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Proceedings of the National Academy of... Oct 2017The ciliary localization of odorant receptors (ORs) is evolutionary conserved and essential for olfactory transduction. However, how the transport of ORs is regulated in...
The ciliary localization of odorant receptors (ORs) is evolutionary conserved and essential for olfactory transduction. However, how the transport of ORs is regulated in mammalian olfactory sensory neurons is poorly understood. Here we demonstrate that odorant responsiveness and OR transport is regulated by the Hedgehog pathway. OR transport is inhibited by conditional gene inactivation of the Hedgehog signal mediator Smoothened (Smo) as well as by systemic administration of the Smo inhibitor vismodegib, a clinically used anticancer drug reported to distort smell perception in patients. The ciliary phenotype of Smo inhibition is haploinsufficient, cell autonomous, and correlates with the accumulation of OR-containing putative transport vesicles in the cytosol. The Smo-dependent OR transport route works in parallel with a low basal transport of vesicle containing both ORs and other olfactory transduction components. These findings both define a physiological function of Hedgehog signaling in olfaction and provide an important evolutionary link between olfaction and the requirement of a ciliary compartment for Hedgehog signaling.
Topics: Anilides; Animals; Cilia; Cytosol; Hedgehog Proteins; Mice; Mice, Inbred C57BL; Protein Transport; Pyridines; Receptors, Odorant; Signal Transduction; Smoothened Receptor
PubMed: 29078327
DOI: 10.1073/pnas.1708321114 -
Scientific Reports Nov 2021Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been...
Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.
Topics: Adaptation, Physiological; Animals; Cytosol; Desiccation; Proteins; Tardigrada
PubMed: 34737320
DOI: 10.1038/s41598-021-00724-6 -
Medecine Sciences : M/S 2019Cyclic GMP-AMP synthase (cGAS) is a universal cytosolic DNA sensor that detects nucleic acids of pathogens. Upon DNA sensing, cGAS triggers the formation of the second... (Review)
Review
Cyclic GMP-AMP synthase (cGAS) is a universal cytosolic DNA sensor that detects nucleic acids of pathogens. Upon DNA sensing, cGAS triggers the formation of the second intracellular messenger, the cyclic GMP-AMP (cGAMP), which activates the adaptor STING. STING engagement induces the secretion of cytokines and type I interferons that contribute to pathogen clearance. However, there is emerging evidence that cGAS is activated by self DNA in cancer cells and in antigen-presenting cells to trigger an antitumoral response. In this review, we will highlight the current understanding of self DNA sensing by cGAS in the context of cancer.
Topics: Cytosol; DNA; Humans; Immunity, Innate; Membrane Proteins; Neoplasms; Nucleotides, Cyclic; Pathogen-Associated Molecular Pattern Molecules; Signal Transduction
PubMed: 31274082
DOI: 10.1051/medsci/2019095 -
Biochemical and Biophysical Research... Sep 2018ALIS are large, transient, cytosolic aggregates that serve as storage compartments for ubiquitin-tagged defective ribosomal products. We determined the importance of the...
ALIS are large, transient, cytosolic aggregates that serve as storage compartments for ubiquitin-tagged defective ribosomal products. We determined the importance of the protein p62 in the formation of ALIS and demonstrated that two domains of p62-PB1 and UBA-are essential for ALIS assembly. Those two major binding domains of p62, also known as sequestosome 1, were shown to play a critical role in the formation of autophagosomes or cytoplasmic aggregates. Specifically, the PB1 domain is essential for self-oligomerization, and the UBA domain allows p62 to bind to polyubiquitin chains or ubiquitinated proteins. After stimulation of RAW 264.7 macrophages with lipopolysaccharide, we observed a significant decrease in the number of cells with ALIS. Importantly, cells overexpressing either a PB1 mutant or UBA-deleted p62 construct also exhibited a substantially diminished number of cells containing ALIS. Since both p62 and ubiquitin are found in ALIS, we evaluated the dynamics of YFP-tagged p62 in ALIS. In contrast to the findings of a previous study that evaluated GFP-tagged ubiquitin motility in ALIS, we determined that YFP-tagged p62 has very limited mobility. Lastly, we determined that GST-tagged full-length p62 binds to Lys-63-linked polyubiquitin chains but not to Lys-48-linked chains. Overall, our findings provide insight on the essential role that p62, particularly its PB1 and UBA domains, has in the formation of ALIS.
Topics: Animals; Cytosol; Humans; Lipopolysaccharides; Mice; Polyubiquitin; Protein Aggregates; Protein Binding; Protein Domains; RAW 264.7 Cells; Sequestosome-1 Protein; Ubiquitin
PubMed: 29966650
DOI: 10.1016/j.bbrc.2018.06.153 -
American Journal of Physiology. Lung... Jul 2021During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca concentration ([Ca]), which leads to a...
During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca concentration ([Ca]), which leads to a contractile response [excitation-contraction (E-C) coupling]. Previously, the sensitivity of the contractile response of ASM to elevated [Ca] (Ca sensitivity) was assessed as the ratio of maximum force to maximum [Ca]. However, this static assessment of Ca sensitivity overlooks the dynamic nature of E-C coupling in ASM. In this study, we simultaneously measured [Ca] and isometric force responses to three concentrations of ACh (1, 2.6, and 10 μM). Both maximum [Ca] and maximum force responses were ACh concentration dependent, but force increased disproportionately, thereby increasing static Ca sensitivity. The dynamic properties of E-C coupling were assessed in several ways. The temporal delay between the onset of ACh-induced [Ca] and onset force responses was not affected by ACh concentration. The rates of rise of the ACh-induced [Ca] and force responses increased with increasing ACh concentration. The integral of the phase-loop plot of [Ca] and force from onset to steady state also increased with increasing ACh concentration, whereas the rate of relaxation remained unchanged. Although these results suggest an ACh concentration-dependent increase in the rate of cross-bridge recruitment and in the rate of rise of [Ca], the extent of regulatory myosin light-chain (rMLC) phosphorylation was not dependent on ACh concentration. We conclude that the dynamic properties of [Ca] and force responses in ASM are dependent on ACh concentration but reflect more than changes in the extent of rMLC phosphorylation.
Topics: Animals; Calcium; Cholinergic Agents; Cytosol; Female; Male; Muscle Contraction; Muscle, Smooth; Respiratory System; Swine
PubMed: 33908264
DOI: 10.1152/ajplung.00596.2020 -
Anais Da Academia Brasileira de Ciencias 2015Bacteria are able to synchronize the population behavior in order to regulate gene expression through a cell-to-cell communication mechanism called quorum sensing. This... (Review)
Review
Bacteria are able to synchronize the population behavior in order to regulate gene expression through a cell-to-cell communication mechanism called quorum sensing. This phenomenon involves the production, detection and the response to extracellular signaling molecules named autoinducers, which directly or indirectly regulate gene expression in a cell density-dependent manner. Quorum sensing may control a wide range of biological processes in bacteria, such as bioluminescence, virulence factor production, biofilm formation and antibiotic resistance. The autoinducers are recognized by specific receptors that can either be membrane-bound histidine kinase receptors, which work by activating cognate cytoplasmic response regulators, or cytoplasmic receptors acting as transcription factors. In this review, we focused on the cytosolic quorum sensing regulators whose three-dimensional structures helped elucidate their mechanisms of action. Structural studies of quorum sensing receptors may enable the rational design of inhibitor molecules. Ultimately, this approach may represent an effective alternative to treat infections where classical antimicrobial therapy fails to overcome the microorganism virulence.
Topics: Bacterial Physiological Phenomena; Cytosol; Quorum Sensing; Signal Transduction
PubMed: 26247154
DOI: 10.1590/0001-3765201520140482 -
Environmental Microbiology Sep 2010Upon entry of Francisella tularensis to macrophages, the Francisella-containing phagosome (FCP) is trafficked into an acidified late endosome-like phagosome with limited...
Upon entry of Francisella tularensis to macrophages, the Francisella-containing phagosome (FCP) is trafficked into an acidified late endosome-like phagosome with limited fusion to the lysosomes followed by rapid escape into the cytosol where the organism replicates. Although the Francisella Pathogenicity Island (FPI), which encodes a type VI-like secretion apparatus, is required for modulation of phagosome biogenesis and escape into the cytosol, the mechanisms involved are not known. To decipher the molecular bases of modulation of biogenesis of the FCP and bacterial escape into the macrophage cytosol, we have screened a comprehensive mutant library of F. tularensis ssp. novicida for their defect in proliferation within human macrophages, followed by characterization of modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data show that at least 202 genes are required for intracellular proliferation within macrophages. Among the 125 most defective mutants in intracellular proliferation, we show that the FCP of at least 91 mutants colocalize persistently with the late endosomal/lysosomal marker LAMP-1 and fail to escape into the cytosol, as determined by fluorescence-based phagosome integrity assays and transmission electron microscopy. At least 34 genes are required for proliferation within the cytosol but do not play a detectable role in modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data indicate a tremendous adaptation and metabolic reprogramming by F. tularensis to adjust to the micro-environmental and nutritional cues within the FCP, and these adjustments play essential roles in modulation of phagosome biogenesis and escape into the cytosol of macrophages as well as proliferation in the cytosol. The plethora of the networks of genes that orchestrate F. tularensis-mediated modulation of phagosome biogenesis, phagosomal escape and bacterial proliferation within the cytosol is novel, complex and involves an unusually large portion of the genome of an intracellular pathogen.
Topics: Cell Line; Cytosol; Francisella tularensis; Genomic Islands; Humans; Macrophages; Mutagenesis, Insertional; Phagosomes; Tularemia
PubMed: 20482590
DOI: 10.1111/j.1462-2920.2010.02229.x -
STAR Protocols Jun 2021Here, we describe an immunofluorescence (IF) microscopy-based approach to quantify cytosolic double-stranded DNA molecules in cultured eukaryotic cells upon the...
Here, we describe an immunofluorescence (IF) microscopy-based approach to quantify cytosolic double-stranded DNA molecules in cultured eukaryotic cells upon the selective and specific permeabilization of plasma membranes. This technique is compatible with widefield microscopy coupled with automated image analysis for mid- to high-throughput applications and high-resolution confocal microscopy for subcellular assessments and co-localization studies. In addition to enabling single-cell and subcellular resolution, this approach circumvents most constraints associated with alternative approaches based on subcellular fractionation. For complete use and execution of this protocol, please refer to Yamazaki et al. (2020).
Topics: Animals; Cell Line; Cytosol; DNA; Mice; Microscopy, Fluorescence; Single-Cell Analysis
PubMed: 34041502
DOI: 10.1016/j.xpro.2021.100488 -
The Journal of Biological Chemistry Feb 2001Most mammalian cells package neutral lipids into droplets that are surrounded by a monolayer of phospholipids and a specific set of proteins including the adipose...
Most mammalian cells package neutral lipids into droplets that are surrounded by a monolayer of phospholipids and a specific set of proteins including the adipose differentiation-related protein (ADRP; also called adipophilin), which is found in a wide array of cell types, and the perilipins, which are restricted to adipocytes and steroidogenic cells. TIP47 was initially identified in a yeast two-hybrid screen for proteins that interact with the cytoplasmic tail of the mannose 6-phosphate receptor, yet its sequence is highly similar to the lipid droplet protein, ADRP, and more distantly related to perilipins. Hence, we hypothesized that TIP47 might be associated with lipid droplets. In HeLa cells grown in standard low lipid-containing culture media, immunofluorescence microscopy revealed that the cells had few lipid droplets; however, TIP47 and ADRP were found on the surfaces of the small lipid droplets present. When the cells were grown in media supplemented with physiological levels of fatty acids, the amount of neutral lipid stored in lipid droplets increased dramatically, as did the staining of TIP47 and ADRP surrounding these droplets. TIP47 was found primarily in the cytosolic fractions of HeLa cells and murine MA10 Leydig cells grown in low lipid-containing culture medium, while ADRP was undetectable in these fractionated cell homogenates. When HeLa and MA10 Leydig cells were lipid-loaded, significant levels of ADRP were found in the floating lipid droplet fractions and TIP47 levels remained constant, but the distribution of a significant portion of TIP47 shifted from the cytosolic fractions to the lipid droplet fractions. Thus, we conclude that TIP47 associates with nascent lipid droplets and can be classified as a lipid droplet-associated protein.
Topics: Antibodies; Brefeldin A; Cell Compartmentation; Cytosol; DNA-Binding Proteins; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; K562 Cells; Lipid Metabolism; Perilipin-3; Pregnancy Proteins; Protein Transport; RNA, Messenger; Subcellular Fractions; Triglycerides; U937 Cells; Vesicular Transport Proteins
PubMed: 11084026
DOI: 10.1074/jbc.M006775200 -
Cellular Physiology and Biochemistry :... 2016The analkaloid drug quinine is utilized mainly for the chemoprophylaxis of malaria. The multiple side effects of quinine include hemolytic anemia and hemolytic uremic...
BACKGROUND/AIMS
The analkaloid drug quinine is utilized mainly for the chemoprophylaxis of malaria. The multiple side effects of quinine include hemolytic anemia and hemolytic uremic syndrome, disorders involving suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide and D4476 sensitive casein kinase. The present study explored the putative effect of quinine on eryptosis and elucidated cellular mechanisms involved.
METHODS
Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies.
RESULTS
A 48 hours exposure of human erythrocytes to quinine (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells without significantly affecting forward scatter. Quinine significantly increased Fluo3-fluorescence, DCF fluorescence and ceramide abundance. The effect of quinine on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of D4476 (10 µM).
CONCLUSIONS
Quinine triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, ceramide and D4476 sensitive casein kinase.
Topics: Calcium; Ceramides; Cytosol; Erythrocyte Membrane; Humans; Phosphatidylserines; Quinine; Reactive Oxygen Species
PubMed: 27898421
DOI: 10.1159/000452578