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Nature Mar 2020Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies. Mitochondrial malfunction needs to be...
Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies. Mitochondrial malfunction needs to be relayed to the cytosol, where an integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) in mammalian cells. eIF2α phosphorylation is mediated by the four eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress. However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown. Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease.
Topics: Cytosol; Enzyme Activation; Eukaryotic Initiation Factor-2; Genome, Human; Humans; Metalloendopeptidases; Mitochondria; Mitochondrial Proteins; Phosphorylation; Protein Binding; Stress, Physiological; Transcription Factor CHOP; eIF-2 Kinase
PubMed: 32132706
DOI: 10.1038/s41586-020-2076-4 -
Molecular Biology of the Cell Jul 2020The spatial structure and physical properties of the cytosol are not well understood. Measurements of the material state of the cytosol are challenging due to its...
The spatial structure and physical properties of the cytosol are not well understood. Measurements of the material state of the cytosol are challenging due to its spatial and temporal heterogeneity. Recent development of genetically encoded multimeric nanoparticles (GEMs) has opened up study of the cytosol at the length scales of multiprotein complexes (20-60 nm). We developed an image analysis pipeline for 3D imaging of GEMs in the context of large, multinucleate fungi where there is evidence of functional compartmentalization of the cytosol for both the nuclear division cycle and branching. We applied a neural network to track particles in 3D and then created quantitative visualizations of spatially varying diffusivity. Using this pipeline to analyze spatial diffusivity patterns, we found that there is substantial variability in the properties of the cytosol. We detected zones where GEMs display especially low diffusivity at hyphal tips and near some nuclei, showing that the physical state of the cytosol varies spatially within a single cell. Additionally, we observed significant cell-to-cell variability in the average diffusivity of GEMs. Thus, the physical properties of the cytosol vary substantially in time and space and can be a source of heterogeneity within individual cells and across populations.
Topics: Cytoplasm; Cytosol; Eremothecium; Image Processing, Computer-Assisted; Machine Learning; Nanoparticles; Orientation, Spatial; Single Molecule Imaging
PubMed: 32401664
DOI: 10.1091/mbc.E20-03-0210 -
Journal of Leukocyte Biology Jul 2019Proinflammatory immune responses to Gram-negative bacterial lipopolysaccharides (LPS) are crucial to innate host defenses but can also contribute to pathology. How host... (Review)
Review
Proinflammatory immune responses to Gram-negative bacterial lipopolysaccharides (LPS) are crucial to innate host defenses but can also contribute to pathology. How host cells sensitively detect structural features of LPS was a mystery for years, especially given that a portion of the molecule essential for its potent proinflammatory properties-lipid A-is buried in the bacterial membrane. Studies of responses to extracellular and vacuolar LPS revealed a crucial role for accessory proteins that specifically bind LPS-rich membranes and extract LPS monomers to generate a complex of LPS, MD-2, and TLR4. These insights provided means to understand better both the remarkable host sensitivity to LPS and the means whereby specific LPS structural features are discerned. More recently, the noncanonical inflammasome, consisting of caspases-4/5 in humans and caspase-11 in mice, has been demonstrated to mediate responses to LPS that has reached the host cytosol. Precisely how LPS gains access to cytosolic caspases-and in what form-is not well characterized, and understanding this process will provide crucial insights into how the noncanonical inflammasome is regulated during infection. Herein, we briefly review what is known about LPS detection by cytosolic caspases-4/5/11, focusing on lessons derived from studies of the better-characterized TLR4 system that might direct future mechanistic questions.
Topics: Animals; Caspases; Cytosol; Humans; Inflammasomes; Lipopolysaccharides; Lymphocyte Antigen 96; Toll-Like Receptor 4
PubMed: 30694581
DOI: 10.1002/JLB.3MIR1118-434R -
The EMBO Journal Aug 2016Cells contain numerous, molecularly distinct cellular compartments that are not enclosed by lipid bilayers. These compartments are implicated in a wide range of cellular... (Review)
Review
Cells contain numerous, molecularly distinct cellular compartments that are not enclosed by lipid bilayers. These compartments are implicated in a wide range of cellular activities, and they have been variously described as bodies, granules, or organelles. Recent evidence suggests that a liquid-liquid phase separation (LLPS) process may drive their formation, possibly justifying the unifying term "droplet organelle". A veritable deluge of recent publications points to the importance of low-complexity proteins and RNA in determining the physical properties of phase-separated structures. Many of the proteins linked to such structures are implicated in human diseases, such as amyotrophic lateral sclerosis (ALS). We provide an overview of the organizational principles that characterize putative "droplet organelles" in healthy and diseased cells, connecting protein biochemistry with cell physiology.
Topics: Cell Physiological Phenomena; Cytosol; Macromolecular Substances; Multienzyme Complexes
PubMed: 27357569
DOI: 10.15252/embj.201593517 -
Current Opinion in Plant Biology Aug 2016Plants have the ability to sense volatile organic compounds (VOCs) so as to efficiently adapt to their environment. The mechanisms underlying such plant 'olfactory'... (Review)
Review
Plants have the ability to sense volatile organic compounds (VOCs) so as to efficiently adapt to their environment. The mechanisms underlying such plant 'olfactory' systems are largely unknown. Here I would like to propose that the metabolism of VOCs in plant tissues is one of the mechanisms by which plants sense VOCs. During the gas-exchange that is essential for photosynthesis, VOCs in the atmosphere are taken into the intercellular spaces of leaves. Each VOC is partitioned between the gas phase (intercellular space) and liquid phase (cell wall) at a certain ratio determined by Henry's law. The VOCs in the cell wall diffuse through the plasma membrane to the cytosol depending on their oil/water partition coefficients. Plants detoxify some VOCs, especially those that are oxidized, through glycosylation, glutathionylation, and reduction. These metabolic processes lower the concentration of VOCs in the cytosol, which facilitates further cytosolic uptake. As a result, vigorous metabolism of VOCs in the cytosol can lead to a substantial accumulation of VOC metabolites and the depletion of glutathione or NADPH. One such metabolite (a VOC glycoside) is known to mount a direct defense against herbivores, whilst deprivation of glutathione and NADPH can fortify plants with responses similar to the oxidative stress response.
Topics: Cytosol; Plant Leaves; Plants; Volatile Organic Compounds
PubMed: 27281633
DOI: 10.1016/j.pbi.2016.05.005 -
PloS One 2023Messenger RNA processing bodies (P-bodies) are cytoplasmic membrane-free organelles that contain proteins involved in mRNA silencing, storage and decay. The mechanism by...
Messenger RNA processing bodies (P-bodies) are cytoplasmic membrane-free organelles that contain proteins involved in mRNA silencing, storage and decay. The mechanism by which P-body components interact and the factors that regulate the stability of these structures are incompletely understood. In this study, we used a fluorescence-based, two-hybrid assay to investigate interactions between P-body components that occur inside the cell. LSm14a, PATL1, XRN1, and NBDY were found to interact with the N-terminal, WD40-domain-containing portion of EDC4. The N-terminus of full-length PATL1 was required to mediate the interaction between EDC4 and DDX6. The C-terminal, alpha helix-domain- containing portion of EDC4 was sufficient to mediate interaction with DCP1a and CCHCR1. In the absence of endogenous P-bodies, caused by depletion of LSm14a or DDX6, expression of the portion of EDC4 that lacked the N-terminus retained the ability to form cytoplasmic dots that were indistinguishable from P-bodies at the level of UV light microscopy. Despite the absence of endogenous P-bodies, this portion of EDC4 was able to recruit DCP1a, CCHCR1 and EDC3 to cytoplasmic dots. The results of this study permit the development of a new model of P-body formation and suggest that the N-terminus of EDC4 regulates the stability of these structures.
Topics: Animals; Processing Bodies; Cell Membrane; Cytoplasm; Cytosol; Mammals; RNA, Messenger
PubMed: 36877681
DOI: 10.1371/journal.pone.0282496 -
Toxins Mar 2010Cholera toxin (CT), an AB(5)-subunit toxin, enters host cells by binding the ganglioside GM1 at the plasma membrane (PM) and travels retrograde through the trans-Golgi... (Review)
Review
Cholera toxin (CT), an AB(5)-subunit toxin, enters host cells by binding the ganglioside GM1 at the plasma membrane (PM) and travels retrograde through the trans-Golgi Network into the endoplasmic reticulum (ER). In the ER, a portion of CT, the enzymatic A1-chain, is unfolded by protein disulfide isomerase and retro-translocated to the cytosol by hijacking components of the ER associated degradation pathway for misfolded proteins. After crossing the ER membrane, the A1-chain refolds in the cytosol and escapes rapid degradation by the proteasome to induce disease by ADP-ribosylating the large G-protein Gs and activating adenylyl cyclase. Here, we review the mechanisms of toxin trafficking by GM1 and retro-translocation of the A1-chain to the cytosol.
Topics: Cell Membrane; Cholera Toxin; Cytosol; Endoplasmic Reticulum; G(M1) Ganglioside; Humans; Protein Conformation; Protein Transport
PubMed: 22069586
DOI: 10.3390/toxins2030310 -
Advances in Experimental Medicine and... 2020Measuring free Ca concentration ([Ca]) in the cytosol or organelles is routine in many fields of research. The availability of membrane permeant forms of indicators... (Review)
Review
Measuring free Ca concentration ([Ca]) in the cytosol or organelles is routine in many fields of research. The availability of membrane permeant forms of indicators coupled with the relative ease of transfecting cell lines with biological Ca sensors have led to the situation where cellular and subcellular [Ca] is examined by many non-specialists. In this chapter, we evaluate the most used Ca indicators and highlight what their major advantages and disadvantages are. We stress the potential pitfalls of non-ratiometric techniques for measuring Ca and the clear advantages of ratiometric methods. Likely improvements and new directions for Ca measurement are discussed.
Topics: Animals; Calcium; Cytological Techniques; Cytosol; Humans; Organelles
PubMed: 31646505
DOI: 10.1007/978-3-030-12457-1_2 -
Journal of the American Chemical Society Mar 2021Intracellular protein delivery enables selective regulation of cellular metabolism, signaling, and development through introduction of defined protein quantities into...
Intracellular protein delivery enables selective regulation of cellular metabolism, signaling, and development through introduction of defined protein quantities into the cell. Most applications require that the delivered protein has access to the cytosol, either for protein activity or as a gateway to other organelles such as the nucleus. The vast majority of delivery vehicles employ an endosomal pathway however, and efficient release of entrapped protein cargo from the endosome remains a challenge. Recent research has made significant advances toward efficient cytosolic delivery of proteins using polymers, but the influence of polymer architecture on protein delivery is yet to be investigated. Here, we developed a family of dendronized polymers that enable systematic alterations of charge density and structure. We demonstrate that while modulation of surface functionality has a significant effect on overall delivery efficiency, the endosomal release rate can be highly regulated by manipulating polymer architecture. Notably, we show that large, multivalent structures cause slower sustained release, while rigid spherical structures result in rapid burst release.
Topics: Animals; Cell Line; Cytosol; Humans; Mice; Molecular Structure; Polymers; Protein Engineering; Proteins
PubMed: 33705125
DOI: 10.1021/jacs.1c00258 -
Current Opinion in Virology Feb 2017The ability to recognize invading viral pathogens and to distinguish their components from those of the host cell is critical to initiate the innate immune response. The... (Review)
Review
The ability to recognize invading viral pathogens and to distinguish their components from those of the host cell is critical to initiate the innate immune response. The efficiency of this detection is an important factor in determining the susceptibility of the cell to viral infection. Innate sensing of viruses is, therefore, an indispensable step in the line of defense for cells and organisms. Recent discoveries have uncovered novel sensors of viral components and hallmarks of infection, as well as mechanisms by which cells discriminate between self and non-self. This review highlights the mechanisms used by cells to detect viral pathogens in the cytosol, and recent advances in the field of cytosolic sensing of viruses.
Topics: Animals; Cytosol; Humans; Immunity, Innate; Receptors, Immunologic; Viruses
PubMed: 27951430
DOI: 10.1016/j.coviro.2016.11.012