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Live cell labeling of various organelles is of great demand in the research field of cell biology. However, current approaches often lack an optimal balance between efficiency and versatility. We took advantage of chemical modified mRNA to express various organelle located fluorescent proteins. This approach facilitated highly efficient multi-organelle labeling across diverse cell types, both in vitro and in vivo. The application of mRNA-based organelle markers offers faster and more efficient transfection and expression compared to plasmids. When expressing fluorescent proteins in neuronal cells, it is faster and safer compared to viral vectors. Notably, this approach excels in rapidly labeling organelles, making it highly effective for dynamic live cell imaging applications. By leveraging this tool, we unveiled the unique behaviors of mitochondria and the endoplasmic reticulum during spike protein-mediated cell fusion, a critical event in SARS-CoV-2 viral entry. Thus, our results demonstrate the potency of mRNA-encoded fluorescent proteins as powerful tools for advancing biological research., (© 2024 Federation of American Societies for Experimental Biology.)

Organelle-specific protein translocation systems are essential for organelle biogenesis and maintenance in eukaryotes but thought to be absent from prokaryotic organelles. Here, we demonstrate that MamF-like proteins are crucial for the formation and functionality of bacterial magnetosome organelles. Deletion of mamF-like genes in the Alphaproteobacterium Magnetospirillum gryphiswaldense results in severe defects in organelle positioning, biomineralization, and magnetic navigation. These phenotypic defects result from the disrupted targeting of a subset of magnetosomal proteins that contain C-terminal glycine-rich integral membrane domains. Phylogenetic analyses reveal an ancient evolutionary link between MamF-like proteins and plastidial Tic20. Our findings redefine the molecular roles of MamF-like proteins and suggest that organelle-specific protein targeting systems also play a role in bacterial organelle formation., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

40 years ago, organelle genomes were assumed to be streamlined and, perhaps, unexciting remnants of their prokaryotic past. However, the field of organelle genomics has exposed an unparallel diversity in genome architecture (i.e. genome size, structure, and content). The transcription of these eccentric genomes can be just as elaborate - organelle genomes are pervasively transcribed into a plethora of RNA types. However, while organelle protein-coding genes are known to produce polycistronic transcripts that undergo heavy posttranscriptional processing, the nature of organelle noncoding transcriptomes is still poorly resolved. Here, we review how wet-lab experiments and second-generation sequencing data (i.e. short reads) have been useful to determine certain types of organelle RNAs, particularly noncoding RNAs. We then explain how third-generation (long-read) RNA-Seq data represent the new frontier in organelle transcriptomics. We show that public repositories (e.g. NCBI SRA) already contain enough data for inter-phyla comparative studies and argue that organelle biologists can benefit from such data. We discuss the prospects of using publicly available sequencing data for organelle-focused studies and examine the challenges of such an approach. We highlight that the lack of a comprehensive database dedicated to organelle genomics/transcriptomics is a major impediment to the development of a field with implications in basic and applied science., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The malaria-causing parasite, P. falciparum, replicates through schizogony, a tightly orchestrated process where numerous daughter parasites are formed simultaneously. Proper division and segregation of one-per-cell organelles, like the mitochondrion and apicoplast, are essential, yet remain poorly understood. We developed a new reporter parasite line that allows visualization of the mitochondrion in blood and mosquito stages. Using high-resolution 3D imaging, we found that the mitochondrion orients in a cartwheel structure, prior to stepwise, non-geometric division during last-stage schizogony. Analysis of focused ion beam scanning electron microscopy data confirmed these mitochondrial division stages. Furthermore, these data allowed us to elucidate apicoplast division steps, highlighted its close association with the mitochondrion, and showed putative roles of the centriolar plaques in apicoplast segregation. These observations form the foundation for a new detailed mechanistic model of mitochondrial and apicoplast division and segregation during P. falciparum schizogony and pave the way for future studies into the proteins and protein complexes involved in organelle division and segregation., (© 2024 Verhoef et al.)

Objective: This study analyzed organelle toxicities of nanometals applied as free formulations or titanium rod-coating materials in rats., Methods: All materials were injected intraperitoneally, including the physiological saline applied to the control group. The first experimental group was implanted with nanosilver-coated titanium rods, and the second, third, and fourth groups received free nanosilver at rising levels. The fifth group was implanted with nanosilver, nanocopper, and nanozinc-coated titanium rods, and the sixth group received the same nanometals as free formulations. Light and electron microscopy and ICP-Mass Spectrometry were utilized to determine the neural, hepatic, and renal toxicities and tissue metal levels., Results: In brains, neuropil, myelin, and cellular damages occurred, especially in groups receiving high-dose nanosilver or nanometal combinations. Histiocyte accumulation and dark mitochondria within hepatocytes were discernible in the liver. Kidneys were the organs that were most severely affected by nanometal toxicity. The nephrotoxicity was apparent with the perturbations of the membrane infoldings and mitochondrial damage in the proximal and distal convoluted epithelia. Large angular peroxisomes developed inside the mesangial cells, and Golgi bodies increased in epithelial cells. Systemic metal levels increased on the thirtieth and prominently dropped on the sixtieth day., Conclusion: These results provide insights into the extent of injury and organelle targets of nanometals and will guide optimizing the nanomaterials and implants used in the surgical practice., Competing Interests: Conflict of Interest None to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Biomolecular condensates (BMCs), play significant roles in organizing cellular functions in the absence of membranes through phase separation events involving RNA, proteins, and RNA-protein complexes. These membrane-less organelles form dynamic multivalent weak interactions, often involving intrinsically disordered proteins or regions (IDPs/IDRs). However, the nature of these crucial interactions, how most of these organelles are organized and are functional, remains unknown. Aberrant condensates have been implicated in neurodegenerative diseases and various cancers, presenting novel therapeutic opportunities for small molecule condensate modulators. Recent advancements in optogenetic technologies, particularly Corelet, enable precise manipulation of BMC dynamics within living cells, facilitating high-throughput screening for small molecules that target these complex structures. By elucidating the molecular mechanisms governing BMC formation and function, this innovative approach holds promise to unlock therapeutic strategies against previously "undruggable" protein targets, paving the way for effective interventions in disease., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘All authors are employees of Nereid Therapeutics’., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Migrasomes are newly identified cellular organelles with a pomegranate-like structure and specifically generated by migrating cells. The mechanisms underlying migrasome biogenesis and methods for their isolation are gradually being elucidated. These organelles are pivotal in cell-to-cell communication and the maintenance of mitochondrial homeostasis. Their involvement in a diverse array of physiological and pathological processes is increasingly recognized. Despite these advancements, research on migrasomes is still in its early stages. It is imperative to delve deeper into the intricate mechanisms and functions of migrasomes to fully understand their role in physiopathology. In this review, we summarize the current research progress on migrasomes, including their distribution, biogenesis, purification and identification techniques, biological functionalities, and roles in physiological and pathological processes. We particularly highlight their potentials in disease diagnosis and therapy and point out the challenges facing us, aiming to provide novel insights into the emerging organelles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tumor-associated macrophages (TAMs) represent the majority of the immune cells present in the tumor microenvironment. These macrophages exhibit an anti-inflammatory (M2)-like physiological state and execute immune-suppressive and tumor-supporting properties. With TAMs being plastic, there is a growing interest in reprogramming them toward a pro-inflammatory (M1)-like phenotype that exhibits anti-tumoral properties. Recent studies have demonstrated that both engineered vesicles derived from macrophages and endogenous extracellular vesicles produced by macrophages can be programmed to alter macrophage phenotype. Here it is demonstrated that pro-inflammatory macrophage-engineered subcellular vesicles (MEVs) have differential properties based on their organelle of origin. Endoplasmic reticulum specific MEVs (erMEVs) treated M2 macrophages exhibit enhanced pro-inflammatory cytokine production compared to plasma membrane specific MEVs (pmMEVs) treated M2 macrophages. In addition, under in vitro co-culture conditions, erMEVs elicit superior efficacy in suppressing the viability of cancer cells compared to the same concentration of pmMEVs. Furthermore, erMEVs and pmMEVs maintain differences in their membrane proteins, that regulate the repolarization efficacy of M2 macrophages toward an M1-like phenotype. In addition, The M2 to M1 repolarizing efficacy of MEVs can be altered by changing the activity of the membrane proteins present on erMEVs or pmMEVs., (© 2024 Wiley‐VCH GmbH.)

Molecular basis of rice aroma formation is sparsely known and developmental programs driving biochemical pathways towards aroma is in infancy. Here, discovery and targeted proteo-metabolome of non-aromatic and aromatic rice seeds across developmental stages identified a total of 442 aroma-responsive proteins (ARPs) and 824 aroma-responsive metabolites (ARMs) involved in metabolism, calcium and G-protein signaling. Biochemical examination revealed ARM/Ps were linked to 2-acetylpyrrolidine, γ-aminobutyrate, anthocyanin, tannins, flavonoids and related enzymes. Pairwise correlation and clustering showed positive correlation among ARM/Ps. Consistent with aroma-related QTLs, ARPs were mapped on chromosomes 3,4,5,8 and were mainly compartmentalized in cytoplasm and mitochondria. ARM/P-correlation network identified associations related to metabolism and signaling. Multiple reaction monitoring (MRM) confirmed role of catechins, quinic acid and quercetin in aroma formation. Pathway enrichment, multivariate analysis and qRT-PCR validated that calcium and G-protein signaling, aromatic/branched-chain aminoacid, 2-acetylpyrrolidine, oxylipin, melvonate and prenylpyrophosphate pathways, indole, phenylacetate, flavonoid, cinnamoic ester govern aroma formation in rice., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Intracellular compartmentalization enhances biological reactions, crucial for cellular function and survival. An example is the carboxysome, a bacterial microcompartment for CO 2 fixation. The carboxysome uses a polyhedral protein shell made of hexamers, pentamers, and trimers to encapsulate Rubisco, increasing CO 2 levels near Rubisco to enhance carboxylation. Despite their role in the global carbon cycle, the molecular mechanisms behind carboxysome shell assembly remain unclear. Here, we present a structural characterization of α-carboxysome shells generated from recombinant systems, which contain all shell proteins and the scaffolding protein CsoS2. Atomic-resolution cryo-electron microscopy of the shell assemblies, with a maximal size of 54 nm, unveil diverse assembly interfaces between shell proteins, detailed interactions of CsoS2 with shell proteins to drive shell assembly, and the formation of heterohexamers and heteropentamers by different shell protein paralogs, facilitating the assembly of larger empty shells. Our findings provide mechanistic insights into the construction principles of α-carboxysome shells and the role of CsoS2 in governing α-carboxysome assembly and functionality.

In cellular environments, certain synthetic molecules can form nanostructures via self-assembly, impacting molecular imaging, and biomedical applications. Control over the formation of these self-assembled nanostructures in subcellular organelle is challenging. By the action of stimuli, either present in the cellular environment or applied externally, in situ generation of molecular precursors can lead to accumulation and supramolecular nanostructure formation, resulting in efficient bioimaging. Here, we summarize smart fluorophore-based ordered nanostructure preparation at specific organelles for efficient bioimaging and therapeutic application towards cancer theranostics. We also present challenges and an outlook regarding intercellular self-assembly for theranostics application. Altogether, smart nanostructured materials with fluorescence read-outs at specific subcellular compartments would be beneficial in synthetic biology and precision therapeutics., Competing Interests: Declarations. Conflicts of Interest: There are no conflicts to declare. Ethics Approval and Consent to Participate: There are no human and animal subjects in this review and informed consent is not applicable., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Viruses are ubiquitous entities that infect organisms across the kingdoms of life. While viruses can infect a range of cells, tissues, and organisms, this aspect is often not explored in cell culture analyses. There is limited information about which infection-induced changes are shared or distinct in different cellular environments. The prevalent pathogen human cytomegalovirus (HCMV) remodels the structure and function of subcellular organelles and their interconnected networks formed by membrane contact sites (MCSs). A large portion of this knowledge has been derived from fibroblasts infected with a lab-adapted HCMV strain. Here, we assess strain- and cell type-specific alterations in MCSs and organelle remodeling induced by HCMV. Integrating quantitative mass spectrometry, super-resolution microscopy, and molecular virology assays, we compare infections with lab-adapted and low-passage HCMV strains in fibroblast and epithelial cells. We determine that, despite baseline proteome disparities between uninfected fibroblast and epithelial cells, infection induces convergent changes and is remarkably similar. We show that hallmarks of HCMV infection in fibroblasts, mitochondria-endoplasmic reticulum (ER) encapsulations and peroxisome proliferation, are also conserved in infected epithelial and macrophage-like cells. Exploring cell type-specific differences, we demonstrate that fibroblasts rely on endosomal cholesterol transport while epithelial cells rely on cholesterol from the Golgi. Despite these mechanistic differences, infections in both cell types result in phenotypically similar cholesterol accumulation at the viral assembly complex. Our findings highlight the adaptability of HCMV, in that infections can be tailored to the initial cell state by inducing both shared and unique proteome alterations, ultimately promoting a unified pro-viral environment.IMPORTANCEHuman cytomegalovirus (HCMV) establishes infections in diverse cell types throughout the body and is connected to a litany of diseases associated with each of these tissues. However, it is still not fully understood how HCMV replication varies in distinct cell types. Here, we compare HCMV replication with lab-adapted and low-passage strains in two primary sites of infection, lung fibroblasts and retinal epithelial cells. We discover that, despite displaying disparate protein compositions prior to infection, these cell types undergo convergent alterations upon HCMV infection, reaching a more similar cellular state late in infection. We find that remodeling of the subcellular landscape is a pervasive feature of HCMV infection, through alterations to both organelle structure-function and the interconnected networks they form via membrane contact sites. Our findings show how HCMV infection in different cell types induces both shared and divergent changes to cellular processes, ultimately leading to a more unified state., Competing Interests: The authors declare no conflict of interest.

Peroxynitrite (ONOO - ), as an important reactive nitrogen species, plays a pivotal role in the regulation of intracellular redox homeostasis, signal transduction, cell growth and metabolism, and other physiological processes. Organelles are important for regulating ONOO - , and the dysregulation of ONOO - in organelles is closely related to various diseases. Therefore, it is essential to monitor ONOO - in cellular organelles, including mitochondria, lysosome, endoplasmic reticulum (ER), Golgi apparatus, and lipid droplets. However, the latest advances in organelle-targeting ONOO - fluorescent probes have not been reviewed systematically. In this review, we focus on the design, sensing mechanism, and organelle-targeting imaging applications of ONOO - fluorescent probes that were reported since 2018. This review will help to facilitate the comprehension of organelle-targeting fluorescent probes for the detection of ONOO - .

Egress represents a crucial process employed by Neospora caninum in the establishment of infection. Dense granule proteins (GRAs), secreted by the dense granule, play significant roles in modifying the parasitophorous vacuole, maintenance of morphology, and regulating host-cell interactions. However, their precise involvement in tachyzoite egress remains inadequately characterized. In this study, we identified a homologous gene, Ncgra41, corresponding to the dense granule protein 41 (GRA41) of Toxoplasma gondii, which is associated with egress, utilizing NCBI and ToxoDB databases. NcGRA41 is localized extracellularly within dense granules and intracellularly within parasitic vacuoles. Deletion of NcGRA41 did not affect tachyzoites invasion or proliferation but significantly reduced egress capacity and pathogenicity in mice. The phenotypic characteristics were restored in a complementary strain. Further investigation revealed that the absence of NcGRA41 reduced gliding motility and the transcription level of the subtilisin-like protein (SUB1). A microneme secretion assay demonstrated a significant decrease in NcMIC1 secretion, along with reduced expression levels of NcMIC1, NcMIC4, and NcMIC8. These findings demonstrate that NcGRA41, a novel dense granule protein in N. caninum, modulates tachyzoites egress and influences pathogenicity by regulating microneme secretion., Competing Interests: Declarations Ethics approval The mice used in this study were handled in compliance with the recommendations outlined in the “Guidelines for the Care and Use of Laboratory Animals” of the Ministry of Science and Technology of China. Additionally, our research protocol received approval from the Laboratory Animal Welfare and Animal Experimental Ethical Committee of China Agricultural University (Certificate No.: CAU-AW31901202-2–1). Consent to participate Done and confirmed. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Once considered unconventional cellular structures, membraneless organelles (MLOs), cellular substructures involved in biological processes or pathways under physiological conditions, have emerged as central players in cellular dynamics and function. MLOs can be formed through liquid-liquid phase separation (LLPS), resulting in the creation of condensates. From neurodegenerative disorders, cardiovascular diseases, aging, and metabolism to cancer, the influence of MLOs on human health and disease extends widely. This review discusses the underlying mechanisms of LLPS, the biophysical properties that drive MLO formation, and their implications for cellular function. We highlight recent advances in understanding how the physicochemical environment, molecular interactions, and post-translational modifications regulate LLPS and MLO dynamics. This review offers an overview of the discovery and current understanding of MLOs and biomolecular condensate in physiological conditions and diseases. This article aims to deliver the latest insights on MLOs and LLPS by analyzing current research, highlighting their critical role in cellular organization. The discussion also covers the role of membrane-associated condensates in cell signaling, including those involving T-cell receptors, stress granules linked to lysosomes, and biomolecular condensates within the Golgi apparatus. Additionally, the potential of targeting LLPS in clinical settings is explored, highlighting promising avenues for future research and therapeutic interventions., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Throughout metazoa, germ cells assemble RNA-protein organelles (germ granules). In Drosophila ovaries, perinuclear nuage forms in the nurse cells, while compositionally similar polar granules form in the oocyte. A similar system appears to exist in the distantly related (∼350 million years) wasp Nasonia, with some surprising divergences. Nuage is similarly formed in Nasonia, except that anterior nurse cells accumulate significantly more nuage, in association with high levels of DNA double-strand breaks, suggesting that increased transposon activity anteriorly is silenced by high nuage levels. In the oocyte, the germ plasm forms a single granule that is 40 times larger than a homologous Drosophila polar granule. While conserved germ granule proteins are recruited to the oosome, they show unusual localization: Tudor protein forms a shell encapsulating the embryonic oosome, while small Oskar/Vasa/Aubergine granules coalesce interiorly. Wasp Vasa itself is unusual since it has an alternative splice form that includes a previously unreported nucleoporin-like phenylalanine-glycine repeat domain. Our work is consistent with the high degree of evolutionary plasticity of membraneless organelles, and describes a new experimental model and resources for studying biomolecular condensates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

In Toxoplasma gondii , the conoid comprises a cone with spiraling tubulin fibers, preconoidal rings, and intraconoidal microtubules. This dynamic organelle undergoes extension and retraction through the apical polar ring (APR) during egress, gliding, and invasion. The forces involved in conoid extrusion are beginning to be understood, and its role in directing F-actin flux to the pellicular space, thereby controlling parasite motility, has been proposed. However, the contribution of the APR and its interactions with the conoid remain unclear. To gain insight into the APR architecture, ultrastructure expansion microscopy was applied to pinpoint known and newly identified APR proteins (APR2 to APR7). Our results revealed that the APR is constructed as a fixed multilayered structure. Notably, conditional depletion of APR2 resulted in significant impairments in motility and invasion. Electron microscopy and cryoelectron tomography revealed that depletion of APR2 alters APR integrity, affecting conoid extrusion and causing cytosolic leakage of F-actin. These findings implicate the APR structure in directing the apico-basal flux of F-actin to regulate parasite motility and invasion., Competing Interests: Competing interests statement:The authors declare no competing interest.

Classical G-protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap, we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial-trans Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments., Competing Interests: WJ, KS, GF, SL, NL No competing interests declared, (© 2024, Jang et al.)

Each organelle referring to a complex multiorder architecture executes respective biological processes via its distinct spatial organization and internal microenvironment. As the assembly of biomolecules is the structural basis of living cells, creating synthetic nanoassemblies with specific physicochemical and morphological properties in living cells to interfere or couple with the natural organelle architectures has attracted great attention in precision therapeutics of cancers. In this review, we give an overview of the latest advances in the synthetic nanoassemblies for precise organelle regulation, including the formation mechanisms, triggering strategies, and biomedical applications in precision therapeutics. We summarize the emerging material systems, including polymers, peptides, and deoxyribonucleic acids (DNAs), and their respective intermolecular interactions for intercellular synthetic nanoassemblies, and highlight their design principles in constructing precursors that assemble into synthetic nanoassemblies targeting specific organelles in the complex cellular environment. We further showcase the developed intracellular synthetic nanoassemblies targeting specific organelles including mitochondria, the endoplasmic reticulum, lysosome, Golgi apparatus, and nucleus and describe their underlying mechanisms for organelle regulation and precision therapeutics for cancer. Last, the essential challenges in this field and prospects for future precision therapeutics of synthetic nanoassemblies are discussed. This review should facilitate the rational design of organelle-targeting synthetic nanoassemblies and the comprehensive recognition of organelles by materials and contribute to the deep understanding and application of the synthetic nanoassemblies for precision therapeutics.

Acidocalcisomes of Trypanosoma brucei are membrane-bounded organelles characterized by their acidity and high content of polyphosphate and cations, like calcium and magnesium. They have important roles in cation and phosphorus storage, osmoregulation, autophagy initiation, calcium signaling, and virulence. Acidocalcisomes of T. brucei possess several membrane transporters, pumps, and channels, some of which were identified by proteomic and immunofluorescence analyses and validated as acidocalcisome proteins by their colocalization with the acidocalcisome marker vacuolar proton pyrophosphatase (VP1). Here, we report that a set of membrane transporters and enzymes, which were proposed to be present in acidocalcisomes by the morphological appearance of tagged proteins, colocalize with VP1, validating their character as acidocalcisome proteins., Importance: Acidocalcisomes are acidic organelles rich in polyphosphate and calcium present in a variety of eukaryotes and important for osmoregulation and calcium signaling. Several proteins were postulated to localize to acidocalcisomes based on their morphological characteristics. We provide validation of the localization of ten10 acidocalcisome proteins by their co-localization with enzymatic markers. These findings reveal the roles of acidocalcisomes in the storage of toxic metals, and the presence of enzymes involved in palmitoylation and polyphosphate metabolism., Competing Interests: The authors declare no conflict of interest.

The carboxysome is a natural proteinaceous organelle for carbon fixation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble to form a polyhedral shell structure to sequester cargo enzymes, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), and carbonic anhydrases. How these protein components assemble to construct a functional carboxysome is a central question in not only understanding carboxysome structure and function but also synthetic engineering of carboxysomes for biotechnological applications. Here, we determined the structure of the chaperone protein CcmS, which has recently been identified to be involved in β-carboxysome assembly, and its interactions with β-carboxysome proteins. The crystal structure at 1.99 Å resolution reveals CcmS from Nostoc sp. PCC 7120 forms a homodimer, and each CcmS monomer consists of five α-helices and four β-sheets. Biochemical assays indicate that CcmS specifically interacts with the C-terminal extension of the carboxysome shell protein CcmK1, but not the shell protein homolog CcmK2 or the carboxysome scaffolding protein CcmM. Moreover, we solved the structure of a stable complex of CcmS and the C-terminus of CcmK1 at 1.67 Å resolution and unveiled how the CcmS dimer interacts with the C-terminus of CcmK1. These findings allowed us to propose a model to illustrate CcmS-mediated β-carboxysome assembly by interacting with CcmK1 at the outer shell surface. Collectively, our study provides detailed insights into the accessory factors that drive and regulate carboxysome assembly, thereby improving our knowledge of carboxysome structure, function, and bioengineering., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) protein that plays a crucial role in cytosolic DNA-mediated innate immunity. Both STING agonists and antagonists have demonstrated their ability to enhance mouse survival against coronavirus, however, the physiological role of endogenous STING in coronavirus infection remains unclear. Our research unveils that STING inhibits coronavirus replication by impeding the formation of the ER-derived double-membrane vesicles (DMVs), the organelles in which coronavirus replicates. We found that STING was still capable of inhibiting coronavirus OC43 infection in cells, regardless of the knockout of cGAS or MAVS, or blocking type I interferon receptor. Moreover, STING disrupted the interaction between two crucial proteins, NSP4 and NSP6, involved in DMV formation, leading to the disruption of DMV formation. Taken together, our study sheds light on a novel antiviral role of STING in coronavirus infection, elucidating how it disrupts the formation of viral replication organelles, thereby impeding the replication process., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Asymmetric cell division in Saccharomyces cerevisiae involves class V myosin-dependent transport of organelles along the polarised actin cytoskeleton to the emerging bud. Vac17 is the vacuole/lysosome-specific myosin receptor. Its timely breakdown terminates transport and results in the proper positioning of vacuoles in the bud. Vac17 breakdown is controlled by the bud-concentrated p21-activated kinase Cla4, and the E3-ubiquitin ligase Dma1. We found that the spindle position checkpoint kinase Kin4 and, to a lesser extent, its paralog Frk1 contribute to successful vacuole transport by preventing the premature breakdown of Vac17 by Cla4 and Dma1. Furthermore, Kin4 and Cla4 contribute to the regulation of peroxisome transport. We conclude that Kin4 antagonises the Cla4/Dma1 pathway to coordinate spatiotemporal regulation of organelle transport., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

To clarify the synergistic pathogenic mechanism of Nicotiana benthamiana double infection with alfalfa mosaic virus (AMV) and white clover mosaic virus (WCMV), AMV and WCMV co-inoculation of N. benthamiana as treatment and single inoculation of AMV or WCMV and phosphate buffer solution (pH 7.0, PBS) as control, respectively. The concentrations and the relative expression of AMV and WCMV coat proteins were determined by a double antibody sandwich enzyme-linked immune sorbent assay (DAS-ELISA) and real-time fluorescence quantitative PCR (RT-qPCR) in a double infection of N. benthamiana with AMV and WCMV. Meanwhile, virion morphology, ultrastructure morphology, and chlorophyll content were observed and determined by electron microscopy. The results showed that the diseased symptoms were more serious, and virus concentration and relative expression of AMV and WCMV coat proteins were also higher in N. benthamiana double infection with AMV and WCMV than in AMV or WCMV single infection. The main symptoms manifested as severe mottle mosaic, shrinkage, and chlorosis. The concentrations of AMV and WCMV were 182.23 pg/mL and 148.77 pg/mL of double infection with AMV and WCMV, which were 1.75-fold and 1.62-fold than AMV and WCMV single infection, respectively. The relative expression of AMV and WCMV coat proteins was 4.25-fold and 2.50-fold than the single virus infection, respectively. Electron microscopy also observed that chloroplast malformation, cell membrane deformation, contents dissolution, grana lamella disorder, fat granules increased and enlarged, starch granules enlarged, and mitochondria were seriously malformed in a double infection of N. benthamiana with AMV and WCMV. The chlorophyll content was significantly lower for double infection with AMV and WCMV than for AMV or WCMV single-infected and CK, reduced by 31.52 %, 22.83 %, and 76.09 %, respectively. This is the first report of a double infection of N. benthamiana with AMV and WCMV that increases both virus concentrations and synergistically changes both host organelle ultrastructure and chlorophyll content., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Liang qiaolan reports financial support was provided by National Natural Science Foundation of China. Liang qiaolan reports a relationship with National Natural Science Foundation of China that includes: consulting or advisory, funding grants, paid expert testimony, speaking and lecture fees, and travel reimbursement. Liang qiaolan, chen ying e has patent pending to liang qiaolan. The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Benzene occurs naturally and is widely applied in the production process of petrochemical products. It is mainly exposed through the respiratory tract and dermal and metabolized in the liver, leading to systemic health effects, and 1,2,4-trihydroxybenzene (THB) is a benzene metabolite used as a hair dye ingredient in some countries. In an effort to identify a toxic mechanism of THB, we first analyzed the hair of consumers who used a shampoo containing THB, and contrary to our expectations, THB was not persistent in the hair. Following, we treated THB to human keratinocytes and HeLa Chang liver cells. Membrane damage was observed in both cell lines, which was more notable in HeLa Chang liver cells than in keratinocytes. Thus, we decided on HeLa Chang liver cells as target cells for further study. Cell viability decreased sharply between 20 μg/ml and 40 μg/mL, inducing G2/M phase arrest and non-apoptotic cell death. The expression of carcinogenesis-, DNA damage-, and transcriptional dysregulation-related genes were notably up-regulated, and the structure and function of mitochondria were disrupted. The volume of the ER and acidic compartments decreased, and intracellular ROS and calcium ion levels increased. More interestingly, we found that THB formed unique structures within the cells, especially around the nuclear membrane, and that those structures seemed to dig into the nucleus over time. A reverse docking analysis also showed that SULT1A1, CYP2E1, and CAT, known to play a significant role in protecting cells from harmful factors, might be potential target proteins for THB. Taken together, we suggest that THB induces non-apoptotic cell death via structural damage of intracellular organelles, especially the nuclear membrane., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Condensation of RNA and proteins is central to cellular functions, and the ability to program it would be valuable in synthetic biology and synthetic cell science. Here we introduce a modular platform for engineering synthetic RNA condensates from tailor-made, branched RNA nanostructures that fold and assemble co-transcriptionally. Up to three orthogonal condensates can form simultaneously and selectively accumulate fluorophores through embedded fluorescent light-up aptamers. The RNA condensates can be expressed within synthetic cells to produce membrane-less organelles with a controlled number and relative size, and showing the ability to capture proteins using selective protein-binding aptamers. The affinity between otherwise orthogonal nanostructures can be modulated by introducing dedicated linker constructs, enabling the production of bi-phasic RNA condensates with a prescribed degree of interphase mixing and diverse morphologies. The in situ expression of programmable RNA condensates could underpin the spatial organization of functionalities in both biological and synthetic cells., Competing Interests: Competing interests E.F., S.L., A.A.T., G.F. and L.D.M., through the Regents of University of California, have filed a patent application in the US Patent and Trademark Office, which includes disclosure of inventions described in this paper, provisional application serial number 63/588,142, filed on 5 October 2023, and entitled ‘Single stranded RNA motifs for in vitro co-transcriptional production of orthogonal phase separated condensates’. The other authors declare no competing interests., (© 2024. The Author(s).)

Given that crop yields are strongly limited by nitrogen, engineering crop plants with self-nitrogen-fertilization capacity holds great promise for sustainable agriculture. Recently, a nitrogen-fixing organelle has been characterized in the unicellular marine microalgae Braarudosphaera bigelowii. Engineering a nitrogen-fixing organelle into the non-nitrogen-fixing crops could benefit both environmental sustainability and global food security., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Site-specific incorporation of noncanonical amino acids (ncAAs) can be realized by genetic code expansion (GCE) technology. Different orthogonal tRNA synthetase/tRNA (RS/tRNA) pairs have been developed to introduce a ncAA at the desired site, delivering a wide variety of functionalities that can be installed into selected proteins. Cytoplasmic expression of RS/tRNA pairs can cause a problem with background ncAA incorporation into host proteins. The application of orthogonally translating organelles (OTOs), inspired by the concept of phase separation, provides a solution for this issue in mammalian cells, allowing site-specific and protein-selective ncAA incorporation. So far, only Methanosarcina mazei (Mm) pyrrolysyl-tRNA synthetase (PylRS) has been used within OTOs, limiting the method's potential. Here, we explored the implementation of four other widely used orthogonal RS/tRNA pairs with OTOs, which, to our surprise, were unsuccessful in generating mRNA-selective GCE. Next, we tested several experimental solutions and developed a new chimeric phenylalanyl-RS/tRNA pair that enables ncAA incorporation in OTOs in a site-specific and protein-selective manner. Our work reveals unaccounted design constraints in the spatial engineering of enzyme functions using designer organelles and presents a strategy to overcome those in vivo. We then discuss current limitations and future directions of in-cell engineering in general and protein engineering using GCE specifically., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: “E.A.L. holds patents related to OTOs. M.E.S. and M.J. declare no competing interests.”., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Hydrogel-based tissue expansion combined with mass spectrometry (MS) offers an emerging spatial proteomics approach. Here, we present a filter-aided expansion proteomics (FAXP) strategy for spatial proteomics analysis of archived formalin-fixed paraffin-embedded (FFPE) specimens. Compared to our previous ProteomEx method, FAXP employed a customized tip device to enhance both the stability and throughput of sample preparation, thus guaranteeing the reproducibility and robustness of the workflow. FAXP achieved a 14.5-fold increase in volumetric resolution. It generated over 8 times higher peptide yield and a 255% rise in protein identifications while reducing sample preparation time by 50%. We also demonstrated the applicability of FAXP using human colorectal FFPE tissue samples. Furthermore, for the first time, we achieved bona fide single-subcellular proteomics under image guidance by integrating FAXP with laser capture microdissection., (© 2024. The Author(s).)

Plant senescence is an integrated programme of plant development that aims to remobilize nutrients and energy from senescing tissues to developing organs under developmental and stress-induced conditions. Upstream in the regulatory network, a small family of single-stranded DNA/RNA-binding proteins known as WHIRLYs occupy a central node, acting at multiple regulatory levels and via trans-localization between the nucleus and organelles. In this review, we summarize the current progress on the role of WHIRLY members in plant development and stress-induced senescence. WHIRLY proteins can be traced back in evolution to green algae. WHIRLY proteins trade off the balance of plant developmental senescence and stress-induced senescence through maintaining organelle genome stability via R-loop homeostasis, repressing the transcription at a configuration condition, and recruiting RNA to impact organelle RNA editing and splicing, as evidenced in several species. WHIRLY proteins also act as retrograde signal transducers between organelles and the nucleus through protein modification and stromule or vesicle trafficking. In addition, WHIRLY proteins interact with hormones, reactive oxygen species and environmental signals to orchestrate cell fate in an age-dependent manner. Finally, prospects for further research and promotion to improve crop production under environmental constraints are highlighted., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Solvatochromic fluorescent probes are crucial molecular tools to investigate and aggregate proteins' fold, visualize fine structures in biomembranes, and label different organelles in dual emission colors. However, solvatochromic fluorogens often displayed a weak emission at high polarity, hindering their bioimaging applications. To resolve this problem, herein, we propose an intramolecular charge transfer (ICT) inhibition strategy. The probe was designed with a single electronic donor and two acceptors in order to split and inhibit the ICT procedure. As a result, the probe displayed an intense emission at both low and high polarities and showed a large emission shift (84 nm) upon polarity change. Using the probe, we successfully imaged lipid droplets and the endoplasmic reticulum in different fluorescence colors. Moreover, the different degrees of lipid accumulation by oleic acid, stearic acid, and cholesterol (oleic acid > stearic acid > cholesterol) have been revealed. The lipid accumulation induced by the three lipids could be rapidly consumed under lipid-less conditions, and the lipids with stearic acid were the most difficult to be consumed. The biological results could facilitate the understanding and treatment of lipid accumulation and obesity. Furthermore, utilizing the polarity increase of diethylamine after the reaction with CO 2 , the ratiometric detection of CO 2 has been achieved for the first time with the probe.

The ultrastructure of human oocytes has been described only qualitatively. To offer a precise organelle spatial distribution and organelle volume during the main maturation stages, we previously conducted stereological studies on prophase-I (GV) and metaphase-I (MI) oocytes, and here we present results on metaphase-II (MII) oocytes. Five donor oocytes from different donors were processed for transmission electron microscopy, and quantification of organelle distribution was performed using point-counting stereology. Statistical tests compared the means of the relative volumes occupied by organelles among oocyte regions. The most abundant organelles were elements of the smooth endoplasmic reticulum (SER), such as SER small vesicles, SER medium vesicles, SER large vesicles and SER isolated tubules, along with mitochondria, followed by SER tubular aggregates, cortical vesicles and lysosomes. Significant differences between oocyte regions were found for lysosomes, cortical vesicles and SER large vesicles. Comparisons of MII oocytes to previous findings in GV and MI oocytes evidenced specific patterns of organelle distribution and relative volumes. This final evaluation thus enables to track organelle spatial reorganization across oocyte stages, which, in addition to gathered knowledge, may be useful to assist in improvements of stimulation protocols, in-vitro maturation media and cryopreservation techniques., (© 2024. The Author(s).)

Membrane-less organelles, formed by the condensation of biomolecules, play a pivotal role in eukaryotes. Artificial membrane-less organelles and condensates are effective tools for the creation of new cellular functions. However, it is poorly understood how to control the properties that affect condensate function, particularly in plants. Here, we report the construction of model artificial condensates using the condensation-prone proteins OsJAZ2 and AtFCA in a transient assay using rice (Oryza sativa) cells, and how condensate properties, such as subcellular localization, protein mobility, and size can be altered. We showed that proteins of interest can be recruited to condensates using nanobodies or chemically induced dimerization. Furthermore, by combining two types of condensation-prone proteins, we demonstrated that artificial hybrid condensates with heterogeneous material properties could be constructed. Finally, we showed that modified artificial condensates can be constructed in transgenic Arabidopsis thaliana plants. These results provide a framework for the basic design of synthetic membrane-less organelles in plants., (© 2024. The Author(s).)

RNA modifications play a pivotal role in the regulation of RNA chemistry within cells. Several technologies have been developed with the goal of using RNA modifications to regulate cellular biochemistry selectively, but achieving selective and precise modifications remains a challenge. Here, we show that by using designer organelles, we can modify mRNA with pseudouridine in a highly selective and guide-RNA-dependent manner. We use designer organelles inspired by concepts of phase separation, a central tenet in developing artificial membraneless organelles in living mammalian cells. In addition, we use circular guide RNAs to markedly enhance the effectiveness of targeted pseudouridinylation. Our studies introduce spatial engineering through optimized RNA editing organelles (OREO) as a complementary tool for targeted RNA modification, providing new avenues to enhance RNA modification specificity., (© 2024. The Author(s).)

The major organelles of the endomembrane system were in place by the time of the last eukaryotic common ancestor (LECA) (~1.5 billion years ago). Their acquisitions were defining milestones during eukaryogenesis. Comparative cell biology and evolutionary analyses show multiple instances of homology in the protein machinery controlling distinct interorganelle trafficking routes. Resolving these homologous relationships allows us to explore processes underlying the emergence of additional, distinct cellular compartments, infer ancestral states predating LECA, and explore the process of eukaryogenesis itself. Here, we undertake a molecular evolutionary analysis (including providing a transcriptome of the jakobid flagellate Reclinomonas americana ), exploring the origins of the machinery responsible for the biogenesis of lysosome-related organelles (LROs), the Biogenesis of LRO Complexes (BLOCs 1,2, and 3). This pathway has been studied only in animals and is not considered a feature of the basic eukaryotic cell plan. We show that this machinery is present across the eukaryotic tree of life and was likely in place prior to LECA, making it an underappreciated facet of eukaryotic cellular organisation. Moreover, we resolve multiple points of ancient homology between all three BLOCs and other post-endosomal retrograde trafficking machinery (BORC, CCZ1 and MON1 proteins, and an unexpected relationship with the "homotypic fusion and vacuole protein sorting" (HOPS) and "Class C core vacuole/endosomal tethering" (CORVET) complexes), offering a mechanistic and evolutionary unification of these trafficking pathways. Overall, this study provides a comprehensive account of the rise of the LROs biogenesis machinery from before the LECA to current eukaryotic diversity, integrating it into the larger mechanistic framework describing endomembrane evolution., Competing Interests: Competing interests statement:The authors declare no competing interest.

Diatoms are central to the global carbon cycle. At the heart of diatom carbon fixation is an overlooked organelle called the pyrenoid, where concentrated CO 2 is delivered to densely packed Rubisco. Diatom pyrenoids fix approximately one-fifth of global CO 2 , but the protein composition of this organelle is largely unknown. Using fluorescence protein tagging and affinity purification-mass spectrometry, we generate a high-confidence spatially defined protein-protein interaction network for the diatom pyrenoid. Within our pyrenoid interaction network are 10 proteins with previously unknown functions. We show that six of these form a shell that encapsulates the Rubisco matrix and is critical for pyrenoid structural integrity, shape, and function. Although not conserved at a sequence or structural level, the diatom pyrenoid shares some architectural similarities to prokaryotic carboxysomes. Collectively, our results support the convergent evolution of pyrenoids across the two main plastid lineages and uncover a major structural and functional component of global CO 2 fixation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Carboxysomes are proteinaceous organelles featuring icosahedral protein shells that enclose the carbon-fixing enzymes, ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), along with carbonic anhydrase. The intrinsically disordered scaffolding protein CsoS2 plays a vital role in the construction of α-carboxysomes through bridging the shell and cargo enzymes. The N-terminal domain of CsoS2 binds Rubisco and facilitates Rubisco packaging within the α-carboxysome, whereas the C-terminal domain of CsoS2 (CsoS2-C) anchors to the shell and promotes shell assembly. However, the role of the middle region of CsoS2 (CsoS2-M) has remained elusive. Here, we conducted in-depth examinations on the function of CsoS2-M in the assembly of the α-carboxysome shell by generating a series of recombinant shell variants in the absence of cargos. Our results reveal that CsoS2-M assists CsoS2-C in the assembly of the α-carboxysome shell and plays an important role in shaping the α-carboxysome shell through enhancing the association of shell proteins on both the facet-facet interfaces and flat shell facets. Moreover, CsoS2-M is responsible for recruiting the C-terminal truncated isoform of CsoS2, CsoS2A, into α-carboxysomes, which is crucial for Rubisco encapsulation and packaging. This study not only deepens our knowledge of how the carboxysome shell is constructed and regulated but also lays the groundwork for engineering and repurposing carboxysome-based nanostructures for diverse biotechnological purposes., Importance: Carboxysomes are a paradigm of organelle-like structures in cyanobacteria and many proteobacteria. These nanoscale compartments enclose Rubisco and carbonic anhydrase within an icosahedral virus-like shell to improve CO 2 fixation, playing a vital role in the global carbon cycle. Understanding how the carboxysomes are formed is not only important for basic research studies but also holds promise for repurposing carboxysomes in bioengineering applications. In this study, we focuses on a specific scaffolding protein called CsoS2, which is involved in facilitating the assembly of α-type carboxysomes. By deciphering the functions of different parts of CsoS2, especially its middle region, we provide new insights into how CsoS2 drives the stepwise assembly of the carboxysome at the molecular level. This knowledge will guide the rational design and reprogramming of carboxysome nanostructures for many biotechnological applications., Competing Interests: The authors declare no conflict of interest.

Eukaryotic cells are compartmentalized into membrane-bound organelles that must coordinate their responses to stimuli. One way that organelles communicate is via membrane contact sites (MCSs), sites of close apposition between organelles used for the exchange of ions, lipids and information. In this Cell Science at a Glance article and the accompanying poster, we describe an explosion of new methods that have led to exciting progress in this area and discuss key examples of how these methods have advanced our understanding of MCSs. We discuss how diffraction-limited and super-resolution fluorescence imaging approaches have provided important insight into the biology of interorganelle communication. We also describe how the development of multiple proximity-based methods has enabled the detection of MCSs with high accuracy and precision. Finally, we assess how recent advances in electron microscopy (EM), considered the gold standard for detecting MCSs, have allowed the visualization of MCSs and associated proteins in 3D at ever greater resolution., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

As most traditional drugs used to treat central nervous system (CNS) diseases have a single therapeutic target, many of them cannot treat complex diseases or diseases whose mechanism is unknown and cannot effectively reverse the root changes underlying CNS diseases. This raises the question of whether multiple functional components are involved in the complex pathological processes of CNS diseases. Organelles are the core functional units of cells, and the replacement of damaged organelles with healthy organelles allows the multitargeted and integrated modulation of cellular functions. The development of therapies that target independent functional units in the cell, specifically, organelle-based therapies, is rapidly progressing. This article comprehensively discusses the pathogenesis of mitochondrial homeostasis disorders, which involve mitochondria, one of the most important organelles in CNS diseases, and the machanisms of mitochondrion-based therapies, as well as current preclinical and clinical studies on the efficacy of therapies targeting mitochondrial to treat CNS diseases, to provide evidence for use of organelle-based treatment strategies in the future., (© 2024. The Author(s).)

Among various cancer treatment methods, photodynamic therapy has received significant attention due to its non-invasiveness and high efficiency in inhibiting tumour growth. Recently, specific organelle targeting photosensitizers have received increasing interest due to their precise accumulation and ability to trigger organelle-mediated cell death signalling pathways, which greatly reduces the drug dosage, minimizes toxicity, avoids multidrug resistance, and prevents recurrence. In this review, recent advances and representative photosensitizers used in targeted photodynamic therapy on organelles, specifically including the endoplasmic reticulum, Golgi apparatus, mitochondria, nucleus, and lysosomes, have been comprehensively reviewed with a focus on organelle structure and organelle-mediated cell death signalling pathways. Furthermore, a perspective on future research and potential challenges in precision photodynamic therapy has been presented at the end.

How are Rab GTPases regulated during lysosome-related organelle (LRO) biogenesis? Li et al. (https://doi.org/10.1083/jcb.202402016) identify LYSMD proteins as crucial activators of Rab32-family GTPases in LRO development, shedding light on the previously ambiguous mechanisms governing Rab functionality in this process., (© 2024 Shi.)

Key Message: Analysis of the N-terminome of Physcomitrella reveals N-terminal monomethylation of nuclear-encoded, mitochondria-localized proteins. Post- or co-translational N-terminal modifications of proteins influence their half-life as well as mediating protein sorting to organelles via cleavable N-terminal sequences that are recognized by the respective translocation machinery. Here, we provide an overview on the current modification state of the N-termini of over 4500 proteins from the model moss Physcomitrella (Physcomitrium patens) using a compilation of 24 N-terminomics datasets. Our data reveal distinct proteoforms and modification states and confirm predicted targeting peptide cleavage sites of 1,144 proteins localized to plastids and the thylakoid lumen, to mitochondria, and to the secretory pathway. In addition, we uncover extended N-terminal methylation of mitochondrial proteins. Moreover, we identified PpNTM1 (P. patens alpha N-terminal protein methyltransferase 1) as a candidate for protein methylation in plastids, mitochondria, and the cytosol. These data can now be used to optimize computational targeting predictors, for customized protein fusions and their targeted localization in biotechnology, and offer novel insights into potential dual targeting of proteins., (© 2024. The Author(s).)

Understanding the spatiotemporal dynamics of formaldehyde (FA) is crucial for elucidating its pathophysiology. In this study, we designed a series of organelle-resolved probes to investigate FA dynamics. By incorporating various organelle anchors into a coumarin hydrazonate, we developed a series of probes with excellent organelle selectivity and FA specificity, enabling rapid and precise sensing of FA in an organelle-resolved way. Leveraging these probes, we captured the spatiotemporal dynamics of native FA in response to exogenous FA or oxidative stress challenges. In particular, we unveiled the distinct responses of various organelles to identical cellular stressors. Moreover, we observed the dynamic response within individual organelles when cells were exposed to stressors for varying durations. We envision these probes will serve as versatile tools for probing FA pathophysiology.

The cytoophidium is a novel type of membraneless organelle, first observed in the ovaries of Drosophila using fluorescence microscopy. In vitro, purified Drosophila melanogaster CTPS (dmCTPS) can form metabolic filaments under the presence of either substrates or products, and their structures that have been analyzed using cryo-electron microscopy (cryo-EM). These dmCTPS filaments are considered the fundamental units of cytoophidia. However, due to the resolution gap between light and electron microscopy, the precise assembly pattern of cytoophidia remains unclear. In this study, we find that dmCTPS filaments can spontaneously assemble in vitro, forming network structures that reach micron-scale dimensions. Using cryo-electron tomography (cryo-ET), we reconstruct the network structures formed by dmCTPS filaments under substrate or product binding conditions and elucidate their assembly process. The dmCTPS filaments initially form structural bundles, which then further assemble into larger networks. By identifying, tracking, and statistically analyzing the filaments, we observed distinct characteristics of the structural bundles formed under different conditions. This study provides the first systematic analysis of dmCTPS filament networks, offering new insights into the relationship between cytoophidia and metabolic filaments., Competing Interests: Declaration of competing interest The authors declare that no interest exists., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Motivation: Intracellular organelle networks (IONs) such as the endoplasmic reticulum (ER) network and the mitochondrial (MITO) network serve crucial physiological functions. The morphology of these networks plays a critical role in mediating their functions. Accurate image segmentation is required for analyzing the morphology and topology of these networks for applications such as molecular mechanism analysis and drug target screening. So far, however, progress has been hindered by their structural complexity and density., Results: In this study, we first establish a rigorous performance baseline for accurate segmentation of these organelle networks from fluorescence microscopy images by optimizing a baseline U-Net model. We then develop the multi-resolution encoder (MRE) and the hierarchical fusion loss (Lhf) based on two inductive components, namely low-level features and topological self-similarity, to assist the model in better adapting to the task of segmenting IONs. Empowered by MRE and Lhf, both U-Net and Pyramid Vision Transformer (PVT) outperform competing state-of-the-art models such as U-Net++, HR-Net, nnU-Net, and TransUNet on custom datasets of the ER network and the MITO network, as well as on public datasets of another biological network, the retinal blood vessel network. In addition, integrating MRE and Lhf with models such as HR-Net and TransUNet also enhances their segmentation performance. These experimental results confirm the generalization capability and potential of our approach. Furthermore, accurate segmentation of the ER network enables analysis that provides novel insights into its dynamic morphological and topological properties., Availability and Implementation: Code and data are openly accessible at https://github.com/cbmi-group/MRE., (© The Author(s) 2024. Published by Oxford University Press.)

In this study, we introduced a novel dual-laser multi-color imaging system. Integrated with a multi-channel filter wheel, this system compared three spectral decontamination algorithms (nonnegative matrix factorization [NMF], RCAN, and PICASSO) showcasing its efficacy in achieving four-color imaging with only two laser sources. Combined with a reliable image reconstruction algorithm, the spatial resolution of four channels super-resolution four-color images reached 130, 125, 133, and 132 nm, respectively. Lipid droplets, mitochondria, lysosomes, and nuclei from the mouse hepatocytes (AML12), human neuroblastoma cells (SH-SY5Y), mouse hippocampal neuronal cells (HT-22), and immortalized murine bone marrow-derived macrophages were imaged. At the same time, the chromatin condensation, nuclear contraction, DNA fragmentation, apoptotic body formation, as well as the fusion of Mito and Lyso involved in mitochondrial autophagy were observed in HT-22 and SH-SY5Y cells suffering oxidative stress. Our multi-color SIM imaging system establishes a powerful platform for dynamic organelle studies and other high-resolution investigations in live cells., (© 2024 Wiley‐VCH GmbH.)

Liquid-liquid phase separation (LLPS) is responsible for the emergence of intracellular membrane-less organelles and the development of coacervate protocells. Benefitting from the advantages of simplicity, precision, programmability, and noninvasiveness, light has become an effective tool to regulate the assembly dynamics of LLPS, and mediate various biochemical processes associated with LLPS. In this review, recent advances in optically controlling membrane-less organelles within living organisms are summarized, thereby modulating a series of biological processes including irreversible protein aggregation pathologies, transcription activation, metabolic flux, genomic rearrangements, and enzymatic reactions. Among these, the intracellular systems (i.e., optoDroplet, Corelet, PixELL, CasDrop, and other optogenetic systems) that enable the photo-mediated control over biomolecular condensation are highlighted. The design of photoactive complex coacervate protocells in laboratory settings by utilizing photochromic molecules such as azobenzene and diarylethene is further discussed. This review is expected to provide in-depth insights into phase separation-associated biochemical processes, bio-metabolism, and diseases., (© 2024 Wiley‐VCH GmbH.)

The presence of membrane-bound organelles with specific functions is one of the main hallmarks of eukaryotic cells. Organelle membranes are composed of specific lipids that govern their function and interorganelle communication. Discoveries in cell biology using imaging and omic technologies have revealed the mechanisms that drive membrane remodeling, organelle contact sites, and metabolite exchange. The interplay between multiple organelles and their interdependence is emerging as the next frontier for discovery using 3D reconstruction of volume electron microscopy (vEM) datasets. We discuss recent findings on the links between organelles that underlie common functions and cellular pathways. Specifically, we focus on the metabolism of ether glycerophospholipids that mediate organelle dynamics and their communication with each other, and the new imaging techniques that are powering these discoveries., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lamellar bodies (LBs) are tissue-specific lysosome-related organelles in type II alveolar cells that are the main site for the synthesis, storage, and secretion of pulmonary surfactants. Defects in pulmonary surfactants lead to a variety of respiratory and immune-related disorders. LB biogenesis is closely related to their function, but the underlying regulatory mechanism is largely unclear. Here, we found that deficiency of HPS6, a subunit of BLOC-2 (biogenesis of lysosome-related organelles complex-2), led to a reduction of the steady-state concentration of vacuolar-type H + -ATPase and an increase in the luminal pH of LBs. Furthermore, we observed increased LB size, accumulated surfactant proteins, and altered lipid profiling of lung tissue and BAL fluid due to HPS6 deficiency. These findings suggest that HPS6 regulates the distribution of vacuolar-type H + -ATPase on LBs to maintain its luminal acidity and LB homeostasis. This may provide new insights into the LB pathology.

In response to the spatiotemporal coordination of various biochemical reactions and membrane-encapsulated organelles, plants appear to provide another effective mechanism for cellular organization by phase separation that allows the internal compartmentalization of cells to form a variety of membrane-less organelles. Most of the research on phase separation has centralized in various non-plant systems, such as yeast and animal systems. Recent studies have shown a remarkable correlation between the formation of condensates in plant systems and the formation of condensates in these systems. Moreover, the last decade has made new advances in phase separation research in the context of plant biology. Here, we provide an overview of the physicochemical forces and molecular factors that drive liquid-liquid phase separation in plant cells and the biochemical characterization of condensates. We then explore new developments in phase separation research specific to plants, discussing examples of condensates found in green plants and detailing their role in plant growth and development. We propose that phase separation may be a conserved organizational mechanism in plant evolution to help plants respond rapidly and effectively to various environmental stresses as sessile organisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)