Long-read RNA-Seq for the discovery of long noncoding and antisense RNAs in plant organelles.

Plant organelle transcription has been studied for decades. As techniques advanced, so did the fields of mitochondrial and plastid transcriptomics. The current view is that organelle genomes are pervasively transcribed, irrespective of their size, content, structure, and taxonomic origin. However, little is known about the nature of organelle noncoding transcriptomes, including pervasively transcribed noncoding RNAs (ncRNAs). Next-generation sequencing data have uncovered small ncRNAs in the organelles of plants and other organisms, but long ncRNAs remain poorly understood. Here, we argue that publicly available third-generation long-read RNA sequencing data from plants can provide a fine-tuned picture of long ncRNAs within organelles. Indeed, given their bloated architectures, plant mitochondrial genomes are well suited for studying pervasive transcription of ncRNAs. Ultimately, we hope to showcase this new avenue of plant research while also underlining the limitations of the proposed approach.

METAPHOR: Metabolic evaluation through phasor-based hyperspectral imaging and organelle recognition for mouse blastocysts and oocytes.

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology.

Actomyosin organelle functions of SPIRE actin nucleators precede animal evolution.

An important question in cell biology is how cytoskeletal proteins evolved and drove the development of novel structures and functions. Here we address the origin of SPIRE actin nucleators. Mammalian SPIREs work with RAB GTPases, formin (FMN)-subgroup actin assembly proteins and class-5 myosin (MYO5) motors to transport organelles along actin filaments towards the cell membrane. However, the origin and extent of functional conservation of SPIRE among species is unknown. Our sequence searches show that SPIRE exist throughout holozoans (animals and their closest single-celled relatives), but not other eukaryotes. SPIRE from unicellular holozoans (choanoflagellate), interacts with RAB, FMN and MYO5 proteins, nucleates actin filaments and complements mammalian SPIRE function in organelle transport. Meanwhile SPIRE and MYO5 proteins colocalise to organelles in Salpingoeca rosetta choanoflagellates. Based on these observations we propose that SPIRE originated in unicellular ancestors of animals providing an actin-myosin driven exocytic transport mechanism that may have contributed to the evolution of complex multicellular animals.

The Nitroplast and Its Relatives Support a Universal Model of Features Predicting Gene Retention in Endosymbiont and Organelle Genomes.

Endosymbiotic relationships have shaped eukaryotic life. As endosymbionts coevolve with their host, toward full integration as organelles, their genomes tend to shrink, with genes being completely lost or transferred to the host nucleus. Modern endosymbionts and organelles show diverse patterns of gene retention, and why some genes and not others are retained in these genomes is not fully understood. Recent bioinformatic study has explored hypothesized influences on these evolutionary processes, finding that hydrophobicity and amino acid chemistry predict patterns of gene retention, both in organelles across eukaryotes and in less mature endosymbiotic relationships. The exciting ongoing elucidation of endosymbiotic relationships affords an independent set of instances to test this theory. Here, we compare the properties of retained genes in the nitroplast, recently reported to be an integrated organelle, two related cyanobacterial endosymbionts that form "spheroid bodies" in their host cells, and a range of other endosymbionts, with free-living relatives of each. We find that in each case, the symbiont's genome encodes proteins with higher hydrophobicity and lower amino pKa than their free-living relative, supporting the data-derived model predicting the retention propensity of genes across endosymbiont and organelle genomes.

Single-tissue proteomics in Caenorhabditis elegans reveals proteins resident in intestinal lysosome-related organelles.

The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry (MS)-based proteomic analysis of the intestine of the Caenorhabditis elegans and of its gut granules. Overall, we identified approximately 5,000 proteins each in the intestine and the gonad and showed that most of these proteins can be detected in samples extracted from a single worm, suggesting the feasibility of individual-level genetic analysis using proteomes. Comparing proteomes and published transcriptomes of the intestine and the gonad, we identified proteins that appear to be synthesized in the intestine and then transferred to the gonad. To identify gut granule proteins, we compared the proteome of individual intestines deficient in gut granules to the wild type. The identified gut granule proteome includes proteins known to be exclusively localized to the granules and additional putative gut granule proteins. We selected two of these putative gut granule proteins for validation via immunohistochemistry, and our successful confirmation of both suggests that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single-tissue MS-based proteomic analysis in small organisms and in its future utility.

An invariant C-terminal tryptophan in McdB mediates its interaction and positioning function with carboxysomes.

Bacterial microcompartments (BMCs) are widespread, protein-based organelles that regulate metabolism. The model for studying BMCs is the carboxysome, which facilitates carbon fixation in several autotrophic bacteria. Carboxysomes can be distinguished as type α or ß, which are structurally and phyletically distinct. We recently characterized the maintenance of carboxysome distribution (Mcd) systems responsible for spatially regulating α- and ß-carboxysomes, consisting of the proteins McdA and McdB. McdA is an ATPase that drives carboxysome positioning, and McdB is the adaptor protein that directly interacts with carboxysomes to provide cargo specificity. The molecular features of McdB proteins that specify their interactions with carboxysomes, and whether these are similar between α- and ß-carboxysomes, remain unknown. Here, we identify C-terminal motifs containing an invariant tryptophan necessary for α- and ß-McdBs to associate with α- and ß-carboxysomes, respectively. Substituting this tryptophan with other aromatic residues reveals corresponding gradients in the efficiency of carboxysome colocalization and positioning by McdB in vivo. Intriguingly, these gradients also correlate with the ability of McdB to form condensates in vitro. The results reveal a shared mechanism underlying McdB adaptor protein binding to carboxysomes, and potentially other BMCs. Our findings also implicate condensate formation as playing a key role in this association.

Benzo[a]pyrene exposure disrupts the organelle distribution and function of mouse oocytes.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon compound that is generated during combustion processes, and is present in various substances such as foods, tobacco smoke, and burning emissions. BaP is extensively acknowledged as a highly carcinogenic substance to induce multiple forms of cancer, such as lung cancer, skin cancer, and stomach cancer. Recently it is shown to adversely affect the reproductive system. Nevertheless, the potential toxicity of BaP on oocyte quality remains unclear. In this study, we established a BaP exposure model via mouse oral gavage and found that BaP exposure resulted in a notable decrease in the ovarian weight, number of GV oocytes in ovarian, and oocyte maturation competence. BaP exposure caused ribosomal dysfunction, characterized by a decrease in the expression of RPS3 and HPG in oocytes. BaP exposure also caused abnormal distribution of the endoplasmic reticulum (ER) and induced ER stress, as indicated by increased expression of GRP78. Besides, the Golgi apparatus exhibited an abnormal localization pattern, which was confirmed by the GM130 localization. Disruption of vesicle transport processes was observed by the abnormal expression and localization of Rab10. Additionally, an enhanced lysosome and LC3 fluorescence intensity indicated the occurrence of protein degradation in oocytes. In summary, our results suggested that BaP exposure disrupted the distribution and functioning of organelles, consequently affecting the developmental competence of mouse oocytes.

Protocells Featuring Membrane-Bound and Dynamic Membraneless Organelles.

Living cells, especially eukaryotic ones, use multicompartmentalization to regulate intra- and extracellular activities, featuring membrane-bound and membraneless organelles. These structures govern numerous biological and chemical processes spatially and temporally. Synthetic cell models, primarily utilizing lipidic and polymeric vesicles, have been developed to carry out cascade reactions within their compartments. However, these reconstructions often segregate membrane-bound and membraneless organelles, neglecting their collaborative role in cellular regulation. To address this, we propose a structural design incorporating microfluidic-produced liposomes housing synthetic membrane-bound organelles made from self-assembled poly(ethylene glycol)-block-poly(trimethylene carbonate) nanovesicles and synthetic membraneless organelles formed via temperature-sensitive elastin-like polypeptide phase separation. This architecture mirrors natural cellular organization, facilitating a detailed examination of the interactions for a comprehensive understanding of cellular dynamics.

Homeostatic coordination of cellular phosphate uptake and efflux requires an organelle-based receptor for the inositol pyrophosphate IP8.

Phosphate (Pi) serves countless metabolic pathways and is involved in macromolecule synthesis, energy storage, cellular signaling, and bone maintenance. Herein, we describe the coordination of Pi uptake and efflux pathways to maintain mammalian cell Pi homeostasis. We discover that XPR1, the presumed Pi efflux transporter, separately supervises rates of Pi uptake. This direct, regulatory interplay arises from XPR1 being a binding partner for the Pi uptake transporter PiT1, involving a predicted transmembrane helix/extramembrane loop in XPR1, and its hitherto unknown localization in a subset of intracellular LAMP1-positive puncta (named "XLPVs"). A pharmacological mimic of Pi homeostatic challenge is sensed by the inositol pyrophosphate IP8, which functionalizes XPR1 to respond in a temporally hierarchal manner, initially adjusting the rate of Pi efflux, followed subsequently by independent modulation of PiT1 turnover to reset the rate of Pi uptake. These observations generate a unifying model of mammalian cellular Pi homeostasis, expanding opportunities for therapeutic intervention.

The symbiosome-a transient organelle in evolution.

This article comments on: Casaes PA, Ferreira dos Santos JM, Silva VC, Rhem MFK, Teixeira Cota MM, de Faria SM, Rando JG, James EK, Gross E. 2024. The radiation of nodulated Chamaecrista species from the rainforest into more diverse habitats has been accompanied by a reduction in growth form and a shift from fixation threads to symbiosomes. Journal of Experimental Botany 75, 3643-3662.

A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis.

BACKGROUND: Hydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane-associated respiratory chain, and consequently in the generation of minimal inner membrane potential (Δψ), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on Δψ and ATP hydrolysis, while TIM22 requires only Δψ. The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown. RESULTS: We report unprecedented modification of TIM in hydrogenosomes of the human parasite Trichomonas vaginalis (TvTIM). We show that the import of the presequence-containing protein into the hydrogenosomal matrix is mediated by the hybrid TIM22-TIM23 complex that includes three highly divergent core components, TvTim22, TvTim23, and TvTim17-like proteins. The hybrid character of the TvTIM is underlined by the presence of both TvTim22 and TvTim17/23, association with small Tim chaperones (Tim9-10), which in mitochondria are known to facilitate the transfer of substrates to the TIM22 complex, and the coupling with TIM23-specific ATP-dependent presequence translocase-associated motor (PAM). Interactome reconstruction based on co-immunoprecipitation (coIP) and mass spectrometry revealed that hybrid TvTIM is formed with the compositional variations of paralogs. Single-particle electron microscopy for the 132-kDa purified TvTIM revealed the presence of a single ring of small Tims complex, while mitochondrial TIM22 complex bears twin small Tims hexamer. TvTIM is currently the only TIM visualized outside of Opisthokonta, which raised the question of which form is prevailing across eukaryotes. The tight association of the hybrid TvTIM with ADP/ATP carriers (AAC) suggests that AAC may directly supply ATP for the protein import since ATP synthesis is limited in hydrogenosomes. CONCLUSIONS: The hybrid TvTIM in hydrogenosomes represents an original structural solution that evolved for protein import when Δψ is negligible and remarkable example of evolutionary adaptation to an anaerobic lifestyle.

Learning Morphological, Spatial, and Dynamic Models of Cellular Components.

In this chapter, we describe protocols for using the CellOrganizer software on the Jupyter Notebook platform to analyze and model cell and organelle shape and spatial arrangement. CellOrganizer is an open-source system for using microscope images to learn statistical models of the structure of cell components and how those components are organized relative to each other. Such models capture the statistical variation in the organization of cellular components by jointly modeling the distributions of their number, shape, and spatial distributions. These models can be created for different cell types or conditions and compared to reflect differences in their spatial organizations. The models are also generative, in that they can be used to synthesize new cell instances reflecting what a model learned and to provide well-structured cell geometries that can be used for biochemical simulations.

Characterization of Nuclear HIV-Induced Membraneless Organelles Through Fluorescence Microscopy.

The postnuclear entry steps of HIV-1 involve reverse transcription, uncoating, and integration into the host genome. The differential regulation of these steps has a significant impact on HIV overall replication, including integration site selection and viral gene expression. Recently, another important phenomenon has been uncovered as part of HIV interplay with the nuclear environment, specifically involving the cleavage and polyadenylation specific factor 6 (CPSF6) protein. This phenomenon is the formation of nuclear HIV-induced membraneless organelles (HIV-1 MLOs). In this article, we will describe the methods used to assess the composition and liquid-liquid phase separation (LLPS) properties of these organelles using fluorescence microscopy. The study of HIV-1 MLOs represents a new frontier that may reveal previously unknown key players in the fate of HIV-infected cells.

Independent organelle and organelle-organelle interactions: essential mechanisms for malignant gynecological cancer cell survival.

Different eukaryotic cell organelles (e.g., mitochondria, endoplasmic reticulum, lysosome) are involved in various cancer processes, by dominating specific cellular activities. Organelles cooperate, such as through contact points, in complex biological activities that help the cell regulate energy metabolism, signal transduction, and membrane dynamics, which influence survival process. Herein, we review the current studies of mechanisms by which mitochondria, endoplasmic reticulum, and lysosome are related to the three major malignant gynecological cancers, and their possible therapeutic interventions and drug targets. We also discuss the similarities and differences of independent organelle and organelle-organelle interactions, and their applications to the respective gynecological cancers; mitochondrial dynamics and energy metabolism, endoplasmic reticulum dysfunction, lysosomal regulation and autophagy, organelle interactions, and organelle regulatory mechanisms of cell death play crucial roles in cancer tumorigenesis, progression, and response to therapy. Finally, we discuss the value of organelle research, its current problems, and its future directions.

Mitochondrially-associated actin waves maintain organelle homeostasis and equitable inheritance.

First identified in dividing cells as revolving clusters of actin filaments, these are now understood as mitochondrially-associated actin waves that are active throughout the cell cycle. These waves are formed from the polymerization of actin onto a subset of mitochondria. Within minutes, this F-actin depolymerizes while newly formed actin filaments assemble onto neighboring mitochondria. In interphase, actin waves locally fragment the mitochondrial network, enhancing mitochondrial content mixing to maintain organelle homeostasis. In dividing cells actin waves spatially mix mitochondria in the mother cell to ensure equitable partitioning of these organelles between daughter cells. Progress has been made in understanding the consequences of actin cycling as well as the underlying molecular mechanisms, but many questions remain, and here we review these elements. Also, we draw parallels between mitochondrially-associated actin cycling and cortical actin waves. These dynamic systems highlight the remarkable plasticity of the actin cytoskeleton.

Enzyme-cargo encapsulation peptides bind between tessellating tiles of the bacterial microcompartment shell.

Bacterial microcompartments are prokaryotic organelles comprising encapsulated enzymes within a thin protein shell. They facilitate metabolic processing including propanediol, choline, glycerol, and ethanolamine utilization, and they accelerate carbon fixation in cyanobacteria. Enzymes targeted to the inside of the microcompartment frequently possess a cargo-encapsulation peptide, but the site to which the peptide binds is unclear. We provide evidence that the encapsulation peptides bind to the hydrophobic groove formed between tessellating subunits of the shell proteins. In silico docking studies provide a compelling model of peptide binding to this prominent hydrophobic groove. This result is consistent with the now widely accepted view that the convex side of the shell oligomers faces the lumen of the microcompartment. The binding of the encapsulation peptide to the groove between tessellating shell protein tiles explains why it has been difficult to define the peptide binding site using other methods, provides a mechanism by which encapsulation-peptide bearing enzymes can promote shell assembly, and explains how the presence of cargo affects the size and shape of the bacterial microcompartment. This knowledge may be exploited in engineering microcompartments or disease prevention by hampering cargo encapsulation.

Human Umbilical Vein Endothelial Cells as a Versatile Cellular Model System in Diverse Experimental Paradigms: An Ultrastructural Perspective.

Human umbilical vein endothelial cells (HUVECs) are primary cells isolated from the vein of an umbilical cord, extensively used in cardiovascular studies and medical research. These cells, retaining the characteristics of endothelial cells in vivo, serve as a valuable cellular model system for understanding vascular biology, endothelial dysfunction, pathophysiology of diseases such as atherosclerosis, and responses to different drugs or treatments. Transmission electron microscopy (TEM) has been a cornerstone in revealing the detailed architecture of multiple cellular model systems including HUVECs, allowing researchers to visualize subcellular organelles, membrane structures, and cytoskeletal elements. Among them, the endoplasmic reticulum, Golgi apparatus, mitochondria, and nucleus can be meticulously examined to recognize alterations indicative of cellular responses to various stimuli. Importantly, Weibel-Palade bodies are characteristic secretory organelles found in HUVECs, which can be easily distinguished in the TEM. These distinctive structures also dynamically react to different factors through regulated exocytosis, resulting in complete or selective release of their contents. This detailed review summarizes the ultrastructural features of HUVECs and highlights the utility of TEM as a pivotal tool for analyzing HUVECs in diverse research frameworks, contributing valuable insights into the comprehension of HUVEC behavior and enriching our knowledge into the complexity of vascular biology.

The impact of, and expectations for, lipid nanoparticle technology: From cellular targeting to organelle targeting.

The success of mRNA vaccines against COVID-19 has enhanced the potential of lipid nanoparticles (LNPs) as a system for the delivery of mRNA. In this review, we describe our progress using a lipid library to engineer ionizable lipids and promote LNP technology from the viewpoints of safety, controlled biodistribution, and mRNA vaccines. These advancements in LNP technology are applied to cancer immunology, and a potential nano-DDS is constructed to evaluate immune status that is associated with a cancer-immunity cycle that includes the sub-cycles in tumor microenvironments. We also discuss the importance of the delivery of antigens and adjuvants in enhancing the cancer-immunity cycle. Recent progress in NK cell targeting in cancer immunotherapy is also introduced. Finally, the impact of next-generation DDS technology is explained using the MITO-Porter membrane fusion-based delivery system for the organelle targeting of the mitochondria. We introduce a successful example of the MITO-Porter used in a cell therapeutic strategy to treat cardiomyopathy.

From cells to subcellular organelles: Next-generation cancer therapy based on peptide self-assembly.

Due to the editability, functionality, and excellent biocompatibility of peptides, in situ self-assembly of peptides in cells is a powerful strategy for biomedical applications. Subcellular organelle targeting of peptides assemblies enables more precise drug delivery, enhances selectivity to disease cells, and mitigates drug resistance, providing an effective strategy for disease diagnosis and therapy. This reviewer first introduces the triggering conditions, morphological changes, and intracellular locations of self-assembling peptides. Then, the functions of peptide assemblies are summarized, followed by a comprehensive understanding of the interactions between peptide assemblies and subcellular organelles. Finally, we provide a brief outlook and the remaining challenges in this field.