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The Plant Journal : For Cell and... Oct 2020Soybean cyst nematode (SCN; Heterodera glycines) is the largest pathogenic cause of soybean yield loss. The Rhg1 locus is the most used and best characterized SCN...
Soybean cyst nematode (SCN; Heterodera glycines) is the largest pathogenic cause of soybean yield loss. The Rhg1 locus is the most used and best characterized SCN resistance locus, and contains three genes including one encoding an α-SNAP protein. Although the Rhg1 α-SNAP is known to play an important role in vesicle trafficking and SCN resistance, the protein's binding partners and the molecular mechanisms underpinning SCN resistance remain unclear. In this report, we show that the Rhg1 α-SNAP strongly interacts with two syntaxins of the t-SNARE family (Glyma.12G194800 and Glyma.16G154200) in yeast and plants; importantly, the genes encoding these syntaxins co-localize with SCN resistance quantitative trait loci. Fluorescent visualization revealed that the α-SNAP and the two interacting syntaxins localize to the plasma membrane and perinuclear space in both tobacco epidermal and soybean root cells. The two syntaxins and their two homeologs were mutated, individually and in combination, using the CRISPR-Cas9 system in the SCN-resistant Peking and SCN-susceptible Essex soybean lines. Peking roots with deletions introduced into syntaxin genes exhibited significantly reduced resistance to SCN, confirming that t-SNAREs are critical to resisting SCN infection. The results presented here uncover a key step in the molecular mechanism of SCN resistance, and will be invaluable to soybean breeders aiming to develop highly SCN-resistant soybean varieties.
Topics: Animals; Clustered Regularly Interspaced Short Palindromic Repeats; Disease Resistance; Host-Parasite Interactions; Plant Diseases; Plant Proteins; Plant Roots; Plants, Genetically Modified; Qa-SNARE Proteins; Quantitative Trait Loci; SNARE Proteins; Glycine max; Two-Hybrid System Techniques; Tylenchoidea
PubMed: 32645235
DOI: 10.1111/tpj.14923 -
Journal of Virology Apr 2001Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and... (Comparative Study)
Comparative Study
Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and pseudorabies virus (PrV), was investigated by electron microscopy of infected cell lines of different origins. In all virus-cell systems analyzed, similar observations were made concerning the different stages of virion morphogenesis. After intranuclear assembly, nucleocapsids bud at the inner leaflet of the nuclear membrane, resulting in enveloped particles in the perinuclear space that contain a sharply bordered rim of tegument and a smooth envelope surface. Egress from the perinuclear cisterna primarily occurs by fusion of the primary envelope with the outer leaflet of the nuclear membrane, which has been visualized for HSV-1 and EHV-1 for the first time. The resulting intracytoplasmic naked nucleocapsids are enveloped at membranes of the trans-Golgi network (TGN), as shown by immunogold labeling with a TGN-specific antiserum. Virions containing their final envelope differ in morphology from particles within the perinuclear cisterna by visible surface projections and a diffuse tegument. Particularly striking was the addition of a large amount of tegument material to ILTV capsids in the cytoplasm. Extracellular virions were morphologically identical to virions within Golgi-derived vesicles, but distinct from virions in the perinuclear space. Studies with gB- and gH-deleted PrV mutants indicated that these two glycoproteins, which are essential for virus entry and direct cell-to-cell spread, are dispensable for egress. Taken together, our studies indicate that the deenvelopment-reenvelopment process of herpesvirus maturation also occurs in EHV-1, HSV-1, and ILTV and that membrane fusion processes occurring during egress are substantially different from those during entry and direct viral cell-to-cell spread.
Topics: Alphaherpesvirinae; Animals; Cell Line; Cell Nucleus; Gene Deletion; Glycoproteins; Herpesvirus 1, Equid; Herpesvirus 1, Human; Herpesvirus 1, Suid; Microscopy, Electron; Nucleocapsid; Viral Envelope Proteins; Virus Assembly; trans-Golgi Network
PubMed: 11264357
DOI: 10.1128/JVI.75.8.3675-3684.2001 -
EMBO Reports Apr 2019Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the...
Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.
Topics: Animals; Cell Line; Endocytosis; Extracellular Space; Host-Pathogen Interactions; Humans; Immunity, Innate; Interferon Type I; Macrophages; Membrane Proteins; Mice; Models, Molecular; Nucleotides, Cyclic; Nucleotidyltransferases; Protein Binding; Protein Conformation; Protein Multimerization; Second Messenger Systems; Signal Transduction; Structure-Activity Relationship
PubMed: 30872316
DOI: 10.15252/embr.201846293 -
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 -
Cells Sep 2020Cell migration requires reposition and reshaping of the cell nucleus. The nuclear lamina is highly important for migration of both primary and cancer cells. B-type...
Cell migration requires reposition and reshaping of the cell nucleus. The nuclear lamina is highly important for migration of both primary and cancer cells. B-type lamins are important for proper migration of epicardial cells and neurons and increased lamin B to lamin A ratio accelerates cancer cell migration through confined spaces. Moreover, a positive association between lamin B1 levels and tumor formation and progression is found in various cancer types. Still, the molecular mechanism by which B-type lamins promote cell migration is not fully understood. To better understand this mechanism, we tested the effects of lamin B1 on perinuclear actin organization. Here we show that induction of melanoma cell migration leads to the formation of a cytosolic Linker of Nucleoskeleton and Cytoskeleton (LINC) complex-independent perinuclear actin rim, which has not been detected in migrating cells, yet. Significantly, increasing the levels of lamin B1 but not the levels of lamin A prevented perinuclear actin rim formation while accelerated the cellular migration rate. To interfere with the perinuclear actin rim, we generated a chimeric protein that is localized to the outer nuclear membrane and cleaves perinuclear actin filaments in a specific manner without disrupting other cytosolic actin filaments. Using this tool, we found that disruption of the perinuclear actin rim accelerated the cellular migration rate in a similar manner to lamin B1 over-expression. Taken together, our results suggest that increased lamin B1 levels can accelerate cell migration by inhibiting the association of the nuclear envelope with actin filaments that may reduce nuclear movement and deformability.
Topics: Actins; Cell Line, Tumor; Cell Movement; Cell Nucleus; Gelsolin; Humans; Lamin Type B; Melanoma
PubMed: 32987785
DOI: 10.3390/cells9102161 -
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 -
Japanese Journal of Infectious Diseases 2015In this study, G proteins of the rabies virus (RABV) Kyoto strain were detected in the cytoplasm but not distributed at the cell membrane of mouse neuroblastoma (MNA)...
In this study, G proteins of the rabies virus (RABV) Kyoto strain were detected in the cytoplasm but not distributed at the cell membrane of mouse neuroblastoma (MNA) cells. G proteins of CVS-26 were detected in both the cell membrane and perinuclear space of MNA cells. We found that N-glycosylation of street RABV G protein by the insertion of the sequon Asn(204) induced the transfer of RABV G proteins to the cell surface membrane. Fixed RABV budding from the plasma membrane has been found to depend not only on G protein but also on other structural proteins such as M protein. However, the differing N-glycosylation of G protein could be associated with the distinct budding and antigenic features of RABV in street and fixed viruses. Our study of the association of N-glycan of G protein at Asn(204) with the transport of RABV G protein to the cell surface membrane contributes to the understanding of the evolution of fixed virus from street virus, which in turn would help for determine the mechanism underlying RABV budding and enhanced host immune responses.
Topics: Animals; Antigens, Viral; Asparagine; Fluorescent Antibody Technique, Indirect; Glycoproteins; Glycosylation; Humans; Mice; Protein Transport; Rabies; Tumor Cells, Cultured; Viral Envelope Proteins
PubMed: 25766612
DOI: 10.7883/yoken.JJID.2014.533 -
Nucleus (Austin, Tex.) 2010The nuclear envelope (NE) is a barrier that separates nuclear from cytoplasmic processes. It is composed of an inner and outer nuclear membrane (INM, ONM), separated by...
The nuclear envelope (NE) is a barrier that separates nuclear from cytoplasmic processes. It is composed of an inner and outer nuclear membrane (INM, ONM), separated by the perinuclear space (PNS). The ONM is contiguous with the endoplasmic reticulum (ER), and thus, the lumen of the NE and that of the ER constitute one compartment. The lamin B receptor (LBR) is a NE protein that has a central structural role as a linker of the INM, the lamina and chromatin, and a less well characterized functional role as a sterol reductase. In a recent study, we reported that the forced expression of mutant variants of LBR in some cell types induces a separation of the INM from the outer nuclear envelope concomitantly with a separation of ER membranes, whereas in other cells no separation is observed. In this extra view, we speculate about the mechanism that leads to this fundamental disruption of NE and ER structure. Our observations furthermore raise the question to what extent LBR contributes to the establishment or maintenance of the ER and PNS luminal compartment, and how a single mutant protein can so drastically interfere with its regular organization.
Topics: Cell Line; Cell Line, Tumor; Cell Nucleus; Chromatin; Endoplasmic Reticulum; Gene Expression Profiling; HeLa Cells; Humans; Mutation; Nuclear Envelope; Osteochondrodysplasias; Pelger-Huet Anomaly; Phenotype; Receptors, Cytoplasmic and Nuclear; Lamin B Receptor
PubMed: 21327079
DOI: 10.4161/nucl.1.4.11801 -
The Journal of Cell Biology Dec 2019Mammalian cells frequently migrate through tight spaces during normal embryogenesis, wound healing, diapedesis, or in pathological situations such as metastasis. Nuclear...
Mammalian cells frequently migrate through tight spaces during normal embryogenesis, wound healing, diapedesis, or in pathological situations such as metastasis. Nuclear size and shape are important factors in regulating the mechanical properties of cells during their migration through such tight spaces. At the onset of migratory behavior, cells often initiate the expression of vimentin, an intermediate filament protein that polymerizes into networks extending from a juxtanuclear cage to the cell periphery. However, the role of vimentin intermediate filaments (VIFs) in regulating nuclear shape and mechanics remains unknown. Here, we use wild-type and vimentin-null mouse embryonic fibroblasts to show that VIFs regulate nuclear shape and perinuclear stiffness, cell motility in 3D, and the ability of cells to resist large deformations. These changes increase nuclear rupture and activation of DNA damage repair mechanisms, which are rescued by exogenous reexpression of vimentin. Our findings show that VIFs provide mechanical support to protect the nucleus and genome during migration.
Topics: Animals; Cell Movement; Cell Nucleus; Collagen; Cytoskeleton; DNA Damage; Fibroblasts; Intermediate Filament Proteins; Intermediate Filaments; Mice; Microscopy, Atomic Force; Microscopy, Confocal; Necrosis; Vimentin
PubMed: 31676718
DOI: 10.1083/jcb.201902046 -
Redox Biology Aug 2015Carbonylation is a generic term which refers to reactive carbonyl groups present in biomolecules due to oxidative reactions induced by reactive oxygen species....
Carbonylation is a generic term which refers to reactive carbonyl groups present in biomolecules due to oxidative reactions induced by reactive oxygen species. Carbonylated proteins, lipids and nucleic acids have been intensively studied and often associated with onset or progression of oxidative stress related disorders. In order to reveal underlying carbonylation pathways and biological relevance, it is crucial to study their intracellular formation and spatial distribution. Carbonylated species are usually identified and quantified in cell lysates and body fluids after derivatization using specific chemical probes. However, spatial cellular and tissue distribution have been less often investigated. Here, we report coumarin-hydrazide, a fluorescent chemical probe for time- and cost-efficient labeling of cellular carbonyls followed by fluorescence microscopy to evaluate their intracellular formation both in time and space. The specificity of coumarin-hydrazide was confirmed in time- and dose-dependent experiments using human primary fibroblasts stressed with paraquat and compared with conventional DNPH-based immunocytochemistry. Both techniques stained carbonylated species accumulated in cytoplasm with strong perinuclear clustering. Using a complimentary array of analytical methods specificity of coumarin-hydrazide probe towards both protein- and lipid-bound carbonyls has been shown. Additionally, co-distribution of carbonylated species and oxidized phospholipids was demonstrated.
Topics: Animals; Cells, Cultured; Chromatography, Thin Layer; Coumarins; Fluorescent Dyes; Humans; Hydrazines; Immunohistochemistry; Mice; Microscopy, Fluorescence; Oxidative Stress; Paraquat; Phospholipids; Protein Carbonylation; Proteins; Rats; Tandem Mass Spectrometry
PubMed: 25974625
DOI: 10.1016/j.redox.2015.04.006