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Molecular Biology of the Cell Aug 2018Linkers of the nucleoskeleton and cytoskeleton are key molecular complexes that span the nuclear envelope (NE) and provide a direct linkage between the nucleoskeleton...
Linkers of the nucleoskeleton and cytoskeleton are key molecular complexes that span the nuclear envelope (NE) and provide a direct linkage between the nucleoskeleton and cytoskeleton. Two major components of these complexes are members of the SUN and KASH protein families that interact in the perinuclear space to allow the transmission of mechanochemical signals across the NE. Structural details of the mammalian SUN domain protein SUN2 have established that SUN2 must form a trimer to bind to KASH, and that this trimerization is mediated through two predicted coiled-coil regions of the protein, CC1 and CC2, which precede the SUN domain. Recent crystallographic data suggest that CC2-SUN formed an unexpected autoinhibited monomer unable to bind to KASH. These structural insights raise the question of how full-length SUN2 transitions from a monomer to a trimer inside the NE. In this study we used a computational approach to model a fragment of SUN2 containing CC1, CC2, and the SUN domain. We observed the dynamics of these modeled structures using ∼1 μs molecular dynamics simulations and showed that the interplay between CC1 and CC2 may be sufficient for the release of CC2-SUN2 from its autoinhibited state. Additionally, using our models and gel filtration analysis, we show the involvement of an E452 residue on CC1 in the monomer--trimer transition of SUN2. Intriguingly, mutations in this residue have been seen in muscular dystrophy-associated SUN2 variants. Finally, we propose a Ca-dependent monomer-trimer transition of SUN2.
Topics: Amino Acid Sequence; Animals; Calcium; Intracellular Signaling Peptides and Proteins; Ions; Membrane Proteins; Mice; Models, Biological; Models, Molecular; Molecular Dynamics Simulation; Multiprotein Complexes; Mutation; Nuclear Envelope; Nuclear Proteins; Protein Binding; Protein Multimerization; Protein Structure, Secondary; Telomere-Binding Proteins
PubMed: 29995584
DOI: 10.1091/mbc.E18-04-0266 -
PloS One 2022Eosinophilic Esophagitis (EoE) is an antigen-triggered inflammatory condition of the esophageal lining characterized by eosinophilic infiltration. EoE is associated with...
Eosinophilic Esophagitis (EoE) is an antigen-triggered inflammatory condition of the esophageal lining characterized by eosinophilic infiltration. EoE is associated with significant remodeling, and although this remodeling is reversed by current treatment regimens, symptoms of EoE and associated remodeling reappear upon cessation of therapies. We hypothesized that structural remodeling of cell-cell adhesion is a key factor in the pathogenesis of EoE and that epithelial to mesenchymal transition (EMT) was a viable molecular process to lead to this remodeling. Endoscopically obtained biopsy samples from 18 EoE and 18 control pediatric patients were evaluated by transmission electron microscopy to measure intercellular spaces (IS) between cells. Biopsy samples from all groups were analyzed for cellular levels of cell-cell adhesion proteins: E-cadherin, zonula occludens associated protein-1 (ZO-1), and N-cadherin. We also analyzed for cellular levels and localization two of transcription factors, Twist1 and β-catenin, that are associated with promoting EMT. The IS was significantly increased in the EoE group compared to the control. We observed a significant decrease in E-cadherin and ZO-1 levels and a concomitant increase in N-cadherin levels in EoE samples compared to control. Further, while there was no significant change in cellular levels of β-catenin, we observed an altered localization of the protein from the cell membrane in control tissue to a nuclear/perinuclear localization in EoE. We observed higher levels of the transcription factor Twist1 in the EoE group compared to normal which was localized mainly at the nucleus. Our results suggest that the integrity of normally sealed esophageal epithelia is compromised in the EoE patients compared to control subjects, and this is due to alterations in the expression of cell adhesion molecules at the esophageal epithelium. Our data also suggest that EMT, potentially regulated by transcription factors β-catenin and Twist1, may be responsible for the molecular alteration which leads to the remodeling of esophageal epithelia in EoE.
Topics: Cadherins; Child; Eosinophilic Esophagitis; Epithelial-Mesenchymal Transition; Humans; Nuclear Proteins; Twist-Related Protein 1; beta Catenin
PubMed: 35239721
DOI: 10.1371/journal.pone.0264622 -
PloS One 2018Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. However, very little is known about the underlying mechanisms that initiate and...
Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. However, very little is known about the underlying mechanisms that initiate and modulate the associated protein aggregation and deposition. Model systems have been established to investigate these disease-associated processes. One of these systems comprises two 114 amino acid light-chain variable domains of the kappa 4 IgG family, SMA and LEN. Despite high sequence identity (93%), SMA is amyloidogenic in vivo, but LEN adopts a stable dimer, displaying amyloidogenic properties only under destabilising conditions in vitro. We present here a refined and reproducible periplasmic expression and purification protocol for SMA and LEN that improves on existing methods and provides high yields of pure protein (10-50mg/L), particularly suitable for structural studies that demand highly concentrated and purified proteins. We confirm that recombinant SMA and LEN proteins have structure and dimerization capabilities consistent with the native proteins and employ fluorescence to probe internalization and cellular localization within cardiomyocytes. We propose periplasmic expression and simplified chromatographic steps outlined here as an optimized method for production of these and other variable light chain domains to investigate the underlying mechanisms of light chain amyloidosis. We show that SMA and LEN can be internalised within cardiomyocytes and were observed to localise to the perinuclear area, assessed by confocal microscopy as a possible mechanism for underlying cytotoxicity and pathogenesis associated with amyloidosis.
Topics: Animals; Cell Line; Escherichia coli; Humans; Immunoglobulin Light Chains; Immunoglobulin Light-chain Amyloidosis; Immunoglobulin Variable Region; Microscopy, Confocal; Myocytes, Cardiac; Periplasm; Protein Structure, Secondary; Rats; Recombinant Proteins
PubMed: 30347409
DOI: 10.1371/journal.pone.0206167 -
Journal of Virology Oct 2017Herpesvirus capsids assemble in the nucleus, while final virion maturation proceeds in the cytoplasm. This requires that newly formed nucleocapsids cross the nuclear...
Herpesvirus capsids assemble in the nucleus, while final virion maturation proceeds in the cytoplasm. This requires that newly formed nucleocapsids cross the nuclear envelope (NE), which occurs by budding at the inner nuclear membrane (INM), release of the primary enveloped virion into the perinuclear space (PNS), and subsequent rapid fusion with the outer nuclear membrane (ONM). During this process, the NE remains intact, even at late stages of infection. In addition, the spacing between the INM and ONM is maintained, as is that between the primary virion envelope and nuclear membranes. The linker of nucleoskeleton and cytoskeleton (LINC) complex consists of INM proteins with a luminal SUN (Sad1/UNC-84 homology) domain connected to ONM proteins with a KASH (Klarsicht, ANC-1, SYNE homology) domain and is thought to be responsible for spacing the nuclear membranes. To investigate the role of the LINC complex during herpesvirus infection, we generated cell lines constitutively expressing dominant negative (dn) forms of SUN1 and SUN2. Ultrastructural analyses revealed a significant expansion of the PNS and the contiguous intracytoplasmic lumen, most likely representing endoplasmic reticulum (ER), especially in cells expressing dn-SUN2. After infection, primary virions accumulated in these expanded luminal regions, also very distant from the nucleus. The importance of the LINC complex was also confirmed by reduced progeny virus titers in cells expressing dn-SUN2. These data show that the intact LINC complex is required for efficient nuclear egress of herpesviruses, likely acting to promote fusion of primary enveloped virions with the ONM. While the viral factors for primary envelopment of nucleocapsids at the inner nuclear membrane are known to the point of high-resolution structures, the roles of cellular components and regulators remain enigmatic. Furthermore, the machinery responsible for fusion with the outer nuclear membrane is unsolved. We show here that dominant negative SUN2 interferes with efficient herpesvirus nuclear egress, apparently by interfering with fusion between the primary virion envelope and outer nuclear membrane. This identifies a new cellular component important for viral egress and implicates LINC complex integrity in nonconventional nuclear membrane trafficking.
Topics: Active Transport, Cell Nucleus; Animals; Cell Line; Cytoskeleton; Herpesviridae; Membrane Proteins; Microtubule-Associated Proteins; Nuclear Envelope; Nuclear Matrix; Nuclear Proteins; Rabbits; Swine; Virus Assembly; Virus Release
PubMed: 28724767
DOI: 10.1128/JVI.00330-17 -
Biology of Reproduction Jun 2019The perinuclear theca (PT) is a cytosolic protein capsule that surrounds the nucleus of eutherian spermatozoa. Compositionally, it is divided into two regions: the...
The perinuclear theca (PT) is a cytosolic protein capsule that surrounds the nucleus of eutherian spermatozoa. Compositionally, it is divided into two regions: the subacrosomal layer (SAL) and the postacrosomal sheath (PAS). In falciform spermatozoa, a third region of the PT emerges that extends beyond the nuclear apex called the perforatorium. The formation of the SAL and PAS differs, with the former assembling early in spermiogenesis concomitant with acrosome formation, and the latter dependent on manchette descent during spermatid elongation. The perforatorium also forms during the elongation phase of spermiogenesis, suggesting that like the PAS, its assembly is facilitated by the manchette. The temporal similarity in biogenesis between the PAS and perforatorium led us to compare their molecular composition using cell fractionation and immunodetection techniques. Although the perforatorium is predominantly composed of its endemic protein FABP9/PERF15, immunolocalization indicates that it also shares proteins with the PAS. These include WBP2NL/PAWP, WBP2, GSTO2, and core histones, which have been implicated in early fertilization and zygotic events. The compositional homogeny between the PAS and perforatorium supports our observation that their development is linked. Immunocytochemistry indicates that both PAS and perforatorial biogenesis depend on the transport and deposition of cytosolic proteins by the microtubular manchette. Proteins translocated from the manchette pass ventrally along the spermatid head into the apical perforatorial space prior to PAS deposition in the wake of manchette descent. Our findings demonstrate that the perforatorium and PAS share a mechanism of developmental assembly and thereby contain common proteins that facilitate fertilization.
Topics: Acrosome; Animals; Cattle; Cytoskeleton; Humans; Male; Proteins; Rats; Rats, Sprague-Dawley; Semen Analysis; Sperm Head; Spermatogenesis; Spermatozoa; Swine
PubMed: 30939204
DOI: 10.1093/biolre/ioz052 -
Journal of Virology Oct 2017In eukaryotic cells, the oluble -ethylmaleimide-ensitive actor (NSF) ttachment protein ceptor (SNARE) proteins comprise the minimal machinery that triggers fusion of...
In eukaryotic cells, the oluble -ethylmaleimide-ensitive actor (NSF) ttachment protein ceptor (SNARE) proteins comprise the minimal machinery that triggers fusion of transport vesicles with their target membranes. Comparative studies revealed that genes encoding the components of the SNARE system are highly conserved in yeast, insect, and human genomes. Upon infection of insect cells by the virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the transcript levels of most SNARE genes initially were upregulated. We found that overexpression of dominant-negative (DN) forms of NSF or knockdown of the expression of NSF, the key regulator of the SNARE system, significantly affected infectious AcMNPV production. In cells expressing DN NSF, entering virions were trapped in the cytoplasm or transported to the nucleus with low efficiency. The presence of DN NSF also moderately reduced trafficking of the viral envelope glycoprotein GP64 to the plasma membrane but dramatically inhibited production of infectious budded virions (BV). Transmission electron microscopy analysis of infections in cells expressing DN NSF revealed that progeny nucleocapsids were retained in a perinuclear space surrounded by inner and outer nuclear membranes. Several baculovirus conserved (core) proteins (Ac76, Ac78, GP41, Ac93, and Ac103) that are important for infectious budded virion production were found to associate with NSF, and NSF was detected within the assembled BV. Together, these data indicate that the cellular SNARE system is involved in AcMNPV infection and that NSF is required for efficient entry and nuclear egress of budded virions of AcMNPV. Little is known regarding the complex interplay between cellular factors and baculoviruses during viral entry and egress. Here, we examined the cellular SNARE system, which mediates the fusion of vesicles in healthy cells, and its relation to baculovirus infection. Using a DN approach and RNA interference knockdown, we demonstrated that a general disruption of the SNARE machinery significantly inhibited the production of infectious BV of AcMNPV. The presence of a DN NSF protein resulted in low-efficiency entry of BV and the retention of progeny nucleocapsids in the perinuclear space during egress. Combined with these effects, we also found that several conserved (core) baculovirus proteins closely associate with NSF, and these results suggest their involvement in the egress of BV. Our findings are the first to demonstrate that the SNARE system is required for efficient entry of BV and nuclear egress of progeny nucleocapsids of baculoviruses.
Topics: Active Transport, Cell Nucleus; Animals; Cell Line; Cell Nucleus; Cytoplasm; Humans; Microscopy, Electron, Transmission; N-Ethylmaleimide-Sensitive Proteins; Nucleocapsid; Nucleopolyhedroviruses; RNA Interference; SNARE Proteins; Sf9 Cells; Spodoptera; Viral Envelope Proteins; Virion; Virus Assembly; Virus Internalization; Virus Release; Yeasts
PubMed: 28747507
DOI: 10.1128/JVI.01111-17 -
Journal of Microscopy and Ultrastructure 2020The damage of the adrenal gland by snake venoms needs to be clarified. Lethality (LD) of () venom was established by intraperitoneally mice injections. Preparation of...
The damage of the adrenal gland by snake venoms needs to be clarified. Lethality (LD) of () venom was established by intraperitoneally mice injections. Preparation of specimens for transmission electron microscopy samples from cortex adrenal gland biopsies at 3, 6, and 24 h was processed. The quantitative description by the principal component analysis (PCA) of the adrenal gland was as follows: thickening of the capillary endothelium, area of the capillary lumen, cell nucleus area, enlargement of the perinuclear space, number of mitochondria, area of the mitochondria, number of mitochondrial cristae, number of cristae per mitochondrial unit, and tubular diameter of the smooth endoplasmic reticulum (SER). Sections of the adrenal cortex, after 3 h postinjection with venom showed in the cortical cells: mitochondria with tubular cristae and slightly swollen SER cisternae, nucleus with variable heterochromatin content, irregular edges, and swollen nuclear envelope. After 6 h, cells with swollen nucleus envelope, electron dense lipids and mitochondria with loss of their cristae were observed. Myelin figures, close to the microvilli of the cortical cell, multivesicular bodies, swollen profiles of the SER, and electron dense lipid drops were noticed. After 24 h, thickening of the endothelial wall, fenestrae and projections into the capillary lumen, loss of the mitochondrial cristae, destruction of the capillary and the plasma membrane of the cortical cell, multivesicular body, SER loss, and an enlargement of the perinuclear space were detected. In the quantitative PCA, there were significant changes after the venom treatments.
PubMed: 33282685
DOI: 10.4103/JMAU.JMAU_49_19 -
Cell Reports Jun 2017APPL1- and RAB5-positive signaling endosomes play a crucial role in the activation of AKT in response to extracellular stimuli. Myosin VI (MYO6) and two of its cargo...
APPL1- and RAB5-positive signaling endosomes play a crucial role in the activation of AKT in response to extracellular stimuli. Myosin VI (MYO6) and two of its cargo adaptor proteins, GIPC and TOM1/TOM1L2, localize to these peripheral endosomes and mediate endosome association with cortical actin filaments. Loss of MYO6 leads to the displacement of these endosomes from the cell cortex and accumulation in the perinuclear space. Depletion of this myosin not only affects endosome positioning, but also induces actin and lipid remodeling consistent with endosome maturation, including accumulation of F-actin and the endosomal lipid PI(3)P. These processes acutely perturb endosome function, as both AKT phosphorylation and RAC-dependent membrane ruffling were markedly reduced by depletion of either APPL1 or MYO6. These results place MYO6 and its binding partners at a central nexus in cellular signaling linking actin dynamics at the cell surface and endosomal signaling in the cell cortex.
Topics: Actins; Adaptor Proteins, Signal Transducing; Animals; Cell Line; Endosomes; Enzyme Activation; Mice; Myosin Heavy Chains; Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 28591580
DOI: 10.1016/j.celrep.2017.05.048 -
Virology Oct 2018Maize mosaic virus (MMV), similar to other nucleorhabdoviruses, replicates in divergent hosts: plants and insects. To compare MMV protein localization and interactions,...
Maize mosaic virus (MMV), similar to other nucleorhabdoviruses, replicates in divergent hosts: plants and insects. To compare MMV protein localization and interactions, we visualized autofluorescent protein fusions in both cell types. Nucleoprotein (N) and glycoprotein (G) localized to the nucleus and cytoplasm, phosphoprotein (P) was only found in the nucleus, and 3 (movement) and matrix (M) were present in the cytoplasm. This localization pattern is consistent with the model of nucleorhabdoviral replication of N, P, L and viral RNA forming a complex in the nucleus and the subvirion associating with M and then G during budding into perinuclear space. The comparable localization patterns in both organisms indicates a similar replication cycle. Changes in localization when proteins were co-expressed suggested viral proteins interact thus altering organelle targeting. We documented a limited number of direct protein interactions indicating host factors play a role in the virus protein interactions during the infection cycle.
Topics: Amino Acid Sequence; Animals; Cell Line; Cell Nucleus; Cloning, Molecular; Cytosol; Drosophila melanogaster; Glycoproteins; Host Specificity; Host-Pathogen Interactions; Macrophages; Nucleoproteins; Optical Imaging; Phosphoproteins; Plant Cells; Recombinant Fusion Proteins; Rhabdoviridae; Nicotiana; Viral Matrix Proteins; Viral Proteins; Virus Replication
PubMed: 30056212
DOI: 10.1016/j.virol.2018.06.019 -
Proceedings of the National Academy of... Dec 2014GABA-A receptors mediating synaptic or extrasynaptic transmission are molecularly and functionally distinct, and glial cells are known to express a plethora of GABA-A...
GABA-A receptors mediating synaptic or extrasynaptic transmission are molecularly and functionally distinct, and glial cells are known to express a plethora of GABA-A subunits. Here we demonstrate that GFAP(+) cells of the granular layer of cerebellum express GABAρ subunits during early postnatal development, thereby conferring peculiar pharmacologic characteristics to GABA responses. Electron microscopy revealed the presence of GABAρ in the plasma membrane of GFAP(+) cells. In contrast, expression in the adult was restricted to Purkinje neurons and a subset of ependymal cells. Electrophysiological studies in vitro revealed that astrocytes express functional receptors with an EC50 of 52.2 ± 11.8 μM for GABA. The evoked currents were inhibited by bicuculline (100 μM) and TPMPA (IC50, 5.9 ± 0.6 μM), indicating the presence of a GABAρ component. Coimmunoprecipitation demonstrated protein-protein interactions between GABAρ1 and GABAα1, and double immunofluorescence showed that these subunits colocalize in the plasma membrane. Three populations of GABA-A receptors in astrocytes were identified: classic GABA-A, bicuculline-insensitive GABAρ, and GABA-A-GABAρ hybrids. Clusters of GABA-A receptors were distributed in the perinuclear space and along the processes of GFAP(+) cells. Time-lapse microscopy showed GABAρ2-GFP accumulation in clusters located in the soma and along the processes. The clusters were relatively immobile, with mean displacement of 9.4 ± 0.9 μm and a net distance traveled of 1-2 μm, owing mainly to directional movement or simple diffusion. Modulation of GABAρ dynamics may be a novel mechanism of extrasynaptic transmission regulating GABAergic control of GFAP(+) cells during early postnatal development.
Topics: Animals; Astrocytes; Bicuculline; Cell Lineage; Cell Membrane; Cell Movement; Cell Proliferation; Cerebellum; Gene Expression Profiling; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Inhibitory Concentration 50; Male; Mice; Mice, Transgenic; Microscopy, Fluorescence; Neuroglia; Neurons; Neurotransmitter Agents; Phosphinic Acids; Protein Interaction Mapping; Protein Transport; Purkinje Cells; Pyridines; Receptors, GABA-A; Time Factors
PubMed: 25422464
DOI: 10.1073/pnas.1419632111