-
Cells Mar 2020Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the...
Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the cytoplasm. For this, they bud at the inner nuclear membrane resulting in primary enveloped particles in the perinuclear space (PNS) followed by fusion of the primary envelope with the outer nuclear membrane (ONM). While the conserved viral nuclear egress complex orchestrates the first steps, effectors of fusion of the primary virion envelope with the ONM are still mostly enigmatic but might include cellular proteins like SUN2 or ESCRT-III components. Here, we analyzed the influence of the only known AAA+ ATPases located in the endoplasmic reticulum and the PNS, the Torsins (Tor), on nuclear egress of the alphaherpesvirus pseudorabies virus. For this overexpression of wild type and mutant proteins as well as CRISPR/Cas9 genome editing was applied. Neither single overexpression nor gene knockout (KO) of TorA or TorB had a significant impact. However, TorA/B double KO cells showed decreased viral titers at early time points of infection and an accumulation of primary virions in the PNS pointing to a delay in capsid release during nuclear egress.
Topics: ATPases Associated with Diverse Cellular Activities; Active Transport, Cell Nucleus; Animals; Cell Nucleus; Cytoplasm; Herpesvirus 1, Suid; Molecular Chaperones; Nuclear Envelope; Rabbits; Viral Proteins; Virus Release
PubMed: 32192107
DOI: 10.3390/cells9030738 -
Molecular Vision 2016Congenital cataract is a leading cause of childhood blindness. Mutations in the EPHA2 gene are one of the causes of inherited congenital cataract. The EPHA2 gene encodes...
PURPOSE
Congenital cataract is a leading cause of childhood blindness. Mutations in the EPHA2 gene are one of the causes of inherited congenital cataract. The EPHA2 gene encodes a membrane-bound tyrosine kinase receptor and is highly expressed in epithelial cells, including in the ocular lens. Signaling through the EPHA2 receptor plays a pivotal role in epithelial cell homeostasis. The aim of this study was to determine the effect of congenital cataract causing mutations in the EPHA2 gene on the encoded protein in epithelial cells.
METHODS
The effect of five disease-causing mutations, p.P584L (c.1751C>T), p.T940I (c.2819C>T), p.D942fsXC71 (c.2826-9G>A), p.A959T (c.2875G>A), and p.V972GfsX39 (c.2915_2916delTG), on localization of the protein was examined in two in vitro epithelial cell culture systems: Madin-Darby Canine Kidney (MDCK) and human colorectal adenocarcinoma (Caco-2) epithelial cells. Myc-tagged mutant constructs were generated by polymerase chain reaction (PCR)-based mutagenesis. The Myc-tagged wild-type construct was used as a control. The Myc-tagged wild-type and mutant proteins were ectopically expressed and detected by immunofluorescence labeling.
RESULTS
Two of the mutations, p.T940I and p.D942fsXC71, located within the cytoplasmic sterile-α-motif (SAM) domain of EPHA2, led to mis-localization of the protein to the perinuclear space and co-localization with the cis-golgi apparatus, indicating sub-organellar/cellular retention of the mutant proteins. The mutant proteins carrying the remaining three mutations, similar to the wild-type EPHA2, localized to the cell membrane.
CONCLUSIONS
Mis-localization of two of the mutant proteins in epithelial cells suggests that some disease-causing mutations in EPHA2 likely affect lens epithelial cell homeostasis and contribute to cataract. This study suggests that mutations in EPHA2 contribute to congenital cataract through diverse mechanisms.
Topics: Animals; Blotting, Western; Caco-2 Cells; Cataract; Cell Line; DNA Primers; Dogs; Epithelial Cells; Fluorescent Antibody Technique, Indirect; Gene Amplification; Gene Expression Regulation; HEK293 Cells; Humans; Madin Darby Canine Kidney Cells; Mutation; Receptor, EphA2; Transfection
PubMed: 26900323
DOI: No ID Found -
Methods in Molecular Biology (Clifton,... 2018Linkers of nucleoskeleton and cytoskeleton (LINC) complexes are conserved nuclear envelope (NE) spanning molecular bridges which mechanically integrate the nucleus with...
Linkers of nucleoskeleton and cytoskeleton (LINC) complexes are conserved nuclear envelope (NE) spanning molecular bridges which mechanically integrate the nucleus with the cytoskeleton and mediate force transmission into the nucleoplasm. Despite their critical roles in fundamental cellular processes such as meiotic chromosome and nuclear positioning, the mechanism of LINC complex assembly in cells remains unclear. To begin to address this deficit, we recently developed z-scan fluorescence fluctuation spectroscopy (FFS) and brightness analysis as a method for quantifying the oligomeric states of fluorescent protein-tagged NE proteins including nesprins and SUN proteins. Since the homo-oligomerization of SUN2 is critical for its ability to interact with nesprins within the perinuclear space, the knowledge obtained through quantitative brightness experiments reveals important insights into the in vivo mechanisms of LINC complex assembly. Here we describe the procedure we use to determine the brightness of proteins in the NE of living cells. In addition to the measurement procedure, we discuss the instrumentation requirements and present the results of applying this procedure to measure the brightness of nesprin-2 and SUN2.
Topics: Cytoskeleton; Gene Expression; Genes, Reporter; Molecular Imaging; Nuclear Envelope; Nuclear Proteins; Protein Multimerization; Spectrometry, Fluorescence
PubMed: 30141043
DOI: 10.1007/978-1-4939-8691-0_11 -
Breast Cancer Research : BCR May 2022CCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and subsequent endocytosis may identify highly aggressive breast cancer cell...
BACKGROUND
CCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and subsequent endocytosis may identify highly aggressive breast cancer cell subtypes likely to spread into the circulatory system.
METHODS
The MDA-MB-231 cell line was used to model and visualize CCR5 activation by stimulation with RANTES, in an effort to quantify CCR5 endocytosis from the cell surface to the perinuclear space. CCR5 expression was then examined in tumor-associated cells (TACs), consisting of circulating tumor cells and circulating stromal cells, isolated from the peripheral blood of 54 metastatic breast cancer (mBC) patients to evaluate these CCR5 pooling patterns as they relate to progression and survival over 2 years.
RESULTS
In MB231 experiments, it was observed that CCR5 formed ~ 1 micron clusters identified as "CCR5 pools" on the surface of the cell, which in the presence of RANTES were endocytosed and translocated to the cell cytoplasm. When TACs from patients were analyzed, CCR5 pools were observed on the cell surface and translocating to the nuclear area, with CCR5 also having a positive statistical correlation between increased numbers of TACs and increased CCR5 pools on the cells. Further, it was determined that patients with very high numbers of CCR5 (> 10 CCR5 pools), specifically in the circulating stromal cells, were associated with worse progression-free survival (hazard ratio = 4.5, p = 0.002) and worse overall survival (hazard ratio = 3.7, p = 0.014).
CONCLUSIONS
Using a liquid biopsy approach, we evaluated two populations of tumor-associated cells emanating from primary tumors, with data suggesting that upregulation of the motility chemokine CCR5 in TACs provides clinically relevant opportunities for treating and tracking drug targetable receptors in mBC.
Topics: Breast Neoplasms; Chemokine CCL5; Endocytosis; Female; Humans; Neoplastic Cells, Circulating; Receptors, CCR5
PubMed: 35606863
DOI: 10.1186/s13058-022-01528-w -
Journal of Virology Feb 2015Herpes simplex virus 1 (HSV-1) capsids are assembled in the nucleus, where they incorporate the viral genome. They then transit through the two nuclear membranes and are...
UNLABELLED
Herpes simplex virus 1 (HSV-1) capsids are assembled in the nucleus, where they incorporate the viral genome. They then transit through the two nuclear membranes and are wrapped by a host-derived envelope. In the process, several HSV-1 proteins are targeted to the nuclear membranes, but their roles in viral nuclear egress are unclear. Among them, glycoprotein M (gM), a known modulator of virus-induced membrane fusion, is distributed on both the inner and outer nuclear membranes at the early stages of the infection, when no other viral glycoproteins are yet present there. Later on, it is found on perinuclear virions and ultimately redirected to the trans-Golgi network (TGN), where it cycles with the cell surface. In contrast, transfected gM is found only at the TGN and cell surface, hinting at an interaction with other viral proteins. Interestingly, many herpesvirus gM analogs interact with their gN counterparts, which typically alters their intracellular localization. To better understand how HSV-1 gM localization is regulated, we evaluated its ability to bind gN and discovered it does so in both transfected and infected cells, an interaction strongly weakened by the deletion of the gM amino terminus. Functionally, while gN had no impact on gM localization, gM redirected gN from the endoplasmic reticulum (ER) to the TGN. Most interestingly, gN overexpression stimulated the formation of syncytia in the context of an infection by a nonsyncytial strain, indicating that gM and gN not only physically but also functionally interact and that gN modulates gM's activity on membrane fusion.
IMPORTANCE
HSV-1 gM is an important modulator of virally induced cell-cell fusion and viral entry, a process that is likely finely modulated in time and space. Until now, little was known of the proteins that regulate gM's activity. In parallel, gM is found in various intracellular locations at different moments, ranging from nuclear membranes, perinuclear virions, the TGN, cell surface, and mature extracellular virions. In transfected cells, however, it is found only on the TGN and cell surface, hinting that its localization is modulated by other viral proteins. The present study identifies HSV-1 gN as a binding partner for gM, in agreement with their analogs in other herpesviruses, but most excitingly shows that gN modulates gM's impact on HSV-1-induced membrane fusion. These findings open up new research avenues on the viral fusion machinery.
Topics: Animals; Cell Line; Herpesvirus 1, Human; Humans; Membrane Glycoproteins; Protein Interaction Mapping; Protein Multimerization; Viral Matrix Proteins; Viral Proteins; Virus Internalization
PubMed: 25505065
DOI: 10.1128/JVI.03041-14 -
The Journal of Clinical Investigation Aug 2019Deciphering novel pathways regulating liver lipid content has profound implications for understanding the pathophysiology of nonalcoholic fatty liver disease and...
Deciphering novel pathways regulating liver lipid content has profound implications for understanding the pathophysiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Recent evidence suggests that the nuclear envelope is a site of regulation of lipid metabolism but there is limited appreciation of the responsible mechanisms and molecular components within this organelle. We showed that conditional hepatocyte deletion of the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1) caused defective VLDL secretion and steatosis, including intranuclear lipid accumulation. LAP1 binds to and activates torsinA, an AAA+ ATPase that resides in the perinuclear space and continuous main ER. Deletion of torsinA from mouse hepatocytes caused even greater reductions in VLDL secretion and profound steatosis. Both of these mutant mouse lines developed hepatic steatosis and subsequent steatohepatitis on a regular chow diet in the absence of whole-body insulin resistance or obesity. Our results establish an essential role for the nuclear envelope-localized torsinA-LAP1 complex in hepatic VLDL secretion and suggest that the torsinA pathway participates in the pathophysiology of nonalcoholic fatty liver disease.
Topics: Animals; Carrier Proteins; Hepatocytes; Lipid Metabolism; Lipoproteins, VLDL; Membrane Proteins; Mice; Mice, Knockout; Molecular Chaperones; Non-alcoholic Fatty Liver Disease; Nuclear Envelope
PubMed: 31408437
DOI: 10.1172/JCI129769 -
MBio Jun 2017Many viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the...
Many viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the cytoplasm. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein. This yields a primary enveloped virion (PEV) whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. We investigated the associated molecular mechanisms by isolating PEVs from US3-null-infected cells and imaging them by cryo-electron microscopy and tomography. (pUS3 is a viral protein kinase in whose absence PEVs accumulate in the perinuclear space.) Unlike mature extracellular virions, PEVs have very few glycoprotein spikes. PEVs are ~20% smaller than mature virions, and the little space available between the capsid and the NEC layer suggests that most tegument proteins are acquired later in the egress pathway. Previous studies have proposed that NEC is organized as hexamers in honeycomb arrays in PEVs, but we find arrays of heptameric rings in extracts from US3-null-infected cells. In a PEV, NEC contacts the capsid predominantly via the pUL17/pUL25 complexes which are located close to the capsid vertices. Finally, the NEC layer dissociates from the capsid as it leaves the nucleus, possibly in response to pUS3-mediated phosphorylation. Overall, nuclear egress emerges as a process driven by a program of multiple weak interactions. On its maturation pathway, the newly formed HSV-1 nucleocapsid must traverse the nuclear envelope, while respecting the integrity of that barrier. Nucleocapsids (125 nm in diameter) are too large to pass through the nuclear pore complexes that conduct most nucleocytoplasmic traffic. It is now widely accepted that the process involves envelopment/de-envelopment of a key intermediate-the primary enveloped virion. In wild-type infections, PEVs are short-lived, which has impeded study. Using a mutant that accumulates PEVs in the perinuclear space, we were able to isolate PEVs in sufficient quantity for structural analysis by cryo-electron microscopy and tomography. The findings not only elucidate the maturation pathway of an important human pathogen but also have implications for cellular processes that involve the trafficking of large macromolecular complexes.
Topics: Animals; Capsid; Capsid Proteins; Cell Nucleus; Chlorocebus aethiops; Cryoelectron Microscopy; Herpesvirus 1, Human; Nuclear Envelope; Phosphorylation; Vero Cells; Viral Proteins; Virion; Virus Assembly; Virus Release
PubMed: 28611252
DOI: 10.1128/mBio.00825-17 -
ACS Biomaterials Science & Engineering Jan 2019Inhibiting protein aggregation under intra-/extracellular space and clearing protein aggregates from the brain are two critical issues for the treatment of various...
Inhibiting protein aggregation under intra-/extracellular space and clearing protein aggregates from the brain are two critical issues for the treatment of various neurodegenerative diseases. Although a variety of anti-amyloidogenic chemicals/biochemicals have been identified for inhibiting such protein aggregation, clearing protein aggregates is a challenging issue. Here we report a designed biopolymer micelle of 15-30 nm hydrodynamic size that can clear protein aggregates from cells via an up-regulated autophagy process. The polymer has a polyaspartic acid backbone and is functionalized with fatty amine, arginine, and primary amine for inducing self-assembly, enhancing cell uptake, and up-regulating autophagy processes, respectively. The polymer micelle (PM) enters into the cell via lipid raft endocytosis, is transported to the perinuclear region where the protein oligomer/aggregate predominantly localizes, clears aggregated protein from the cell, and enhances the cell's survival against toxic protein aggregates. The designed PM may be used as a drug delivery carrier for anti-amyloidogenic drugs for enhanced efficacy in the treatment of neurodegenerative diseases.
PubMed: 33405873
DOI: 10.1021/acsbiomaterials.8b01196 -
F1000Research 2019Capsids of herpes simplex virus 1 (HSV-1) are assembled in cell nuclei, released into the perinuclear space by budding at the inner nuclear membrane acquiring tegument...
Capsids of herpes simplex virus 1 (HSV-1) are assembled in cell nuclei, released into the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope. Alternatively, capsids gain access to the cytoplasm via dilated nuclear pores. They are enveloped by Golgi membranes. Us3 is a non-essential viral kinase that is involved in nucleus-to-cytoplasm translocation, preventing apoptosis and regulation of phospholipid-biosynthesis. Us3-deletion mutants HSV-1∆Us3) accumulate in the perinuclear space. Nuclear and Golgi membranes proliferate, and homogeneous, proteinaceous structures of unknown identity are deposited in nuclei and cytoplasm. Glycoprotein K (gK), a highly hydrophobic viral protein, is essential for production of infectious progeny virus but, according to the literature, exclusively vital for envelopment of capsids by Golgi membranes. In the absence of Us3, virions remain stuck in the perinuclear space but mature to infectivity without reaching Golgi membranes, suggesting further function of gK than assumed. We constructed a HSV-1∆Us3 mutant designated CK177∆Us3gK-HA, in which gK was hemagglutinin (HA) epitope-tagged in order to localize gK by immunolabeling using antibodies against HA for light and electron microscopy. CK177∆Us3gK-HA-infected Vero cells showed similar alterations as those reported for other HSV-1∆Us3, including accumulation of virions in the perinuclear space, overproduction of nuclear and Golgi membranes containing electron dense material with staining property of proteins. Immunolabeling using antibodies against HA revealed that gK is overproduced and localized at nuclear membranes, perinuclear virions stuck in the perinuclear space, Golgi membranes and on protein deposits in cytoplasm and nuclei. Us3 is involved in proper assembly of membranes needed for envelopment and incorporation of gK. Without Us3, virions derived by budding at nuclear membranes remain stuck in the perinuclear space but incorporate gK into their envelope to gain infectivity.
Topics: Animals; Chlorocebus aethiops; Glycoproteins; Herpesvirus 1, Human; Vero Cells; Viral Proteins; Virion
PubMed: 31448105
DOI: 10.12688/f1000research.19194.1 -
Rheumatology International Jul 2023The objective of this study was to investigate the effects of prolonged exposure to the oxidative agent NaClO on histopathological changes in the lung tissues of...
The objective of this study was to investigate the effects of prolonged exposure to the oxidative agent NaClO on histopathological changes in the lung tissues of laboratory animals. Specifically, the study aimed to examine morphological changes in the pulmonary microcirculation and the level of vascular cell adhesion molecule-1 (VCAM-1) as a functional activity indicator of endothelial cells in animals with induced systemic sclerosis (SSc). A laboratory animal model was used to assess the impact of long-term exposure to NaClO on lung tissues. The animals were divided into three groups: the experimental group (25 rats) was exposed to NaClO, while the control group (20 rats) received an isotonic solution, and the intact group (15 animals) was without any exposure. The concentration of VCAM-1 in the serum of the animals was measured using an enzyme-linked immunosorbent assay. Histopathological analysis of lung tissue specimens was performed using both light and electron microscopy. The concentration of VCAM-1 in the serum of the animals in the experimental group was significantly higher than that of the control group (91.25 [85.63-143.75] vs 19.50 [13.53-22.20], p < 0.05). The histopathological analysis revealed significant abnormalities in the lung tissue specimens from the experimental group, including disruption in the structure of the hemocapillaries of the lungs, narrowing of the microvessel lumen, and perivascular infiltration by polymorphonuclear cells. The electron microscopic analysis showed several ultrastructural changes in the endotheliocytes of the hemocapillaries, including uneven expansion of the perinuclear space, swollen mitochondria, and fragmentation of the membranes of the granular endoplasmic reticulum. Additionally, the basement membrane of hemocapillaries showed uneven thickening with indistinct contours, and the peripheral parts of endotheliocytes were marked by numerous micropinocytotic vesicles and vacuoles. Erythrocyte aggregates and leukocyte adhesion were identified in the lumen of many hemocapillaries, while adhesion and aggregation of platelets were also observed in several hemocapillaries. Long-term exposure to NaClO can cause significant histopathological changes in lung tissues, including damage to the hemocapillaries and disruption in the structure of endotheliocytes.
Topics: Rats; Animals; Endothelial Cells; Vascular Cell Adhesion Molecule-1; Lung; Neutrophils; Scleroderma, Systemic
PubMed: 37071178
DOI: 10.1007/s00296-023-05328-z