Perinuclear Protein Research Articles

The pro-oncogenic noncanonical activity of a RAS•GTP:RanGAP1 complex facilitates nuclear protein export.

Canonical RAS signaling, including PI3K/AKT- and RAF/MEK-dependent activities, results mainly from RAS•GTP interaction with its effectors at the plasma membrane. Here, we identified a fundamental, oncogenic, noncanonical RAS•GTP activity that increases XPO1-dependent export of nuclear protein cargo into the cytoplasm and is independent of PI3K/AKT and RAF/MEK signaling. This RAS-dependent step acts downstream from XPO1 binding to nuclear protein cargo and is mediated by a perinuclear protein complex between RAS•GTP and RanGAP1 that facilitates hydrolysis of Ran•GTP to Ran•GDP, which promotes release of nuclear protein cargo into the cytoplasm. The export of nuclear EZH2, which promotes cytoplasmic degradation of the DLC1 tumor suppressor protein, is a biologically important component of this pro-oncogenic activity. Conversely, preventing nuclear protein export contributes to the antitumor activity of KRAS inhibition, which can be further augmented by reactivating the tumor suppressor activity of DLC1 or potentially combining RAS inhibitors with other cancer treatments.

Reversal of injury-associated retinal ganglion cell gene expression by a phosphodiesterase anchoring disruptor peptide

Loss of retinal ganglion cells (RGCs) is central to the pathogenesis of optic neuropathies such as glaucoma. Increased RGC cAMP signaling is neuroprotective. We have shown that displacement of the cAMP-specific phosphodiesterase PDE4D3 from an RGC perinuclear compartment by expression of the modified PDE4D3 N-terminal peptide 4D3(E) increases perinuclear cAMP and protein kinase A activity in cultured neurons and in vivo RGC survival after optic nerve crush (ONC) injury. To explore mechanisms by which PDE4D3 displacement promotes neuroprotection, in this study mice intravitreally injected with an adeno-associated virus to express an mCherry-tagged 4D3(E) peptide were subjected to ONC injury and analyzed by single cell RNA-sequencing (scRNA-seq). 4D3(E)-mCherry expression was associated with an attenuation of injury-induced changes in gene expression, thereby supporting the hypothesis that enhanced perinuclear PKA signaling promotes neuroprotective RGC gene expression.

Covalent Interactions of Anthocyanins with Proteins: Activity-Based Protein Profiling of Cyanidin-3-O-glucoside.

Anthocyanins are common natural pigments with a variety of physiological activities. Traditional perspectives attribute their molecular mechanism to noncovalent interactions influencing signaling pathways. However, this ignores the nature of its benzopyrylium skeleton, which readily reacts with the electron-rich groups of proteins. Here, we modified cyanidin-3-O-glucoside (C3G) via activity-based protein profiling technology by our previous synthesis route and prepared the covalent binding probe (C3G-Probe) and the noncovalent photoaffinity probe (C3G-Diazirine). The properties of C3G's covalent binding to proteins were also discovered by comparing the labeling of the two probes to the whole HepG2 cell proteome. We further explored its target proteins and enriched pathways in HepG2 and HeLa cells. Western blot analysis further confirmed the covalent binding of C3G to four target proteins: insulin-degrading enzyme, metal cation symporter ZIP14, spermatid perinuclear RNA-binding protein, and Cystatin-B. Pathway analysis showed that covalent targets of C3G were concentrated in metabolic pathways and several ribonucleoprotein complexes that were also coenriched. The results of this study provide new insights into the interaction of the naturally active molecule C3G with proteins.

Effect of drugs regulating acute immune responses on the liver in septic processes

Objective. To study the effect of drugs regulating acute immune responses on liver in septic processes. Materials and methods. The study was conducted on 39 male white rats weighing 250-400 g. The animals were initiated septic process development by intraperitoneal injection of Klebsiella pneumoniae culture suspension with simultaneous intravenous injection of polyvinylpyrrolidone (PVP) and gadolinium in the volume of 0.6 ml. On the 14th day the animals were removed from the experiment by decapitation under light ether anesthesia, blood and liver were taken for biochemical and histological studies. Results. When analyzing the morphometric parameters of liver preparations, an increase in the number of nuclei and a slight increase in the area of nuclei in three experimental groups was established by 1,2 times (p < 0,05) compared to the control group, which reflects the ongoing processes of hepatocyte regeneration due to possible mechanisms, namely hypertrophy nuclei and proliferation with both PVP and gadolinium in systemic inflammatory response and sepsis. Moreover, the use of PVP and gadolinium led to a decrease in the likelihood of perinuclear edema, protein degeneration and large droplet vacuolization (p < 0,05). When using gadolinium, the lumen diameter of the sinusoids was the largest and amounted to 4,47; 3,22-5,63 µm (p<0,05), and in septic shock it did not differ from the group where PVP was used (p > 0,05). Of the laboratory parameters of surviving individuals, the lowest ALaT level was noted in the gadolinium experimental group – 53,7; 51,8-55,1 U/L (p<0,05), while the lowest urea level was observed when using PVP (5,0; 4,99-5,15 mmol/l) (p < 0,05) as part of a systemic inflammatory response. Conclusion. Gadolinium and PVP have a positive effect on the detoxification function of liver. Moreover, the effect of PVP on the morphology and function of the liver differs at the stages of septic process.

In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration.

Oxidative stress represents a hallmark for many degenerative pathologies of the Central Nervous System. Throughout life, the constant pressure of noxious stimuli and/or episodes of traumatic events may expose the brain to a microenvironment where the non-balanced reactive oxygen species inevitably lead to neuronal loss and cognitive decline. HO-1, a 32 kDa heat-shock protein catalyzing the degradation of heme into carbon monoxide (CO), iron and biliverdin/bilirubin is considered one of the main antioxidant defense mechanisms playing pivotal roles in neuroprotection. Restoring the redox homeostasis is the goal of many natural or synthetic antioxidant molecules pursuing beneficial effects on brain functions. Here, we investigated the antioxidant capacity of four selected benzofuran-2-one derivatives in a cellular model of neurodegeneration represented by differentiated SH-SY5Y cells exposed to catechol-induced oxidative stress. Our main results highlight how all the molecules have antioxidant properties, especially compound 9, showing great abilities in reducing intracellular ROS levels and protecting differentiated SH-SY5Y cells from catechol-induced death. This compound above all seems to boost HO-1 mRNA and perinuclear HO-1 protein isoform expression when cells are exposed to the oxidative insult. Our findings open the way to consider benzofuran-2-ones as a novel and promising adjuvant antioxidant strategy for many neurodegenerative disorders.

Deletion of DWORF does not affect cardiac function in aging and in PLN-R14del cardiomyopathy.

The phospholamban (PLN) pathogenic gene variant p.Arg14del causes cardiomyopathy, which is characterized by perinuclear PLN protein clustering and can lead to severe heart failure (HF). Elevated expression of dwarf open reading frame (DWORF), a protein counteracting the function of PLN in the sarcoplasmic reticulum (SR), can delay disease progression in a PLN-R14del mouse model. Here, we evaluated whether deletion of DWORF (DWORF-/-) would have an opposite effect and accelerate age-dependent disease progression in wild-type (WT) mice and mice with a pathogenic PLN-R14del allele (R14Δ/+). We show that DWORF-/- mice maintained a normal left ventricular ejection fraction (LVEF) during aging and no difference with WT control mice could be observed up to 20 mo of age. R14Δ/+ mice maintained a normal cardiac function until 12 mo of age, but at 18 mo of age, LVEF was significantly reduced as compared with WT mice. Absence of DWORF did neither accelerate the R14Δ/+-induced reduction in LVEF nor enhance the increases in gene expression of markers related to cardiac remodeling and fibrosis and did not exacerbate cardiac fibrosis caused by the R14Δ/+ mutation. Together, these results demonstrate that absence of DWORF does not accelerate or exacerbate PLN-R14del cardiomyopathy in mice harboring the pathogenic R14del allele. In addition, our data indicate that DWORF appears to be dispensable for cardiac function during aging.NEW & NOTEWORTHY Although DWORF overexpression significantly delayed heart failure development and strongly prolonged life span in PLN-R14del mice, the current study shows that deletion of DWORF does not accelerate or exacerbate PLN-R14del cardiomyopathy in mice harboring the pathogenic R14del allele. In addition, DWORF appears to be dispensable for cardiac function during aging. Changes in DWORF gene expression are therefore unlikely to contribute to the clinical heterogeneity observed in patients with PLN-R14del cardiomyopathy.

The role of vitamin D through SphK1/S1P in the regulation of MS progression

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-β, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-β, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

A novel gene therapy for familial dilated cardiomyopathy based upon disruption of PP2A -- mAKAP-beta signalosomes

Familial Dilated Cardiomyopathy (DCM) is a common inherited cause of heart failure characterized by progressive left ventricular dilatation and systolic dysfunction. Despite the discovery of novel genetic mutations causing DCM, the downstream mechanisms resulting in heart failure are still unclear, and there remains a great unmet clinical need. Contributing to the phenotype of ventricular dilatation in DCM is the lengthening of cardiac myocytes, which exhibit increased overall length to width ratio. We have shown that increasing the phosphorylation of the transcription factor serine response factor (SRF) at serine residue 103 by displacement of protein phosphatase 2A (PP2A) from the perinuclear scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) can inhibit myocyte lengthening and improve cardiac function following myocardial infarction. Notably, SRF S103 phosphorylation is decreased in both human heart failure and in the hearts of transgenic mice expressing the human DCM α-tropomyosin Glu54Lys (Tm-E54K) mutant allele. We now show that, accordingly, targeting of mAKAPβ-PP2A signalosomes constitutes a new therapeutic strategy for DCM. A self-complementary serotype 9 adeno-associated virus (AAV9sc) conferring cardiac-specific expression of a mAKAPβ fragment comprising the PP2A binding domain (PBD) was used to target the signalosomes in mice. AAV9sc.PBD administration into neonatal Tm-E54K mice provided long term preservation of cardiac function and prevented ventricular dilatation. At 16 weeks of age, ejection fraction was significantly improved 14%[MK1], and wall thickness was increased for AAV9sc.PBD TM-E54K mice when compared with control AAV9sc.GFP treated TM-E54K mice. Morphometry of acutely dissociated adult mouse myocytes showed that AAV9sc.PBD treatment restored myocyte length to width ratio to that of non-transgenic animals, while western blots showed increased SRF S103 phosphorylation. The results in mice have been corroborated by PBD expression in engineered heart tissues (EHT) generated from an induced pluripotent stem cell (iPSC) line for a DCM phospholamban R14del patient. PBD expression increased SRF S103 phosphorylation and improved tissue contractility. Taken together, this study suggests that increasing SRF S103 phosphorylation by disruption of mAKAPβ-PP2A signalosomes can constitute a novel therapeutic strategy for DCM. Funded by Department of NIH Grant R01HL146111 (Dr. Kapiloff), R01 HL139679 and Leducq Foundation CurePlan (Dr. Karakikes), and the NHLBI Gene Therapy Resource Program. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The perinuclear theca protein Calicin helps shape the sperm head and maintain the nuclear structure in mice.

The perinuclear theca (PT) is a cytoskeletal element encapsulating the sperm nucleus; however, our understanding of the physiological roles of PT in sperm is very limited. We show that Calicin interacts with itself and many other PT components, indicating it may serve as an organizing center of the PT assembly. Calicin is detectable first when surrounding the acrosome, then detected around the entire nucleus, and finally translocated to the postacrosomal region of spermatid heads. Intriguingly, loss of Calicin specifically causes surface subsidence of sperm heads in the nuclear condensation stage. Calicin interacts with inner acrosomal membrane (IAM) protein Spaca1 and nuclear envelope (NE) components to form an "IAM-PT-NE" structure. Intriguingly, Ccin-knockout sperm also exhibit DNA damage and failure of fertilization. Our study provides solid animal evidence to suggest that the PT encapsulating sperm nucleus helps shape the sperm head and maintain the nuclear structure.

Distribution of cardiomyocyte-selective adeno-associated virus serotype 9 vectors in swine following intracoronary and intravenous infusion.

Limited reports exist regarding adeno-associated virus (AAV) biodistribution in swine. This study assessed biodistribution following antegrade intracoronary and intravenous delivery of two self-complementary serotype 9 AAV (AAV9sc) biologics designed to target signaling in the cardiomyocyte considered important for the development of heart failure. Under the control of a cardiomyocyte-specific promoter, AAV9sc.shmAKAP and AAV9sc.RBD express a small hairpin RNA for the perinuclear scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) and an anchoring disruptor peptide for p90 ribosomal S6 kinase type 3 (RSK3), respectively. Quantitative PCR was used to assess viral genome (vg) delivery and transcript expression in Ossabaw and Yorkshire swine tissues. Myocardial viral delivery was 2-5 × 105 vg/µg genomic DNA (gDNA) for both infusion techniques at a dose ∼1013 vg/kg body wt, demonstrating delivery of ∼1-3 viral particles per cardiac diploid genome. Myocardial RNA levels for each expressed transgene were generally proportional to dose and genomic delivery, and comparable with levels for moderately expressed endogenous genes. Despite significant AAV9sc delivery to other tissues, including the liver, neither biologic induced toxic effects as assessed using functional, structural, and circulating cardiac and systemic markers. These results indicate successful targeted delivery of cardiomyocyte-selective viral vectors in swine without negative side effects, an important step in establishing efficacy in a preclinical experimental setting.

014 C1ORF162 is specifically expressed by pathogenic Th2 cells in atopic dermatitis

Pathogenic Th2 cells are a recently described subpopulation of allergen-specific Th2 cells that are crucial mediators of type 2 immune diseases such as asthma and allergic dermatitis. Understanding the unique biology of pathogenic Th2 cells holds the promise of identifying specific and novel therapeutic targets. The gene chromosome 1 open reading frame 162 (C1ORF162) has been repeatedly identified to be specifically expressed by pathogenic Th2 cells and its expression correlates with disease severity in atopic dermatitis. Yet, its function is completely unknown. Here, we characterized the cellular expression of C1ORF162 at the mRNA and protein level in primary human T cells. RNA sequencing and qPCR analysis confirmed that both in-vivo- and in-vitro- primed Th2 express higher levels of C1ORF162 than Th1 and Th17 cells. In addition, C1ORF162 expression in Th2 cells correlated with the expression of cytokines that are specifically expressed by pathogenic Th2 cells, such as IL-9 and IL-5. These findings were confirmed at the protein level by Western Blot and FACS analysis. Initial protein localization experiments and functional analyses suggest that C1ORF162 is a perinuclear protein that is rapidly downregulated by IL-2 signaling. Finally, C1ORF162 was found to be expressed by T cells infiltrating lesional skin of atopic dermatitis. In conclusion, C1ORF162 is consistently expressed in pathogenic Th2 cells and associated with disease severity in atopic dermatitis. Future experiments aim to further define the identity and function of C1ORF162 in human Th2 cells. Based on the close association of C1ORF162 with pathogenic Th2 cells, these studies may have therapeutic implications for type 2-driven disease in the skin and beyond.

Sir4 Deficiency Reverses Cell Senescence by Sub-Telomere Recombination.

Telomere shortening results in cellular senescence and the regulatory mechanisms remain unclear. Here, we report that the sub-telomere regions facilitate telomere lengthening by homologous recombination, thereby attenuating senescence in yeast Saccharomyces cerevisiae. The telomere protein complex Sir3/4 represses, whereas Rif1 promotes, the sub-telomere Y′ element recombination. Genetic disruption of SIR4 increases Y′ element abundance and rescues telomere-shortening-induced senescence in a Rad51-dependent manner, indicating a sub-telomere regulatory switch in regulating organismal senescence by DNA recombination. Inhibition of the sub-telomere recombination requires Sir4 binding to perinuclear protein Mps3 for telomere perinuclear localization and transcriptional repression of the telomeric repeat-containing RNA TERRA. Furthermore, Sir4 repression of Y′ element recombination is negatively regulated by Rif1 that mediates senescence-evasion induced by Sir4 deficiency. Thus, our results demonstrate a dual opposing control mechanism of sub-telomeric Y′ element recombination by Sir3/4 and Rif1 in the regulation of telomere shortening and cell senescence.

The Autophagy-Initiating Protein Kinase ULK1 Phosphorylates Human Cytomegalovirus Tegument Protein pp28 and Regulates Efficient Virus Release.

Autophagy is a catabolic process contributing to intrinsic cellular defense by degrading viral particles or proteins; however, several viruses hijack this pathway for their own benefit. The role of autophagy during human cytomegalovirus (HCMV) replication has not been definitely clarified yet. Utilizing small interfering RNA (siRNA)-based screening, we observed that depletion of many autophagy-related proteins resulted in reduced virus release, suggesting a requirement of autophagy-related factors for efficient HCMV replication. Additionally, we could show that the autophagy-initiating serine/threonine protein kinase ULK1 as well as other constituents of the ULK1 complex were upregulated at early times of infection and stayed upregulated throughout the replication cycle. We demonstrate that indirect interference with ULK1 through inhibition of the upstream regulator AMP-activated protein kinase (AMPK) impaired virus release. Furthermore, this result was verified by direct abrogation of ULK1 kinase activity utilizing the ULK1-specific kinase inhibitors SBI-0206965 and ULK-101. Analysis of viral protein expression in the presence of ULK-101 revealed a connection between the cellular kinase ULK1 and the viral tegument protein pp28 (pUL99), and we identified pp28 as a novel viral substrate of ULK1 by in vitro kinase assays. In the absence of ULK1 kinase activity, large pp28- and pp65-positive structures could be detected in the cytoplasm at late time points of infection. Transmission electron microscopy demonstrated that these structures represent large perinuclear protein accumulations presumably representing aggresomes. Our results indicate that HCMV manipulates ULK1 and further components of the autophagic machinery to ensure the efficient release of viral particles.IMPORTANCE The catabolic program of autophagy represents a powerful immune defense against viruses that is, however, counteracted by antagonizing viral factors. Understanding the exact interplay between autophagy and HCMV infection is of major importance since autophagy-related proteins emerged as promising targets for pharmacologic intervention. Our study provides evidence for a proviral role of several autophagy-related proteins suggesting that HCMV has developed strategies to usurp components of the autophagic machinery for its own benefit. In particular, we observed strong upregulation of the autophagy-initiating protein kinase ULK1 and further components of the ULK1 complex during HCMV replication. In addition, both siRNA-mediated depletion of ULK1 and interference with ULK1 protein kinase activity by two chemically different inhibitors resulted in impaired viral particle release. Thus, we propose that ULK1 kinase activity is required for efficient HCMV replication and thus represents a promising novel target for future antiviral drug development.

Predicted gene 31453 (Gm31453) and the gene encoding carboxypeptidase A5 (Cpa5) are not essential for spermatogenesis and male fertility in the mouse.

Numerous long non-coding (lnc) RNAs are highly enriched or exclusively expressed in the mammalian testis, even in spermatids. Spermatid perinuclear RNA-binding protein (STRBP) can bind many RNAs, and loss of STRBP impairs male fertility. However, the functions of lncRNAs interacting with STRBP are unknown. In this study, the roles of one STRBP-interacting lncRNA, namely predicted gene 31453 (Gm31453), and its potential target gene encoding carboxypeptidase A5 (Cpa5) in spermatogenesis were determined using gene-knockout (KO) mice. Gm31453 and Cpa5 are located adjacent to each other on the same chromosome and are highly expressed in the testis. Gm31453 and Cpa5 are primarily expressed from secondary spermatocytes to elongated spermatids, implying their involvement in spermiogenesis. Although deletion of Gm31453 disturbed the expression of both its target and interacting gene, as indicated by decreased Cpa5 and increased Strbp mRNA levels, both Gm31453- and Cpa5-KO mice showed normal spermatogenesis and fertility, and had no detectable abnormalities in terms of testicular and epididymal development, sperm production morphology or motility, pregnancy rate or litter size. Thus, Gm31453 and Cpa5 are dispensable for spermatogenesis and male fertility in mice. Their involvement in spermatogenesis may be a fine-tuning role, regulating gene expression at the molecular level.

Folliculin variants linked to Birt-Hogg-Dubé syndrome are targeted for proteasomal degradation.

Germline mutations in the folliculin (FLCN) tumor suppressor gene are linked to Birt-Hogg-Dubé (BHD) syndrome, a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. Most BHD-linked FLCN variants include large deletions and splice site aberrations predicted to cause loss of function. The mechanisms by which missense variants and short in-frame deletions in FLCN trigger disease are unknown. Here, we present an integrated computational and experimental study that reveals that the majority of such disease-causing FLCN variants cause loss of function due to proteasomal degradation of the encoded FLCN protein, rather than directly ablating FLCN function. Accordingly, several different single-site FLCN variants are present at strongly reduced levels in cells. In line with our finding that FLCN variants are protein quality control targets, several are also highly insoluble and fail to associate with the FLCN-binding partners FNIP1 and FNIP2. The lack of FLCN binding leads to rapid proteasomal degradation of FNIP1 and FNIP2. Half of the tested FLCN variants are mislocalized in cells, and one variant (ΔE510) forms perinuclear protein aggregates. A yeast-based stability screen revealed that the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones regulate the turnover of the FLCN variants. Lowering the temperature led to a stabilization of two FLCN missense proteins, and for one (R362C), function was re-established at low temperature. In conclusion, we propose that most BHD-linked FLCN missense variants and small in-frame deletions operate by causing misfolding and degradation of the FLCN protein, and that stabilization and resulting restoration of function may hold therapeutic potential of certain disease-linked variants. Our computational saturation scan encompassing both missense variants and single site deletions in FLCN may allow classification of rare FLCN variants of uncertain clinical significance.

Granule Cell Ensembles in Mouse Dentate Gyrus Rapidly Upregulate the Plasticity-Related Protein Synaptopodin after Exploration Behavior.

The plasticity-related protein Synaptopodin (SP) has been implicated in neuronal plasticity. SP is targeted to dendritic spines and the axon initial segment, where it organizes the endoplasmic reticulum (ER) into the spine apparatus and the cisternal organelle, respectively. Here, we report an inducible third localization of SP in the somata of activated granule cell ensembles in mouse dentate gyrus. Using immunofluorescence and fluorescence in situ hybridization, we observed a subpopulation of mature granule cells (~1–2%) exhibiting perinuclear SP protein and a strong somatic SP mRNA signal. Double immunofluorescence labeling for Arc demonstrated that ~ 75% of these somatic SP-positive cells are also Arc-positive. Placement of mice into a novel environment caused a rapid (~2–4 h) induction of Arc, SP mRNA, and SP protein in exploration-induced granule cell ensembles. Lesion experiments showed that this induction requires input from the entorhinal cortex. Somatic SP colocalized with α-Actinin2, a known binding partner of SP. Finally, ultrastructural analysis revealed SP immunoprecipitate on dense plates linking cytoplasmic and perinuclear ER cisterns; these structures were absent in granule cells of SP-deficient mice. Our data implicate SP in the formation of contextual representations in the dentate gyrus and the behaviorally induced reorganization of cytoplasmic and perinuclear ER.

CRISPR-PIN: Modifying gene position in the nucleus via dCas9-mediated tethering

Spatial organization of DNA within the nucleus is important for controlling DNA replication and repair, genetic recombination, and gene expression. Here, we present CRISPR-PIN, a CRISPR/dCas9-based tool that allows control of gene Position in the Nucleus for the yeast Saccharomyces cerevisiae. This approach utilizes a cohesin-dockerin interaction between dCas9 and a perinuclear protein. In doing so, we demonstrate that a single gRNA can enable programmable interaction of nuclear DNA with the nuclear periphery. We demonstrate the utility of this approach for two applications: the controlled segregation of an acentric plasmid and the re-localization of five endogenous loci. In both cases, we obtain results on par with prior reports using traditional, more cumbersome genetic systems. Thus, CRISPR-PIN offers the opportunity for future studies of chromosome biology and gene localization.

Charcot-Leyden crystal formation is closely associated with eosinophil extracellular trap cell death

AbstractProtein crystallization in human tissue rarely occurs. Charcot-Leyden crystals (CLCs) were described in various eosinophilic diseases >150 years ago, but our understanding of CLC formation still remains limited. In this study, we demonstrate that CLCs observed in varied inflamed human tissues are closely associated with eosinophil cell-free granules and nuclear envelope/plasma membrane disintegration with release of filamentous chromatin (extracellular traps), typical morphologies of a regulated pathway of extracellular trap cell death (ETosis). During the process of eosinophil ETosis, eccentrically localized cytoplasmic and perinuclear CLC protein (galectin-10) is homogeneously redistributed in the cytoplasm. Rapid (1-2 minutes) formation of intracytoplasmic CLCs was observed using time-lapse imaging. Plasma membrane rupture enabled the release of both intracellularly formed CLCs and soluble galectin-10 that further contributed to formation of CLCs extracellularly, in parallel with the expulsion of free intact granules and extracellular traps. CLC formation and galectin-10 release were dependent on nicotinamide adenine dinucleotide phosphate oxidase activation. To our knowledge, this is the first demonstration of natural formation of CLCs in association with an active physiological process (ie, ETosis). These results indicate that dynamic changes in intracellular localization and release of galectin-10 contribute to CLC formation in vivo and suggest that CLC/galectin-10 might serve as an indicator of ETosis.

Molecular characterization and expression analysis of strbp in Chinese tongue sole (Cynoglossus semilaevis)

Spermatid perinuclear RNA-binding protein (Strbp) is a kind of double-stranded (ds) RNA specific binding protein that plays important roles in mammalian spermatogenesis. In this study, we have isolated and characterized the strbp gene of Chinese tongue sole, Cynoglossus semilaevis, termed as CS-strbp. The CS-strbp genomic sequence contained fifteen exons and fourteen introns. Its cDNA was 2655 bp in length, encoding a 666-amino-acid protein with two conserved ds binding motifs. Using quantitative PCR, we found that CS-strbp mRNA exhibited sex-biased and tissue-specific distribution, predominantly expressed in the fertile male testis, though the expression levels varied throughout different developmental stages. Comparison of methylation profile in different sexual genotypes demonstrated the low methylation level of CS-strbp promoter in male and pseudo-male, which is consistent with the high expression levels in those genotypes. In situ hybridization revealed that CS-strbp mRNA mainly localized in male germ cells, especially in spermatids and spermatozoa. Given these findings, we postulate that CS-strbp might function in spermatogenesis of Chinese tongue sole.

Metabolic stress directs WNK kinases to aggresomes for pooling and degradation in the distal convoluted tubule of the kidney

ObjectiveRenal salt retention due to excessive function of the renal NaCl cotransporter (NCC) along the distal convoluted tubule (DCT) plays a central role in the pathogenesis of hypertension. The Western type diet with high salt and low potassium content (HS/LK) has been shown to increase NCC activity by stimulating its phosphorylation via a kinase cascade comprising with‐no‐lysine kinases (WNKs) and a Ste20‐related kinase (SPAK). WNKs and SPAK have been shown to accumulate in perinuclear aggregates in response to potassium depletion, but the functional significance of these aggregates remained obscure. These structures may in part reflect a degradative pathway but may serve pooling functions as well.MethodsMice received HS/LK or control diets for 10 days. WNK1‐transfected DCT cells were exposed to vehicle, hyperosmotic stress, or potassium depletion. WNK1, WNK4, SPAK, phospho‐S383 SPAK, and various autophagy markers were studied by immunofluorescence and immunoblotting. Electron microscopy was applied for ultrastructural analysis.ResultsHS/LK diet induced accumulation of WNKs and SPAK in the form of perinuclear protein aggregates within DCT cells. These aggregates were rare in DCT of mice treated with control diet and virtually absent after administration of high potassium diet. The autophagy marker ATG5 was present in the aggregates and the 20S proteasome signal was intensified adjacently, whereas standard lysosomal markers showed no significant association with the punctate WNK/SPAK signal. Ultrastructural analysis of DCT in kidneys of mice treated with HS/LK revealed abundant aggresomes adjacent to rough endoplasmic reticulum and clathrin‐coated vesicles. Evaluation of cultured DCT cells transfected with WNK1 or KS‐WNK1 revealed formation of similar aggresomes in response to potassium depletion, whereas hyperosmotic stress induced formation of autophagosomes. Pretreatment of cells with proteasome inhibitor MG‐132 or autophagy/lysosome inhibitor bafilomycin A1 prior to potassium depletion substantially augmented the size of WNK1‐containing aggresomes.ConclusionChallenging of DCT epithelium induces the formation of perinuclear aggresomes which pool the components of the kinase cascade controlling NCC activity. From there, WNKs and SPAK may either be degraded via proteasomes or lysosomes, or reintroduced into the signal chain.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.