophenolate counter-anions, where it functions as an unparalleled enantioselective nuclear DNA imaging reagent especially suitable for correlative light and electron microscopy (CLEM) studies in both living and fixed cells, which can clearly visualize chromosome aggregation and decondensation during mitosis simultaneously. We propose that the chiral Os(II) polypyridyl complexes can be used as a distinctive group of enantioselective high-resolution CLEM imaging probes for live-cell nuclear DNA studies.

Spatial distribution of IL4 controls iNKT cell-DC crosstalk in tumors.

The spatiotemporal distribution of cytokines orchestrates immune responses in vivo, yet the underlying mechanisms remain to be explored. We showed here that the spatial distribution of interleukin-4 (IL4) in invariant natural killer T (iNKT) cells regulated crosstalk between iNKT cells and dendritic cells (DCs) and controlled iNKT cell-mediated T-helper type 1 (Th1) responses. The persistent polarization of IL4 induced by strong lipid antigens, that is, α-galactosylceramide (αGC), caused IL4 accumulation at the immunological synapse (IS), which promoted the activation of the IL4R-STAT6 (signal transducer and activator of transcription 6) pathway and production of IL12 in DCs, which enhanced interferon-γ (IFNγ) production in iNKT cells. Conversely, the nonpolarized secretion of IL4 induced by Th2 lipid antigens with a short or unsaturated chain was incapable of enhancing this iNKT cell-DC crosstalk and thus shifted the immune response to a Th2-type response. The nonpolarized secretion of IL4 in response to Th2 lipid antigens was caused by the degradation of Cdc42 in iNKT cells. Moreover, reduced Cdc42 expression was observed in tumor-infiltrating iNKT cells, which impaired IL4 polarization and disturbed iNKT cell-DC crosstalk in tumors.

Author Correction: Molecular resolution imaging by repetitive optical selective exposure.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular resolution imaging by repetitive optical selective exposure.

We introduce an interferometric single-molecule localization method for super-resolution fluorescence microscopy. Fluorescence molecules are located by the intensities of multiple excitation patterns of an interference fringe, providing around a twofold improvement in the localization precision compared with the conventional imaging with the same photon budget. We demonstrate this technique by resolving nanostructures down to 5 nm in size over a large 25 × 25 µm2 field of view.

Presynaptic Endosomal Cathepsin D Regulates the Biogenesis of GABAergic Synaptic Vesicles.

Presynaptic endosomes reportedly participate in synaptic vesicle (SV) recycling. However, it remains unclear whether they differentially regulate SV biogenesis and synaptic transmission in different types of synapses and how they are implicated in diseases. Using cryo-electron tomography and endocytic tracing, we uncover different endocytic modes and dynamics associated with distinct SV morphology between glutamatergic and GABAergic synapses. We further find that cathepsin D (CatD), a lysosomal storage disease (LSD) protein, is selectively located in GABAergic presynaptic endosomes. Inactivation of CatD results in enlarged presynaptic endosomes, reduces the readily releasable pool, and impairs synaptic transmission in GABAergic, but not glutamatergic, synapses. Moreover, CatD-deficient mice exhibit hyperactivity and increased sensitivity to seizure, mimicking epileptic behavior in CatD-related LSD patients. These data reveal an important role for presynaptic endosomal CatD in regulating GABAergic SV biogenesis and provide mechanistic insights for understanding the synaptic pathology and behavioral defects in CatD-associated LSD.

Rab5-dependent autophagosome closure by ESCRT.

In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17-Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17-Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.

A coumarin analogue NFA from endophytic Aspergillus fumigatus improves drought resistance in rice as an antioxidant.

BACKGROUND: Drought and its resulting oxidative damage are the major yield limiting factors for crops in arid and semi-arid regions. Recent studies have found that endophytic fungi coexisting in plants can alleviate biotic or abiotic damage to plant growth and development. In order to screen for the endophytes associated with drought stress, 12 strains of endophytic fungi with high antioxidant activity isolated from riparian plants Myricaria laxiflora were evaluated for their effects in rice by the crude extracts. RESULTS: Of the 12 endophytic fungi, Aspergillus fumigatus SG-17 functioned most effectively, with the crude extract exhibiting relatively higher antioxidant capacity both in vivo and in vitro. The subsequent MS and NMR analysis showed that the primary substance responsible for the antioxidant activity in the extract was (Z)-N-(4-hydroxystyryl) formamide (NFA), an analogue of coumarin. Enzyme activity assay in nerve cells SH-SY5Y showed that NFA could maintain the membrane integrity and regulate the antioxidase activity under oxidative stress. In rice suffering drought stress, NFA effectively alleviated the harm by regulating the contents of NADPH oxidases, antioxidants and heat shock proteins, all of which are closely related with the reactive oxygen species pathway. CONCLUSION: These findings indicated that some endophytes from plants often subjected to flooding and oxidative stress could enhance drought resistance by producing compounds such as NFA to regulate the oxidative pathway.

An Upconversion Nanoparticle Enables Near Infrared-Optogenetic Manipulation of the Caenorhabditis elegans Motor Circuit.

Near-infrared (NIR) light penetrates tissue deeply, but its application to motor behavior stimulation has been limited by the lack of known genetic NIR light-responsive sensors. We designed and synthesized a Yb3+/Er3+/Ca2+-based lanthanide-doped upconversion nanoparticle (UCNP) that effectively converts 808 nm NIR light to green light emission. This UCNP is compatible with Chrimson, a cation channel activated by green light; as such, it can be used in the optogenetic manipulation of the motor behaviors of Caenorhabditis elegans. We show that this UCNP effectively activates Chrimson-expressing, inhibitory GABAergic motor neurons, leading to reduced action potential firing in the body wall muscle and resulting in locomotion inhibition. The UCNP also activates the excitatory glutamatergic DVC interneuron, leading to potentiated muscle action potential bursts and active reversal locomotion. Moreover, this UCNP exhibits negligible toxicity in neural development, growth, and reproduction, and the NIR energy required to elicit these behavioral and physiological responses does not activate the animal's temperature response. This study shows that UCNP provides a useful integrated optogenetic toolset, which may have wide applications in other experimental systems.