Membrane-bound Organelles Research Articles

Convergence of autism proteins at the cilium.

Hundreds of high confidence autism genes have been identified, yet the relevant etiological mechanisms remain unclear. Gene ontology analyses have repeatedly identified enrichment of proteins with annotated functions in gene expression regulation and neuronal communication. However, proteins are often pleiotropic and these annotations are inherently incomplete. Our recent autism functional genetics work has suggested that these genes may share a common mechanism at the cilium, a membrane-bound organelle critical for neurogenesis, brain patterning, and neuronal activity-all processes strongly implicated in autism. Moreover, autism commonly co-occurs with conditions that are known to involve ciliary-related pathologies, including congenital heart disease, hydrocephalus, and blindness. However, the role of autism genes at the cilium has not been systematically investigated. Here we demonstrate that autism proteins spanning disparate functional annotations converge in expression, localization, and function at cilia, and that patients with pathogenic variants in these genes have cilia-related co-occurring conditions and biomarkers of disrupted ciliary function. This degree of convergence among genes spanning diverse functional annotations strongly suggests that cilia are relevant to autism, as well as to commonly co-occurring conditions, and that this organelle should be explored further for therapeutic potential.

Bacterial Organelles in Iron Physiology.

Bacteria were once thought to be simple organisms, lacking the membrane-bound organelles found in eukaryotic cells. However, recent advancements in microscopy have changed this view, revealing a diverse array of organelles within bacterial cells. These organelles, surrounded by lipid bilayers, protein-lipid monolayers, or proteinaceous shells, play crucial roles in facilitating biochemical reactions and protecting cells from harmful byproducts. Unlike eukaryotic organelles, which are universally present, bacterial organelles are species-specific and induced only under certain conditions. This review focuses on the bacterial organelles that contain iron, an essential micronutrient for all life forms but potentially toxic when present in excess. To date, three types of iron-related bacterial organelles have been identified: two membrane-bound organelles, magnetosomes and ferrosomes, and one protein-enclosed organelle, the encapsulated ferritin-like proteins. This article provides an updated overview of the genetics, biogenesis, and physiological functions of these organelles. Furthermore, we discuss how bacteria utilize these specialized structures to adapt, grow, and survive under various environmental conditions.

P53 engagement is a hallmark of an unfolded protein response in the nucleus of mammalian cells.

Exposure to exogenous and endogenous stress is associated with the intracellular accumulation of aberrant unfolded and misfolded proteins. In eukaryotic cells, protein homeostasis within membrane-bound organelles is regulated by specialized signaling pathways, with the unfolded protein response in the endoplasmic reticulum serving as a foundational example. Yet, it is unclear if a similar surveillance mechanism exists in the nucleus. Here we leveraged engineered proteins called destabilizing domains to acutely expose mammalian cells to nuclear- or cytosolic- localized unfolded protein. We show that the appearance of unfolded protein in either compartment engages a common transcriptional response associated with the transcription factors Nrf1 and Nrf2. Uniquely, only in the nucleus does unfolded protein activate a robust p53-driven transcriptional response and a transient p53-independent cell cycle delay. These studies highlight the distinct effects of localized protein folding stress and the unique protein quality control environment of the nucleus.

Bacterial toxins induce non-canonical migracytosis to aggravate acute inflammation.

Migracytosis is a recently described cellular process that generates and releases membrane-bound pomegranate-like organelles called migrasomes. Migracytosis normally occurs during cell migration, participating in various intercellular biological functions. Here, we report a new type of migracytosis induced by small GTPase-targeting toxins. Unlike classic migracytosis, toxin-induced migrasome formation does not rely on cell migration and thus can occur in both mobile and immobile cells. Such non-canonical migracytosis allows the cells to promptly respond to microbial stimuli such as bacterial toxins and effectors and release informative cellular contents in bulk. We demonstrated that C. difficile TcdB3 induces liver endothelial cells and Kupffer cells to produce migrasomes in vivo. Moreover, the migracytosis-defective Tspan9‒/‒ mice show less acute inflammation and lower lethality rate in the toxin challenge assay. Therefore, we propose that the non-canonical migracytosis acts as a new mechanism for mammalian species to sense and exacerbate early immune response upon microbial infections.

Ciliopathy organoid models: a comprehensive review.

Cilia are membrane-bound organelles found on the surface of most mammalian cell types and play numerous roles in human physiology and development, including osmo- and mechanosensation, as well as signal transduction. Ciliopathies are a large group of, usually rare, genetic disorders resulting from abnormal ciliary structure or ciliary dysfunction that have a high collective prevalence. Autosomal dominant or recessive polycystic kidney disease (ADPKD/ARPKD), Bardet-Biedl-Syndrome, and primary ciliary dyskinesia (PCD) are the most frequent etiologies. Rodent and zebrafish models have improved the understanding of ciliopathy pathophysiology. Yet, the limitations of these genetically modified animal strains include the inability to fully replicate the phenotypic heterogeneity found in humans, including variable multiorgan involvement. Organoids, self-assembled three-dimensional cell-based models derived from human induced pluripotent stem cells (iPSCs) or primary tissues, can recapitulate certain aspects of the development, architecture, and function of the target organ "in the dish." The potential of organoids to model patient-specific genotype-phenotype correlations has increased their popularity in ciliopathy research and led to the first preclinical organoid-based ciliopathy drug screens. This review comprehensively summarizes and evaluates current ciliopathy organoid models, focusing on kidney, airway, liver, and retinal organoids, as well as the specific methodologies used for their cultivation and for interrogating ciliary dysfunction.

Novel structure and composition of the unusually large germline determinant of the wasp Nasonia vitripennis.

Specialized, maternally derived ribonucleoprotein (RNP) granules play an important role in specifying the primordial germ cells in many animal species. Typically, these germ granules are small (~100 nm to a few microns in diameter) and numerous; in contrast, a single, extremely large granule called the oosome plays the role of germline determinant in the wasp Nasonia vitripennis. The organizational basis underlying the form and function of this unusually large membraneless RNP granule remains an open question. Here we use a combination of super-resolution and transmission electron microscopy to investigate the composition and morphology of the oosome. We show that the oosome has properties of a viscous liquid or elastic solid. The most prominent feature of the oosome is a branching mesh-like network of high abundance mRNAs that pervades the entire structure. Homologs of the core polar granule proteins Vasa and Oskar do not appear to nucleate this network, but rather are distributed adjacently as separate puncta. Low abundance RNAs appear to cluster in puncta that similarly do not overlap with the protein puncta. Several membrane-bound organelles, including lipid droplets and rough ER-like vesicles, are incorporated within the oosome, whereas mitochondria are nearly entirely excluded. Our findings show that the remarkably large size of the oosome is reflected in a complex sub-granular organization and suggest that the oosome is a powerful model for probing interactions between membraneless and membrane-bound organelles, structural features that contribute to granule size, and the evolution of germ plasm in insects.

Peroxisome inter-organelle cooperation in Drosophila

Many cellular functions are compartmentalized within the optimized environments of organelles. However, processing or storage of metabolites from the same pathway can occur in multiple organelles. Thus, spatially separated organelles need to cooperate functionally. Coordination between organelles in different specialized cells is also needed, with shared metabolites passed via circulation. Peroxisomes are membrane-bounded organelles responsible for cellular redox and lipid metabolism in eukaryotic cells. Peroxisomes coordinate with other organelles including mitochondria, endoplasmic reticulum, lysosomes, and lipid droplets. This functional coordination requires, or is at least enhanced by, direct contact between peroxisomes and other organelles. Peroxisome dysfunction in humans leads to multiorgan effects including neurological, metabolic, developmental, and age-related diseases. Thus, increased understanding of peroxisome coordination with other organelles, especially cells in various organs is essential. Drosophila melanogaster (fruit fly) has emerged recently as an effective animal model for understanding peroxisomes. Here we review current knowledge of pathways regulating coordination between peroxisomes with other organelles in flies, speculating about analogous roles for conserved Drosophila genes encoding proteins with known organelle coordinating roles in other species.

Distinct Hippocampal Expression Profiles of lncRNAs in Obese Type 2 Diabetes Mice Exhibiting Cognitive Impairment.

Cognitive dysfunction has been accepted as a possible complication of type 2 diabetes (T2D), but few studies revealed the potential roles of Long non‑coding RNAs (lncRNAs) in cognitive dysfunction in T2D. The current research aims to demonstrate the specific expression patterns of lncRNA-mRNA in the hippocampi of T2D db/db mice exhibiting cognitive impairment. In this study, the results from behavioral tests showed that T2D db/db mice displayed short-term and spatial working memory deficits compared to db/m mice. Furthermore, western blot analysis demonstrated that compared with db/m mice, p-GSK3β (ser9) protein levels were markedly elevated in T2D db/db mice (P < 0.01). In addition, though not statistically significant, the ratio of p-Tau (Ser396) to Tau 46, α-Synuclein expression, and p-GSK3α (ser21) expression were also relatively higher in T2D db/db mice than in db/m mice. The microarray profiling revealed that 75 lncRNAs and 26 mRNAs were dysregulated in T2D db/db mice (> 2.0 fold change, P < 0.05). GO analysis demonstrated that the differentially expressed mRNAs participated in immune response, extracellular membrane-bounded organelle, and extracellular region. KEGG analysis revealed that the differentially expressed mRNAs were mainly involved in one carbon pool by folate, glyoxylate and dicarboxylate metabolism, autophagy, glycine, serine and threonine metabolism, and B cell receptor signaling pathway. A lncRNA‑mRNA coexpression network containing 71 lncRNAs and 26 mRNAs was built to investigate the interaction between lncRNA and mRNA. Collectively, these results revealed the differential hippocampal expression profiles of lncRNAs in T2D mice with cognitive dysfunction, and the findings from this study provide new clues for exploring the potential roles of lncRNAs in the pathogenesis of cognitive dysfunction in T2D.

Stress granules and organelles: Coordinating cellular responses in health and disease.

Membrane-bound organelles and membraneless organelles (MLOs) coordinate various biological processes within eukaryotic cells. Among these, stress granules (SGs) are significant cytoplasmic MLOs that form in response to cellular stress, exhibiting liquid-like properties alongside stable substructures. SGs interact with diverse organelles, thereby influencing cellular pathways that are critical in both health and disease contexts. This review discusses the interplay between SGs and organelles and explores the methodologies employed to analyze interactions between SGs and other MLOs. Furthermore, it highlights the pivotal roles SGs play in regulating cellular responses and the pathogenesis of ALS. Gaining insights into these interactions is essential for deciphering the mechanisms underlying both physiological processes and pathological conditions.

A transcriptionally active lipid vesicle encloses the injected Chimalliviridae genome in early infection.

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses 1-3 . Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA 4-10 . Previously, we observed lipid membrane-bound vesicles in cells infected by Chimalliviridae , but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a bona fide stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART 11 in combination with fluorescence microscopy and cryo-electron tomography, we show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. We demonstrate that early phage genes are transcribed by the virion-associated RNA polymerase from the genome within the compartment, making the EPI vesicle the first known example of a lipid membrane-bound organelle that separates transcription from translation in prokaryotes. Further, we show that the phage nucleus is essential for the phage life cycle, with genome replication only beginning after the injected DNA is transferred from the EPI vesicle to the newly assembled phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

Imaging interorganelle contacts at a glance.

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.

Phthalate monoesters affect membrane fluidity and cell-cell contacts in endometrial stromal adherent cell lines and spheroids

Phthalate monoesters have been identified as endocrine disruptors in a variety of models, yet understanding of their exact mechanisms of action and molecular targets in cells remains incomplete. Here, we set to determine whether epidemiologically relevant mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) can affect biological processes by altering cell plasma membrane fluidity or formation of cell-cell contacts. As a model system, we chose endometrial stromal cell lines, one of which was previously used in a transcriptomic study with MEHHP or MEHHP-containing mixtures. A short-term exposure (1 h) of membrane preparations to endocrine disruptors was sufficient to induce changes in membrane fluidity/rigidity, whereas different mixtures showed different effects at various depths of the bilayer. A longer exposure (96 h) affected the ability of cells to form spheroids and highlighted issues with membrane integrity in loosely assembled spheroids. Finally, in spheroids assembled from T-HESC cells, MEHHP interfered with the formation of cell-cell contacts as indicated by the immunostaining of zonula occludens 1 protein. Overall, this study emphasized the need to consider plasma membrane, membrane-bound organelles, and secretory vesicles as possible biological targets of endocrine disruptors and offered an explanation for a multitude of endocrine disruptor roles documented earlier.

Biomolecular Condensates are Characterized by Interphase Electric Potentials.

Biomolecular condensates form via processes that combine phase separation and reversible associations of multivalent macromolecules. Condensates can be two- or multiphase systems defined by coexisting dense and dilute phases. Here, we show that solution ions partition asymmetrically across coexisting phases defined by condensates formed by intrinsically disordered proteins or homopolymeric RNA molecules. Our findings were enabled by direct measurements of the activities of cations and anions within coexisting phases of protein and RNA condensates. Asymmetries in ion partitioning between coexisting phases vary with protein sequence, macromolecular composition, salt concentration, and ion type. The Donnan equilibrium set up by the asymmetrical partitioning of solution ions generates interphase electric potentials known as Donnan and Nernst potentials. Our measurements show that the interphase potentials of condensates are of the same order of magnitude as membrane potentials of membrane-bound organelles. Interphase potentials quantify the degree to which microenvironments of coexisting phases are different from one another. Importantly, and based on condensate-specific interphase electric potentials, we reason that condensates are akin to capacitors that store charge. Interphase potentials should lead to electric double layers at condensate interfaces, thereby explaining recent observations of condensate interfaces being electrochemically active.

Molecular mechanics studies of factors affecting overall rate in cascade reactions: Multi-enzyme colocalization and environment.

Millions of years of evolution have optimized many biosynthetic pathways by use of multi-step catalysis. In addition, multi-step metabolic pathways are commonly found in and on membrane-bound organelles in eukaryotic biochemistry. The fundamental mechanisms that facilitate these reaction processes provide strategies to bioengineer metabolic pathways in synthetic chemistry. Using Brownian dynamics simulations, here we modeled intermediate substrate transportation of colocalized yeast-ester biosynthesis enzymes on the membrane. The substrate acetate ion traveled from the pocket of aldehyde dehydrogenase to its target enzyme acetyl-CoA synthetase, then the substrate acetyl CoA diffused from Acs1 to the active site of the next enzyme, alcohol-O-acetyltransferase. Arranging two enzymes with the smallest inter-enzyme distance of 60 Å had the fastest average substrate association time as compared with anchoring enzymes with larger inter-enzyme distances. When the off-target side reactions were turned on, most substrates were lost, which suggests that native localization is necessary for efficient final product synthesis. We also evaluated the effects of intermolecular interactions, local substrate concentrations, and membrane environment to bring mechanistic insights into the colocalization pathways. The computation work demonstrates that creating spatially organized multi-enzymes on membranes can be an effective strategy to increase final product synthesis in bioengineering systems.

Membrane and organelle rearrangement during ascospore formation in budding yeast.

SUMMARYIn ascomycete fungi, sexual spores, termed ascospores, are formed after meiosis. Ascospore formation is an unusual cell division in which daughter cells are created within the cytoplasm of the mother cell by de novo generation of membranes that encapsulate each of the haploid chromosome sets created by meiosis. This review describes the molecular events underlying the creation, expansion, and closure of these membranes in the budding yeast, Saccharomyces cerevisiae. Recent advances in our understanding of the regulation of gene expression and the dynamic behavior of different membrane-bound organelles during this process are detailed. While less is known about ascospore formation in other systems, comparison to the distantly related fission yeast suggests that the molecular events will be broadly similar throughout the ascomycetes.

Magnetotactic bacteria and magnetosomes – an overview

Magnetotactic bacteria are a diverse group of prokaryotes that create intracellular, membrane-bound organelles containing magnetic minerals known as magnetosomes. These minerals have a sdpecialized purpose with the use of mineral crystals such as magnetite (Fe3O4) or greigite (Fe3S4). Due to the stability of these crystal structures, the bacteria can orient themselves with the Earth’s geomagnetic field lines, functioning as compass needles to help them locate suitable environments to inhabit. The objective of this review, based on previous progressions in this area of study, is focused on presenting a comprehensive summary of MTB, its abundance and magnetosome formation. Moreover, the effects of certain environmental factors, as well as their significance and applications in material science, medicine, and biotechnology, are elucidated.

Cell-type specific methylation changes in the newborn child associated to obstetric pain relief

Although it is widely known that various pharmaceuticals affect the methylome, the knowledge of the effects from anesthesia is limited, and nearly nonexistent regarding the effects of obstetric anesthesia on the newborn child. Using sequencing based-methylation data and a reference-based statistical deconvolution approach we performed methylome-wide association studies (MWAS) of neonatal whole blood, and for each cell-type specifically, to detect methylation variations that are associated with the pain relief administered to the mother during delivery. Significant findings were replicated in a different dataset and followed-up with gene ontology analysis to pinpoint biological functions of potential relevance to these neonatal methylation alterations. The MWAS analyses detected methylome-wide significant (q&lt;0.1) alterations in the newborn for laughing gas in granulocytes (two CpGs, p&lt;5.50x10-9, q = 0.067), and for pudendal block in monocytes (five CpGs across three loci, p&lt;1.51 x10-8, q = 0.073). Suggestively significant findings (p&lt;1.00x10-6) were detected for both treatments for bulk and all cell-types, and replication analyses showed consistent significant enrichment (odds ratios ranging 3.47–39.02; p&lt;4.00×10−4) for each treatment, suggesting our results are robust. In contrast, we did not observe any overlap across treatments, suggesting that the treatments are associated with different alterations of the neonatal blood methylome. Gene ontology analyses of the replicating suggestively significant results indicated functions related to, for example, cell differentiation, intracellular membrane-bound organelles and calcium transport. In conclusion, for the first time, we investigated and detected effect of obstetric pain-relief on the blood methylome in the newborn child. The observed differences suggest that anesthetic treatment, such as laughing gas or pudendal block, may alter the neonatal methylome in a cell-type specific manner. Some of the observed alterations are part of gene ontology terms that previously have been suggested in relation to anesthetic treatment, supporting its potential role also in obstetric anesthesia.

Intracellular morphogenesis of diatom silica is guided by local variations in membrane curvature

Silica cell-wall formation in diatoms is a showcase for the ability of organisms to control inorganic mineralization. The process of silicification by these unicellular algae is tightly regulated within a membrane-bound organelle, the silica deposition vesicle (SDV). Two opposing scenarios were proposed to explain the tight regulation of this intracellular process: a template-mediated process that relies on preformed scaffolds, or a template-independent self-assembly process. The present work points to a third scenario, where the SDV membrane is a dynamic mold that shapes the forming silica. We use in-cell cryo-electron tomography to visualize the silicification process in situ, in its native-state, and with a nanometer-scale resolution. This reveals that the plasma membrane interacts with the SDV membrane via physical tethering at membrane contact sites, where the curvature of the tethered side of the SDV membrane mirrors the intricate silica topography. We propose that silica growth and morphogenesis result from the biophysical properties of the SDV and plasma membranes.

Iron-depleting nutritional immunity controls extracellular bacterial replication in Legionella pneumophila infections

The accidental human pathogen Legionella pneumophila (Lp) is the etiological agent for a severe atypical pneumonia known as Legionnaires’ disease. In human infections and animal models of disease alveolar macrophages are the primary cellular niche that supports bacterial replication within a unique intracellular membrane-bound organelle. The Dot/Icm apparatus—a type IV secretion system that translocates ~300 bacterial proteins within the cytosol of the infected cell—is a central virulence factor required for intracellular growth. Mutant strains lacking functional Dot/Icm apparatus are transported to and degraded within the lysosomes of infected macrophages. The early foundational work from Dr. Horwitz’s group unequivocally established that Legionella does not replicate extracellularly during infection—a phenomenon well supported by experimental evidence for four decades. Our data challenges this paradigm by demonstrating that macrophages and monocytes provide the necessary nutrients and support robust Legionella extracellular replication. We show that the previously reported lack of Lp extracellular replication is not a bacteria intrinsic feature but rather a result of robust restriction by serum-derived nutritional immunity factors. Specifically, the host iron-sequestering protein Transferrin is identified here as a critical suppressor of Lp extracellular replication in an iron-dependent manner. In iron-overload conditions or in the absence of Transferrin, Lp bypasses growth restriction by IFNγ-primed macrophages though extracellular replication. It is well established that certain risk factors associated with development of Legionnaires’ disease, such as smoking, produce a chronic pulmonary environment of iron-overload. Our work indicates that iron-overload could be an important determinant of severe infection by allowing Lp to overcome nutritional immunity and replicate extracellularly, which in turn would circumvent intracellular cell intrinsic host defenses. Thus, we provide evidence for nutritional immunity as a key underappreciated host defense mechanism in Legionella pathogenesis.

The partitioning of fatty acids between membrane and storage lipids controls ER membrane expansion.

The biogenesis of membrane-bound organelles involves the synthesis, remodelling and degradation of their constituent phospholipids. How these pathways regulate organelle size, remains still poorly understood. Here we demonstrate that a lipid degradation pathway inhibits the expansion of the endoplasmic reticulum (ER) membrane. Phospholipid diacylglycerol acyltransferases (PDATs) use endogenous phospholipids as fatty acyl donors to generate triglyceride stored in lipid droplets. The significance of this non-canonical triglyceride biosynthetic pathway has remained elusive. We find that the activity of the yeast PDAT Lro1 is regulated by a membrane-proximal domain facing the luminal side of the ER bilayer. To reveal the biological roles of PDATs, we engineered an Lro1 variant with derepressed activity. We show that active Lro1 mediates the retraction of ER membrane expansion driven by phospholipid synthesis. Furthermore, the subcellular distribution and membrane turnover activity of Lro1 are controlled by diacylglycerol, produced by the activity of Pah1, a conserved member of the lipin family. Collectively, our findings reveal a lipid metabolic network that regulates endoplasmic reticulum biogenesis by converting phospholipids into storage lipids.