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Phosphatidylserine (PS) is an important anionic phospholipid that is synthesized within the endoplasmic reticulum (ER). While PS shows the highest enrichment and serves important functional roles in the plasma membrane (PM) but its role in the nucleus is poorly explored. Using three orthogonal approaches, we found that PS is also uniquely enriched in the inner nuclear membrane (INM) and the nuclear reticulum (NR). Nuclear PS is critical for supporting the translocation of CCTα and Lipin1α, two key enzymes important for phosphatidylcholine (PC) biosynthesis, from the nuclear matrix to the INM and NR in response to oleic acid treatment. We identified the PS-interacting regions within the M-domain of CCTα and M-Lip domain of Lipin1α, and show that lipid droplet formation is altered by manipulations of nuclear PS availability. Our studies reveal an unrecognized regulatory role of nuclear PS levels in the regulation of key PC synthesizing enzymes within the nucleus. Synopsis: Phosphatidylserine (PS) is a phospholipid mainly enriched in the inner leaflet of the plasma membrane; however, its role in the nucleus remains unclear. This study describes the presence of PS in the inner nuclear membrane and nuclear reticulum, where it promotes nuclear membrane association of two key enzymes of phosphatidylcholine (PC) biosynthesis in response to oleic acid treatment. PS biosensors show its enrichment in the inner nuclear membrane (INM) and the nuclear reticulum (NR). PS in the inner nuclear membrane is synthesized at the endoplasmic reticulum. Nuclear PS interacts with two PC biosynthetic enzymes, CCTα and Lipin1α, promoting their translocation to the inner nuclear membrane. Nuclear PS biases PC biosynthesis toward membrane expansion rather than lipid droplet formation. Phosphatidylserine supports inner nuclear membrane enrichment of phosphatidylcholine biosynthetic enzymes, CCTα and Lipin1α, for nuclear membrane expansion. [ABSTRACT FROM AUTHOR]

Triglycerides constitute an inert storage form for fatty acids deposited in lipid droplets and are mobilized to provide metabolic energy or membrane building blocks. The biosynthesis of triglycerides is highly conserved within eukaryotes and normally involves the sequential esterification of activated fatty acids with a glycerol backbone. Some eukaryotes, however, can also use cellular membrane lipids as direct fatty acid donors for triglyceride synthesis. The biological significance of a pathway that generates triglycerides at the expense of organelle membranes has remained elusive. Here we review current knowledge on how cells use membrane lipids as fatty acid donors for triglyceride synthesis and discuss the hypothesis that a primary function of this pathway is to regulate membrane lipid remodeling and organelle function. [ABSTRACT FROM AUTHOR]

Eukaryotic cells organize their genome within the nucleus with a double-layered membrane structure termed the nuclear envelope (NE) as the physical barrier. The NE not only shields the nuclear genome but also spatially separates transcription from translation. Proteins of the NE including nucleoskeleton proteins, inner nuclear membrane proteins, and nuclear pore complexes have been implicated in interacting with underlying genome and chromatin regulators to establish a higher-order chromatin architecture. Here, I summarize recent advances in the knowledge of NE proteins that are involved in chromatin organization, gene regulation, and coordination of transcription and mRNA export. These studies support an emerging view of plant NE as a central hub that contributes to chromatin organization and gene expression in response to various cellular and environmental cues. [ABSTRACT FROM AUTHOR]

ABSTRACTEukaryotic cells organize their genome within the nucleus with a double-layered membrane structure termed the nuclear envelope (NE) as the physical barrier. The NE not only shields the nuclear genome but also spatially separates transcription from translation. Proteins of the NE including nucleoskeleton proteins, inner nuclear membrane proteins, and nuclear pore complexes have been implicated in interacting with underlying genome and chromatin regulators to establish a higher-order chromatin architecture. Here, I summarize recent advances in the knowledge of NE proteins that are involved in chromatin organization, gene regulation, and coordination of transcription and mRNA export. These studies support an emerging view of plant NE as a central hub that contributes to chromatin organization and gene expression in response to various cellular and environmental cues.

Nucleus is at the center stage of cellular drama orchestrated in the life of a cell and the nucleoplasm is surrounded by a double membranous compartment constituting the Nuclear membrane/envelope (NE) that separates it from the cytoplasm in nucleated cells. The initial understanding of the NE was that of a border security entity between the nucleus and the cytoplasm, separating gene regulation and transcription in the nucleus from translation in the cytoplasm. However, the discovery of a wide array of inherited diseases caused by mutations in genes encoding proteins that reside or interact with NE diverted the interest into deciphering the lipid-protein-rich environment of the NE. Today, the NE is considered a dynamic organelle which forms a functional linkage between the nucleus and the rest of the cell. The exposure of NE to constant mechanical constraints by its connectivity to the large polymer network of the lamina and chromatin on one side, and to the cytoskeleton on the other side results, in a variety of shape changes. We discuss two such deformation, the formation of nuclear blebs and nucleoplasmic reticulum (NER). Although the protein and the lipid composition of NE comprises a small fraction of the total lipid-protein load of the cell, the ability to define the lipid-protein composition of Inner nuclear membrane (INM) and Outer nuclear membrane (ONM) with precision is crucial for obtaining a deeper mechanistic understanding of their lipid-protein interaction and the various signaling pathways that are triggered by them. In addition, this allows us to further understand the direct and indirect roles of NE machinery in the chromosomal organization and gene regulation. [ABSTRACT FROM AUTHOR]

An emerging pathophysiology associated with the neurodegenerative Alzheimer's disease (AD) is the impairment of nucleocytoplasmic transport (NCT). The impairment can originate from damage to the nuclear pore complex (NPC) or other factors involved in NCT. The phenylalanine-glycine nucleoporins (FG-Nups) form a crucial component of the NPC, which is central to NCT. Recent discoveries have highlighted that the neuropathological protein tau is involved in direct interactions with the FG-Nups and impairment of the NCT process. Targeting such interactions may lead to the identification of novel interaction inhibitors and offer new therapeutic alternatives for the treatment of AD. This review highlights recent findings associated with impaired NCT in AD and the interaction between tau and the FG-Nups. [ABSTRACT FROM AUTHOR]

The electrokinetic potential of isolated cell nuclei obtained from wheat seedlings irradiated with nonionizing electromagnetic radiation of extremely high frequencies has been studied. Electrokinetic potential is an important factor in many biological processes and, perhaps, plays an important role in the formation of the response of biological organisms to electromagnetic waves. An increase in the absolute values of the electrokinetic potential of isolated cell nuclei of wheat seedlings from –7.7 to –131.6 mV in the electric field gradient under the influence of electromagnetic radiation of extremely high frequencies, in the frequency range of 46.0–50.3 GHz, was shown. Unidirectional changes in the electrokinetic potential under the influence of such radiation have been revealed; it was explained by an increase in the potential difference between the nuclear membrane and the nuclear matrix. It was also shown that there were sharp differences between the electrokinetic potential of isolated cell nuclei treated with electromagnetic radiation of extremely high frequencies under in vivo and in vitro conditions. It was found that the magnitude of changes in the electrokinetic potential of nuclei induced by exposure to electromagnetic radiation of extremely high frequencies under in vivo conditions far exceeded the magnitude of the electrokinetic potential of isolated nuclei treated with electromagnetic radiation of the same frequency under in vitro conditions; this confirmed the prolonged effect of electromagnetic radiation of extremely high frequencies. The authors discuss the modulation of the electrokinetic potential depending on the change in the difference between the content of anionic phospholipids in the nuclear membrane and the soluble nuclear fraction arising under the effect of electromagnetic radiation of extremely high frequencies, which leads to a change in the surface charge of the nuclear membrane, the electrokinetic potential of the nuclei, and the formation of a more compact double electric layer. [ABSTRACT FROM AUTHOR]

The architecture of eukaryotic cells is defined by extensive membrane-delimited compartments, which entails separate metabolic processes that would otherwise interfere with each other, leading to functional differences between cells. G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors, and their signal transduction is traditionally viewed as a chain of events initiated from the plasma membrane. Furthermore, their intracellular trafficking, internalization, and recycling were considered only to regulate receptor desensitization and cell surface expression. On the contrary, accumulating data strongly suggest that GPCRs also signal from intracellular compartments. GPCRs localize in the membranes of endosomes, nucleus, Golgi and endoplasmic reticulum apparatuses, mitochondria, and cell division compartments. Importantly, from these sites they have shown to orchestrate multiple signals that regulate different cell pathways. In this review, we summarize the current knowledge of this fascinating phenomenon, explaining how GPCRs reach the intracellular sites, are stimulated by the endogenous ligands, and their potential physiological/pathophysiological roles. Finally, we illustrate several mechanisms involved in the modulation of the compartmentalized GPCR signaling by drugs and endogenous ligands. Understanding how GPCR signaling compartmentalization is regulated will provide a unique opportunity to develop novel pharmaceutical approaches to target GPCRs and potentially lead the way towards new therapeutic approaches. [ABSTRACT FROM AUTHOR]

The eukaryotic cells can change the size of the cell nucleus, including the genomic DNA, depending on the environment surrounding the cell nucleus. How cells can scale the nuclear size by sensing the environment has remained an enigma for over a century in the field of cell biology. Here, we found a general scaling rule of nuclear size to cell size among eukaryotes and another new aspect for size scaling to the chromatin status inside the nucleus. In this review, I describe the known mechanisms underlying these size scaling characteristics and discuss putative physiological significances for the size scaling. [ABSTRACT FROM AUTHOR]

Objective: Fungi are invasive species responsible for infections in many people around the world and which severely affect the immune system. The opportunistic pathogenic species, such as Candida species and Aspergillus fumigatus, can cause death in people with weakened immune systems. Natural medicines derived from plants are often used to treat fungal diseases. In connection with our efforts to unearth possible cellular targets of antimicrobial agents, in this study, we aimed to determine the functional consequences of benzyl alcohol treatment on the nuclear membrane. Materials and Methods: We analysed the nuclear membrane distortions caused by benzyl alcohol in Saccharomyces cerevisiae cells using Nup49-GFP reporter strain. We also studied cellular distributions of various fluorescently tagged nuclearcytoplasmic shuttling proteins to determine any functional disturbances in nuclear pore complexes upon benzyl alcohol treatment. Localization of 51.5 kDa protein LexA-NES-GFP and 61.8 kDa protein Pho4(Δ157-164)-GFP to the nucleus in yeast cells was key for evaluating the effect upon diffusion limit of pores. Results: By analyzing the distribution of fluorescently tagged nuclear localization signal or nuclear export signals bearing reporter proteins between the nucleus and cytoplasm, we have shown that the nuclear membrane becomes leaky upon benzyl alcohol treatment. Conclusion: The diffusion limit across the nuclear membrane in yeast cells is increased upon benzyl alcohol treatment. We believe that these findings not only increase our understanding of the mode of action of benzyl alcohol bearing antifungal agents, but also help widening their use. [ABSTRACT FROM AUTHOR]

Craniotubular dysplasia , Ikegawa type (OMIM #619727) denotes the autosomal recessive skeletal disease identified in 2021 featuring blindness acquired in childhood. Five young members of four Indian families harbored a homozygous indel within TMEM53 (OMIM *619722), the gene that encodes transmembrane protein 53 (TMEM53). When intact, TMEM53 spans the nuclear envelope of osteoprogenitor cells, dampens BMP-SMAD signaling, and thereby slows bone formation. Consequently, defective TMEM53 accelerates osteogenesis. Herein, an American boy is compound heterozygous for a novel deletion and a novel missense mutation within TMEM53. His vision and sensorineural hearing became impaired. Radiographic survey revealed diploic thickening of his skull, broad calvarial and facial bones, skeletal modeling errors, vertebral body flattening, wide ribs, and osteopenia of expanded bones. DXA areal bone density (gm/cm2) Z -scores were low. His optic, auditory, and spinal canals were narrow. Mineral metabolism was intact. Serum alkaline phosphatase and osteocalcin levels were normal yet CTX was high. Iliac crest histomorphometry documented accelerated bone formation. His acute vision loss briefly improved following prednisone administration, optic canal decompression, and optic nerve sheath fenestration, but then progressed despite further surgeries and zoledronate treatment aimed to suppress bone turnover. Next generation sequencing of genes associated with elevated skeletal mass, including from high bone turnover, did not suggest an etiology. Whole genome sequencing then revealed within TMEM53 : i) a paternally transmitted 54-base deletion, which included the mRNA splice acceptor site for exon 2 as well as 31 bases of exonic sequence (c. 62-23_92del), and ii) a maternally transmitted missense variant (c.650C > T, p.Ser217Leu: NM_024587.4/NP_078863.2) which is extremely rare in gnomAD (frequency = 0.000036), replaces Ser217 highly conserved across species, and is scored as damaging by SIFT and Mutation Taster. We call this new osteopathy TMEM53 craniotubular dysplasia. • TMEM53 craniotubular dysplasia (OMIM # 619727) was identified in 2021. • In four Indian families, a homozygous TMEM53 indel enhanced BMP/SMAD signaling. • An affected American boy is compound heterozygous for novel mutations of TMEM53. • Iliac crest histomorphometry documented accelerated bone formation. • Blindness progressed despite foraminal surgeries and steroid/zoledronate therapy. [ABSTRACT FROM AUTHOR]

The nuclear envelope (NE) is a permeable barrier that maintains nuclear-cytoplasmic compartmentalization and ensures nuclear function; however, it ruptures in various situations such as mechanical stress and mitosis. Although the protein components for sealing a ruptured NE have been identified, the mechanism by which lipid components are involved in this process remains to be elucidated. Here, we found that an inner nuclear membrane (INM) protein Bqt4 directly interacts with phosphatidic acid (PA) and serves as a platform for NE maintenance in the fission yeast Schizosaccharomyces pombe. The intrinsically disordered region (IDR) of Bqt4, proximal to the transmembrane domain, binds to PA and forms a solid aggregate in vitro. Excessive accumulation of Bqt4 IDR in INM results in membrane overproliferation and lipid droplet formation in the nucleus, leading to centromere dissociation from the NE and chromosome missegregation. Our findings suggest that Bqt4 IDR controls nuclear membrane homeostasis by recruiting PA to the INM, thereby maintaining the structural integrity of the NE., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The cell nucleus is best known as the container of the genome. Its envelope provides a barrier for passive macromolecule diffusion, which enhances the control of gene expression. As its largest and stiffest organelle, the nucleus also defines the minimal space requirements of a cell. Internal or external pressures that deform a cell to its physical limits cause a corresponding nuclear deformation. Evidence is consolidating that the nucleus, in addition to its genetic functions, serves as a physical sensing device for critical cell body deformation. Nuclear mechanotransduction allows cells to adapt their acute behaviors, mechanical stability, paracrine signaling, and fate to their physical surroundings. This review summarizes the basic chemical and mechanical properties of nuclear components, and how these properties are thought to be utilized for mechanosensing. [ABSTRACT FROM AUTHOR]

Large-conductance cation channels (LCC-channels) were found in both (inner and outer) nuclear membranes of cerebellar Purkinje neurons. They are the most common type of intracellular spontaneously active ion channels among other identified. Their structure and physiological functions are still unknown, but the previous findings confirmed their sensitivity to a number of agonists/antagonists of nicotinic acetylcholine receptors. The purpose of the investigation was to estimate the effect of other regulators of the N-cholinoreceptors functioning – neostigmine, hexamethonium, and methyllycaconitine (MLA) on the LCC-channels in the nuclear membrane of cerebellar Purkinje neurons of rats. The effect of the agents was estimated based on changes in the following biophysical parameters: current amplitude, Po, channel flickering effect. Ion currents through single channels were registered using the patch-clamp technique in a nucleus-attached mode in voltage-clamp configuration. Among the studied substances, only MLA and hexamethonium influenced the LCC-channels functioning. Hexamethonium at a concentration of 2 mM reduced the Po of the LCC-channels by 46%. Under the influence of MLA, a slight effect of channel flickering was observed ("Poisson surprise" was 2.14 in the control and 3.81 under the influence of 200 μM of the substance respectively). No significant change of the biophysical characteristics of the LCC-channels under the influence of neostigmine was detected. Despite the low efficiency as LCC-channels blockers, the lack or only slight effect is a strong argument in favor of the substance usage in medicine due to their wide therapeutic potential. The severity of their effects is necessary for a comprehensive analysis of the effect patterns of the abovementioned substances on the molecular dynamics of the studied channels. The results will also be important for the identification or synthesis of new and more effective inhibitors of the LCC-channels. [ABSTRACT FROM AUTHOR]

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1‐CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1‐CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1‐CTDNEP1 downregulation also restores nuclear pore membrane fusion in post‐mitotic dTorsinKO fat body cells. However, dTorsin‐associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel‐like structures in place of mature nuclear pores. We conclude that the NEP1R1‐CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid‐dependent and lipid‐independent mechanisms, explaining some of the pleiotropic effects of Torsins. SYNOPSIS: The molecular processes that insert the nuclear pore complex (NPC) into the intact nuclear double membranes during interphase are poorly described. The nuclear envelope NEP1R1‐CTDNEP1 phosphatase complex connects Torsin ATPases to lipid metabolism and membrane remodeling for NPC insertion in Drosophila and mammalian cells. Drosophila Torsin (dTorsin) function in fat body cells depends on homo‐oligomerization rather than ATPase activity.dTorsin knock‐out (KO) fat body cells have abnormal lipid metabolism and nuclear pore membrane fusion.The lipid metabolism and nuclear pore defects in dTorsinKO cells are independent of each other, but both are prevented by downregulation of the NEP1R1‐CTDNEP1 complex.Additionally, NEP1R1/CTDNEP1/Lipin pathway hyperactivity decreases nuclear pore membrane curvature. [ABSTRACT FROM AUTHOR]

The architecture of eukaryotic cells is defined by extensive membrane-delimited compartments, which entails separate metabolic processes that would otherwise interfere with each other, leading to functional differences between cells. G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors, and their signal transduction is traditionally viewed as a chain of events initiated from the plasma membrane. Furthermore, their intracellular trafficking, internalization, and recycling were considered only to regulate receptor desensitization and cell surface expression. On the contrary, accumulating data strongly suggest that GPCRs also signal from intracellular compartments. GPCRs localize in the membranes of endosomes, nucleus, Golgi and endoplasmic reticulum apparatuses, mitochondria, and cell division compartments. Importantly, from these sites they have shown to orchestrate multiple signals that regulate different cell pathways. In this review, we summarize the current knowledge of this fascinating phenomenon, explaining how GPCRs reach the intracellular sites, are stimulated by the endogenous ligands, and their potential physiological/pathophysiological roles. Finally, we illustrate several mechanisms involved in the modulation of the compartmentalized GPCR signaling by drugs and endogenous ligands. Understanding how GPCR signaling compartmentalization is regulated will provide a unique opportunity to develop novel pharmaceutical approaches to target GPCRs and potentially lead the way towards new therapeutic approaches.

Electroporation has been widely used in biology, medicine, and the food industry as a means to transport various molecules through the cell membrane. The phenomenon of electroporation is the result of cell membrane damage caused by the application of an electric field. In order to understand more precisely how cells function, we established a dielectric model of a spherical cell and analyzed its characteristics by the finite element method. The effects of altering different electrical parameters were determined. The results showed that the electric field strength was positively related to the transmembrane voltage (TMV) and pore density. There was a minimum electric field strength necessary to induce a critical TMV for the formation of pores. Pulse width also had to be long enough to charge the cell membrane, compared with the normal membrane charging time constant of about 1 μs. When the pulse width was shorter than the charging time constant, it was necessary to increase pulse frequency to create a high enough TMV. The rise-time of the electric pulse also affected electroporation: a fast rise-time pulse not only allowed penetration of the plasma membrane but also the organelle membrane. With slow rise-time pulse, the organelle was shielded from electroporation. This study defines the response characteristics of electrical parameters on the electric load cell and establishes the specificity of parameters for different purposes. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), can be clinically heterogeneous which may be explained by the co-inheritance of multiple genetic variants that modify the clinical course. In this study we examine variants in three genes in a family with one individual presenting with ALS and lipodystrophy. Sequencing revealed a p.Gly602Ser variant in LMNA, and two additional variants, one each in SETX (g.intron10-13delCTT) and FUS (p.Gly167_Gly168del). These latter genes have been linked to ALS. All family members were genotyped and each variant, and each combination of variants detected, were functionally evaluated in vitro regarding effects on cell survival, expression patterns and cellular phenotype. Muscle biopsy retrieved from the individual with ALS showed leakage of chromatin from the nucleus, a phenotype that was recapitulated in vitro with expression of all three variants simultaneously. Individually expressed variants gave cellular phenotypes there were unremarkable. Interestingly the FUS variant appears to be protective against the effects of the SETX and the LMNA variants on cell viability and may indicate loss of interaction of FUS with SETX and/or R-loops. We conclude that these findings support genetic modifications as an explanation of the clinical heterogeneity observed in human disease. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are integral membrane proteins that transduce a wide array of inputs including light, ions, hormones, and neurotransmitters into intracellular signaling responses which underlie complex processes ranging from vision to learning and memory. Although traditionally thought to signal primarily from the cell surface, GPCRs are increasingly being recognized as capable of signaling from intracellular membrane compartments, including endosomes, the Golgi apparatus, and nuclear membranes. Remarkably, GPCR signaling from these membranes produces functional effects that are distinct from signaling from the plasma membrane, even though often the same G protein effectors and second messengers are activated. In this review, we will discuss the emerging idea of a "spatial bias" in signaling. We will present the evidence for GPCR signaling through G protein effectors from intracellular membranes, and the ways in which this signaling differs from canonical plasma membrane signaling with important implications for physiology and pharmacology. We also highlight the potential mechanisms underlying spatial bias of GPCR signaling, including how intracellular membranes and their associated lipids and proteins affect GPCR activity and signaling. [ABSTRACT FROM AUTHOR]

Regenerative therapeutic approaches involving the transplantation of stem cells differentiated into insulin‐producing cells are being studied in patients with rapidly progressing severe diabetes. Adipose‐derived mesenchymal stem cells have been reported to have varied cellular characteristics depending on the biological environment of the location from which they were harvested. However, the characteristics of mesenchymal stem cells in type II diabetes have not been clarified. In this study, we observed the organelles of mesenchymal stem cells from patients with type II diabetes under a transmission electron microscope to determine the structure of stem cells in type II diabetes. Transmission electron microscopic observation of mesenchymal stem cells from healthy volunteers (N‐ADSC) and those from patients with type II diabetes (T2DM‐ADSC) revealed enlarged nuclei and degenerated mitochondrial cristae in T2DM‐ADSCs. Moreover, T2DM‐ADSCs were shown to exhibit a lower expression of Emerin, a constituent protein of the nuclear membrane, and a decreased level of mitochondrial enzyme activity. In this study, we successfully demonstrated the altered structure of nuclear membrane and the decreased mitochondrial enzyme activity in adipose‐derived mesenchymal cells from patients with type II diabetes. These findings have contributed to the understanding of type II diabetes‐associated changes in mesenchymal stem cells used for regenerative therapy. [ABSTRACT FROM AUTHOR]

Activity of the endo-beta-glucuronidase heparanase, capable of cleaving heparan sulfate (HS), is most often elevated in many types of tumors, associating with increased tumor metastasis and decreased patients’ survival. Heparanase is therefore considered to be a valid drug target, and heparanase inhibitors are being evaluated clinically in cancer patients. Heparanase 2 (Hpa2) is a close homolog of heparanase that gained very little attention, likely because it lacks HS-degrading activity typical of heparanase. The role of Hpa2 in cancer was not examined in detail. In head and neck cancer, high levels of Hpa2 are associated with decreased tumor cell dissemination to regional lymph nodes and prolonged patients’ survival, suggesting that Hpa2 functions to attenuate tumor growth. Here, we examined the role of Hpa2 in normal thyroid tissue and in benign thyroid tumor, non-metastatic, and metastatic papillary thyroid carcinoma (PTC) utilizing immunostaining in correlation with clinicopathological parameters. Interestingly, we found that Hpa2 staining intensity does not significantly change in the transition from normal thyroid gland to benign, non-metastatic, or metastatic thyroid carcinoma. Remarkably, we observed that in some biopsies, Hpa2 is accumulating on the membrane (envelop) of the nucleus and termed this cellular localization NM (nuclear membrane). Notably, NM localization of Hpa2 occurred primarily in metastatic PTC and was associated with an increased number of positive (metastatic) lymph nodes collected at surgery. These results describe for the first time unrecognized localization of Hpa2 to the nuclear membrane, implying that in PTC, Hpa2 functions to promote tumor metastasis.

Activity of the endo-beta-glucuronidase heparanase, capable of cleaving heparan sulfate (HS), is most often elevated in many types of tumors, associating with increased tumor metastasis and decreased patients' survival. Heparanase is therefore considered to be a valid drug target, and heparanase inhibitors are being evaluated clinically in cancer patients. Heparanase 2 (Hpa2) is a close homolog of heparanase that gained very little attention, likely because it lacks HS-degrading activity typical of heparanase. The role of Hpa2 in cancer was not examined in detail. In head and neck cancer, high levels of Hpa2 are associated with decreased tumor cell dissemination to regional lymph nodes and prolonged patients' survival, suggesting that Hpa2 functions to attenuate tumor growth. Here, we examined the role of Hpa2 in normal thyroid tissue and in benign thyroid tumor, non-metastatic, and metastatic papillary thyroid carcinoma (PTC) utilizing immunostaining in correlation with clinicopathological parameters. Interestingly, we found that Hpa2 staining intensity does not significantly change in the transition from normal thyroid gland to benign, non-metastatic, or metastatic thyroid carcinoma. Remarkably, we observed that in some biopsies, Hpa2 is accumulating on the membrane (envelop) of the nucleus and termed this cellular localization NM (nuclear membrane). Notably, NM localization of Hpa2 occurred primarily in metastatic PTC and was associated with an increased number of positive (metastatic) lymph nodes collected at surgery. These results describe for the first time unrecognized localization of Hpa2 to the nuclear membrane, implying that in PTC, Hpa2 functions to promote tumor metastasis. [ABSTRACT FROM AUTHOR]

Transport of proteins, transcription factors, and other signaling molecules between the nucleus and cytoplasm is necessary for signal transduction. The study of these transport phenomena is particularly challenging in neurons because of their highly polarized structure. The bidirectional exchange of molecular cargoes across the nuclear envelope (NE) occurs through nuclear pore complexes (NPCs), which are aqueous channels embedded in the nuclear envelope. The NE and NPCs regulate nuclear transport but are also emerging as relevant regulators of chromatin organization and gene expression. The alterations in nuclear transport are regularly identified in affected neurons associated with human neurodegenerative diseases. This review presents insights into the roles played by nuclear transport defects in neurodegenerative disease, focusing primarily on NE proteins and NPCs. The subcellular mislocalization of proteins might be a very desirable means of therapeutic intervention in neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Background: Endothelial cells are constantly exposed to mechanical forces in the form of fluid shear stress, extracellular stiffness, and cyclic strain. The mechanoresponsive activity of YAP (Yes-associated protein) and its role in vascular development are well described; however, whether changes to transcription or epigenetic regulation of YAP are involved in these processes remains unanswered. Furthermore, how mechanical forces are transduced to the nucleus to drive transcriptional reprogramming in endothelial cells is poorly understood. The YAP target gene, AmotL2 (angiomotin-like 2), is a junctional mechanotransducer that connects cell-cell junctions to the nuclear membrane via the actin cytoskeleton., Methods: We applied mechanical manipulations including shear flow, stretching, and substrate stiffness to endothelial cells to investigate the role of mechanical forces in modulating YAP transcription. Using in vitro and in vivo endothelial depletion of AmotL2, we assess nuclear morphology, chromatin organization (using transposase-accessible chromatin sequencing), and whole-mount immunofluorescent staining of the aorta to determine the regulation and functionality of YAP. Finally, we use genetic and chemical inhibition to uncouple the nuclear-cytoskeletal connection to investigate the role of this pathway on YAP transcription., Results: Our results reveal that mechanical forces sensed at cell-cell junctions by the YAP target gene AmotL2 are directly involved in changes in global chromatin accessibility and activity of the histone methyltransferase EZH2, leading to modulation of YAP promotor activity. Functionally, shear stress-induced proliferation of endothelial cells in vivo was reliant on AmotL2 and YAP/TAZ (transcriptional coactivator with PDZ-binding motif) expression. Mechanistically, uncoupling of the nuclear-cytoskeletal connection from junctions and focal adhesions led to altered nuclear morphology, chromatin accessibility, and YAP promotor activity., Conclusions: Our findings reveal a role for AmotL2 and nuclear-cytoskeletal force transmission in modulating the epigenetic and transcriptional regulation of YAP to maintain a mechano-enforced positive feedback loop of vascular homeostasis. These findings may offer an explanation as to the proinflammatory phenotype that leads to aneurysm formation observed in AmotL2 endothelial deletion models., Competing Interests: Disclosures None.

For seven years of researching the transport systems of nuclear membranes using the patch-clamp method, we observed a certain pattern: in winter, this method's efficiency significantly decreased. Since different seasons are characterized by different light and temperature indicators, we decided to pay attention to the latter's possible impact on the success of the research. Therefore, the purpose of this work was to test the influence of seasonal factors such as changes in daylight hours, temperature, atmospheric pressure, precipitation, and cloudiness on the quality of patchclamp recordings of ion currents through the LCC channels of the nuclear membrane of cardiomyocytes and cerebellar Purkinje neurons. We assumed that with decreasing daylength and decreasing temperature, the patch-clamp registrations' qualitative and quantitative indicators also decrease. We applied Pearson's correlation analysis with initial data on daylight hours, meteorological conditions, and calculated progress of registrations (%) for a specific day to test this assumption. Based on the results of this analysis, we found out that there is a direct pronounced linear dependence of the quality and number of registrations on the length of daylight hours (r = 0.6) and temperature (r = 0.6), as well as a weak inverse dependence on cloudiness (r = 0.3). Analysis of variance (ANOVA) also confirmed a significantly greater success of registrations performed in the summer compared to the winter of the same year. The obtained results can become the basis for optimizing the research activities of working groups studying intracellular transport systems' functioning by electrophysiological methods, in particular, patch-clamp. [ABSTRACT FROM AUTHOR]

The regulation of lipid homeostasis is essential for normal cell physiology, and its disruption can lead to disease. Lipid droplets (LDs) are ubiquitous organelles dedicated to storing nonpolar lipids that are used for metabolic energy production or membrane biogenesis. LDs normally emerge from, and associate with, the endoplasmic reticulum and interact with other cytoplasmic organelles to deliver the stored lipids. Recently, LDs were found to reside also at the inner side of the nuclear envelope and inside the nucleus in yeast and mammalian cells. This unexpected finding raises fundamental questions about the nature of the inner nuclear membrane, its connection with the endoplasmic reticulum and the pathways of LD formation. In this viewpoint, we will highlight recent developments relating to these questions and discuss possible roles of LDs in nuclear physiology. [ABSTRACT FROM AUTHOR]

Bidirectional molecular movements between the nucleus and cytoplasm take place through nuclear pore complexes (NPCs) embedded in the nuclear membrane. These macromolecular structures are composed of several nucleoporins, which form seven different subcomplexes based on their biochemical affinity. These nucleoporins are integral components of the complex, not only allowing passive transport but also interacting with importin, exportin, and other molecules that are required for transport of protein in various cellular processes. Transport of different proteins is carried out either dependently or independently on transport receptors. As well as facilitating nucleocytoplasmic transport, nucleoporins also play an important role in cell differentiation, possibly by their direct gene interaction. This review will cover the general role of nucleoporins (whether its dependent or independent) and nucleocytoplasmic transport receptors in cell differentiation.

Purpose. To determine the contribution of the real contact spots distribution in the total conductivity of the conductors contact. Methodology. The electrical contact resistance research was carried out on models. The experimental part of this work was done on paper with a graphite layer with membranes (the first type) and conductive liquids with discrete partitions (the second type). Findings. It is shown that the contact electrical resistance is mainly determined by the real area of metal contact. The experimental dependence of the electrical resistance of the second type model on the distance between the electrodes and the potential distribution along the sample surface for the first type model were obtained. The theoretical model based on the principle of electric field superposition was considered. The dependences obtained experimentally and calculated by using the theoretical model are in good agreement. Originality. The regularity of the electrical contact resistance formation on a large number of membranes was researched for the first time. A new model of discrete electrical contact based on the liquid as the conducting environment with nuclear membrane partitions was developed. The conclusions of the additivity of contact and bulk electrical resistance were done. Practical value. Based on these researches, a new experimental method of kinetic macroidentation that as a parameter of the metal surface layer deformation uses the real contact area was developed. This method allows to determine the value of average contact stresses, yield point, change of the stress on the depth of deformation depending on the surface treatment.

The centromere and telomere are universal heterochromatic domains; however, the proper positioning of those domains in nuclear space during the mitotic interphase differs among eukaryotes. Consequently, the question arises how and why this difference occurs. Studies over the past 2 decades have identified several nuclear membrane proteins, nucleolar proteins, and the structural maintenance of a chromosome complex as factors involved in the positional control of centromeres and/or telomeres during the mitotic interphase in yeasts, animals, and plants. In this review, with a primary focus on plants, the roles of those factors are summarized, and the biological significance of proper centromere and telomere positionings during the mitotic interphase is discussed in an effort to provide guidance for this question. [ABSTRACT FROM AUTHOR]

Hutchinson-Gilford progeria syndrome (or Progeria) is an exceptionally rare genetic disorder in children. It is caused by a rare point mutation in the lamin gene. It encodes lamin A protein, resulting in the de-shaping of nuclear membrane. This altered structure of the nuclear membrane renders the nucleus unstable. The shortened lifespan of the nucleus makes the cell liable for rapid ageing. Children are healthy by appearance when they are born but the signs appear after 12-24 months of age. Cardiovascular system is greatly affected which became a reason for the death of most of the patients of progeria. Stiffened joints disturb the bone movements; and alopecia affects the appearance of the patient. Rate of occurrence of the disease is one per four hundred thousand of people, though both sexes are equally affected. [ABSTRACT FROM AUTHOR]

Bidirectional molecular movements between the nucleus and cytoplasm take place through nuclear pore complexes (NPCs) embedded in the nuclear membrane. These macromolecular structures are composed of several nucleoporins, which form seven different subcomplexes based on their biochemical affinity. These nucleoporins are integral components of the complex, not only allowing passive transport but also interacting with importin, exportin, and other molecules that are required for transport of protein in various cellular processes. Transport of different proteins is carried out either dependently or independently on transport receptors. As well as facilitating nucleocytoplasmic transport, nucleoporins also play an important role in cell differentiation, possibly by their direct gene interaction. This review will cover the general role of nucleoporins (whether its dependent or independent) and nucleocytoplasmic transport receptors in cell differentiation. [ABSTRACT FROM AUTHOR]

In eukaryotes, chromosome ends (telomeres) are tethered to the inner nuclear membrane. During the early stages of meiosis, telomeres move along the nuclear membrane and gather near the spindle-pole body, resulting in a bouquet-like arrangement of chromosomes. This chromosomal configuration appears to be widely conserved among eukaryotes, and is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In fission yeast, the Bqt1–Bqt2 protein complex plays a key role in tethering the telomere to the inner nuclear membrane. However, the structural details of the complex required to clarify how telomeres are gathered near the spindle-pole body remain enigmatic. Previously, we devised a preparation procedure for the Schizosaccharomyces japonicus Bqt1–Bqt2 complex, in which a SUMO tag was fused to the N-terminus of the Bqt1 protein. This allowed us to purify the Bqt1–Bqt2 complex from the soluble fraction. In the present study, we found that a maltose-binding protein homolog, Athe_0614, served as a better fusion partner than the SUMO protein, resulting in the marked increase in the solubility of the Bqt1–Bqt2 complex. The Athe_0614 fusion partner may open up new avenues for X-ray crystallographic analyses of the structure of the Bqt1–Bqt2 complex. [ABSTRACT FROM AUTHOR]

Baculovirus nucleocapsids egress from the nuclear membrane during infection. However, details of alternation of nuclear membrane structure during baculovirus egress are unknown. In this study, we examined the changes of lamin B receptor (LBR), a main inner nuclear membrane component, during Autographa californica nucleopolyhedrovirus (AcMNPV) infection. Firstly, the open reading frame (Orf) of Sf9 lbr was cloned by reverse transcription PCR, and the distribution of LBR in Sf9 cells were observed by fusing LBR with the red fluorescence protein mcherry. Besides, the amount of endogenous LBR during AcMNPV infection was detected by western blotting. Moreover, the distribution of LBR after AcMNPV infection was observed under the confocal fluorescence microscopy. Furthermore, the effects of protein kinase C (PKC) inhibitor on stability of LBR and release of budded virus (BVs) were determined. The results showed that Sf9 lbr contains an Orf of 2040 nucleotides (NTs), which encodes a predicted protein of 679 amino acids (AAs). Fluorescence microscopy showed that LBR is localized to the nuclear membrane. Western blotting result showed that the amount of endogenous LBR is significantly reduced after AcMNPV infection. Transfection and infection assay demonstrated that the fluorescence of LBR nearly completely disappeared after viral infection. PKC inhibitor can suppress the degradation of LBR induced by AcMNPV, resulting in the reduction of viral titer of progeny viruses. The electron microscopy analysis demonstrated that PKC inhibitor did not influence virion entry, uncoating, and assembly, but may partially protect the nuclear membrane from disruption by AcMNPV. Taken together, AcMNPV infection can distort the expression of LBR, which may promote the egress of nucleocapsids. [ABSTRACT FROM AUTHOR]

Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aquaporin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we used CRISPR-Cas9 to delete the MLCK gene (Mylk) to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphoproteomics using stable isotope labeling with amino acids in cell culture for quantification. Immunocytochemistry and electron microscopy demonstrated a defect in the processing of AQP2-containing early endosomes to late endosomes. The phosphoproteomics experiments revealed that, of the 1,743 phosphopeptides quantified over multiple replicates, 107 were changed in abundance by MLCK deletion (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain. Network analysis indicated that targeted phosphoproteins clustered into distinct structural/functional groups: actomyosin, signaling, nuclear envelope, gene transcription, mRNA processing, energy metabolism, intermediate filaments, adherens junctions, and tight junctions. There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. The presence of multiple proteins in the actomyosin category prompted experiments showing that MLCK deletion inhibits the normal effect of vasopressin to depolymerize F-actin, providing a potential explanation for the AQP2 trafficking defect. Changes in phosphorylation of multiple proteins in the nuclear envelope prompted measurement of nuclear size, showing a significant increase in average nuclear volume. We conclude that MLCK is part of a multicomponent signaling pathway in both the cytoplasm and nucleus that includes much more than simple regulation of conventional nonmuscle myosins through myosin regulatory light chain phosphorylation. [ABSTRACT FROM AUTHOR]

Large conductance cationic channels (LCC-channels) are highly expressed in the nuclear envelope of neurons and cardiomyocytes. Using electrophysiological registration of ion currents through the inner nuclear membrane of cardiomyocytes and cerebellar Purkinje neurons, we have tested the effect of n-choline receptor modulators (activators and inhibitors) on LCC-channels. Among the substances tested, rocuronium- and pipecuronium-bromide and nicotine proved to be effective. The pharmacological sensitivity of LCC-channels in Purkinje neuron and cardiomyocyte nuclear membranes was almost identical, indicating existence of a single population of these channels in the tissues studied. At a concentration of 0.2 mM nicotine reduced in half the current through LCC-channels, while rocuronium- and pipecuroniumbromide at the same concentration inhibited these channels by 7 and 12 % respectively. Therefore, among the listed substances, only the effect of nicotine on LCC-channels was close to the effect of previously studied tubocurarine, that's why tubocurarine and nicotine remain the most promising tools for further investigation of the physiological role of these channels. [ABSTRACT FROM AUTHOR]

HIV-1 enters the nucleus of non-dividing cells through the nuclear pore complex where it integrates into the host genome. The mechanism of HIV-1 nuclear import remains poorly understood. A powerful means to investigate the docking of HIV-1 at the nuclear pore and nuclear import of viral complexes is through single virus tracking in live cells. This approach necessitates fluorescence labeling of HIV-1 particles and the nuclear envelope, which may be challenging, especially in the context of primary cells. Here, we leveraged a deep neural network model for label-free visualization of the nuclear envelope using transmitted light microscopy. A training image set of cells with fluorescently labeled nuclear Lamin B1 (ground truth), along with the corresponding transmitted light images, was acquired and used to train our model to predict the morphology of the nuclear envelope in fixed cells. This protocol yielded accurate predictions of the nuclear membrane and was used in conjunction with virus infection to examine the nuclear entry of fluorescently labeled HIV-1 complexes. Analyses of HIV-1 nuclear import as a function of virus input yielded identical numbers of fluorescent viral complexes per nucleus using the ground truth and predicted nuclear membrane images. We also demonstrate the utility of predicting the nuclear envelope based on transmitted light images for multicolor fluorescence microscopy of infected cells. Importantly, we show that our model can be adapted to predict the nuclear membrane of live cells imaged at 37 °C, making this approach compatible with single virus tracking. Collectively, these findings demonstrate the utility of deep learning approaches for label-free imaging of cellular structures during early stages of virus infection. • 3-dimensional nuclear membrane contours are predicted from transmitted light images of cells using a deep learning approach. • The predicted nuclear membrane contours enable the measurements of nuclear import of fluorescently labeled HIV-1 complexes. • The applicability of this label-free nuclear membrane prediction approach to live cell imaging is demonstrated. [ABSTRACT FROM AUTHOR]

In order to tackle the study of DNA repair pathways, the physical and chemical agents creating DNA damage, the genotoxins, are frequently employed. Despite their utility, their effects are rarely restricted to DNA, and therefore simultaneously harm other cell biomolecules. Methyl methanesulfonate (MMS) is an alkylating agent that acts on DNA by preferentially methylating guanine and adenine bases. It is broadly used both in basic genome stability research and as a model for mechanistic studies to understand how alkylating agents work, such as those used in chemotherapy. Nevertheless, MMS exerts additional actions, such as oxidation and acetylation of proteins. In this work, we introduce the important notion that MMS also triggers a lipid stress that stems from and affects the inner nuclear membrane. The inner nuclear membrane plays an essential role in virtually all genome stability maintenance pathways. Thus, we want to raise awareness that the relative contribution of lipid and genotoxic stresses when using MMS may be difficult to dissect and will matter in the conclusions drawn from those studies.

The ability of iPSCs (induced pluripotent stem cells) to generate any cell type in the body makes them valuable tools for cell replacement therapies. However, differentiation of iPSCs can be demanding, slow and variable. During differentiation chromatin is re-organized and silent dense heterochromatin becomes tethered to the nuclear periphery by processes involving the nuclear lamina and proteins of the INM (inner nuclear membrane). The INM protein, Samp1 (Spindle Associated Membrane Protein 1) interacts with Lamin A/C and the INM protein Emerin, which has a chromatin binding LEM (Lap2-Emerin-Man1)-domain. In this paper we investigate if Samp1 can play a role in the differentiation of iPSCs. Samp1 levels increased as differentiating iPSCs started to express Lamin A/C. Interestingly, even under pluripotent culturing conditions, ectopic expression of Samp1 induced a rapid differentiation of iPSCs, of which some expressed the neuronal marker βIII-tubulin already after 6 days. This suggests that Samp1 is involved in early differentiation of iPSCs and could potentially be explored as a tool to promote progression of the differentiation process.

Oral cancer is in approximately 30% of all cancers in India. This study was conducted to evaluate the cytotoxic activity of ethanolic extract of Acacia catechu bark (ACB) against human squamous cell carcinoma cell line-25 (SCC-25). Cytotoxic effect of ACB extract was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide assay. A. catechu extract was treated SCC-25 cells with 25 and 50 μg/mL for 24 h. Apoptosis markers such as caspases-8 and 9, bcl-2, bax, and cytochrome c (Cyt-c) were done by RT-PCR. Morphological changes of ACB treated cells were evaluated using acridine orange/ethidium bromide (AO/EB) dual staining. Nuclear morphology and DNA fragmentation were evaluated using propidium iodide (PI) staining. Further, cell cycle analysis was performed using flow cytometry. A. catechu treatment caused cytotoxicity in SCC-25 cells with an IC50 of 52.09 μg/mL. Apoptotic marker gene expressions were significantly increased on ACB treatment. Staining with AO/EB and PI shows membrane blebbing and nuclear membrane distortion, respectively, and it confirms the apoptosis induction in SCC-25 cells. These results suggest that ACB extract can be used as a modulating agent in oral squamous cell carcinoma.

The investigation of pharmacological sensitivity of the cationic channels in nuclear membrane to the influence of anesthetics and natural venoms is relevant since it was shown that some modulators of N-cholinoreceptors (dithylinum, atracurium) affecting the large conductance cation channels (LCC-channels) functional activity are used in medicine during surgery. In addition, some injectable forms of toxins from the snake venom are used as drugs with an analgesic effect. Therefore, the aim of the study was to investigate the pharmacological sensitivity of the LCC-channels to the muscle relaxants, anaesthetics (mydocalm, diprofol) and natural venoms (neurotoxin II, α-Cobratoxin). The influence of these substances was evaluated based on changes in biophysical parameters of functioning of the LCC-channels of nuclear membrane of the cardiomyocytes and cerebellar Purkinje neurons. Ion currents through these channels were registered in the nucleus-attached or excised patch configuration and the voltage-clamp mode of the patch-clamp technique. We found that mydocalm (2 mM), diprofol (2 mM) and α-Cobratoxin (1 mM) reduced several times the probability of the channels being in the open state. Under the influence of mydocalm and α-Kobratoxin in high concentrations (1-2 mM) the effect of channels flickering was observed which indicates the channel pore blocking in its open state. At the same time, the average amplitude of the K+ current through the LCC-channels decreased by 13 % under the influence of NT II (25 μM). The results will be the basis for identification of new, more effective inhibitors of the LCC-channels that will be promising for the physiological relevance and structure of the channels investigation. [ABSTRACT FROM AUTHOR]

Cilia are microtubule-based organelles that protrude from the surface of eukaryotic cells to generate motility and to sense and respond to environmental cues. In order to carry out these functions, the complement of proteins in the cilium must be specific for the organelle. Regulation of protein entry into primary cilia has been shown to utilize mechanisms and components of nuclear gating, including nucleoporins of the nuclear pore complex (NPC). We show that nucleoporins also localize to the base of motile cilia on the surface of trachea epithelial cells. How nucleoporins are anchored at the cilium base has been unclear as transmembrane nucleoporins, which anchor nucleoporins at the nuclear envelope, have not been found to localize at the cilium. Here we use the directed yeast two-hybrid assay to identify direct interactions between nucleoporins and nephronophthisis proteins (NPHPs) which localize to the cilium base and contribute to cilium assembly and identity. We validate NPHP-nucleoporin interactions in mammalian cells using the knocksideways assay and demonstrate that the interactions occur at the base of the primary cilium using bimolecular fluorescence complementation. We propose that NPHP proteins anchor nucleoporins at the base of primary cilia to regulate protein entry into the organelle. [ABSTRACT FROM AUTHOR]

Purpose: To explore the relationships between vessel density (VD) in the retinal vascular plexuses with the thickness and structural changes of their corresponding retinal layers in patients with diabetic retinopathy (DR). Methods: Retrospective analysis of 17 eyes of 17 Type 1 diabetes (T1D) patients with severe non-proliferative or proliferative DR and no current or past macular edema. Seventeen age- and sex-matched healthy subjects were included as controls. Using optical coherence tomography (OCT) and OCT-angiography (OCTA), VD was measured in the superficial vascular plexus (SVP) and deep vascular complex (DVC) that includes the intermediate (ICP) and deep capillary plexuses (DCP), and compared to the retinal thickness (RT) of the inner (from the inner limiting membrane to the inner plexiform layer) and intermediate (inner nuclear and outer plexiform layer) retinal layers. The correlation between the inner and intermediate RT and the VD of the corresponding vascular networks (SVP and DVC, respectively) was assessed. All OCT and OCTA examinations were performed using the RTVue XR Avanti (Optovue, Fremont, CA). Results: The inner RT and VD in all plexuses were significantly reduced in T1D patients compared to healthy subjects. The capillary drop-out patterns were polygonal and well-defined in the SVP while the ICP and DCP showed a more diffuse capillary rarefaction and a VD that varied in the same proportion. The inner RT significantly correlated with VD in the SVP (r = 0.71 in healthy subjects and r = 0.62 in T1D patients, p <0.01). The intermediate RT did not significantly correlate with VD in the DVC. Conclusions: In T1D subjects, OCTA allowed observing different capillary drop-out patterns in the SVP and in the ICP-DCP, with different structural changes in the corresponding retinal layers, suggesting that they should be considered as distinct anatomical and functional entities. [ABSTRACT FROM AUTHOR]

TMEM16K, a membrane protein carrying 10 transmembrane regions, has phospholipid scramblase activity. TMEM16K is localized to intracellular membranes, but whether it actually scrambles phospholipids inside cells has not been demonstrated, due to technical difficulties in studying intracellular lipid distributions. Here, we developed a freeze-fracture electron microscopy method that enabled us to determine the phosphatidylserine (PtdSer) distribution in the individual leaflets of cellular membranes. Using this method, we found that the endoplasmic reticulum (ER) of mammalian cells harbored abundant PtdSer in its cytoplasmic leaflet and much less in the luminal leaflet, whereas the outer and inner nuclear membranes (NMs) had equivalent amounts of PtdSer in both leaflets. The ER and NMs of budding yeast also harbored PtdSer in their cytoplasmic leaflet, but asymmetrical distribution in the ER was not observed. Treating mouse embryonic fibroblasts with the Ca2+ ionophore A23187 compromised the cytoplasmic leaflet-dominant PtdSer asymmetry in the ER and increased PtdSer in the NMs, especially in the nucleoplasmic leaflet of the inner NM. This Ca2+-induced PtdSer redistribution was not observed in TMEM16K-null fibroblasts, but was recovered in these cells by reexpressing TMEM16K. These results indicate that, similar to the plasma membrane, PtdSer in the ER of mammalian cells is predominantly localized to the cytoplasmic leaflet, and that TMEM16K directly or indirectly mediates Ca2+-dependent phospholipid scrambling in the ER. [ABSTRACT FROM AUTHOR]

Evidence is rapidly mounting that transposable element (TE) expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging, such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by the use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, reporter constructs demonstrating engineered long interspersed nuclear element (LINE-1; L1) mobilization have been in use for quite some time, and strategies similar to L1 retrotransposition reporter assays have been developed to report replication of Ty1 elements in yeast and mouse intracisternal A particle (IAP) long terminal repeat (LTR) retrotransposons in cultivated cells. We describe a novel approach termed cellular labeling of endogenous retrovirus replication (CLEVR), which reports replication of the gypsy element within specific cells in vivo in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA (siRNA) silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR retrotransposons, endogenous retroviruses (ERVs), and exogenous retroviruses. [ABSTRACT FROM AUTHOR]

Macroautophagy (hereafter autophagy) is a conserved intracellular degradation mechanism required for cell survival. A double-membrane structure, the phagophore, is generated to sequester cytosolic cargos destined for degradation in the vacuole. The mechanism involved in the biogenesis of the phagophore is still an open question. We focused on 4 autophagy-related (Atg) proteins (Atg2, Atg9, Atg14, and Atg18), which are involved in the formation of the phagophore in order to gain a more complete understanding of the membrane dynamics that occur during formation of the autophagosome. The corresponding mutants, while defective in autophagy, nonetheless generate the membrane-bound form of Atg8, allowing us to use this protein as a marker for the nascent autophagosome precursor membrane. Using electron microscopy (EM), we discovered in these atg mutants a novel single-membrane structure (~120 to 150 nm in size). Electron tomography revealed that this structure originates from a part of the nuclear membrane, and we have named it the alphasome. Our data suggest that the alphasome is associated with Atg8, and sequesters selective cargo, the Cvt complex, during autophagy. Abbreviations: 3D: three-dimensional; AB: autophagic body; AP: autophagosome; Atg: autophagy-related; Cvt: cytoplasm-to-vacuole targeting; EM: electron microscopy; IEM: immunoelectron microscopy; L: lipid droplet; N: nucleus; NM: nuclear membrane; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor aminopeptidase I; rER: rough endoplasmic reticulum; TEM: transmission electron microscopy; V: vacuole; VLP: virus-like particle; [ABSTRACT FROM AUTHOR]