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American Journal of Physiology. Renal... Feb 2023Vasopressin (VP)-regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water...
Vasopressin (VP)-regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water homeostasis. VP affects AQP2 phosphorylation at several serine residues in the COOH-terminus; among them, serine 256 (S256) appears to be a major regulator of AQP2 trafficking. Mutation of this serine to aspartic acid, which mimics phosphorylation, induces constitutive membrane expression of AQP2. However, the intracellular location(s) at which S256 phosphorylation occurs remains elusive. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells and monitored VP-stimulated phosphorylation of S256 at these sites by immunofluorescence and Western blot analysis with phospho-specific antibodies. Using methyl-β-cyclodextrin, cold block or bafilomycin, and taxol, we blocked AQP2 at the plasma membrane, in the perinuclear -Golgi network, and in scattered cytoplasmic vesicles, respectively. Regardless of its cellular location, VP induced a significant increase in S256 phosphorylation, and this effect was not dependent on a functional microtubule cytoskeleton. To further investigate whether protein kinase A (PKA) was responsible for S256 phosphorylation in these cellular compartments, we created PKA-null cells and blocked AQP2 trafficking using the same procedures. We found that S256 phosphorylation was no longer increased compared with baseline, regardless of AQP2 localization. Taken together, our data indicate that AQP2 S256 phosphorylation can occur at the plasma membrane, in the -Golgi network, or in cytoplasmic vesicles and that this event is dependent on the expression of PKA in these cells. Phosphorylation of aquaporin-2 by PKA at serine 256 (S256) occurs in various subcellular locations during its recycling itinerary, suggesting that the protein complex necessary for AQP2 S256 phosphorylation is present in these different recycling stations. Furthermore, we showed, using PKA-null cells, that PKA activity is required for vasopressin-induced AQP2 phosphorylation. Our data reveal a complex spatial pattern of intracellular AQP2 phosphorylation at S256, shedding new light on the role of phosphorylation in AQP2 membrane accumulation.
Topics: Animals; Aquaporin 2; LLC-PK1 Cells; Phosphorylation; Serine; Swine; Vasopressins; Intracellular Space
PubMed: 36454701
DOI: 10.1152/ajprenal.00123.2022 -
BioRxiv : the Preprint Server For... Jun 2023Mutations in the gene encoding nuclear lamins A/C cause a diverse array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite...
UNLABELLED
Mutations in the gene encoding nuclear lamins A/C cause a diverse array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the molecular perturbations emanating from mutations, an integrative understanding of the pathogenesis leading to cardiac dysfunction remains elusive. Using a novel cell-type specific deletion mouse model capable of translatome profiling, we found that cardiomyocyte-specific deletion in adult mice led to rapid cardiomyopathy with pathological remodeling. Prior to the onset of cardiac dysfunction, lamin A/C-depleted cardiomyocytes displayed nuclear envelope deterioration, golgi dilation/fragmentation, and CREB3-mediated golgi stress activation. Translatome profiling identified upregulation of Med25, a transcriptional co-factor that can selectively dampen UPR axes. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the golgi or inducing nuclear damage by increased matrix stiffness. Systemic administration of pharmacological modulators of autophagy or ER stress significantly improved the cardiac function. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the development of cardiomyopathy.
TEASER
Interplay of stress responses underlying the development of cardiomyopathy.
PubMed: 36824975
DOI: 10.1101/2023.02.14.528563 -
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 -
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 -
Cells Apr 2021Retinal degeneration is a leading cause of blindness. The unfolded protein response (UPR) is an endoplasmic reticulum (ER) stress response that affects cell survival and...
Retinal degeneration is a leading cause of blindness. The unfolded protein response (UPR) is an endoplasmic reticulum (ER) stress response that affects cell survival and death and GRP78 forms a representative protective response. We aimed to determine the exact localization of GRP78 in an animal model of light-induced retinal degeneration. Dark-adapted mice were exposed to blue light-emitting diodes and retinas were obtained at 24 h and 72 h after exposure. In the normal retina, we found that GRP78 was rarely detected in the photoreceptor cells while it was expressed in the perinuclear space of the cell bodies in the inner nuclear and ganglion cell layers. After injury, the expression of GRP78 in the outer nuclear and inner plexiform layers increased in a time-dependent manner. However, an increased GRP78 expression was not observed in damaged photoreceptor cells in the outer nuclear layer. GRP78 was located in the perinuclear space and ER lumen of glial cells and the ER developed in glial cells during retinal degeneration. These findings suggest that GRP78 and the ER response are important for glial cell activation in the retina during photoreceptor degeneration.
Topics: Animals; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Light; Male; Mice; Mice, Inbred BALB C; Retina; Retinal Degeneration
PubMed: 33922686
DOI: 10.3390/cells10050995 -
Stress Biology Jun 2024In eukaryotes, the nuclear membrane that encapsulates genomic DNA is composed of an inner nuclear membrane (INM), an outer nuclear membrane (ONM), and a perinuclear...
In eukaryotes, the nuclear membrane that encapsulates genomic DNA is composed of an inner nuclear membrane (INM), an outer nuclear membrane (ONM), and a perinuclear space. SUN proteins located in the INM and KASH proteins in the ONM form the SUN-KASH NM-bridge, which functions as the junction of the nucleocytoplasmic complex junction. Proteins containing the SUN domain showed the highest correlation with differentially accumulated proteins (DAPs) in the wheat response to fungal stress. To understand the characteristics of SUN and its associated proteins in wheat responding to pathogen stress, here we investigated and comprehensive analyzed SUN- and KASH-related proteins among the DAPs under fungi infection based on their conserved motifs. In total, four SUN proteins, one WPP domain-interacting protein (WIP), four WPP domain-interacting tail-anchored proteins (WIT), two WPP proteins and one Ran GTPase activating protein (RanGAP) were identified. Following transient expression of Nicotiana benthamiana, TaSUN2, TaRanGAP2, TaWIT1 and TaWIP1 were identified as nuclear membrane proteins, while TaWPP1 and TaWPP2 were expressed in both the nucleus and cell membrane. RT-qPCR analysis demonstrated that the transcription of TaSUN2, TaRanGAP2 and TaWPP1 were strongly upregulated in response to fungal infection. Furthermore, using the bimolecular fluorescence complementation, the luciferase complementation and a nuclear and split-ubiquitin-based membrane yeast two-hybrid systems, we substantiated the interaction between TaSUN2 and TaWIP1, as well as TaWIP1/WIT1 and TaWPP1/WPP2. Silencing of TaSUN2, TaRanGAP2 and TaWPP1 in wheat leaves promoted powdery mildew infection and hyphal growth, and reduced the expression of TaBRI1, TaBAK1 and Ta14-3-3, indicating that these NM proteins play a positive role in resistance to fungal stress. Our study reveals the characteristics of NM proteins and propose the preliminary construction of SUN-WIP-WPP-RanGAP complex in wheat, which represents a foundation for detail elucidating their functions in wheat in future.
PubMed: 38861095
DOI: 10.1007/s44154-024-00163-z -
Journal of Virology Nov 2019During nuclear egress of nascent progeny herpesvirus nucleocapsids, the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane of...
During nuclear egress of nascent progeny herpesvirus nucleocapsids, the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane of infected cells into the perinuclear space between the inner and outer nuclear membranes. Herpes simplex virus 1 (HSV-1) U34 and U31 proteins form a nuclear egress complex (NEC) and play critical roles in this budding process, designated primary envelopment. To clarify the role of NEC binding to progeny nucleocapsids in HSV-1 primary envelopment, we established an assay system for HSV-1 NEC binding to nucleocapsids and capsid proteins Using this assay system, we showed that HSV-1 NEC bound to nucleocapsids and to capsid protein U25 but not to the other capsid proteins tested (i.e., VP5, VP23, and U17) and that HSV-1 NEC binding of nucleocapsids was mediated by the interaction of NEC with U25. U31 residues arginine-281 (R281) and aspartic acid-282 (D282) were required for efficient NEC binding to nucleocapsids and U25. We also showed that alanine substitution of U31 R281 and D282 reduced HSV-1 replication, caused aberrant accumulation of capsids in the nucleus, and induced an accumulation of empty vesicles that were similar in size and morphology to primary envelopes in the perinuclear space. These results suggested that NEC binding via U31 R281 and D282 to nucleocapsids, and probably to U25 in the nucleocapsids, has an important role in HSV-1 replication by promoting the incorporation of nucleocapsids into vesicles during primary envelopment. Binding of HSV-1 NEC to nucleocapsids has been thought to promote nucleocapsid budding at the inner nuclear membrane and subsequent incorporation of nucleocapsids into vesicles during nuclear egress of nucleocapsids. However, data to directly support this hypothesis have not been reported thus far. In this study, we have present data showing that two amino acids in the membrane-distal face of the HSV-1 NEC, which contains the putative capsid binding site based on the solved NEC structure, were in fact required for efficient NEC binding to nucleocapsids and for efficient incorporation of nucleocapsids into vesicles during primary envelopment. This is the first report showing direct linkage between NEC binding to nucleocapsids and an increase in nucleocapsid incorporation into vesicles during herpesvirus primary envelopment.
Topics: Active Transport, Cell Nucleus; Binding Sites; Capsid Proteins; Cell Nucleus; Herpes Simplex; Herpesvirus 1, Human; Humans; Nuclear Proteins; Nucleocapsid; Protein Binding; Viral Proteins; Virion; Virus Assembly; Virus Release
PubMed: 31391274
DOI: 10.1128/JVI.01290-19 -
Small (Weinheim An Der Bergstrasse,... Dec 2019The migration of cells through constricting spaces or along fibrous tracks in tissues is important for many biological processes and depends on the mechanical properties...
The migration of cells through constricting spaces or along fibrous tracks in tissues is important for many biological processes and depends on the mechanical properties of a cytoskeleton made up of three different filaments: F-actin, microtubules, and intermediate filaments. The signaling pathways and cytoskeletal structures that control cell motility on 2D are often very different from those that control motility in 3D. Previous studies have shown that intermediate filaments can promote actin-driven protrusions at the cell edge, but have little effect on overall motility of cells on flat surfaces. They are however important for cells to maintain resistance to repeated compressive stresses that are expected to occur in vivo. Using mouse embryonic fibroblasts derived from wild-type and vimentin-null mice, it is found that loss of vimentin increases motility in 3D microchannels even though on flat surfaces it has the opposite effect. Atomic force microscopy and traction force microscopy experiments reveal that vimentin enhances perinuclear cell stiffness while maintaining the same level of acto-myosin contractility in cells. A minimal model in which a perinuclear vimentin cage constricts along with the nucleus during motility through confining spaces, providing mechanical resistance against large strains that could damage the structural integrity of cells, is proposed.
Topics: Animals; Biomechanical Phenomena; Capillaries; Cell Movement; Collagen; Cytoskeleton; Hydrogels; Mice; Myosin Type II; NIH 3T3 Cells; Vimentin
PubMed: 31721440
DOI: 10.1002/smll.201903180 -
European Review For Medical and... Feb 2020The aim of this study was to explore the influence of hydrogen sulfide (H2S) on cardiomyocyte apoptosis in rats with myocardial ischemia-reperfusion injury via the c-Jun...
OBJECTIVE
The aim of this study was to explore the influence of hydrogen sulfide (H2S) on cardiomyocyte apoptosis in rats with myocardial ischemia-reperfusion injury via the c-Jun N-terminal kinase (JNK) pathway.
MATERIALS AND METHODS
A total of 60 normal female Sprague-Dawley (SD) rats aged 38 weeks were divided into 3 groups, including the sham operation group (n=20), ischemia group (n=20) and ischemia + sodium hydrosulfide (NaHS) group (n=20). Subsequently, differences in cardiac function, the morphology of myocardial tissues, protein expression of JNK2, the content of plasma H2S and malondialdehyde (MDA), the activity of superoxide dismutase (SOD), cystathionine-γ-lyase (CSE) and glutathione peroxidase (GSH-Px) were analyzed among rats in all groups.
RESULTS
Left ventricular diastolic pressure (LVDP) and maximum rate of pressure rise/fall (± dP/dtmax) were the highest in of rats of the sham operation group and the lowest in the ischemia group. Meanwhile, they were significantly elevated in the ischemia + NaHS group compared with those in the ischemia group (p<0.01). Left ventricular end-diastolic pressure (LVEDP) was the lowest in rats of the sham operation group and the highest in the ischemia group. Similarly, it decreased markedly in the ischemia + NaHS group compared with the ischemia group (p<0.01). Compared with the sham operation group, the perinuclear space in the myocardium was gradually larger, the arrangement of fibers became significantly more disordered, and the damage of mitochondrial cristae and membrane was remarkably more severe in rats in the ischemia group. Compared with the ischemia group, the above-mentioned conditions of rat cardiomyocytes were markedly improved (p<0.01). Meanwhile, the content of H2S and activity of CSE in the cardiomyocytes were altered in rats of the ischemia + NaHS group. Western blotting results indicated that, compared with the sham operation group, both the ischemia group and ischemia + NaHS group showed significantly up-regulated protein expression level of phosphorylated JNK2, with the highest level in the ischemia group. The content of MDA in rat myocardial tissues was markedly higher in the ischemia group than that of the ischemia + NaHS group, with the lowest level in the sham operation group (p<0.01). Additionally, the activity of SOD and GSH-Px in rat myocardial tissues was remarkably worse in the ischemia group than that of the ischemia + NaHS group, and it was the strongest in the sham operation group (p<0.01).
CONCLUSIONS
H2S inhibits the activity of the JNK pathway, decreases its phosphorylation level and down-regulates the protein expression level of JNK2, thereby protecting against myocardial ischemia-reperfusion injury.
Topics: Animals; Female; Hydrogen Sulfide; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Myocardial Reperfusion Injury; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley
PubMed: 32141574
DOI: 10.26355/eurrev_202002_20383 -
Acta Histochemica Et Cytochemica Jun 2022Epithelial protein lost in neoplasm (EPLIN) is an actin-associated cytoskeletal protein that plays an important role in epithelial cell adhesion. EPLIN has two isoforms:...
Epithelial protein lost in neoplasm (EPLIN) is an actin-associated cytoskeletal protein that plays an important role in epithelial cell adhesion. EPLIN has two isoforms: EPLINα and EPLINβ. In this study, we investigated the role of EPLINβ in osteoblasts using EPLINβ-deficient ( ) mice. The skeletal phenotype of mice is indistinguishable from the wildtype (WT), but bone properties and strength were significantly decreased compared with WT littermates. Histomorphological analysis revealed altered organization of bone spicules and osteoblast cell arrangement, and decreased alkaline phosphatase activity in mouse bones. Transmission electron microscopy revealed wider intercellular spaces between osteoblasts in mice, suggesting aberrant cell adhesion. In osteoblasts, α- and β-catenins and F-actin were observed at the cell membrane, but OB-cadherin was localized at the perinuclear region, indicating that cadherin-catenin complexes were not formed. EPLINβ knockdown in MC3T3-e1 osteoblast cells showed similar results as in calvaria cell cultures. Bone formation markers, such as , , , and mRNA were reduced in EPLINβ knockdown cells, suggesting an important role for EPLINβ in osteoblast formation. In conclusion, we propose that EPLINβ is involved in the assembly of cadherin-catenin complexes in osteoblasts and affects bone formation.
PubMed: 35821749
DOI: 10.1267/ahc.22-00027