Potential vs challenges of expanding the protein universe with genetic code expansion in eukaryotic cells.

Following decades of innovation and perfecting, genetic code expansion has become a powerful tool for in vivo protein modification. Some of the major hurdles that had to be overcome include suboptimal performance of GCE-specific translational components in host systems, competing cellular processes, unspecific modification of the host proteome and limited availability of codons for reassignment. Although strategies have been developed to overcome challenges, there is critical need for further improvement. Here we discuss the current state-of-the-art in genetic code expansion technology and the issues that still need to be addressed to unleash the full potential of this method in eukaryotic cells.

Cell-cycle-dependent mRNA localization in P-bodies.

Understanding the dynamics of RNA targeting to membraneless organelles is essential to disentangle their functions. Here, we investigate how P-bodies (PBs) evolve during cell-cycle progression in HEK293 cells. PB purification across the cell cycle uncovers widespread changes in their RNA content, partly uncoupled from cell-cycle-dependent changes in RNA expression. Single-molecule fluorescence in situ hybridization (FISH) shows various mRNA localization patterns in PBs peaking in G1, S, or G2, with examples illustrating the timely capture of mRNAs in PBs when their encoded protein becomes dispensable. Rather than directly reflecting absence of translation, cyclic mRNA localization in PBs can be controlled by RBPs, such as HuR in G2, and by RNA features. Indeed, while PB mRNAs are AU rich at all cell-cycle phases, they are specifically longer in G1, possibly related to post-mitotic PB reassembly. Altogether, our study supports a model where PBs are more than a default location for excess untranslated mRNAs.

Artificial Compartments Encapsulating Enzymatic Reactions: Towards the Construction of Artificial Organelles.

Cells have used compartmentalization to implement complex biological processes involving thousands of enzyme cascade reactions. Enzymes are spatially organized into the cellular compartments to carry out specific and efficient reactions in a spatiotemporally controlled manner. These compartments are divided into membrane-bound and membraneless organelles. Mimicking such cellular compartment systems has been a challenge for years. A variety of artificial scaffolds, including liposomes, polymersomes, proteins, nucleic acids, or hybrid materials have been used to construct artificial membrane-bound or membraneless compartments. These artificial compartments may have great potential for applications in biosynthesis, drug delivery, diagnosis and therapeutics, among others. This review first summarizes the typical examples of cellular compartments. In particular, the recent studies on cellular membraneless organelles (biomolecular condensates) are reviewed. We then summarize the recent advances in the construction of artificial compartments using engineered platforms. Finally, we provide our insights into the construction of biomimetic systems and the applications of these systems. This review article provides a timely summary of the relevant perspectives for the future development of artificial compartments, the building blocks for the construction of artificial organelles or cells.

MX2 forms nucleoporin-comprising cytoplasmic biomolecular condensates that lure viral capsids.

Human myxovirus resistance 2 (MX2) can restrict HIV-1 and herpesviruses at a post-entry step through a process requiring an interaction between MX2 and the viral capsids. The involvement of other host cell factors, however, remains poorly understood. Here, we mapped the proximity interactome of MX2, revealing strong enrichment of phenylalanine-glycine (FG)-rich proteins related to the nuclear pore complex as well as proteins that are part of cytoplasmic ribonucleoprotein granules. MX2 interacted with these proteins to form multiprotein cytoplasmic biomolecular condensates that were essential for its anti-HIV-1 and anti-herpes simplex virus 1 (HSV-1) activity. MX2 condensate formation required the disordered N-terminal region and MX2 dimerization. Incoming HIV-1 and HSV-1 capsids associated with MX2 at these dynamic cytoplasmic biomolecular condensates, preventing nuclear entry of their viral genomes. Thus, MX2 forms cytoplasmic condensates that likely act as nuclear pore decoys, trapping capsids and inducing premature viral genome release to interfere with nuclear targeting of HIV-1 and HSV-1.

Transgenic sensors reveal compartment-specific effects of aggregation-prone proteins on subcellular proteostasis during aging.

Loss of proteostasis is a hallmark of aging that underlies many age-related diseases. Different cell compartments experience distinctive challenges in maintaining protein quality control, but how aging regulates subcellular proteostasis remains underexplored. Here, by targeting the misfolding-prone FlucDM luciferase to the cytoplasm, mitochondria, and nucleus, we established transgenic sensors to examine subcellular proteostasis in Drosophila. Analysis of detergent-insoluble and -soluble levels of compartment-targeted FlucDM variants indicates that thermal stress, cold shock, and pro-longevity inter-organ signaling differentially affect subcellular proteostasis during aging. Moreover, aggregation-prone proteins that cause different neurodegenerative diseases induce a diverse range of outcomes on FlucDM insolubility, suggesting that subcellular proteostasis is impaired in a disease-specific manner. Further analyses with FlucDM and mass spectrometry indicate that pathogenic tauV337M produces an unexpectedly complex regulation of solubility for different FlucDM variants and protein subsets. Altogether, compartment-targeted FlucDM sensors pinpoint a diverse modulation of subcellular proteostasis by aging regulators.

Melatonin decreases excessive polyspermy for single oocyte in pigs through the MT2 receptor.

Melatonin supplementation during in vitro maturation (IVM) improves porcine oocyte maturation and embryonic development by exerting antioxidative effects. Nevertheless, the mechanism by which melatonin prevents polyspermy after in vitro fertilization (IVF) remains unclear. Here, we examined the effects of melatonin on cytoplasmic maturation and the incidence of polyspermic penetration in porcine oocytes. No statistically significant difference was observed in the rate of first polar body formation between the groups (Control, Melatonin, Melatonin + Luzindole, and Melatonin + 4-P-PDOT). Interestingly, melatonin supplementation significantly improved the cytoplasmic maturation of porcine oocytes by enhancing the normal distribution of organelles (Golgi apparatus, endoplasmic reticulum and mitochondria) and upregulating organelle-related gene expressions (P < 0.05). However, these promotional effects were counteracted by melatonin antagonists, suggesting that melatonin enhances cytoplasmic maturation through its receptors in porcine oocytes. Melatonin supplementation also significantly improved the rate of diploid and blastocyst formation after IVF by promoting the normal distribution of cortical granules (P < 0.05). In conclusion, melatonin supplementation during in vitro maturation of porcine oocyte improves fertilization efficiency and embryonic developmental competence by enhancing cytoplasmic maturation.

Organelle Interactions in Plant Cells.

The sequestration of enzymes and associated processes into sub-cellular domains, called organelles, is considered a defining feature of eukaryotic cells. However, what leads to specific outcomes and allows a eukaryotic cell to function singularly is the interactivity and exchanges between discrete organelles. Our ability to observe and assess sub-cellular interactions in living plant cells has expanded greatly following the creation of fluorescent fusion proteins targeted to different organelles. Notably, organelle interactivity changes quickly in response to stress and reverts to a normal less interactive state as homeostasis is re-established. Using key observations of some of the organelles present in a plant cell, this chapter provides a brief overview of our present understanding of organelle interactions in plant cells.

Liquid-liquid phase inclusion bodies in acute and persistent parainfluenaza virus type 5 infections.

Cytoplasmic inclusion bodies (IBs) are a common feature of single-stranded, non-segmented, negative-strand RNA virus (Mononegavirales) infections and are thought to be regions of active virus transcription and replication. Here we followed the dynamics of IB formation and maintenance in cells infected with persistent and lytic/acute variants of the paramyxovirus, parainfluenza virus type 5 (PIV5). We show that there is a rapid increase in the number of small inclusions bodies up until approximately 12 h post-infection. Thereafter the number of inclusion bodies decreases but they increase in size, presumably due to the fusion of these liquid organelles that can be disrupted by osmotically shocking cells. No obvious differences were observed at these times between inclusion body formation in cells infected with lytic/acute and persistent viruses. IBs are also readily detected in cells persistently infected with PIV5, including in cells in which there is little or no ongoing virus transcription or replication. In situ hybridization shows that genomic RNA is primarily located in IBs, whilst viral mRNA is more diffusely distributed throughout the cytoplasm. Some, but not all, IBs show incorporation of 5-ethynyl-uridine (5EU), which is integrated into newly synthesized RNA, at early times post-infection. These results strongly suggest that, although genomic RNA is present in all IBs, IBs are not continuously active sites of virus transcription and replication. Disruption of IBs by osmotically shocking persistently infected cells does not increase virus protein synthesis, suggesting that in persistently infected cells most of the virus genomes are in a repressed state. The role of IBs in PIV5 replication and the establishment and maintenance of persistence is discussed.

Multiple roles of arsenic compounds in phase separation and membraneless organelles formation determine their therapeutic efficacy in tumors.

Arsenic compounds are widely used for the therapeutic intervention of multiple diseases. Ancient pharmacologists discovered the medicinal utility of these highly toxic substances, and modern pharmacologists have further recognized the specific active ingredients in human diseases. In particular, Arsenic trioxide (ATO), as a main component, has therapeutic effects on various tumors (including leukemia, hepatocellular carcinoma, lung cancer, etc.). However, its toxicity limits its efficacy, and controlling the toxicity has been an important issue. Interestingly, recent evidence has pointed out the pivotal roles of arsenic compounds in phase separation and membraneless organelles formation, which may determine their toxicity and therapeutic efficacy. Here, we summarize the arsenic compounds-regulating phase separation and membraneless organelles formation. We further hypothesize their potential involvement in the therapy and toxicity of arsenic compounds, highlighting potential mechanisms underlying the clinical application of arsenic compounds.

Construction of Liposome-Based Extracellular Artificial Organelles on Individual Living Cells.

Integration of living cells with extrinsic functional entities gives rise to bioaugmented nanobiohybrids, which hold tremendous potential across diverse fields such as cell therapy, biocatalysis, and cell robotics. This study presents a biocompatible method for incorporating multilayered functional liposomes onto the cell surface, creating extracellular artificial organelles. The introduction of various extrinsic functionalities to cells is achieved without comprising their viabilities. The integration of extrinsic enzymatic reactions is exemplified through the cascade reaction involving glucose oxidase and horseradish peroxidase. Furthermore, our protocol offers the design flexibility to customize liposome compositions, thereby providing effective cell modification. The versatility of the liposome-based exorganelle approach establishes an advanced chemical tool, empowering cells with novel functionalities that surpass or are complementary to their innate capabilities.

The progress and future of the treatment of Candida albicans infections based on nanotechnology.

Systemic infection with Candida albicans poses a significant risk for people with weakened immune systems and carries a mortality rate of up to 60%. However, current therapeutic options have several limitations, including increasing drug tolerance, notable off-target effects, and severe adverse reactions. Over the past four decades, the progress in developing drugs to treat Candida albicans infections has been sluggish. This comprehensive review addresses the limitations of existing drugs and summarizes the efforts made toward redesigning and innovating existing or novel drugs through nanotechnology. The discussion explores the potential applications of nanomedicine in Candida albicans infections from four perspectives: nano-preparations for anti-biofilm therapy, innovative formulations of "old drugs" targeting the cell membrane and cell wall, reverse drug resistance therapy targeting subcellular organelles, and virulence deprivation therapy leveraging the unique polymorphism of Candida albicans. These therapeutic approaches are promising to address the above challenges and enhance the efficiency of drug development for Candida albicans infections. By harnessing nano-preparation technology to transform existing and preclinical drugs, novel therapeutic targets will be uncovered, providing effective solutions and broader horizons to improve patient survival rates.

[Construction of Aspergillus oryzae strains with organelles labeled with fluorescence and observation of organelle morphology].

The organelles in the multi-nucleated filamentous fungus Aspergillus oryzae present polymorphism. To observe the organelle morphology in A. oryzae and provide references for the localization prediction of unknown proteins and the disclosure of biological reaction pathways in A. oryzae, we fused different subcellular localization signals with green fluorescent protein (GFP) to obtain different subcellular localization vectors, which were then transferred into A. oryzae by Agrobacterium tumefaciens-mediated transformation. The A. oryzae reporter strains with fluorescence-labeled nuclei, mitochondria, endoplasmic reticulum, vacuole, lipid droplets, peroxisome, and Golgi apparatus were successfully constructed. Furthermore, staining with small-molecule specific dyes was carried out to validate the co-localization of fluorescence-labeled mitochondria, nuclei, and lipid droplets in the reporter strains, which further confirmed that the reporter strains were successfully constructed. The distribution and morphology of fluorescence-labeled organelles were observed at different growth stages and under different culture conditions. The constructed reporter strains provide basic tools for studying the organelle morphology, localization of unknown target proteins, and subcellular localization in A. oryzae.

Indirect ELISAs with sucrose subcellular fractions of Neospora caninum as antigens for diagnosis of neosporosis in cattle.

Neosporosis is one of the major causes of abortion in cattle, and it is responsible for significant economic losses in those animals. Thus, this study aimed to evaluate indirect ELISA using subcellular fractions of Neospora caninum obtained via sucrose gradient separation. Eighty-five sera from dairy cattle previously tested using indirect immunofluorescence assay (IFA) were used. Three distinct bands were separated at 1.0 M, 1.4 M, 1.6 M, and the pellet at 1.8 M, which were identified as fractions one (F1), two (F2), three (F3), and four (F4), respectively. These fractions showed parasite membranes in the F1, rhoptry and conoids in the F2, mitochondria in the F3, and tachyzoite ghosts remain in F4. Indirect ELISAs for IgM, and IgG were performed. Additionally, sensitivity, specificity, and kappa values were defined considering the IFA as the gold standard. The highest and lowest specificities were observed for F1 (76 %) and F3 (16 %), respectively. F2 and F4 showed the highest sensitivity (93.3 %), kappa agreement (0.46), and Negative Preventive Value (NPV) (73 %) respectively. It was possible to standardize indirect ELISAs using whole soluble antigen and subcellular fractions of N. caninum, and F2 and F4 showed higher sensitivity (93.3 %), kappa (0.41), and NPV values (75 %) than F1, and F3, which could be used for epidemiology studies such as screening.

TDP-43 nuclear condensation and neurodegenerative proteinopathies.

RNA-binding proteins (RBPs) can undergo phase separation and form condensates, processes that, in turn, can be critical for their functionality. In a recent study, Huang, Ellis, and colleagues show that cellular stress can trigger transient alterations in nuclear TAR DNA-binding protein 43 (TDP-43), leading to changes crucial for proper neuronal function. These findings have implications for understanding neurological TDP-43 proteinopathies.

Positive-strand RNA virus replication organelles at a glance.

Membrane-bound replication organelles (ROs) are a unifying feature among diverse positive-strand RNA viruses. These compartments, formed as alterations of various host organelles, provide a protective niche for viral genome replication. Some ROs are characterised by a membrane-spanning pore formed by viral proteins. The RO membrane separates the interior from immune sensors in the cytoplasm. Recent advances in imaging techniques have revealed striking diversity in RO morphology and origin across virus families. Nevertheless, ROs share core features such as interactions with host proteins for their biogenesis and for lipid and energy transfer. The restructuring of host membranes for RO biogenesis and maintenance requires coordinated action of viral and host factors, including membrane-bending proteins, lipid-modifying enzymes and tethers for interorganellar contacts. In this Cell Science at a Glance article and the accompanying poster, we highlight ROs as a universal feature of positive-strand RNA viruses reliant on virus-host interplay, and we discuss ROs in the context of extensive research focusing on their potential as promising targets for antiviral therapies and their role as models for understanding fundamental principles of cell biology.

Mild Heat Stress Alters the Physical State and Structure of Membranes in Triacylglycerol-Deficient Fission Yeast, Schizosaccharomyces pombe.

We investigated whether the elimination of two major enzymes responsible for triacylglycerol synthesis altered the structure and physical state of organelle membranes under mild heat shock conditions in the fission yeast, Schizosaccharomyces pombe. Our study revealed that key intracellular membrane structures, lipid droplets, vacuoles, the mitochondrial network, and the cortical endoplasmic reticulum were all affected in mutant fission yeast cells under mild heat shock but not under normal growth conditions. We also obtained direct evidence that triacylglycerol-deficient cells were less capable than wild-type cells of adjusting their membrane physical properties during thermal stress. The production of thermoprotective molecules, such as HSP16 and trehalose, was reduced in the mutant strain. These findings suggest that an intact system of triacylglycerol metabolism significantly contributes to membrane protection during heat stress.

Redefining drug therapy: innovative approaches using catalytic compartments.

INTRODUCTION: Rapid excretion of drug derivatives often results in short drug half-lives, necessitating frequent administrations. Catalytic compartments, also known as nano- and microreactors, offer a solution by providing confined environments for in situ production of therapeutic agents. Inspired by natural compartments, polymer-based catalytic compartments have been developed to improve reaction efficiency and enable site-specific therapeutic applications. AREAS COVERED: Polymer-based compartments provide stability, permeability control, and responsiveness to stimuli, making them ideal for generating localized compounds/signals. These sophisticated systems, engineered to carry active compounds and enable selective molecular release, represent a significant advancement in pharmaceutical research. They mimic cellular functions, creating controlled catalytic environments for bio-relevant processes. This review explores the latest advancements in synthetic catalytic compartments, focusing on design approaches, building blocks, active molecules, and key bio-applications. EXPERT OPINION: Catalytic compartments hold transformative potential in precision medicine by improving therapeutic outcomes through precise, on-site production of therapeutic agents. While promising, challenges like scalable manufacturing, biodegradability, and regulatory hurdles must be addressed to realize their full potential. Addressing these will be crucial for their successful application in healthcare.

Lysosome-related organelle integrity suppresses TIR-1 aggregation to restrain toxic propagation of p38 innate immunity.

Innate immunity in bacteria, plants, and animals requires the specialized subset of Toll/interleukin-1/resistance gene (TIR) domain proteins that are nicotinamide adenine dinucleotide (NAD+) hydrolases. Aggregation of these TIR proteins engages their enzymatic activity, but it is unknown how this protein multimerization is regulated. Here, we discover that TIR oligomerization is controlled to prevent immune toxicity. We find that p38 propagates its own activation in a positive feedback loop, which promotes the aggregation of the lone enzymatic TIR protein in the nematode C. elegans (TIR-1, homologous to human sterile alpha and TIR motif-containing 1 [SARM1]). We perform a forward genetic screen to determine how the p38 positive feedback loop is regulated. We discover that the integrity of the specific lysosomal subcompartment that expresses TIR-1 is actively maintained to limit inappropriate TIR-1 aggregation on the membranes of these organelles, which restrains toxic propagation of p38 innate immunity. Thus, innate immunity in C. elegans intestinal epithelial cells is regulated by specific control of TIR-1 multimerization.

Intestinal immunity in C. elegans is activated by pathogen effector-triggered aggregation of the guard protein TIR-1 on lysosome-related organelles.

Toll/interleukin-1/resistance (TIR)-domain proteins with enzymatic activity are essential for immunity in plants, animals, and bacteria. However, it is not known how these proteins function in pathogen sensing in animals. We discovered that the lone enzymatic TIR-domain protein in the nematode C. elegans (TIR-1, homolog of mammalian sterile alpha and TIR motif-containing 1 [SARM1]) was strategically expressed on the membranes of a specific intracellular compartment called lysosome-related organelles. The positioning of TIR-1 on lysosome-related organelles enables intestinal epithelial cells in the nematode C. elegans to survey for pathogen effector-triggered host damage. A virulence effector secreted by the bacterial pathogen Pseudomonas aeruginosa alkalinized and condensed lysosome-related organelles. This pathogen-induced morphological change in lysosome-related organelles triggered TIR-1 multimerization, which engaged its intrinsic NAD+ hydrolase (NADase) activity to activate the p38 innate immune pathway and protect the host against microbial intoxication. Thus, TIR-1 is a guard protein in an effector-triggered immune response, which enables intestinal epithelial cells to survey for pathogen-induced host damage.

Advances in multi-enzyme co-localization strategies for the construction of microbial cell factory.

Biomanufacturing, driven by technologies such as synthetic biology, offers significant potential to advance the bioeconomy and promote sustainable development. It is anticipated to transform traditional manufacturing and become a key industry in future strategies. Cell factories are the core of biomanufacturing. The advancement of synthetic biology and growing market demand have led to the production of a greater variety of natural products and increasingly complex metabolic pathways. However, this progress also presents challenges, notably the conflict between natural product production and chassis cell growth. This conflict results in low productivity and yield, adverse side effects, metabolic imbalances, and growth retardation. Enzyme co-localization strategies have emerged as a promising solution. This article reviews recent progress and applications of these strategies in constructing cell factories for efficient natural product production. It comprehensively describes the applications of enzyme-based compartmentalization, metabolic pathway-based compartmentalization, and synthetic organelle-based compartmentalization in improving product titers. The article also explores future research directions and the prospects of combining multiple strategies with advanced technologies.