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Viruses Nov 2021Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by... (Review)
Review
Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by the nuclear egress complex (NEC) proteins UL31/34. At that stage of egress, enveloped virions are found for a short time in the perinuclear space. In the second step of nuclear egress, perinuclear enveloped virions (PEVs) fuse with the outer nuclear membrane (ONM) delivering capsids into the cytoplasm. Once in the cytoplasm, capsids undergo re-envelopment in the Golgi/trans-Golgi apparatus producing mature virions. This second step of nuclear egress is known as de-envelopment and is the focus of this review. Compared with herpesvirus envelopment at the INM, much less is known about de-envelopment. We propose a model in which de-envelopment involves two phases: (i) fusion of the PEV membrane with the ONM and (ii) expansion of the fusion pore leading to release of the viral capsid into the cytoplasm. The first phase of de-envelopment, membrane fusion, involves four herpes simplex virus (HSV) proteins: gB, gH/gL, gK and UL20. gB is the viral fusion protein and appears to act to perturb membranes and promote fusion. gH/gL may also have similar properties and appears to be able to act in de-envelopment without gB. gK and UL20 negatively regulate these fusion proteins. In the second phase of de-envelopment (pore expansion and capsid release), an alpha-herpesvirus protein kinase, US3, acts to phosphorylate NEC proteins, which normally produce membrane curvature during envelopment. Phosphorylation of NEC proteins reverses tight membrane curvature, causing expansion of the membrane fusion pore and promoting release of capsids into the cytoplasm.
Topics: Capsid; Cell Nucleus; Cytoplasm; Herpesviridae; Herpesviridae Infections; Humans; Membrane Fusion; Nuclear Envelope; Phosphorylation; Simplexvirus; Viral Envelope; Viral Fusion Proteins; Virion; trans-Golgi Network
PubMed: 34960625
DOI: 10.3390/v13122356 -
Nucleic Acids Research Dec 2021G-quadruplexes (G4s) are secondary structures forming in G-rich nucleic acids. G4s are assumed to play critical roles in biology, nonetheless their detection in cells is...
G-quadruplexes (G4s) are secondary structures forming in G-rich nucleic acids. G4s are assumed to play critical roles in biology, nonetheless their detection in cells is still challenging. For tracking G4s, synthetic molecules (G4 ligands) can be used as reporters and have found wide application for this purpose through chemical functionalization with a fluorescent tag. However, this approach is limited by a low-labeling degree impeding precise visualization in specific subcellular regions. Herein, we present a new visualization strategy based on the immuno-recognition of 5-bromo-2'-deoxyuridine (5-BrdU) modified G4 ligands, functionalized prior- or post-G4-target binding by CuAAC. Remarkably, recognition of the tag by antibodies leads to the detection of the modified ligands exclusively when bound to a G4 target both in vitro, as shown by ELISA, and in cells, thereby providing a highly efficient G4-ligand Guided Immunofluorescence Staining (G4-GIS) approach. The obtained signal amplification revealed well-defined fluorescent foci located in the perinuclear space and RNase treatment revealed the preferential binding to G4-RNA. Furthermore, ligand treatment affected significantly BG4 foci formation in cells. Our work headed to the development of a new imaging approach combining the advantages of immunostaining and G4-recognition by G4 ligands leading to visualization of G4/ligands species in cells with unrivaled precision and sensitivity.
Topics: A549 Cells; Bromodeoxyuridine; Cell Line; Click Chemistry; Enzyme-Linked Immunosorbent Assay; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique; G-Quadruplexes; Humans; Ligands
PubMed: 34875077
DOI: 10.1093/nar/gkab1166 -
Acta Crystallographica. Section D,... Dec 2021Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell...
Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell motility. Visualizing actin filaments has so far relied on fluorescence microscopy and electron microscopy/tomography. The former lacks the capacity to capture the overall local ultrastructure, while the latter requires rigorous sample preparation that can lead to potential artefacts, and only delivers relatively small volumes of imaging data at the thinnest areas of a cell. In this work, a correlative approach utilizing in situ super-resolution fluorescence imaging and cryo X-ray tomography was used to image bundles of actin filaments deep inside cells under near-native conditions. In this case, fluorescence 3D imaging localized the actin bundles within the intracellular space, while X-ray tomograms of the same areas provided detailed views of the local ultrastructure. Using this new approach, actin trails connecting vesicles in the perinuclear area and hotspots of actin presence within and around multivesicular bodies were observed. The characteristic prevalence of filamentous actin in cytoplasmic extensions was also documented.
Topics: Actins; Animals; Cell Line, Tumor; Cryoelectron Microscopy; Humans; Lasers; Protein Conformation; X-Rays
PubMed: 34866605
DOI: 10.1107/S2059798321010329 -
Open Biology Nov 2021The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (ER) modulator that cooperates with the nuclear pore complex (NPC)...
The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (ER) modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here, we identified a short amphipathic α-helix (AH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an () version in which AH is disrupted show NPC biogenesis and NE integrity defects, without impacting Apq12-ah topology or NE/ER localization. Overexpression of but not triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE possibly during NPC biogenesis.
Topics: Membrane Proteins; Mutation; Nuclear Envelope; Nuclear Pore; Phosphatidic Acids; Protein Conformation, alpha-Helical; Protein Domains; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34814743
DOI: 10.1098/rsob.210250 -
Journal of Virology Jan 2022During the nuclear export of nascent nucleocapsids of herpesviruses, the nucleocapsids bud through the inner nuclear membrane (INM) by acquiring the INM as a primary...
During the nuclear export of nascent nucleocapsids of herpesviruses, the nucleocapsids bud through the inner nuclear membrane (INM) by acquiring the INM as a primary envelope (primary envelopment). We recently reported that herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), which consists of UL34 and UL31, interacts with an endosomal sorting complex required for transport III (ESCRT-III) adaptor ALIX and recruits ESCRT-III machinery to the INM for efficient primary envelopment. In this study, we identified a cluster of six arginine residues in the disordered domain of UL34 as a minimal region required for the interaction with ALIX, as well as the recruitment of ALIX and an ESCRT-III protein CHMP4B to the INM in HSV-1-infected cells. Mutations in the arginine cluster exhibited phenotypes similar to those with ESCRT-III inhibition reported previously, including the mislocalization of NEC, induction of membranous invagination structures containing enveloped virions, aberrant accumulation of enveloped virions in the invaginations and perinuclear space, and reduction of viral replication. We also showed that the effect of the arginine cluster in UL34 on HSV-1 replication was dependent primarily on ALIX. These results indicated that the arginine cluster in the disordered domain of UL34 was required for the interaction with ALIX and the recruitment of ESCRT-III machinery to the INM to promote primary envelopment. Herpesvirus UL34 homologs contain conserved amino-terminal domains that mediate vesicle formation through interactions with UL31 homologs during primary envelopment. UL34 homologs also comprise other domains adjacent to their membrane-anchoring regions, which differ in length, are variable in herpesviruses, and do not form distinguished secondary structures. However, the role of these disordered domains in infected cells remains to be elucidated. In this study, we present data suggesting that the arginine cluster in the disordered domain of HSV-1 UL34 mediates the interaction with ALIX, thereby leading to the recruitment of ESCRT-III machinery to the INM for efficient primary envelopment. This is the first study to report the role of the disordered domain of a UL34 homolog in herpesvirus infections.
Topics: Arginine; Calcium-Binding Proteins; Cell Cycle Proteins; Endosomal Sorting Complexes Required for Transport; HeLa Cells; Herpesvirus 1, Human; Humans; Morphogenesis; Mutation; Nuclear Envelope; Nucleocapsid; Phosphorylation; Viral Proteins; Virion; Virus Release; Virus Replication
PubMed: 34730397
DOI: 10.1128/JVI.01704-21 -
Cellular and Molecular Life Sciences :... Dec 2021Astronauts on board the International Space Station (ISS) are exposed to the damaging effects of microgravity and cosmic radiation. One of the most critical and...
Astronauts on board the International Space Station (ISS) are exposed to the damaging effects of microgravity and cosmic radiation. One of the most critical and sensitive districts of an organism is the eye, particularly the retina, and > 50% of astronauts develop a complex of alterations designated as spaceflight-associated neuro-ocular syndrome. However, the pathogenesis of this condition is not clearly understood. In the current study, we aimed to explore the cellular and molecular effects induced in the human retinal pigment ARPE-19 cell line by their transfer to and 3-day stay on board the ISS in the context of an experiment funded by the Agenzia Spaziale Italiana. Treatment of cells on board the ISS with the well-known bioenergetic, antioxidant, and antiapoptotic coenzyme Q10 was also evaluated. In the ground control experiment, the cells were exposed to the same conditions as on the ISS, with the exception of microgravity and radiation. The transfer of ARPE-19 retinal cells to the ISS and their living on board for 3 days did not affect cell viability or apoptosis but induced cytoskeleton remodeling consisting of vimentin redistribution from the cellular boundaries to the perinuclear area, underlining the collapse of the network of intermediate vimentin filaments under unloading conditions. The morphological changes endured by ARPE-19 cells grown on board the ISS were associated with changes in the transcriptomic profile related to the cellular response to the space environment and were consistent with cell dysfunction adaptations. In addition, the results obtained from ARPE-19 cells treated with coenzyme Q10 indicated its potential to increase cell resistance to damage.
Topics: Apoptosis; Cell Proliferation; DNA Damage; Gene Expression Profiling; Gene Expression Regulation; Humans; Retinal Pigment Epithelium; Space Flight; Ubiquinone; Weightlessness
PubMed: 34714361
DOI: 10.1007/s00018-021-03989-2 -
Pharmaceutics Oct 2021The nucleolin-binding G-quadruplex AS1411 aptamer has been widely used for cancer therapy and diagnosis and linked to nanoparticles for its selective targeting activity....
The nucleolin-binding G-quadruplex AS1411 aptamer has been widely used for cancer therapy and diagnosis and linked to nanoparticles for its selective targeting activity. We applied a computational and experimental integrated approach to study the effect of engineering AS1411 aptamer on an octahedral truncated DNA nanocage to obtain a nanostructure able to combine selective cancer-targeting and anti-tumor activity. The nanocages functionalized with one aptamer molecule (Apt-NC) displayed high stability in serum, were rapidly and selectively internalized in cancer cells through an AS1411-dependent mechanism, and showed over 200-fold increase in anti-cancer activity when compared with the free aptamer. Comparison of Apt-NCs and free AS1411 intracellular distribution showed that they traffic differently inside cells: Apt-NCs distributed through the endo-lysosomal pathway and were never found in the nuclei, while the free AS1411 was mostly found in the perinuclear region and in nucleoli. Molecular dynamics simulations indicated that the aptamer, when linked to the nanocage, sampled a limited conformational space, more confined than in the free state, which is characterized by a large number of metastable conformations. A different intracellular trafficking of Apt-NCs compared with free aptamer and the confined aptamer conformations induced by the nanocage were likely correlated with the high cytotoxic enhancement, suggesting a structure-function relationship for the AS1411 aptamer activity.
PubMed: 34683964
DOI: 10.3390/pharmaceutics13101671 -
Biochimica Et Biophysica Acta.... Jan 2022Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to...
Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.
Topics: Actin Cytoskeleton; Animals; Cells, Cultured; Chick Embryo; Intrinsically Disordered Proteins; Lysosomes; Microscopy, Electron, Transmission; Mitochondria; Nuclear Envelope; Proteome; Zebrafish
PubMed: 34655689
DOI: 10.1016/j.bbamcr.2021.119161 -
Frontiers in Endocrinology 2021Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants that have become globally ubiquitous in humans and the environment. PFAS exposure is...
BACKGROUND
Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants that have become globally ubiquitous in humans and the environment. PFAS exposure is associated with neurodevelopmental effects; however, the mechanism is poorly understood. Brain-derived neurotrophic factor (BDNF) signaling is critical to fetal neurodevelopment during pregnancy and maintains important regulatory roles later in life. This study aims to characterize placental BDNF signaling and investigate whether PFAS exposure disrupts the signaling pathway in placental trophoblast cells.
METHODS
The expression and localization of BDNF receptors-p75 and TrkB-in first trimester and term human placentas and trophoblast cells were investigated by immunofluorescence staining. To assess the effects of PFAS exposure on the BDNF pathway, BeWo cells were treated with PFAS mixtures that mimicked blood levels in a highly exposed population and major PFAS compounds in the mixture at 0.01, 0.1, 1, and 10 µM concentrations. Changes in pro-BDNF levels and phosphorylation of TrkB receptors were examined by Western blot.
RESULTS
In first trimester human placentas, TrkB and p75 receptors were primarily localized to syncytiotrophoblast and cytotrophoblast cells. At term, TrkB and p75 receptors were primarily observed in the placental villous stroma. TrkB receptor staining in trophoblasts was reduced at term, while p75 receptor staining was negative. TrkB receptors were confined to the nuclear and perinuclear spaces, and phosphorylation occurred at the Tyr816 residue in BeWo cells. Exposure to PFOS, PFOA, PFBS, and the six-PFAS mixture did not significantly affect BDNF levels or activation (phosphorylation) of TrkB. Treating cells with 1 μM and 10 μM of PFNA resulted in increased TrkB phosphorylation compared to unexposed controls, but BDNF levels were unchanged.
CONCLUSIONS
BDNF receptors are present in different regions of human placental villi, indicating diverse functions of BDNF signaling in placental development. Our findings suggest that the BDNF pathway in placental trophoblast cells is not disrupted by exposures to PFOS, PFOA, PFBS, and a PFAS mixture, but may be affected by PFNA exposures. Further investigation is needed on how PFAS affects other critical signaling pathways during fetal neurodevelopment.
Topics: Brain-Derived Neurotrophic Factor; Female; Fluorocarbons; Humans; Maternal Exposure; Membrane Glycoproteins; Phosphorylation; Placenta; Placentation; Pregnancy; Pregnancy Trimester, First; Receptor, trkB; Signal Transduction; Trophoblasts; Tumor Cells, Cultured
PubMed: 34394001
DOI: 10.3389/fendo.2021.694885 -
Biochemical and Biophysical Research... Sep 2021ALS2, a product of the causative gene for familial amyotrophic lateral sclerosis (ALS) type 2, plays a pivotal role in the regulation of endosome dynamics by activating...
ALS2, a product of the causative gene for familial amyotrophic lateral sclerosis (ALS) type 2, plays a pivotal role in the regulation of endosome dynamics by activating small GTPase Rab5 via its intrinsic guanine nucleotide-exchange factor activity. Previously, we have reported that the N-terminal region of ALS2 has crucial roles in its endosomal localization and self-oligomerization, both of which are indispensable for the cellular function of ALS2. The N-terminus of ALS2 contains the regulator of chromosome condensation 1-like domain (RLD), which is predicted to form a seven-bladed β-propeller structure. Interestingly, the RLD is interrupted by the intrinsically disordered region (IDR), within which there are several amino acid residues which undergo phosphorylation. In this study, we sought to investigate as to whether and how the IDR as well as phosphorylation at either Ser483, Ser492 or Thr510 affect the intracellular localization and self-oligomerization of ALS2. All phospho- and dephospho-mimetic ALS2 mutants that were transiently expressed in HeLa cells were diffusely distributed throughout the cytosol with a partial localization to early endosomes. When expressed under Rac1-activating conditions, these mutants were localized to membrane ruffles as well as enlarged endosomes. Further, gel-filtration analysis revealed that these mutants primarily existed as a tetramer in cells. However, all these phenotypes were indistinguishable from those of wild-type ALS2. On the other hand, IDR-deleted ALS2 mutant was exclusively present in perinuclear aggregates colocalizing with the autophagy-related protein SQSTM1. Moreover, IDR-deleted ALS2 mutant formed an abnormally high molecular weight complex compared to wild-type ALS2. These results indicate that the IDR of ALS2 plays a crucial role not only in the regulation of intracellular localization but also in the self-oligomerization of ALS2 in cells, whereas phosphorylation of certain residues within the IDR exerts limited effects on such phenotypes.
Topics: Amyotrophic Lateral Sclerosis; Blotting, Western; Endosomes; Genetic Predisposition to Disease; Guanine Nucleotide Exchange Factors; HeLa Cells; Humans; Intracellular Space; Intrinsically Disordered Proteins; Microscopy, Fluorescence; Mutation; Phosphorylation; Protein Binding; Protein Multimerization; Protein Transport; Sequestosome-1 Protein; rab5 GTP-Binding Proteins
PubMed: 34243065
DOI: 10.1016/j.bbrc.2021.06.095