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Journal of Lipid Research Aug 2018Neutrophils form neutrophil extracellular traps (NETs), which have been implicated in microcirculatory plugging. NET formation (NETosis) involves the fusion of granule...
Neutrophils form neutrophil extracellular traps (NETs), which have been implicated in microcirculatory plugging. NET formation (NETosis) involves the fusion of granule and nuclear contents, which are then released in the extracellular space. Myeloperoxidase (MPO) plays a major role in NETosis leading to the dissociation of DNA from histones. During neutrophil activation, MPO is released and activated to convert hydrogen peroxide and chloride to hypochlorous acid (HOCl). HOCl targets plasmalogens leading to the production of the chlorinated lipids, 2-chlorofatty aldehyde and 2-chlorofatty acid (2-ClFA). Here, we tested the hypothesis that 2-ClFAs are important lipid mediators of NETosis. Human neutrophils treated with physiological levels of 2-ClFAs formed NETs, characterized by MPO association with DNA and neutrophil elastase (NE) redistribution to the perinuclear area. 2-ClFA-induced NETs reduced colony forming units. 2-ClFA-induced NETosis is calcium- and protein arginine deiminase 4-dependent. Interestingly, unlike PMA, 2-ClFA initiates the NETosis process without neutrophil activation and degranulation. Furthermore, 2-ClFA elicits NETosis in bone-marrow derived neutrophils from MPO-deficient mice. Taken together, these findings suggest 2-ClFA as an MPO product that triggers the NETosis pathway following neutrophil activation.
Topics: Calcium; DNA; Extracellular Traps; Fatty Acids; Healthy Volunteers; Humans; Lipid Metabolism; Neutrophils; Protein-Arginine Deiminase Type 4; Protein-Arginine Deiminases
PubMed: 29739865
DOI: 10.1194/jlr.M084731 -
Journal of the Royal Society, Interface Nov 2016Angiogenesis, the formation of blood vessels from pre-existing ones, is a key event in pathology, including cancer progression, but also in homeostasis and regeneration....
Angiogenesis, the formation of blood vessels from pre-existing ones, is a key event in pathology, including cancer progression, but also in homeostasis and regeneration. As the phenotype of endothelial cells (ECs) is continuously regulated by local biomechanical forces, studying endothelial behaviour in altered gravity might contribute to new insights towards angiogenesis modulation. This study aimed at characterizing EC behaviour after hypergravity exposure (more than 1), with special focus on cytoskeleton architecture and capillary-like structure formation. Herein, human umbilical vein ECs (HUVECs) were cultured under two-dimensional and three-dimensional conditions at 3 and 10 for 4 and 16 h inside the large diameter centrifuge at the European Space Research and Technology Centre (ESTEC) of the European Space Agency. Although no significant tendency regarding cytoskeleton organization was observed for cells exposed to high 's, a slight loss of the perinuclear localization of β-tubulin was observed for cells exposed to 3 with less pronounced peripheral bodies of actin when compared with 1 control cells. Additionally, hypergravity exposure decreased the assembly of HUVECs into capillary-like structures, with a 10 level significantly reducing their organization capacity. In conclusion, short-term hypergravity seems to affect EC phenotype and their angiogenic potential in a time and -level-dependent manner.
Topics: Actins; Human Umbilical Vein Endothelial Cells; Humans; Hypergravity; Neovascularization, Physiologic; Tubulin
PubMed: 28334696
DOI: 10.1098/rsif.2016.0688 -
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 -
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 -
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 -
Journal of Virology Aug 2018Nuclear egress of herpesvirus capsids is mediated by the conserved nuclear egress complex (NEC), composed of the membrane-anchored pUL34 and its nucleoplasmic...
Nuclear egress of herpesvirus capsids is mediated by the conserved nuclear egress complex (NEC), composed of the membrane-anchored pUL34 and its nucleoplasmic interaction partner, pUL31. The recently solved crystal structures of the NECs from different herpesviruses show a high structural similarity, with the pUL34 homologs building a platform recruiting pUL31 to the inner nuclear membrane. Both proteins possess a central globular fold, while the conserved N-terminal portion of pUL31 forms an extension reaching around the core of pUL34. However, the extreme N terminus of the pUL31 homologs, which is highly variable in length and amino acid composition, had to be removed for crystallization. Several pUL31 homologs contain a classical nuclear localization signal (NLS) within this part mediating efficient nuclear import. In addition, membrane-binding activity, blocking premature interaction with pUL34, nucleocapsid trafficking, and regulation of NEC assembly and disassembly via phosphorylation were assigned to the extreme pUL31 N terminus. To test the functional importance in the alphaherpesvirus pseudorabies virus (PrV) pUL31, N-terminal truncations and site-specific mutations were generated, and the resulting proteins were tested for intracellular localization, interaction with pUL34, and functional complementation of PrV-ΔUL31. Our data show that neither the bipartite NLS nor the predicted phosphorylation sites are essential for pUL31 function during nuclear egress. Moreover, nearly the complete variable N-terminal part was dispensable for function as long as a stretch of basic amino acids was retained. Phosphorylation of this domain controls efficient nucleocapsid release from the perinuclear space. Nuclear egress of herpesvirus capsids is a unique vesicle-mediated nucleocytoplasmic transport. Crystal structures of the heterodimeric NECs from different herpesviruses provided important details of this viral nuclear membrane deformation and scission machinery but excluded the highly variable N terminus of the pUL31 component. We present here a detailed mutagenesis study of this important portion of pUL31 and show that basic amino acid residues within this domain play an essential role for proper targeting, complex formation, and function during nuclear egress, while phosphorylation modulates efficient release from the perinuclear space. Thus, our data complement previous structure-function assignments of the nucleocapsid-interacting component of the NEC.
Topics: Animals; Cell Line; Crystallography, X-Ray; Herpesvirus 1, Suid; Humans; Phosphorylation; Protein Domains; Viral Proteins; Virus Release
PubMed: 29793954
DOI: 10.1128/JVI.00566-18