Rapid phagosome isolation enables unbiased multiomic analysis of human microglial phagosomes.

Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis-and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions. Here, we developed a method for rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various in vitro conditions, and from human brain biopsies, for unbiased multiomic analysis. Phagosome profiling revealed that microglial phagosomes were equipped to sense minute changes in their environment and were highly dynamic. We detected proteins involved in synapse homeostasis, or implicated in brain pathologies, and identified the phagosome as the site where quinolinic acid was stored and metabolized for de novo nicotinamide adenine dinucleotide (NAD+) generation in the cytoplasm. Our findings highlight the central role of phagosomes in microglial functioning in the healthy and diseased brain.

Development of a Signal-integrating Reporter to Monitor Mitochondria-ER Contacts.

Mitochondria-endoplasmic reticulum contact sites (MERCS) serve as hotspots for important cellular processes, including calcium homeostasis, phospholipid homeostasis, mitochondria dynamics, and mitochondrial quality control. MERCS reporters based on complementation of green fluorescent proteins (GFP) fragments have been designed to visualize MERCS in real-time, but we find that they do not accurately respond to changes in MERCS content. Here, we utilize split LacZ complementing fragments to develop the first MERCS reporter system (termed SpLacZ-MERCS) that continuously integrates the MERCS information within a cell and generates a fluorescent output. Our system exhibits good organelle targeting, no artifactual tethering, and effective, dynamic tracking of the MERCS level in single cells. The SpLacZ-MERCS reporter was validated by drug treatments and genetic perturbations known to affect mitochondria-ER contacts. The signal-integrating nature of SpLacZ-MERCS may enable systematic identification of genes and drugs that regulate mitochondria-ER interactions. Our successful application of the split LacZ complementation strategy to study MERCS may be extended to study other forms of interorganellar crosstalk.

Mapping autophagy-related membrane contact site proteins and complexes with AutoMCS Navigator.

In eukaryotic cells, membrane contact sites (MCSs) mediate interactions and communication between organelles by bringing their membranes into close proximity without fusion. These sites play crucial roles in intracellular transport, signal transduction, and the regulation of organelle functions. In a recent study, we compiled data on MCS proteins and complexes from publications to create the MCSdb database. During data compilation, we discovered that many MCSs, their associated proteins, and complexes are highly relevant to macroautophagy/autophagy. To elucidate the role of MCSs in autophagy, we reorganized the autophagy-related MCS proteins and complexes from MCSdb, creating a data map called AutoMCS Navigator. The current version of this map includes 30 complexes and 84 proteins, covering 13 different MCSs and 7 species. Meanwhile, we embedded a dedicated webpage for AutoMCS Navigator on the MCSdb website. This webpage features an orchestrated visual guide that hierarchically displays MCS proteins and complexes involved in autophagy. In summary, our research has developed a user-friendly visual map for querying, browsing, and visualizing detailed information on autophagy-related MCS proteins and complexes. This tool offers researchers easy access to understand autophagy-related MCS structure, assembly, functions, and therapeutic strategies for related diseases. AutoMCS Navigator is freely available at https://cellknowledge.com.cn/mcsdb/autophagy.html.

Extracellular Vesicle Isolation and Mass Spectrometry-Based Proteomic Analysis in Arabidopsis thaliana.

Extracellular vesicles (EVs) can transport various biological materials, including proteins, lipids, nucleic acids, and metabolites, through the unconventional protein secretion (UPS) pathway. Plant EVs can be classified into at least three major types: tetraspanin 8 (TET8)-positive EVs, penetration 1 (PEN1)-positive EVs, and exocyst-positive organelle (EXPO)-derived EVs. However, the research progress of plant EVs has been hindered due to the limitations inherent in EV isolation techniques. Moreover, since previous research on plant EVs has primarily focused on the interaction between plants and microbes, the biogenesis, transport, and secretion of plant EVs remain unexplored. Recent advances in centrifugation methods for extraction of apoplastic wash fluids, combined with mass spectrometry-based proteomic analysis, provide approaches to identify regulators and cargoes of plant EVs and thus serve as an important step for future studies on the biogenesis and function of plant EVs. Here, we illustrate detailed methods of EV isolation and mass spectrometry-based proteomic analysis in Arabidopsis.

Vacuum-Based Agroinfiltration for Transient Gene Expression in Arabidopsis Seedlings.

Agrobacterium-mediated transient expression is a flexible and efficient technique for introducing genes into plants, allowing for rapid and temporary gene expression. Agroinfiltration of Arabidopsis seedlings is a newly developed Agrobacterium-based transient expression system. The expression of target genes and the localization of relevant proteins can be observed within 3 days using this method. In this chapter, we present the detailed protocol for transient transformation in Arabidopsis thaliana seedlings utilizing vacuum infiltration of Agrobacterium. This procedure enables rapid and temporary gene expression by introducing exogenous DNA into Arabidopsis seedlings, particularly in easily accessible tissues such as cotyledons. This protocol provides a detailed description of experimental procedures, including Arabidopsis seedlings cultivation, the preparation of Agrobacterium suspensions, and subsequent steps leading to confocal microscope observation. Through this protocol, researchers can efficiently investigate gene function and subcellular localization in Arabidopsis cotyledons within 8 days in total.

Overexpression of the transcription factor ANAC017 results in a genomes uncoupled phenotype under lincomycin.

Over-expression (OE) lines for the ER-tethered NAC transcription factor ANAC017 displayed de-repression of gun marker genes when grown on lincomycin (lin). RNA-seq revealed that ANAC017OE2 plants constitutively expressed greater than 40% of the genes induced in wild-type with lin treatment, including plastid encoded genes ycf1.2 and the gene cluster ndhH-ndhA-ndhI-ndhG-ndhE-psaC-ndhD, documented as direct RNA targets of GUN1. Genes encoding components involved in organelle translation were enriched in constitutively expressed genes in ANAC017OE2. ANAC017OE resulted in constitutive location in the nucleus and significant constitutive binding of ANAC017 was detected by ChIP-Seq to target genes. ANAC017OE2 lines maintained the ability to green on lin, were more ABA sensitive, did not show photo-oxidative damage after exposure of de-etiolated seedlings to continuous light and the transcriptome response to lin were as much as 80% unique compared to gun1-1. Both double mutants, gun1-1:ANAC017OE and bzip60:ANAC017OE (but not single bzip60), have a gun molecular gene expression pattern and result in variegated and green plants, suggesting that ANAC017OE may act through an independent pathway compared to gun1. Over-expression of ANAC013 or rcd1 did not produce a GUN phenotype or green plants on lin. Thus, constitutive ANAC017OE2 establishes an alternative transcriptional program that likely acts through a number of pathways, that is, maintains plastid gene expression, and induction of a variety of transcription factors involved in reactive oxygen species metabolism, priming plants for lin tolerance to give a gun phenotype.

MpR2R3-MYB2 is a key regulator of oil body formation in Marchantia polymorpha.

MAIN CONCLUSION: MpMYB02, a regulator of marchantin accumulation, also acts as a key regulator of oil body formation. MpMYB02 induces the expression of MpSYP12B and promotes oil body formation, subsequently leading to marchantin accumulation. The oil body observed in Marchantia polymorpha is a cellular organelle surrounded by a unit membrane, accumulating various secondary metabolites such as marchantins and terpenes. We observed that oil body formation is regulated by MpMYB02, a key regulator of marchantin accumulation. In the Mpmyb02 mutant, no oil bodies were observed, although idioblast-like cells were present in the gemma. We introduced MpMYB02-glucocorticoid receptor (GR), a steroid-inducible transcriptional activator, into Mpmyb02 and assessed the effect of dexamethasone (DEX) on oil body formation. Following DEX treatment, transformed liverworts began forming oil bodies within 12 h. During the initial stages of oil body development, we observed the aggregation of small globular structures. DEX treatment upregulated several genes implicated in oil body formation, including MpSYP12B. Our findings underscore that MpMYB02 plays a crucial role not only in marchantin accumulation but also in oil body formation.

Artificial targeting of autophagy components to mitochondria reveals both conventional and unconventional mitophagy pathways.

Macroautophagy/autophagy enables lysosomal degradation of a diverse array of intracellular material. This process is essential for normal cellular function and its dysregulation is implicated in many diseases. Given this, there is much interest in understanding autophagic mechanisms of action in order to determine how it can be best targeted therapeutically. In mitophagy, the selective degradation of mitochondria via autophagy, mitochondria first need to be primed with signals that allow the recruitment of the core autophagy machinery to drive the local formation of an autophagosome around the target mitochondrion. To determine how the recruitment of different core autophagy components can drive mitophagy, we took advantage of the mito-QC mitophagy assay (an outer mitochondrial membrane-localized tandem mCherry-GFP tag). By tagging autophagy proteins with an anti-mCherry (or anti-GFP) nanobody, we could recruit them to mitochondria and simultaneously monitor levels of mitophagy. We found that targeting ULK1, ATG16L1 and the different Atg8-family proteins was sufficient to induce mitophagy. Mitochondrial recruitment of ULK1 and the Atg8-family proteins induced a conventional mitophagy pathway, requiring RB1CC1/FIP200, PIK3C3/VPS34 activity and ATG5. Surprisingly, the mitophagy pathway upon recruitment of ATG16L1 proceeded independently of ATG5, although it still required RB1CC1 and PIK3C3/VPS34 activity. In this latter pathway, mitochondria were alternatively delivered to lysosomes via uptake into early endosomes.

Organelle-Targeting Polymer Dots Exhibiting Thermally Activated Delayed Fluorescence for Subcellular Imaging.

Selective imaging of specific subcellular structures provides valuable information about the cellular microenvironment. Materials exhibiting thermally activated delayed fluorescence (TADF) are rapidly emerging as metal-free probes with long-lived emission for intracellular time-gated imaging applications. Polymers incorporating TADF emitters can self-assemble into luminescent nanoparticles, termed polymer dots (Pdots), and this strategy enables them to circumvent the limitations of commercial organelle trackers and small molecule TADF emitters. In this study, diblock copolymers comprised of a hydrophilic block containing organelle-targeting monomers and a hydrophobic TADF-active block were synthesized by ring-opening metathesis polymerization (ROMP). Oxanorbornene-based monomers incorporating morpholine and triphenylphosphonium groups for lysosome and mitochondria targeting, respectively, were also synthesized. ROMP by sequential addition yielded well-defined diblock copolymers with dispersities <1.28. To analyze the effect of tuning the hydrophilic corona on cellular viability and uptake, we prepared Pdots with poly(ethylene glycol) (PEG) and bis-guanidinium (BGN) coronas, resulting in limited and efficient cellular uptake, respectively. Red-emissive Pdots with BGN-based coronas and organelle-targeting functionality were obtained with quantum yields up to 12% in water under air. Colocalization analysis confirmed that lysosome and mitochondria labeling in live HeLa cells was accomplished within 2 h of incubation, affording Pearson's correlation coefficients of 0.37 and 0.70, respectively. The potential application of these Pdots for time-resolved imaging is highlighted by a proof of concept using time-gated spectroscopy, which effectively separates the delayed emission of the TADF Pdots from the background autofluorescence of biological serum.

Metabolic Engineering of Candida tropicalis for the De Novo Synthesis of ß-Ionone.

ß-ionone, a norisoprenoid, is a natural aromatic compound derived from plants, which displays various biological activities including anticancer, antioxidant and deworming properties. Due to its large biomass and strong environmental tolerance, the nonconventional oleaginous yeast Candida tropicalis was selected to efficiently synthesize ß-ionone. We initially investigated the capacity of the cytoplasm and subcellular compartments to synthesize ß-ionone independently. Subsequently, through adaptive screening of enzymes, functional identification of subcellular localization signal peptides and subcellular compartment combination strategies, a titer of 152.4 mg/L of ß-ionone was achieved. Finally, directed evolution of rate-limiting enzyme and overexpression of key enzymes were performed to enhance ß-ionone production. The resulting titer was 400.5 mg/L in shake flasks and 730 mg/L in a bioreactor. This study demonstrates the first de novo synthesis of ß-ionone in C. tropicalis, providing a novel cellular chassis for terpenoid fragrances with considerable industrial potential.

Mitochondria-derived vesicles: potential nano-batteries to recharge the cellular powerhouse.

Mitochondria dysfunction is increasingly recognized as a critical factor in various pathogenic processes. The mechanism governing mitochondrial quality control serves as an adaptive response, ensuring the preservation of mitochondrial morphology, quantity, and overall function, crucial for cell survival. The generation of mitochondria-derived vesicles (MDVs) is one of the processes of mitochondrial quality control. Recent literature has suggested MDV heterogeneity; however, the detailed characteristics of various MDV subtypes still need to be studied better. Recent studies have shown that MDVs also play a role in inter-organelle communication for mitochondria besides quality control. For instance, Hazan et al. demonstrated that functional mitochondria from Saccharomyces cerevisiae release vesicles independent of the fission machinery. These vesicles, falling within the typical size range of MDVs, were selectively loaded with mitochondrial proteins, especially with functional ATP synthase subunits. Intriguingly, these MDVs maintained membrane potential and could generate ATP. Moreover, MDVs could fuse with naïve mitochondria, transferring their ATP generation machinery. Lastly, this study revealed a potential delivery mechanism of ATP-producing vesicles, presenting a promising avenue to rejuvenate ATP-deficient mitochondria. Overall, this study unveils a novel mechanism for inter-organelle communication by vesicles, which is crucial for maintaining cellular homeostasis and could also be important in pathological conditions.

Biomolecular condensates with liquid properties formed during viral infections.

During a viral infection, several membraneless compartments with liquid properties are formed. They can be of viral origin concentrating viral proteins and nucleic acids, and harboring essential stages of the viral cycle, or of cellular origin containing components involved in innate immunity. This is a paradigm shift in our understanding of viral replication and the interaction between viruses and innate cellular immunity.

Visualization and Quantification of Organelle Axonal Transport in Cultured Neurons.

The specialized function and extreme geometry of neurons necessitates a unique reliance upon long-distance microtubule-based transport. Appropriate trafficking of axonal cargos by motor proteins is essential for establishing circuitry during development and continuing function throughout a lifespan. Visualizing and quantifying cargo movement provides valuable insight into how axonal organelles are replenished, recycled, and degraded during the dynamic dance of outgoing and incoming axonal traffic. Long-distance axonal trafficking is of particular importance as it encompasses a pathway commonly disrupted in developmental and degenerative disease states. Here, we describe neuronal organelles and outline methods for live imaging and quantifying their movement throughout the axon via transient expression of fluorescently labeled organelle markers. This resource provides recommendations for target proteins/domains and appropriate acquisition time scales for visualizing distinct neuronal cargos in cultured neurons derived from human induced pluripotent stem cells (iPSCs) and primary rat neurons.

Intermediate filaments spatially organize intracellular nanostructures to produce the bright structural blue of ribbontail stingrays across ontogeny.

In animals, pigments but also nanostructures determine skin coloration, and many shades are produced by combining both mechanisms. Recently, we discovered a new mechanism for blue coloration in the ribbontail stingray Taeniura lymma, a species with electric blue spots on its yellow-brown skin. Here, we characterize finescale differences in cell composition and architecture distinguishing blue from non-blue regions, the first description of elasmobranch chromatophores and the nanostructures responsible for the stingray's novel structural blue, contrasting with other known mechanisms for making nature's rarest color. In blue regions, the upper dermis comprised a layer of chromatophore units -iridophores and melanophores entwined in compact clusters framed by collagen bundles- this structural stability perhaps the root of the skin color's robustness. Stingray iridophores were notably different from other vertebrate light-reflecting cells in having numerous fingerlike processes, which surrounded nearby melanophores like fists clenching a black stone. Iridophores contained spherical iridosomes enclosing guanine nanocrystals, suspended in a 3D quasi-order, linked by a cytoskeleton of intermediate filaments. We argue that intermediate filaments form a structural scaffold with a distinct optical role, providing the iridosome spacing critical to produce the blue color. In contrast, black-pigmented melanosomes within melanophores showed space-efficient packing, consistent with their hypothesized role as broadband-absorbers for enhancing blue color saturation. The chromatophore layer's ultrastructure was similar in juvenile and adult animals, indicating that skin color and perhaps its ecological role are likely consistent through ontogeny. In non-blue areas, iridophores were replaced by pale cells, resembling iridophores in some morphological and nanoscale features, but lacking guanine crystals, suggesting that the cell types arise from a common progenitor cell. The particular cellular associations and structural interactions we demonstrate in stingray skin suggest that pigment cells induce differentiation in the progenitor cells of iridophores, and that some features driving color production may be shared with bony fishes, although the lineages diverged hundreds of millions of years ago and the iridophores themselves differ drastically.

Actin cytoskeleton and associated myosin motors within the renal epithelium.

This review highlights the complex membrane architectures and organelles observed along the renal tubular segments through careful review of ultrastructural and physiological studies published over the past several decades. We also showcase the vital role(s) played by the actin cytoskeleton and actin associated myosin motor proteins in regulating cell type-specific physiological functions within cells of the renal epithelium. The purpose of this review is to provide a fresh conceptual framework to explain the structure-function relationships that exist between the actin cytoskeleton, organelle structure, and cargo transport within the mammalian kidney. We believe that with recent advances in technologies to visualize the actin cytoskeleton and associated proteins within intact kidneys, it is imperative to reimagine the functional role(s) for these proteins in situ, which will provide a rationale for their unique, cell type specific function(s), necessary to build and maintain complex physiological processes.

De novo assembly and comprehensive analysis of the mitochondrial genome of Taxus wallichiana reveals different repeats mediate recombination to generate multiple conformations.

Taxus plants are the exclusive source of paclitaxel, an anticancer drug with significant medicinal and economic value. Interspecies hybridization and gene introgression during evolution have obscured distinctions among Taxus species, complicating their phylogenetic classification. While the chloroplast genome of Taxus wallichiana, a widely distributed species in China, has been sequenced, its mitochondrial genome (mitogenome) remains uncharacterized.We sequenced and assembled the T. wallichiana mitogenome using BGI short reads and Nanopore long reads, facilitating comparisons with other gymnosperm mitogenomes. The T. wallichiana mitogenome spanning 469,949 bp, predominantly forms a circular configuration with a GC content of 50.51%, supplemented by 3 minor configurations mediated by one pair of LRs and two pairs of IntRs. It includes 32 protein-coding genes, 7 tRNA genes, and 3 rRNA genes, several of which exist in multiple copies.We detailed the mitogenome's structure, codon usage, RNA editing, and sequence migration between organelles, constructing a phylogenetic tree to elucidate evolutionary relationships. Unlike typical gymnosperm mitochondria, T. wallichiana shows no evidence of mitochondrial-plastid DNA transfer (MTPT), highlighting its unique genomic architecture. Synteny analysis indicated extensive genomic rearrangements in T. wallichiana, likely driven by recombination among abundant repetitive sequences. This study offers a high-quality T. wallichiana mitogenome, enhancing our understanding of gymnosperm mitochondrial evolution and supporting further cultivation and utilization of Taxus species.

An innovative dual-organelle targeting NIR fluorescence probe for detecting hydroxyl radicals in biosystem and inflammation models.

The hydroxyl radical (OH) is highly reactive and plays a significant role in a number of physiological and pathological processes within biosystems. Aberrant changes in the level of hydroxyl radical are associated with many disorders including tumor, inflammatory and cardiovascular diseases. Thus, detecting reactive oxygen species (ROS) in biological systems and imaging them is highly significant. In this work, a novel fluorescent probe (HR-DL) has been developed, targeting two organelles simultaneously. The probe is based on a coumarin-quinoline structure and exhibits high selectivity and sensitivity towards hydroxyl radicals (OH). When reacting with OH, the hydrogen abstraction process released its long-range π-conjugation and ICT processes, leading to a substantial red-shift in wavelength. This probe has the benefits of good water solubility (in its oxidative state), short response time (within 10 s), and unique dual lysosome/mitochondria targeting capabilities. It has been applied for sensing OH in biosystem and inflammation mice models.

Folic Acid-Functionalized ß-Cyclodextrin for Delivery of Organelle-Targeted Peptide Chemotherapeutics in Cancer.

Recent emphasis on the design of drug delivery systems typically involves the effective transport of a pharmaceutical substance to the disease site with the desired therapeutic efficacy and minimal cytotoxicity. Organelle-targeted peptides have become an integral part of designing an important class of prodrug/prodrug assemblies for new supramolecular therapeutics owing to their favorable biocompatibility, synthetic ease, tunability of their aggregation behavior, and desired functionalization for site-specificity. However, it is still limited due to the low selectivity. We designed a folic acid-functionalized ß-cyclodextrin (FA-CD) as a delivery platform for specific and selective delivery of organelle-targeted (such as microtubule, lysosome, and mitochondria) peptide chemotherapeutics to the folate receptor (FR) overexpressing cancer cell lines. Low toxicity was found for the FA-CD and organelle-targeted peptide inclusion complex in FR-negative normal cells, but superior inhibition of tumor growth with no in vivo toxicity was found for the inclusion complex in the xenograft tumor model.

Assembly and comparative analysis of the complete mitochondrial genome of Vaccinium carlesii Dunn.

Vaccinium L. is an important fruit tree with nutritional, medicinal, and ornamental values. However, the mitochondrial (mt) genome of Vaccinium L. remains largely unexplored. Vaccinium carlesii Dunn is an endemic wild resource in China, which is crucial for blueberry breeding. The V. carlesii mt genomes were sequenced using Illumina and Nanopore, which total length was 636,904 bp with 37 protein coding genes, 20 tRNA genes, and three rRNA genes. We found four pairs of long repeat fragments homologous recombination mediated the generation of substructures in the V. carlesii mt genome. We predicted 383 RNA editing sites, all converting cytosine (C) to uracil (U). According to the phylogenetic analysis, V. carlesii and V. macrocarpon of the Ericaceae exhibited the closest genetic relationship. This study provides a theoretical basis for understanding the evolution of higher plants, species classification and identification, and will also be useful for further utilization of Vaccinium germplasm resources.

HT-2 toxin impairs porcine oocyte in vitro maturation through disruption of endomembrane system.

HT-2 toxin is a type of mycotoxin which is shown to affect gastric and intestinal lesions, hematopoietic and immunosuppressive effects, anorexia, lethargy, nausea. Recently, emerging evidences indicate that HT-2 also disturbs the reproductive system. In this study, we investigated the impact of HT-2 toxin exposure on the organelles of porcine oocytes. Our results found that the abnormal distribution of endoplasmic reticulum increased after HT-2 treatment, with the perturbation of ribosome protein RPS3 and GRP78 expression; Golgi apparatus showed diffused localization pattern and GM130 localization was also impaired, thereby affecting the Rab10-based vesicular transport; Due to the impairment of ribosomes, ER, and Golgi apparatus, the protein supply to lysosomes was hindered, resulting in lysosomal damage, which further disrupted the LC3-based autophagy. Moreover, the results indicated that the function and distribution of mitochondria were also affected by HT-2 toxin, showing with fragments of mitochondria, decreased TMRE and ATP level. Taken together, our study suggested that HT-2 toxin exposure induces damage to the organelles for endomembrane system, which further inhibited the meiotic maturation of porcine oocytes.