This service exclusively searches for literature that cites resources. Please be aware that the total number of searchable documents is limited to those containing RRIDs and does not include all open-access literature.
Protein misfolding is usually deleterious for the cell, either as a consequence of the loss of protein function or the buildup of insoluble and toxic aggregates. The aggregation behavior of a given polypeptide is strongly influenced by the intrinsic properties encoded in its sequence. This has allowed the development of effective computational methods to predict protein aggregation propensity.
Knowledge about the abundance of molecular components is an important prerequisite for building quantitative predictive models of cellular behavior. Proteins are central components of these models, since they carry out most of the fundamental processes in the cell. Thus far, protein concentrations have been difficult to measure on a large scale, but proteomic technologies have now advanced to a stage where this information becomes readily accessible.
Cells have been described under the microscope as organelles containing cytoplasm and the nucleus. However, an unnoted structure exists between the cytoplasm and the nucleoplasm of eukaryotic cells. In addition to the nuclear envelope, there exists a perinuclear region (PNR or perinucleus) with unknown composition and function. Until now, an investigation of the role of the perinucleus has been restricted by the absence of a PNR isolation method. This manuscript describes a perinucleus isolation technique on the basis of its unique compact organization. The perinucleus was found to contain approximately 15 to 18% of the total proteins of the mammalian cell, almost half of the proteins of nuclei. Using four different normal and cancer cell lines, it was shown that the composition of PNR is highly dynamic. Application of the method showed that translocation of the p53 tumor-suppressor protein to the perinucleus in immortalized MEF cells is correlated with the translocation of p53-stabilizing protein, nucleophosmin (B23), to the PNR. Herein, the concept of the perinuclear region is advanced as a formal, identifiable structure. The roles of the perinucleus in maintaining genome integrity, regulation of gene expression and understanding of malignant transformation are discussed.
The inhibitor of apoptosis protein (IAP) family has been implicated in immune regulation, but the mechanisms by which IAP proteins contribute to immunity are incompletely understood. We show here that X-linked IAP (XIAP) is required for innate immune control of Listeria monocytogenes infection. Mice deficient in XIAP had a higher bacterial burden 48 h after infection than wild-type littermates, and exhibited substantially decreased survival. XIAP enhanced NF-kappaB activation upon L. monocytogenes infection of activated macrophages, and prolonged phosphorylation of Jun N-terminal kinase (JNK) specifically in response to cytosolic bacteria. Additionally, XIAP promoted maximal production of pro-inflammatory cytokines upon bacterial infection in vitro or in vivo, or in response to combined treatment with NOD2 and TLR2 ligands. Together, our data suggest that XIAP regulates innate immune responses to L. monocytogenes infection by potentiating synergy between Toll-like receptors (TLRs) and Nod-like receptors (NLRs) through activation of JNK- and NF-kappaB-dependent signaling.
The concept that heat stress (HS) causes a large accumulation of reactive oxygen species (ROS) is widely accepted. However, the intracellular compartmentation of ROS accumulation has been poorly characterized. We therefore used redox-sensitive green fluorescent protein (roGFP2) to provide compartment-specific information on heat-induced redox changes of the nuclei and cytosol of Arabidopsis leaf epidermal and stomatal guard cells. We show that HS causes a large increase in the degree of oxidation of both compartments, causing large shifts in the glutathione redox potentials of the cells. Heat-induced increases in the levels of the marker transcripts, heat shock protein (HSP)101, and ascorbate peroxidase (APX)2 were maximal after 15 min of the onset of the heat treatment. RNAseq analysis of the transcript profiles of the control and heat-treated seedlings revealed large changes in transcripts encoding HSPs, mitochondrial proteins, transcription factors, and other nuclear localized components. We conclude that HS causes extensive oxidation of the nucleus as well as the cytosol. We propose that the heat-induced changes in the nuclear redox state are central to both genetic and epigenetic control of plant responses to HS.
Secretory and membrane (glyco)proteins are subject to quality control in the endoplasmic reticulum (ER) to ensure that only functional proteins reach their destination. Proteins deemed terminally misfolded and hence functionally defective may be dislocated to the cytosol, where the proteasome degrades them. What we know about this process stems mostly from overexpression of tagged misfolded proteins, or from situations where viruses have hijacked the quality control machinery to their advantage. We know of only very few endogenous substrates of ER quality control, most of which are degraded as part of a signaling pathway, such as Insig-1, but such examples do not necessarily represent terminally misfolded proteins. Here we show that endogenous dislocation clients are captured specifically in association with the cytosolic chaperone BAG6, or retrieved en masse via their glycan handle.
Many biological toxins are known to attack specific cell types, delivering their enzymatic payloads to the cytosol. This process can be manipulated by molecular engineering of chimeric toxins. Using toxins with naturally unlinked components as a starting point is advantageous because it allows for the development of payloads separately from the binding/translocation components. Here the Clostridium botulinum C2 binding/translocation domain was retargeted to neural cell populations by deleting its non-specific binding domain and replacing it with a C. botulinum neurotoxin binding domain. This fusion protein was used to deliver fluorescently labeled payloads to Neuro-2a cells. Intracellular delivery was quantified by flow cytometry and found to be dependent on artificial enrichment of cells with the polysialoganglioside receptor GT1b. Visualization by confocal microscopy showed a dissociation of payloads from the early endosome indicating translocation of the chimeric toxin. The natural Clostridium botulinum C2 toxin was then delivered to human glioblastoma A172 and synchronized HeLa cells. In the presence of the fusion protein, native cytosolic enzymatic activity of the enzyme was observed and found to be GT1b-dependent. This retargeted toxin may enable delivery of therapeutics to peripheral neurons and be of use in addressing experimental questions about neural physiology.
In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain-of-cytosolic functions-a phenomenon we name ER to Cytosol Signaling (ERCYS).
After the COVID-19 pandemic, society has become more aware of the importance of some basic hygienic habits to avoid exposure to pathogens transmitted via hands. Given that a high frequency of touching mucous membranes can lead to a high risk of infection, it is essential to establish strategies to reduce this behavior as a preventive measure against contagion. This risk can be extrapolated to a multitude of health scenarios and transmission of many infectious diseases. #RedPingüiNO was designed as an intervention to prevent the transmission of SARS-CoV-2 and other pathogens through the reduction of facial self-touches by thoughtfully engaging participants in a serious game.
The Bcl-2 family member Bax translocates from the cytosol to mitochondria, where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by prosurvival Bcl-2 proteins, but how they inhibit Bax remains controversial because they neither colocalize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-x(L) in detergents and cell-free MOMP activity but unexpectedly induce Bax accumulation on mitochondria. Fluorescence loss in photobleaching (FLIP) reveals constant retrotranslocation of WT Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on prosurvival Bcl-2 family proteins, and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-x(L) inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax.
Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.
Hundreds of nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. However, the early processes associated with mitochondrial protein targeting remain poorly understood. Here, we show that in Saccharomyces cerevisiae, the cytosol has the capacity to transiently store mitochondrial matrix-destined precursors in dedicated deposits that we termed MitoStores. Competitive inhibition of mitochondrial protein import via clogging of import sites greatly enhances the formation of MitoStores, but they also form during physiological cell growth on nonfermentable carbon sources. MitoStores are enriched for a specific subset of nucleus-encoded mitochondrial proteins, in particular those containing N-terminal mitochondrial targeting sequences. Our results suggest that MitoStore formation suppresses the toxic potential of aberrantly accumulating mitochondrial precursor proteins and is controlled by the heat shock proteins Hsp42 and Hsp104. Thus, the cytosolic protein quality control system plays an active role during the early stages of mitochondrial protein targeting through the coordinated and localized sequestration of mitochondrial precursor proteins.
Undiluted cytoplasmic extract prepared from unfertilized Xenopus laevis eggs by low-speed centrifugation (CSF extracts) is useful for reconstitution of egg microtubule dynamics and meiosis-II spindle organization, but it suffers limitations for biochemical analysis due to abundant particulates. Here, we describe preparation and the use of fully clarified, undiluted mitotic cytosol derived from CSF extract. Addition of glycogen improves the ability of this cytosol to reconstitute microtubule organization, in part through improved energy metabolism. Using fully clarified, glycogen-supplemented mitotic cytosol, we reconstituted (i) stimulation of microtubule polymerization by Ran.GTP (Groen, Coughlin, & Mitchison, 2011; Ohba, Nakamura, Nishitani, & Nishimoto, 1999) and (ii) self-organization of highly regular bipolar arrays of taxol-stabilized microtubules that we termed "pineapples" (Mitchison, Nguyen, Coughlin, & Groen, 2013). Both systems will be useful for biochemical dissection of spindle assembly mechanisms. We also describe reliable small-scale methods for preparing fluorescent antibody probes that can be used for live imaging in egg extract systems as well as standard immunofluorescence.
Bax, a member of the Bcl-2 protein family, accelerates apoptosis by an unknown mechanism. Bax has been recently reported to be an integral membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 family member localization in living cells, the green fluorescent protein (GFP) was fused to the NH2 termini of Bax, Bcl-2, and Bcl-XL. Confocal microscopy performed on living Cos-7 kidney epithelial cells and L929 fibroblasts revealed that GFP-Bcl-2 and GFP-Bcl-XL had a punctate distribution and colocalized with a mitochondrial marker, whereas GFP-Bax was found diffusely throughout the cytosol. Photobleaching analysis confirmed that GFP-Bax is a soluble protein, in contrast to organelle-bound GFP-Bcl-2. The diffuse localization of GFP-Bax did not change with coexpression of high levels of Bcl-2 or Bcl-XL. However, upon induction of apoptosis, GFP-Bax moved intracellularly to a punctate distribution that partially colocalized with mitochondria. Once initiated, this Bax movement was complete within 30 min, before cellular shrinkage or nuclear condensation. Removal of a COOH-terminal hydrophobic domain from GFP-Bax inhibited redistribution during apoptosis and inhibited the death-promoting activity of both Bax and GFP-Bax. These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death.
The key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are starved of glucose. FBPase is targeted from the cytosol to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. Several vid mutants defective in the glucose-induced degradation of FBPase in the vacuole have been isolated. In some vid mutants, FBPase is found in punctate structures in the cytoplasm. When extracts from these cells are fractionated, a substantial amount of FBPase is sedimentable in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these vid mutants. In this paper we investigated whether FBPase association with intracellular structures also existed in wild-type cells. We report the purification of novel FBPase-associated vesicles from wild-type cells to near homogeneity. Kinetic studies indicate that FBPase association with these vesicles is stimulated by glucose and occurs only transiently, suggesting that these vesicles are intermediate in the FBPase degradation pathway. Fractionation analysis demonstrates that these vesicles are distinct from known organelles such as the vacuole, ER, Golgi, mitochondria, peroxisomes, endosomes, COPI, or COPII vesicles. Under EM, these vesicles are 30-40 nm in diam. Proteinase K experiments indicate that the majority of FBPase is sequestered inside the vesicles. We propose that FBPase is imported into these vesicles before entering the vacuole.
Either caspase-1 or caspase-11 can cleave gasdermin D to cause pyroptosis, eliminating intracellular replication niches. We previously showed that macrophages detect Burkholderia thailandensis via NLRC4, triggering the release of interleukin (IL)-18 and driving an essential interferon (IFN)-γ response that primes caspase-11. We now identify the IFN-γ-producing cells as a mixture of natural killer (NK) and T cells. Although both caspase-1 and caspase-11 can cleave gasdermin D in macrophages and neutrophils, we find that NLRC4-activated caspase-1 triggers pyroptosis in macrophages, but this pathway does not trigger pyroptosis in neutrophils. In contrast, caspase-11 triggers pyroptosis in both macrophages and neutrophils. This translates to an absolute requirement for caspase-11 in neutrophils during B. thailandensis infection in mice. We present an example of inflammasome sensors causing diverging outcomes in different cell types. Thus, cell fates are dictated not simply by the pathogen or inflammasome, but also by how the cell is wired to respond to detection events.
Five yeast enzymes synthesizing various glycerophospholipids belong to the CDP-alcohol phosphatidyltransferase (CAPT) superfamily. They only share the so-called CAPT motif, which forms the active site of all these enzymes. Bioinformatic tools predict the CAPT motif of phosphatidylinositol synthase Pis1 as either ER luminal or cytosolic. To investigate the membrane topology of Pis1, unique cysteine residues were introduced into either native or a Cys-free form of Pis1 and their accessibility to the small, membrane permeating alkylating reagent N-ethylmaleimide (NEM) and mass tagged, non-permeating maleimides, in the presence and absence of non-denaturing detergents, was monitored. The results clearly point to a cytosolic location of the CAPT motif. Pis1 is highly sensitive to non-denaturing detergent, and low concentrations (0.05%) of dodecylmaltoside change the accessibility of single substituted Cys in the active site of an otherwise cysteine free version of Pis1. Slightly higher detergent concentrations inactivate the enzyme. Removal of the ER retrieval sequence from (wt) Pis1 enhances its activity, again suggesting an influence of the lipid environment. The central 84% of the Pis1 sequence can be aligned and fitted onto the 6 transmembrane helices of two recently crystallized archaeal members of the CAPT family. Results delineate the accessibility of different parts of Pis1 in their natural context and allow to critically evaluate the performance of different cysteine accessibility methods. Overall the results show that cytosolically made inositol and CDP-diacylglycerol can access the active site of the yeast PI synthase Pis1 from the cytosolic side and that Pis1 structure is strongly affected by mild detergents.
The protozoan parasite Trypanosoma cruzi invades a wide variety of vertebrate cells, by a mechanism distinct from phagocytosis. No pseudopods or other host cell surface alterations are observed during trypanosome entry, and invasion is enhanced after actin filaments are disrupted with cytochalasin D. These observations created a puzzle; what is the origin of the membrane required to form the intracellular vacuoles, if it is not originated from the host cell plasma membrane? Recent observations provided the answer: during cell invasion T. cruzi recruits host lysosomes, which gradually fuse with the plasma membrane at the site of parasite entry. The membrane of the parasitophorous vacuole is, therefore, very similar if not identical to the membrane of lysosomes. Lgps, major glycoproteins from mammalian lysosomes, are desialylated by a glycosylphosphatidylinositol (GPI)-anchored trans-sialidase present on the surface of trypomastigote forms. Lack of sialic acid on lgps facilitates membrane lysis by a parasite-secreted molecule, Tc-TOX, that has membrane pore-forming activity at acidic pH. We propose a sequential model in which these trypanosome products would promote parasite entry into host cells and their subsequent liberation into the cytosol.
The role of myristoylation in the localization and catalytic activity of Src at focal adhesions was investigated by live-cell imaging and site-directed mutagenesis. Although the majority of activated Src molecules are localized at focal adhesions, it is unclear how activated Src molecules are recruited to focal adhesions. Because Src is activated at the cell membrane, translocation of Src to cell membranes is considered to be essential for its recruitment to focal adhesions. Membrane-targeting-deficient Src mutant SrcG2A localizes at focal adhesions, indicating direct recruitment of Src from cytosol to focal adhesions. Furthermore, directly recruited Src molecules are shown to enhance paxillin dynamics at focal adhesions. These results reveal that the regulation of Src activation and translocation is more complex than previously suggested.
Welcome to the FDI Lab - SciCrunch.org Resources search. From here you can search through a compilation of resources used by FDI Lab - SciCrunch.org and see how data is organized within our community.
You are currently on the Community Resources tab looking through categories and sources that FDI Lab - SciCrunch.org has compiled. You can navigate through those categories from here or change to a different tab to execute your search through. Each tab gives a different perspective on data.
If you have an account on FDI Lab - SciCrunch.org then you can log in from here to get additional features in FDI Lab - SciCrunch.org such as Collections, Saved Searches, and managing Resources.
Here is the search term that is being executed, you can type in anything you want to search for. Some tips to help searching:
You can save any searches you perform for quick access to later from here.
We recognized your search term and included synonyms and inferred terms along side your term to help get the data you are looking for.
If you are logged into FDI Lab - SciCrunch.org you can add data records to your collections to create custom spreadsheets across multiple sources of data.
Here are the facets that you can filter your papers by.
From here we'll present any options for the literature, such as exporting your current results.
If you have any further questions please check out our FAQs Page to ask questions and see our tutorials. Click this button to view this tutorial again.
Year:
Count: