Showing papers on "Cellular compartment published in 2022"
TL;DR: It is discussed that highly specific interactions, rather than unspecific ones, appear to be the main driver of biogenesis of subcellular compartments, while phase separation may be harnessed locally in selected instances to generate material properties tailored for specific functions, as exemplified by nucleocytoplasmic transport.
Abstract: Molecular mechanistic biology has ushered us into the world of life’s building blocks, revealing their interactions in macromolecular complexes and inspiring strategies for detailed functional interrogations. The biogenesis of membraneless cellular compartments, functional mesoscale subcellular locales devoid of strong internal order and delimiting membranes, is among mechanistic biology’s most demanding current challenges. A developing paradigm, biomolecular phase separation, emphasizes solvation of the building blocks through low‐affinity, weakly adhesive unspecific interactions as the driver of biogenesis of membraneless compartments. Here, I discuss the molecular underpinnings of the phase separation paradigm and demonstrate that validating its assumptions is much more challenging than hitherto appreciated. I also discuss that highly specific interactions, rather than unspecific ones, appear to be the main driver of biogenesis of subcellular compartments, while phase separation may be harnessed locally in selected instances to generate material properties tailored for specific functions, as exemplified by nucleocytoplasmic transport.
64 citations
TL;DR: In this paper , the authors present a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions, which predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate.
Abstract: When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource re-allocation under cellular constraints. Eukaryal cells contain metabolically active organelles such as mitochondria, competing for cytosolic space and resources, and the nature of the relevant cellular constraints remain to be determined for such cells. Here, we present a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions. The model predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate. Comparing model predictions with quantitative experimental data suggests that under glucose limitation, a mitochondrial constraint limits growth at the onset of ethanol formation-known as the Crabtree effect. Under sugar excess, however, a constraint on total cytosolic volume dictates overflow metabolism. Our comprehensive model thus identifies condition-dependent and compartment-specific constraints that can explain metabolic strategies and protein expression profiles from growth rate optimisation, providing a framework to understand metabolic adaptation in eukaryal cells.
30 citations
TL;DR: In this paper , the authors discuss current and potential methods to fabricate artificial cells for sequential enzymatic reactions, which are inspired by mechanisms and metabolic pathways developed by living cells.
13 citations
TL;DR: Although the separation of transcription and translation, mediated by the nuclear envelope, is the defining characteristic of Eukaryotes, the barrier between the nuclear and cytoplasmic compartments needs to be semipermeable to enable material to be moved between them.
Abstract: Although the separation of transcription and translation, mediated by the nuclear envelope, is the defining characteristic of Eukaryotes, the barrier between the nuclear and cytoplasmic compartments needs to be semipermeable to enable material to be moved between them. Moreover, each compartment needs to have a distinctive complement of macromolecules to mediate specific functions and so movement between them needs to be controlled. This is achieved through the selective active transport of macromolecules through the nuclear pores that stud the nuclear envelope, and which serve as a conduit between these compartments. Nuclear pores are huge cylindrical macromolecular assemblies and are constructed from the order of 30 different proteins called nucleoporins. Nuclear pores have a central transport channel that is filled with a dense network of natively unfolded portions of many different nuclear pore proteins (nucleoporins or nups). This network generates a barrier that impedes, but does not entirely prevent, the diffusion of many macromolecules through the pores. The rapid movement of a range of proteins and RNAs through the pores is mediated by a range of transport factors that bind their cargo in one compartment and release it in the other. However, although as their size increases the diffusion of macromolecules through nuclear pores is progressively impaired, additional mechanisms, including the binding of some macromolecules to immobile components of each compartment and also the active removal of macromolecules from the inappropriate compartment, are needed to fully maintain the distinctive compositions of each compartment.
12 citations
TL;DR: A better understanding of ATP dynamics is crucial to revealing the differences in cellular metabolic processes across various cell types and conditions, and requires innovative methodologies to record real-time spatiotemporal ATP changes in subcellular regions of living cells.
Abstract: Adenosine 5′-triphosphate, or ATP, is the primary molecule for storing and transferring energy in cells. ATP is mainly produced via oxidative phosphorylation in mitochondria, and to a lesser extent, via glycolysis in the cytosol. In general, cytosolic glycolysis is the primary ATP producer in proliferative cells or cells subjected to hypoxia. On the other hand, mitochondria produce over 90% of cellular ATP in differentiated cells under normoxic conditions. Under pathological conditions, ATP demand rises to meet the needs of biosynthesis for cellular repair, signaling transduction for stress responses, and biochemical processes. These changes affect how mitochondria and cytosolic glycolysis function and communicate. Mitochondria undergo remodeling to adapt to the imbalanced demand and supply of ATP. Otherwise, a severe ATP deficit will impair cellular function and eventually cause cell death. It is suggested that ATP from different cellular compartments can dynamically communicate and coordinate to adapt to the needs in each cellular compartment. Thus, a better understanding of ATP dynamics is crucial to revealing the differences in cellular metabolic processes across various cell types and conditions. This requires innovative methodologies to record real-time spatiotemporal ATP changes in subcellular regions of living cells. Over the recent decades, numerous methods have been developed and utilized to accomplish this task. However, this is not an easy feat. This review evaluates innovative genetically encoded biosensors available for visualizing ATP in living cells, their potential use in the setting of human disease, and identifies where we could improve and expand our abilities.
7 citations
TL;DR: In this article , the authors reviewed the discovery and structure of the mitoferrins and the significance of these proteins in maintaining cytosolic and mitochondrial iron homeostasis for the prevention of cancer and many other diseases.
Abstract: Iron is essential for many cellular processes, but cellular iron homeostasis must be maintained to ensure the balance of cellular signaling processes and prevent disease. Iron transport in and out of the cell and cellular organelles is crucial in this regard. The transport of iron into the mitochondria is particularly important, as heme and the majority of iron-sulfur clusters are synthesized in this organelle. Iron is also required for the production of mitochondrial complexes that contain these iron-sulfur clusters and heme. As the principal iron importers in the mitochondria of human cells, the mitoferrins have emerged as critical regulators of cytosolic and mitochondrial iron homeostasis. Here, we review the discovery and structure of the mitoferrins, as well as the significance of these proteins in maintaining cytosolic and mitochondrial iron homeostasis for the prevention of cancer and many other diseases.
7 citations
TL;DR: In this article , the authors used soft X-ray tomography to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells, and they used a non-disruptive sample preparation technique involving rapid cryopreservation, and a fluorescent reporter virus to facilitate correlation of structural changes with the stage of infection in individual cells.
Abstract: Herpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus and its assembly in the cell is a complex multi-step process during which viral particles interact with numerous cellular compartments such as the nucleus and organelles of the secretory pathway. Transmission electron microscopy and fluorescence microscopy are commonly used to study HSV-1 infection. However, 2D imaging limits our understanding of the 3D geometric changes to cellular compartments that accompany infection and sample processing can introduce morphological artefacts that complicate interpretation. In this study, we used soft X-ray tomography to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells. To protect the near-native structure of cellular compartments we used a non-disruptive sample preparation technique involving rapid cryopreservation, and a fluorescent reporter virus was used to facilitate correlation of structural changes with the stage of infection in individual cells. We observed viral capsids and assembly intermediates interacting with nuclear and cytoplasmic membranes. Additionally, we observed differences in the morphology of specific organelles between uninfected and infected cells. The local concentration of cytoplasmic vesicles at the juxtanuclear compartment increased and their mean width decreased as infection proceeded, and lipid droplets transiently increased in size. Furthermore, mitochondria in infected cells were elongated and highly branched, suggesting that HSV-1 infection alters the dynamics of mitochondrial fission/fusion. Our results demonstrate that high-resolution 3D images of cellular compartments can be captured in a near-native state using soft X-ray tomography and have revealed that infection causes striking changes to the morphology of intracellular organelles.
5 citations
TL;DR: Genetically encoded and modified variants of phot that localize only to the cytosol or the PM are produced and determined that only PM-associated phot induced cold avoidance in the liverwort Marchantia polymorpha.
Abstract: Abstract Plant cells perceive cold temperatures and initiate cellular responses to protect themselves against cold stress, but which cellular compartment mediates cold sensing has been unknown. Chloroplasts change their position in response to cold to optimize photosynthesis in plants in a process triggered by the blue-light photoreceptor phototropin (phot), which thus acts as a cold-sensing molecule. However, phot in plant cells is present in multiple cellular compartments, including the plasma membrane (PM), cytosol, Golgi apparatus, and chloroplast periphery, making it unclear where phot perceives cold and activates this cold-avoidance response. Here, we produced genetically encoded and modified variants of phot that localize only to the cytosol or the PM and determined that only PM-associated phot-induced cold avoidance in the liverwort Marchantia polymorpha. These results indicate that the phot localized to the PM constitutes a cellular compartment for cold sensing in plants.
4 citations
TL;DR: It is demonstrated that SubcellulaRVis precisely describes the subcellular localisation of gene lists whose locations have been previously ascertained, and will be useful for experimental biologists with limited bioinformatics expertise who want to analyse data related to protein (re)localisation and location-specific modules within the intracellular protein network.
Abstract: Abstract Cells contain intracellular compartments, including membrane-bound organelles and the nucleus, and are surrounded by a plasma membrane. Proteins are localised to one or more of these cellular compartments; the correct localisation of proteins is crucial for their correct processing and function. Moreover, proteins and the cellular processes they partake in are regulated by relocalisation in response to various cellular stimuli. High-throughput ‘omics experiments result in a list of proteins or genes of interest; one way in which their functional role can be understood is through the knowledge of their subcellular localisation, as deduced through statistical enrichment for Gene Ontology Cellular Component (GOCC) annotations or similar. We have designed a bioinformatics tool, named SubcellulaRVis, that compellingly visualises the results of GOCC enrichment for quick interpretation of the localisation of a group of proteins (rather than single proteins). We demonstrate that SubcellulaRVis precisely describes the subcellular localisation of gene lists whose locations have been previously ascertained. SubcellulaRVis can be accessed via the web (http://phenome.manchester.ac.uk/subcellular/) or as a stand-alone app (https://github.com/JoWatson2011/subcellularvis). SubcellulaRVis will be useful for experimental biologists with limited bioinformatics expertise who want to analyse data related to protein (re)localisation and location-specific modules within the intracellular protein network.
4 citations
TL;DR: Analysis is presented for energy-dependent reactions localized in the mitochondrial matrix using data obtained from both isolated mitochondria and intact tissues and it is concluded that the energy state ([ATP]f/[ADP] f[Pi]f] is not different from the energyState in the cytoplasm.
Abstract: Maintaining a robust, stable source of energy for doing chemical and physical work is essential to all living organisms. In eukaryotes, metabolic energy (ATP) production and consumption occurs in two separate compartments, the mitochondrial matrix and the cytosol. As a result, understanding eukaryotic metabolism requires knowledge of energy metabolism in each compartment and how metabolism in the two compartments is coordinated. Central to energy metabolism is the adenylate energy state ([ATP]/[ADP][Pi]). ATP is synthesized by oxidative phosphorylation (mitochondrial matrix) and glycolysis (cytosol) and each compartment provides the energy to do physical work and to drive energetically unfavorable chemical syntheses. The energy state in the cytoplasmic compartment has been established by analysis of near equilibrium metabolic reactions localized in that compartment. In the present paper, analysis is presented for energy-dependent reactions localized in the mitochondrial matrix using data obtained from both isolated mitochondria and intact tissues. It is concluded that the energy state ([ATP]f/[ADP]f[Pi]) in the mitochondrial matrix, calculated from the free (unbound) concentrations, is not different from the energy state in the cytoplasm. Corollaries are: (1) ADP in both the cytosol and matrix is selectively bound and the free concentrations are much lower than the total measured concentrations; and (2) under physiological conditions, the adenylate energy states in the mitochondrial matrix and cytoplasm are not substantially different.
4 citations
TL;DR: In this article , the structural roles and signalling of inter-related phosphoinositide lipids and their derivative, diacylglycerol, in the regulation of nuclear envelope biogenesis and other subcellular compartments such as the nucleoplasmic reticulum were investigated.
Abstract: The general segregation of a molecular class, lipids, from the pathways of cellular communication, via endo-membranes, has resulted in the over-simplification and misconceptions in deciphering cell signalling mechanisms. Mechanisms in signal transduction and protein activation require targeting of proteins to membranous compartments with a specific localised morphology and dynamics that are dependent on their lipid composition. Many posttranslational events define cellular behaviours and without the active role of membranous compartments these events lead to various dysregulations of the signalling pathways. We summarise the key findings, using tools such as the rapalogue dimerisation, in the structural roles and signalling of the inter-related phosphoinositide lipids and their derivative, diacylglycerol, in the regulation of nuclear envelope biogenesis and other subcellular compartments such as the nucleoplasmic reticulum.
TL;DR: In this paper , a combination of yeast genetics, subcellular fractionation, and inductively coupled plasma-mass spectrometry-based metal measurements was used to determine the specificity of elesclomol and the ES-Cu complex in delivering Cu to cuproenzymes in different intracellular compartments.
TL;DR: In this paper, the structural roles and signalling of inter-related phosphoinositide lipids and their derivative, diacylglycerol, in the regulation of nuclear envelope biogenesis and other subcellular compartments such as the nucleoplasmic reticulum were investigated.
TL;DR: In this paper , the authors examined the emerging regulatory functions of the citrate/acetyl-CoA pathway and the specific role of the endoplasmic reticulum (ER) acetylation machinery in the maintenance of intracellular crosstalk and homeostasis.
Abstract: Key cellular metabolites reflecting the immediate activity of metabolic enzymes as well as the functional metabolic state of intracellular organelles can act as powerful signal regulators to ensure the activation of homeostatic responses. The citrate/acetyl-CoA pathway, initially recognized for its role in intermediate metabolism, has emerged as a fundamental branch of this nutrient-sensing homeostatic response. Emerging studies indicate that fluctuations in acetyl-CoA availability within different cellular organelles and compartments provides substrate-level regulation of many biological functions. A fundamental aspect of these regulatory functions involves Nε-lysine acetylation.Here, we will examine the emerging regulatory functions of the citrate/acetyl-CoA pathway and the specific role of the endoplasmic reticulum (ER) acetylation machinery in the maintenance of intracellular crosstalk and homeostasis. These functions will be analyzed in the context of associated human diseases and specific mouse models of dysfunctional ER acetylation and citrate/acetyl-CoA flux. A primary objective of this review is to highlight the complex yet integrated response of compartment- and organelle-specific Nε-lysine acetylation to the intracellular availability and flux of acetyl-CoA, linking this important post-translational modification to cellular metabolism.The ER acetylation machinery regulates the proteostatic functions of the organelle as well as the metabolic crosstalk between different intracellular organelles and compartments. This crosstalk enables the cell to impart adaptive responses within the ER and the secretory pathway. However, it also enables the ER to impart adaptive responses within different cellular organelles and compartments. Defects in the homeostatic balance of acetyl-CoA flux and ER acetylation reflect different but converging disease states in humans as well as converging phenotypes in relevant mouse models. In conclusion, citrate and acetyl-CoA should not only be seen as metabolic substrates of intermediate metabolism but also as signaling molecules that direct functional adaptation of the cell to both intracellular and extracellular messages. Future discoveries in CoA biology and acetylation are likely to yield novel therapeutic approaches.
TL;DR: Insight is provided into how redox and Cu homeostasis interplay by modulating specific protein expressions at the subcellular levels, shedding light on understanding the effects of Cu-induced redox misregulation on the diseases.
Abstract: Excess intracellular Cu perturbs cellular redox balance and thus causes diseases. However, the relationship between cellular redox status and Cu homeostasis and how such an interplay is coordinated within cellular compartments has not yet been well established. Using combined approaches of organelle-specific redox sensor Grx1-roGFP2 and non-targeted proteomics, we investigate the real-time Cu-dependent antioxidant defenses of mitochondria and cytosol in live HEK293 cells. The Cu-dependent real-time imaging experiments show that CuCl2 treatment results in increased oxidative stress in both cytosol and mitochondria. In contrast, subsequent Cu depletion by BCS, a Cu chelating reagent, lowers oxidative stress in mitochondria but causes even higher oxidative stress in the cytosol. The proteomic data reveal that several mitochondrial proteins, but not cytosolic ones, undergo significant abundance change under Cu treatments. The proteomic analysis also shows that proteins with significant changes are related to mitochondrial oxidative phosphorylation and glutathione synthesis. The differences in redox behaviors and protein profiles in different cellular compartments reveal distinct mitochondrial and cytosolic response mechanisms upon Cu-induced oxidative stress. These findings provide insights into how redox and Cu homeostasis interplay by modulating specific protein expressions at the subcellular levels, shedding light on understanding the effects of Cu-induced redox misregulation on the diseases. Graphical abstract
TL;DR: Subcellular compartmentalization provides cells with tremendous advantages for the operation of cellular metabolism as discussed by the authors , allowing the cell to execute otherwise thermodynamically exclusive reactions simultaneously, thus, the elucidation of compartment-specific metabolic processes has become essential for a thorough understanding of cellular metabolic processes.
01 Jan 2022
TL;DR: A review of the recent progress in subcellular-compartment-focused chemical proteomics approaches, which do not rely on the conventional sub-cellular fractionation method, can be found in this article .
Abstract: Proteins play central roles in numerous biological events in all living organisms. Notably, the subcellular localization, trafficking, and dynamic alteration of proteins have a crucial impact on cellular functions and fates. To date, scientists have explored a large number of strategies to profile protein characteristics under native conditions. Chemical proteomics is one of the most powerful strategies, offering convenient and efficient profiling of a proteome of interest. Herein, we review the recent progress in subcellular-compartment-focused chemical proteomics approaches, which do not rely on the conventional subcellular fractionation method. Recently, methods for proximity labeling using genetically engineered enzymes have been developed for proteome analysis within organelles and even sub-organelles. In parallel, a few powerful chemical proteomics methods have been invented for subcellular-compartment-focused protein profiling. Organelle-localizable reactive molecules have been designed that can selectively chemically tag proteins localized in the nucleus, mitochondria, and endoplasmic reticulum. A photoactivatable proximity labeling method has also been reported for identifying organellar proteomes with high spatiotemporal resolution. Additionally, a conditional proteomics approach has been used to elucidate proteomes involved in specific microenvironments or processes, such as Zn2+ homeostasis and NO-, and H2O2-rich micro-spaces in live cells.
01 Jan 2022
TL;DR: A review of the recent progress in subcellular-compartment-focused chemical proteomics approaches, which do not rely on the conventional sub-cellular fractionation method, can be found in this article.
Abstract: Proteins play central roles in numerous biological events in all living organisms. Notably, the subcellular localization, trafficking, and dynamic alteration of proteins have a crucial impact on cellular functions and fates. To date, scientists have explored a large number of strategies to profile protein characteristics under native conditions. Chemical proteomics is one of the most powerful strategies, offering convenient and efficient profiling of a proteome of interest. Herein, we review the recent progress in subcellular-compartment-focused chemical proteomics approaches, which do not rely on the conventional subcellular fractionation method. Recently, methods for proximity labeling using genetically engineered enzymes have been developed for proteome analysis within organelles and even sub-organelles. In parallel, a few powerful chemical proteomics methods have been invented for subcellular-compartment-focused protein profiling. Organelle-localizable reactive molecules have been designed that can selectively chemically tag proteins localized in the nucleus, mitochondria, and endoplasmic reticulum. A photoactivatable proximity labeling method has also been reported for identifying organellar proteomes with high spatiotemporal resolution. Additionally, a conditional proteomics approach has been used to elucidate proteomes involved in specific microenvironments or processes, such as Zn2+ homeostasis and NO-, and H2O2-rich micro-spaces in live cells.
TL;DR: In this paper , the authors investigated the real-time Cu-dependent antioxidant defenses of mitochondria and cytosol in live HEK293 cells and showed that CuCl2 treatment results in increased oxidative stress in both cytosols and mitochondria.
Abstract: Abstract Excess intracellular Cu perturbs cellular redox balance and thus causes diseases. However, the relationship between cellular redox status and Cu homeostasis and how such an interplay is coordinated within cellular compartments has not yet been well established. Using combined approaches of organelle-specific redox sensor Grx1-roGFP2 and non-targeted proteomics, we investigate the real-time Cu-dependent antioxidant defenses of mitochondria and cytosol in live HEK293 cells. The Cu-dependent real-time imaging experiments show that CuCl2 treatment results in increased oxidative stress in both cytosol and mitochondria. In contrast, subsequent excess Cu removal by bathocuproine sulfonate, a Cu chelating reagent, lowers oxidative stress in mitochondria but causes even higher oxidative stress in the cytosol. The proteomic data reveal that several mitochondrial proteins, but not cytosolic ones, undergo significant abundance change under Cu treatments. The proteomic analysis also shows that proteins with significant changes are related to mitochondrial oxidative phosphorylation and glutathione synthesis. The differences in redox behaviors and protein profiles in different cellular compartments reveal distinct mitochondrial and cytosolic response mechanisms upon Cu-induced oxidative stress. These findings provide insights into how redox and Cu homeostasis interplay by modulating specific protein expressions at the subcellular levels, shedding light on understanding the effects of Cu-induced redox misregulation on the diseases.
TL;DR: In this article, the authors used 3 μm polystyrene beads as a model particle, and assessed the detailed modes of their cellular uptake by non-phagocytic HeLa cells using confocal, scanning electron, and scanning ion conductance microscopy analyses.
Abstract: Extracellular fine particles of various sizes and origins can be taken up by cells, affecting their function. Understanding the cellular uptake processes is crucial for understanding the cellular effects of these particles and the development of means to control their internalization. Although macropinocytosis is a possible pathway for the cellular uptake of particles larger than 0.2 μm, its contribution to cellular uptake in non-phagocytic cells is controversial. Using 3 μm polystyrene beads as a model particle, we aimed to assess the detailed modes of their cellular uptake by non-phagocytic HeLa cells. Cellular uptake was assessed using confocal, scanning electron, and scanning ion conductance microscopy analyses, together with inhibitor studies. Our results revealed that 3 μm beads were taken up by HeLa cells by an actin-, cholesterol-, and membrane protrusions-dependent noncanonical endocytic pathway, different from the canonical macropinocytic and phagocytic pathways. Our work provides a framework for studying the cellular uptake of extracellular fine particles.
TL;DR: This work developed, test, and apply methods that can quantitatively measure families of metabolites within distinct sub‐cellular compartments in eukaryotic cells, and successfully applied these methods to cells and human tissue demonstrating distinct compartmental metabolic changes by pathway.
Abstract: Metabolism in eukaryotes relies on compartmentalization of processes between sub‐cellular compartments. Our objective was to develop, test, and apply methods that can quantitatively measure families of metabolites within distinct sub‐cellular compartments in eukaryotic cells. We created Stable Isotope Labeling of Essential nutrients in Cell culture ‐ Subcellular Fractionation (SILEC‐SF) with the essential precursors of the major cellular coenzymes, Coenzyme A and NAD to incorporate a 13C,15N‐label into the families of each coenzyme present within cells. Using multiple fractionation techniques coupled to liquid chromatography‐high resolution mass spectrometry we quantify distinct cytoplasmic, mitochondrial, and nuclear pools within eukaryotic cells. We successfully applied these methods to cells and human tissue demonstrating distinct compartmental metabolic changes by pathway in genetic models of compartmentalized metabolism, in adipocyte differentiation and in changing oxygen tension. This confirmed orthogonal measurements of subcellular metabolism but revealed unexpected localizations and enrichments of certain metabolite pools.
TL;DR: In this article , the authors characterise a range of OSER compartments and show how the structure of the inducing polyprotein constructs affect the final compartment morphology, with the cytosolic-facing antiparallel oligomerization domain demonstrated to be an essential component to trigger OSER formation.
Abstract: Engineering of subcellular compartmentalisation is one of synthetic biology’s key challenges. Among different approaches, de novo construction of a synthetic compartment is the most coveted but also most difficult option. Restructuring the endoplasmic reticulum (ER), via the introduction of recombinant oligomerising ER-membrane resident proteins, is an alternative starting point for building a new compartment. The presence of such proteins leads to a massive expansion of the ER and the formation of organised smooth endoplasmic reticulum (OSER), a large membranous compartment. However, OSER is poorly characterised and our understanding of its effect on the underlying biology of the plant is limited. Here we characterise a range of OSER compartments and show how the structure of the inducing polyprotein constructs affect the final compartment morphology, with the cytosolic-facing antiparallel oligomerisation domain demonstrated to be an essential component to trigger OSER formation. We show that while OSER retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol. Using high-resolution quantitative image analysis, we also show that the presence of this large compartment does not disrupt the rest of the ER network. Moreover, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally. These properties collectively suggest that OSER could be developed as a plant synthetic biology tool for compartmentalisation, combining the benefits of several existing strategies. Only a single protein construct is necessary to induce its formation, and the compartment retains a delimiting membrane and a diffusional barrier to the rest of the cell.
TL;DR: A simple method that involves the use of a tabletop centrifuge and different detergents to obtain cell fractions enriched in cytosolic (Cyt), plasma membrane (PM), membranous organelle (MO), and nuclear (Nu) proteins and identify the proteins in each fraction is described.
Abstract: Proteins in eukaryotic cells reside in different cell compartments. Many studies require the specific localization of proteins and the detection of any dynamic changes in intracellular protein distribution. There are several methods available for this purpose that rely on the fractionation of the different cell compartments. Fractionation protocols have evolved since the first use of a centrifuge to isolate organelles. In this study, we described a simple method that involves the use of a tabletop centrifuge and different detergents to obtain cell fractions enriched in cytosolic (Cyt), plasma membrane (PM), membranous organelle (MO), and nuclear (Nu) proteins and identify the proteins in each fraction. This method serves to identify transmembrane proteins such as channel subunits as well as PM-embedded or weakly associated proteins. This protocol uses a minute amount of cell material and typical equipment present in laboratories, and it takes approximately 3 h. The process was validated using endogenous and exogenous proteins expressed in the HEK293T cell line that were targeted to each compartment. Using a specific stimulus as a trigger, we showed and quantified the shuttling of a protein channel (ASIC1a, acid sensing ion channel) from the MO fraction to the PM fraction and the shuttling of a kinase from a cytosolic location to a nuclear location.
21 Feb 2022
TL;DR: In this paper , the authors describe regulatory pathways that govern the formation and function of granules with the focus on ribonucleoprotein granules, Stress Granules, in order to study cellular aggregation in live cells.
Abstract: The hypothesis motivating this thesis is that aggregation of proteins in the cytoplasm can have protective functions and can facilitate cell survival. Conversely, the disruption of functional aggregation can lead to cellular dysfunction and death. There is, therefore, a tremendous need to understand the mechanisms and precise sub-cellular architecture of functional aggregates. So far, the multitude of cellular aggregate structures or granules have been proposed to function as enzyme storage compartments, centers of memory retention, signaling hubs, mRNA triage compartments, degradation and protein refolding depots, structural elements, and transport granules. One of the unifying aspects of cytoplasmic granules appears to be stress dependent transient formation. In order to study cellular aggregation in live cells I chose to investigate factors that regulate the formation and clearance of cellular inclusions and their functions in cellular metabolism and signaling pathways. In this thesis I describe regulatory pathways that govern formation and function of granules with the focus on ribonucleoprotein granules, Stress Granules.
TL;DR: In this paper , the peroxidase is physically separated from the POI and only a rapamycin-induced dimerization using the FRB-FKBP12 system brings the two proteins together.
Abstract: Protein-protein interactions are central to most cellular processes and their dysregulation has been related to the development of various diseases. Proximity-based labeling methods are used to identify the endogenous interaction partners of specific proteins of interest (POIs). The POI is fused to promiscuous enzymes, which generate reactive species in vivo and label proteins in close vicinity. APEX-based proximity labeling techniques utilize an engineered ascorbate peroxidase, which in the presence of H2O2 oxidizes biotin-phenol to short lived biotin-phenoxyl radicals that biotinylate nearby proteins. The biotinylated proteins are enriched by biotin affinity capture and identified by mass spectrometry. We devised an advanced method, RAPIDS, in which the peroxidase is physically separated from the POI and only a rapamycin-induced dimerization using the FRB-FKBP12 system brings the two proteins together. RAPIDS improves the specificity of APEX-based interactome analysis by strictly eliminating false positives. In this chapter, we describe this method in detail, with VAPB as a protein of interest and versions of APEX2 with different subcellular localizations. VAPB localizing to different cellular compartments, the endoplasmic reticulum and the inner nuclear membrane, yielded distinct sets of proximity partners as identified by RAPIDS.