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Lamins interact with a host of nuclear membrane proteins, transcription factors, chromatin regulators, signaling molecules, splicing factors, and even chromatin itself to form a nuclear subcompartment, the nuclear lamina, that is involved in a variety of cellular processes such as the governance of nuclear integrity, nuclear positioning, mitosis, DNA repair, DNA replication, splicing, signaling, mechanotransduction and -sensation, transcriptional regulation, and genome organization. Lamins are the primary scaffold for this nuclear subcompartment, but interactions with lamin-associated peptides in the inner nuclear membrane are self-reinforcing and mutually required. Lamins also interact, directly and indirectly, with peripheral heterochromatin domains called lamina-associated domains (LADs) and help to regulate dynamic 3D genome organization and expression of developmentally regulated genes.

Investigation of the intracellular generation of H2O2, one of the most important reactive oxygen species (ROS), is crucial for preventing various diseases since it is closely linked with different physiological and complex cell signaling pathways. Despite the development of various fluorescent probes, the majority of the fluorescent probes cannot move across the nuclear membrane. However, detection of the nuclear level of H2O2 is very important since it can directly cause oxidative DNA damage which ultimately leads to various diseases. Therefore, in this study, p-phenylenediamine based carbon quantum dots (B-PPD CDs) have been synthesized and integrated with 4-formylbenzeneboronic acid as a doping agent for the detection of H2O2. The detection mechanism showed that, upon exposure to H2O2, the fluorescence of the B-PPD CDs was immediately quenched. Further investigation has been done in the in vitro RAW 264.7 cell line by both exogenous and endogenous exposure of H2O2 to demonstrate the feasibility of the method. It is shown successfully that the exogenous presence and endogenous generation of H2O2 in RAW 264.7 cells can be detected using B-PPD CDs. The limit of detection (LOD) was determined to be 0.242 μM. The development of such imaging probes using carbon quantum dots will lead to live-cell imaging as well as ROS detection.

This study investigated the acute toxicity of cadmium (Cd) to the freshwater mussel Anodonta woodiana. The freshwater mussels were exposed to five concentrations of Cd (0 mg/L, 8.43 mg/L, 16.86 mg/L, 33.72 mg/L, and 67.45 mg/L) for up to 96 h. The 24-h, 48-h, 72-h, and 96-h LC50 values for Cd were estimated as 562.3 mg/L, 331.1 mg/L, 182.0 mg/L, and 134.9 mg/L, respectively. Caspase-3, caspase-8, caspase-9, and Ca-ATPase activities; protein and H2O2 levels; DNA fragmentation; and ultrastructure of the gill were also investigated. The activities of caspase-3 and caspase-9 in mussels were increased by Cd in a dose-dependent manner, where higher doses of Cd (33.72 mg/L and 67.45 mg/L) significantly increased the enzyme activities compared to the controls (P

Nuclear factor kappa B (NF-κB) transcriptionally regulates several genes involved in initiating uterine contractions. A key factor controlling NF-κB activity is its translocation to the nucleus. In myometrial smooth muscle cells (MSMCs), this translocation can be stimulated by the inflammatory molecule lipopolysaccharide (LPS) or by blocking the potassium calcium-activated channel subfamily M alpha 1 (KCNMA1 or BKCa) with paxilline (PAX). Here, we sought to determine the mechanism by which blocking BKCa causes NF-κB-p65 translocation to the nucleus in MSMCs. We show that LPS- and PAX-induced NF-κB-p65 translocation are similar in that neither depends on several mitogen-activated protein kinase pathways, but both require increased intracellular calcium (Ca2+). However, the nuclear transport inhibitor wheat germ agglutinin prevented NF-κB-p65 nuclear translocation in response to LPS but not in response to PAX. Blocking BKCa located on the plasma membrane resulted in a transient NF-κB-p65 nuclear translocation that was not sufficient to induce expression of its transcriptional target, suggesting a role for intracellular BKCa. We report that BKCa also localizes to the nucleus and that blocking nuclear BKCa results in an increase in nuclear Ca2+ in MSMCs. Together, these data suggest that BKCa localized on the nuclear membrane plays a key role in regulating nuclear Ca2+ and NF-κB-p65 nuclear translocation in MSMCs.

Nucleophagy, the selective subtype of autophagy that predominantly targets only a selected and (nonessential) portion of the nucleus, and rarely the nucleus in its entirety, for degradation, reinforces the paradigm that nucleophagy recycling is a meticulous and highly delicate process guarded by fail-safe mechanisms. Our goal in this commentary is to encourage autophagy researchers and other scientists to explore nucleophagy blind spots and gain advanced insights into the diverse roles of this process and its selective modality as they pertain to intranuclear quality control and cellular homeostasis. Identifying and deciphering nucleophagic signaling, regulation, molecular mechanism(s) and its mediators, cargo composition and nuclear membrane dynamics under numerous physiological and/or pathological settings will provide important advances in our understanding of this critical type of organelle-selective autophagy. Abbreviations: INM, inner nuclear membrane; LN, late nucleophagy; mRNA, messenger RNA; NE, nuclear envelope; NL, nuclear lamina; NPC(s), nuclear pore complex(es); NVJ(s), nucleus-vacuole junction(s); ONM, outer nuclear membrane; PMN, piecemeal microautophagy of the nucleus; PND, programmed nuclear death; PNuD, programmed nuclear destruction; rDNA/rRNA, ribosomal DNA/RNA

T7 expression system (T7 RNA polymerase / T7 promoter), derived from T7 bacteriophage, is one of the most extensively used protein expression systems, which is also an enabling tool in synthetic biology. However, in eukaryote, most of T7 expression system is transient expression system. This is mainly due to the absence of post-transcriptional processing of mRNAs transcribed by T7RNAP in eukaryotic cells, so they cannot effectively pass through nuclear membrane and enter cytoplasm. In this study, Saccharomyces cerevisiae was selected as host to construct stable T7 expression system, in which HIV-1 viroporin (Vpu) was used to improve the permeability of nuclear membrane. Results of NanoLuc® (Nluc) luciferase expression indicated that Vpu could effectively promote the transport of T7 transcripts and increase the amount of protein synthesized. The method of using viroporin to improve permeability of the nuclear membrane provides an effective tool for constructing a stable T7 expression system in eukaryote. Supplementary Information The online version contains supplementary material available at 10.1007/s12010-021-03665-y.

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.

The nucleus is a dynamic environment containing chromatin, membraneless organelles, and specialized molecular structures at the nuclear membrane. Within the spectrum of DNA repair activities are observations of increased mobility of damaged chromatin and the displacement of DNA lesions to specific nuclear environments. Here, we focus on the role that nuclear-specific filamentous actin plays in mobilizing damaged chromatin in response to DNA double-strand breaks and replication stress. We also examine nuclear pore complexes and promyelocytic leukemia-nuclear bodies as specialized platforms for homology-directed repair. The literature suggests an emerging model where specific types of DNA lesions are subjected to nuclear-derived forces that mobilize damaged chromatin and promote interaction with repair hubs to facilitate specialized repair reactions.

Early and accurate diagnosis of prostate cancer (PCa) is extremely important, as metastatic PCa remains hard to treat. EWI‐2, a member of the Ig protein subfamily, is known to inhibit PCa cell migration. In this study, we found that EWI‐2 localized on both the cell membrane and exosomes regulates the distribution of miR‐3934‐5p between cells and exosomes. Interestingly, we observed that EWI‐2 is localized not only on the plasma membrane but also on the nuclear envelope (nuclear membrane), where it regulates the nuclear translocation of signaling molecules and miRNA. Collectively, these functions of EWI‐2 found in lipid bilayers appear to regulate PCa cell metastasis through the epidermal growth factor receptor‐mitogen‐activated protein kinase‐extracellular‐signal‐regulated kinase (EGFR‐MAPK‐ERK) pathway. Our research provides new insights into the molecular function of EWI‐2 on PCa metastasis, and highlights EWI‐2 as a potential PCa biomarker., Our findings identify EWI‐2 as a potential prostate cancer (PCa) biomarker. EWI‐2 is involved in PCa metastasis through its localization on lipid bilayers where it facilitates nucleocytoplasmic shuttling of EGFR signaling molecules and also regulates microRNA sorting in exosomes. These molecular functions of EWI‐2 result in downstream activation of the epidermal growth factor receptor‐mitogen‐activated protein kinase‐extracellular‐signal‐regulated kinase (EGFR‐MAPK‐ERK) signaling pathway and promote PCa cell migration.

As one of the core framework proteins of nuclear pore complex (NPC), nucleoporin Nupl70 acts as a structural adapter between the nucleolus and nuclear pore membrane and maintains the stability of NPC structure through interaction with other proteins. In this study, we identified a Nup170 protein in the microsporidian Nosema bombycis for the first time and named it as NbNup170. Secondary structure prediction showed that the NbNup170 contains α-helices and random coils. The three-dimensional structure of NbNup170 is elliptical in shape. Phylogenetic analysis based on the Nup170 and homologous sequences showed that N. bombycis clustered together with Vairimorpha ceranae and Vairimorpha apis. The immunofluorescence localization results showed that the NbNup170 was located on the plasma membrane of the dormant spore and transferred to the surface of sporoplasm in a punctate pattern when the dormant spore has finished germination, and that NbNup170 was distributed on the nuclear membrane and both sides of the nuclei of early proliferative phase, and only on the nuclear membrane during sporogonic phase in the N. bombycis. qPCR analysis showed that the relative expression level of NbNup170 maintained at a low level from 30 to 78 h post-infection with N. bombycis, then reached the highest at 102 h, while that of NbNup170 was repressed at a very low level throughout its life cycle by RNA interference. These results suggested that NbNup170 protein is involved in the proliferative phase and active during the sporogonic phase of N. bombycis.

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.

Although nuclei are the defining features of eukaryotes, we still do not fully understand how the nuclear compartment is duplicated and partitioned during division. This is especially the case for organisms that do not completely disassemble their nuclear envelope upon entry into mitosis. In studying this process in Drosophila neural stem cells, which undergo asymmetric divisions, we find that the nuclear compartment boundary persists during mitosis thanks to the maintenance of a supporting nuclear lamina. This mitotic nuclear envelope is then asymmetrically remodeled and partitioned to give rise to two daughter nuclei that differ in envelope composition and exhibit a30-fold difference in volume. The striking difference in nuclear size was found to depend on two consecutive processes: asymmetric nuclear envelope resealing at mitotic exit at sites defined by the central spindle, and differential nuclear growth that appears to depend on the available local reservoir of ER/nuclear membranes, which is asymmetrically partitioned between the two daughter cells. Importantly, these asymmetries in size and composition of the daughter nuclei, and the associated asymmetries in chromatin organization, all become apparent long before the cortical release and the nuclear import of cell fates determinants. Thus, asymmetric nuclear remodeling during stem cell divisions may contribute to the generation of cellular diversity by initiating distinct transcriptional programs in sibling nuclei that contribute to later changes in daughter cell identity and fate.

The issues of brain morphogenesis during the early postnatal period and the effect of perinatal hypoxia thereupon, which can lead to the development of neuropsychic pathology, are among the major medical and social challenges. It is well known that the nucleoli of neocortical neurons synthesize ribosomal subunits and are involved in various morphogenetic processes. When studying the effect of perinatal hypoxia on the developing brain in the model of neonatal encephalopathy, we revealed ultrastructural changes in the nucleoli of neocortical neurons and in the numerical ratio of their types. In control rats, as they develop in the neonatal period, there is an increase in the number of both granular and reticulated nucleolar types, which may be due to ribosomal RNA (rRNA) processing. By the end of the neonatal period, the emergence of granular agglomerates (ribosomal subunits) near the nuclear membrane may be due to nerve cell differentiation and the biogenesis of the rough endoplasmic reticulum. In the present work, we established the fact of changing the numerical ratio of the granular and reticulated nucleolar types in the nuclei of neocortical neurons after perinatal hypoxic exposure of experimental vs. control rats. Our data suggest that the nucleoli in neocortical neurons are the targets for the exposure to perinatal hypoxia, i.e. a link in the pathogenesis of this disease. Post-hypoxic administration of Phenibut (a GABA derivative, nootropic drug) corrected the pathogenetic effect of perinatal hypoxia by restoring the numerical ratio of the nucleolar types in neocortical neurons, which is inherent to control rats. We assume that Phenibut can exert a neuroprotective effect through its ability to affect the nucleolar ultrastructure in neocortical neurons.

To examine the reliability to model cellular S-values for the Auger electron (AE) emitters, 111In, 197Hg and 197mHg with MCNP6 and their relative dose deposition in subcellular targets. A model cel...

Nuclear pore complexes (NPC) are highly conserved mega protein complexes that penetrate the double-layered nuclear membrane and form channels to allow bi-directional transport of macromolecules between the nucleus and the cytosol. Non-passive nucleocytoplasmic transport also requires nuclear transport receptors (NTR), which bind cargo molecules and shuttle them across the NPC. The NPC and NTRs constitute two fundamental layers of regulatory mechanisms that together determine the selective nuclear translocation of signal molecules and play essential roles in activating the precise response of a cell to environmental stimuli. Here we discuss recent findings in the NPC made by advanced structural biology approaches, and dissect distinct functions of different NPC components and NTRs in plants' responses to various biotic and abiotic stresses.

Актуальність. Широке використання наркотичних середників у клінічній практиці з метою отримання знеболювального та протизапального ефектів вимагає комплексного морфологічного дослідження особливостей структурної організації органів за умов впливу опіоїдів. Незважаючи на актуальність проблеми, дії опіоїдів на морфологічний стан жовчних проток присвячені лише поодинокі праці. Мета: встановити особливості змін мікро- та ультраструктурної організації спільної жовчної протоки за умов чотиритижневого введення опіоїду в експерименті. Методи. Застосували гістологічний, електронномікроскопічний, морфометричний та статистичний методами дослідження. Для вимірювання діаметра просвіту та товщини стінки спільної жовчної протоки використали комп’ютерну програму ImageJ. Результати. Через 4 тижні введення налбуфіну спільна жовчна протока експериментальних тварин дилатована, стінка її дезорганізована, пласт епітеліоцитів (холангіоцитів) слизової оболонки протоки деформований, часто без чітких контурів. Цитоплазма холангіоцитів просвітлена, мітохондрії з пошкодженими кристами, зерниста ендоплазматична сітка деструктурована, ядра епітеліальних клітин втрачали характерну для них колоноподібну форму, ставали округлими, містили конденсований хроматин вздовж ядерної оболонки, виявляли ядра в стані апоптозу та каріопікнозу. Власна пластинка слизової оболонки спільної жовчної протоки, розпушена, в стані набряку, залози деструктуровані, зміщені в напрямку епітеліального пласту. Пучки гладких міоцитів м’язової оболонки розшаровані, розділені широкими прошарками сполучної тканини. Мітохондрії міоцитів набряклі, кристи багатьох мітохондрій гомогенізовані, в більшості міоцитів виявлено безструктурні ділянки цитоплазми в стані дрібновакуольної дистрофії. Артеріоли стінки спільної жовчної протоки дилатовані, їхня стінка потовщена, в просвітах відбувалося формування пристінкових тромбів, венули розширені, стінки венул стоншені. Підсумок. Чотиритижневе введення опіоїду «Налбуфін» призводить до глибоких змін мікро- та ультраструктури стінки спільної жовчної протоки експериментальних білих щурів. Відбувається дилатація спільної жовчної протоки, потовщення її стінки, у слизовій оболонці переважають ознаки ексудативно-проліферативного запального процесу, в м’язовій оболонці спільної жовчної протоки розвивалася вакуольна дистрофія міоцитів та фіброзно-склеротичні зміни у сполучнотканинних прошарках., Background. The widespread use of narcotic agents in clinical practice to obtain analgesic and anti-inflammatory effects requires a comprehensive morphological study of the structural organization of organs during the effect of opioids. Despite the urgency of the problem, only a few works have been devoted to the effect of opioids on the morphological state of the bile ducts. Objective: to establish the features of changes in the micro- and ultrastructural organization of the common bile duct during the four-week opioid administration in the experiment. Methods. We used histological, electron microscopic, morphometric, and statistical methods. A computer program ImageJ was used to measure the diameter of the lumen and thickness of the common bile duct wall. Results. After 4 weeks of nalbuphine administration, the common bile duct of experimental animals is dilated, its wall is disorganized, the layer of epitheliocytes (cholangiocytes) of the duct mucous membrane is deformed, often without clear contours. The cytoplasm of cholangiocytes is enlightened, mitochondria have the damaged crystals, a granular endoplasmic reticulum is destructured, the nuclei of epithelial cells lost their characteristic columnar shape, became rounded, contained condensed chromatin along the nuclear membrane, found nuclei in a state of apoptosis and karyopyknosis. Own plate of the mucous membrane in the common bile duct is fluffy and swollen, the glands are destructured and shifted in the direction of the epithelial layer. The bundles of smooth myocytes of the muscular membrane are stratified and separated by wide layers of connective tissue. Mitochondria of myocytes are swollen, cristae of many mitochondria are homogenized; in most of myocytes, there are revealed the unstructured sites of cytoplasm in a condition of small vacuole dystrophy. The arterioles of the common bile duct wall are dilated, their wall is thickened; in lumens, there was a formation of wall thrombi, the venules are expanded, the walls of venules are thinned. Conclusion. Four-week administration of the Nalbuphine opioid leads to profound changes in the micro- and ultrastructure of the common bile duct wall in experimental white rats. There is the dilatation of the common bile duct, thickening of its wall, the signs of exudative-proliferative inflammatory process predominate in the mucous membrane, there are developed the vacuolar dystrophy of myocytes in the muscular membrane of the common bile duct and fibro-sclerotic changes in the connective tissue layers., Актуальность. Широкое использование наркотических веществ в клинической практике с целью получения обезболивающего и противовоспалительного эффектов требует комплексного морфологического исследования особенностей структурной организации органов в условиях воздействия опиоидов. Несмотря на актуальность проблемы, влиянию опиоидов на морфологическое состояние желчных протоков посвящены лишь единичные работы. Цель: установить особенности изменений микро- и ультраструктурной организации общего желчного протока в условиях четырехнедельного воздействия опиоидов в эксперименте. Методы. Использовали гистологический, электронномикроскопический, морфометрический и статистический методы исследования. Для измерения диаметра просвета и толщины стенки общего желчного протока использовали компьютерную программу ImageJ. Результаты. Через 4 недели введение налбуфина общий желчный проток экспериментальных животных дилатирован, стенка его дезорганизована, слой эпителиоцитов (холангиоцитов) слизистой оболочки протока деформирован, без четких контуров. Цитоплазма холангиоцитов просветленная, митохондрии с поврежденными кристами, зернистая эндоплазматическая сеть повреждена, ядра эпителиальных клеток теряли характерную для них овальную форму, становились округлыми, содержали конденсированный хроматин вдоль ядерной оболочки, выявляли ядра в состоянии апоптоза и кариопикноза. Собственная пластинка слизистой оболочки общего желчного протока рыхлая, отечна, железы деструктурированы, смещены в направлении эпителиального слоя. Пучки гладких миоцитов мышечной оболочки расслоенные, разделены широкими слоями соединительной ткани. Митохондрии миоцитов набухшие, кристи многих митохондрий гомогенизированные, в большинстве миоцитов обнаружено бесструктурные участки цитоплазмы в состоянии мелковакуольной дистрофии. Артериолы стенки общего желчного протока дилатированы, их стенка утолщена, в просветах формировались пристеночные тромбы, венулы расширены, стенки венул истонченные. Заключение. Четырехнедельное введение опиоида «Налбуфин» приводит к глубоким изменениям микро- и ультраструктуры стенки общего желчного протока экспериментальных белых крыс. Происходит дилатация общего желчного протока, утолщение ее стенки, в слизистой оболочке преобладают признаки экссудативно-пролиферативного воспалительного процесса, в мышечной оболочке общего желчного протока развивалась вакуольная дистрофия миоцитов и фиброзно-склеротические изменения в соединительнотканных прослойках.

Spatial organization of the genome in the nucleus plays a critical role in development and regulation of transcription. A genomic region that resides at the nuclear periphery is part of the chromatin layer marked with histone H3 lysine 9 dimethyl (H3K9me2), but chromatin reorganization during cell differentiation can cause movement in and out of this nuclear compartment with patterns specific for individual cell fates. Here we describe a CRISPR-based system that allows visualization coupled with forced spatial relocalization of a target genomic locus in live cells. We demonstrate that a specified locus can be tethered to the nuclear periphery through direct binding to a dCas9-Lap2β fusion protein at the nuclear membrane, or via targeting of a histone methyltransferase (HMT), G9a fused to dCas9, that promotes H3K9me2 labeling and localization to the nuclear periphery. The enzymatic activity of the HMT is sufficient to promote this repositioning, while disruption of the catalytic activity abolishes the localization effect. We further demonstrate that dCas9-G9a-mediated localization to the nuclear periphery is independent of nuclear actin polymerization. Our data suggest a function for epigenetic histone modifying enzymes in spatial chromatin organization and provide a system for tracking and labeling targeted genomic regions in live cells.

The major constituent of the nuclear envelope is the nuclear lamina. A fibrous meshwork of lamin filaments spanning underneath the nuclear membrane provides mechanical support to the nucleus. Lamins, type V intermediate filament proteins, are also involved in many other nuclear activities such as DNA repair and transcription. Recent structural studies provide new insight into how lamins assemble into ∼3.5nm thick filaments, which is in contrast with cytoplasmic intermediate filaments. The thinnest component of the cell cytoskeleton exhibits surprising mechanical properties. Here, we critically review and discuss structural and mechanical aspects of single lamin filaments.

The transport of proteins across the nuclear membrane is a highly regulated process, essential for the cell function. This transport is actively mediated by members of the karyopherin family, termed importins, or exportins, depending on the direction of transport. These proteins play an active part in tumorigenesis, through aberrant localization of their cargoes, which include oncogenes, tumor-suppressor genes and mediators of key signal transduction pathways. Overexpression of importins and exportins is reported in many malignancies, with implications in cell growth and viability, differentiation, drug resistance, and tumor microenvironment. Given their broad significance across tumors and pathways, much effort is being put to develop specific inhibitors as a novel anticancer therapeutics. Already, selinexor, a specific inhibitor of exportin-1 (XPO1), is approved for clinical use. This review will focus on the role of importins and exportins in hematological malignancies. We will discuss current preclinical and clinical data on importins and exportins, and demonstrate how our growing understanding of their functions has identified new therapeutic targets.

Apoptosis is the prototype for a regulated form of cell death, but recent studies have revealed other types of regulated forms of cell death, including necroptosis and ferroptosis. The molecular mechanisms underlying the execution of these processes have been intensively investigated, yet the hallmarks of their morphology are not fully understood. Here, we report that electron lucent cytoplasm was a common feature of both necroptosis and ferroptosis, which was consistent with cytoplasmic vacuolization due to a defect in the cytoplasmic membrane integrity. Notably, the perinuclear space was dilated in necroptosis, but such dilation did not occur in ferroptosis. Cells undergoing ferroptosis, but not necroptosis, exhibited an electron lucent nucleus. We previously reported that one of the nuclear danger-associated molecular patterns (DAMPs), high mobility group box (HMGB)1, is rapidly released from the nucleus to the extracellular spaces of cells undergoing necroptosis through the ruptured nuclear and cytoplasmic membrane. Via time-lapse imaging of cells stably expressing HMGB1 fused to a fluorescence protein, we found that HMGB1 was also released from the nucleus to the cytosol, and then eventually released into the extracellular spaces in cells undergoing ferroptosis. Thus, nuclear membrane damage was induced prior to cytoplasmic membrane rupture in ferroptosis. Thus, dilation of the perinuclear space and an electron lucent nucleus may be the hallmarks of necroptosis and ferroptosis, respectively.

The human nuclear membrane is composed of a double bilayer, the inner membrane being linked to the protein lamina network and the outer nuclear membrane continuous with the endoplasmic reticulum. Nuclear membranes can form large invaginations inside the nucleus; their specific roles still remain unknown. Although much of the protein identification has been determined, their lipid composition remains largely undetermined. In order to understand the mechanical and dynamic properties of nuclear membranes we investigated their lipid composition by two quantitative methods, namely, 31P and 1H multidimensional NMR and mass spectrometry, using internal standards. We also developed a nondetergent nuclei extraction protocol allowing to produce milligram quantities of nuclear membrane lipids. We found that the nuclear membrane lipid extract is composed of a complex mixture of phospholipids with different phosphatidylcholine species present in large amounts. Negatively charged lipids, with elevated amounts of phosphatidylinositol (PI), were also present. Mass spectrometry confirmed the phospholipid composition and provided further information on acyl-chain length and unsaturation. Lipid chain lengths ranged between 30 and 38 carbon atoms (two chains summed up) with a high proportion of 34 carbon atom length for most species. PI lipids have high amounts of chain lengths with 36-38 carbons. Independent of the chain length unsaturations were highly elevated with one to two double bonds per lipid species.

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.

Plant cation/proton antiporters can maintain intracellular ion homeostasis and resist ion toxicity. In this study, a gene encoding a maize cation/proton antiporter was cloned and named as ZmNHX7 . The coding sequence of ZmNHX7 was 3411 bp, encoding a protein with 1136 amino acids. ZmNHX7 is ubiquitously expressed in various tissues of maize, With higher expression level in roots and stems at the V7 stage. The expression of ZmNHX7 was induced by NaCl and LiCl stresses. In phylogenetic tree ZmNHX7 showed a close relation with AtNHX7 and AtNHX8 of Arabidopsis thaliana . ZmNHX7 was located in cell membrane and nuclear membrane by confocal laser scanning microscopy analysis of the lower epidermis of tobacco leaves. When ZmNHX7 gene was transformed into the Arabidopsis thaliana T-DNA insertion mutant nhx8 , the transgenic complementary lines could restore the tolerance of nhx8 to Li+. These results indicate that ZmNHX7 encodes a plasma membrane cation/proton antiporter of maize, which plays an important role in reducing the toxicity of Li+ to plants and maintaining intracellular ion homeostasis.

Photodynamic therapy (PDT) has drawn significant attention as a noninvasive cancer treatment. At present, PDT applications are limited in vivo because of the short lifetime and limited diffusion distance of cytotoxic reactive oxygen species (ROS) in the cellular environment. We propose to overcome this challenge by generating limited and short-lived ROS that are localized in key organelles, particularly the nucleus, in tumor cells. We developed Se/N-doped carbon dots (Se/N-CDs) as a photosensitizer for PDT. The Se/N-CDs were shown to bind RNA selectively by digestion experiments, isothermal titration microcalorimetry, and cell dyeing with RNA probes. Se/N-CDs near the nuclear membrane produced ROS that damaged the membrane under light irradiation, permitting the entry of more Se/N-CDs and thereby improving the conversion efficiency of photosensitization. Furthermore, in vitro and in vivo experiments indicated that the Se/N-CDs could inhibit tumor growth more effectively by PDT within the nucleus.

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.

The paper presents the results of a study of the surface and optical properties of the PM-1 polyimide film and the polyimide track membrane. For the study, polyimide (track) nuclear membranes manufactured by ION TRACK TECHNOLOGY FOR INNOVATIVE PRODUCT (it4ip, Belgium)) is used. The pore diameter is 200 nm, the pore density is 5 • 108 cm-1, and the thickness of the polyimide membrane is 25 μm. The data of IR spectroscopy are presented. It is shown that the decrease in the intensity of the peaks of the IR spectra of the membrane compared to the film is a consequence of the oxidative degradation of the polyimide after exposure to heavy ions and chemical etching. The data on the effects of heavy ion irradiation and chemical etching of a polyimide film to obtain a membrane on surface hydrophobicity / hydrophilicity are presented. The optical density of the film and track membranes is determined in the wavelength range of 350-950 nm. The optical density is measured in increments of 50 nm. It is shown that despite the strong difference in the spectral dependence of the optical density of the polyimide film and the track membrane, the optical density of the PM-1 film and the track membrane is quite low in the entire studied wavelength range and does not exceed 0.26 and 0.42, respectively.

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.

The aim of this research work was to study the histological structure of the pancreatic acini by transmission electron microscope in two avian species, duck and pigeon. The specimens were collected and processed for electron microscopic study. The results showed that the acini of the two avian species were two types; the first one was an electron dense and the second one an electron lucent. The light acinar cells were larger in size than the dark cells. These cells contained centrally located ovoid nuclei with prominent nucleoli and abundant euchromatin. The cytoplasm was electron lucent, with many rough endoplasmic reticulum, polymorphic mitochondria. Numerous zymogen granules were distributed in the basal part and around the nucleus, so these cells considered active cells. The dark acinar cells were characterized by an electron dense cytoplasm. The most prominent cell organelle in these cells were the zymogen granules that appeared in different sizes while other organelles as mitochondria, and rough endoplasmic reticulum were inconspicuous or few, so these cells were considered as inactive cells. The nucleus with indented nuclear membrane located centrally with prominent nucleoli and abundant heterochromatin. Prominent intercellular spaces between the individual acinar cells, as well as well-developed basement membrane separating the electron dense cells and the lumen contained the secretion between acinar cells. It could be concluded that the acinar cells in ducks and pigeons were divided into two types, that is, light and dark acinar cells which mainly attributed to the activity of these cells.

The endosomal sorting complex required for transport (ESCRT) pathway is required for efficient egress of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this study, we found that Ac93, a baculovirus core protein, contains a conserved MIM1-like motif. Alanine substitutions for six leucine residues in MIM1-like motif revealed that L142, L145, L146, and L149 are required for association of Ac93 with the MIT domain of Vps4. Mutations of these residues also blocked self-association and the association of Ac93 with ESCRT-III proteins or other viral core proteins Ac76 and Ac103, and resulted in a substantial reduction of infectious virus production, less efficient nuclear egress of progeny nucleocapsids, and the defect of intranuclear microvesicles formation. Combined with the localization of the association of Ac93 with ESCRT-III/Vps4 and other viral proteins at the nuclear membrane, we propose that the coordinated action of these viral proteins and ESCRT-III/Vps4 may be involved in remodeling the nuclear membrane.

The nuclear membranes function as a barrier to separate the cell nucleus from the cytoplasm, but this barrier can be compromised by nuclear membrane ruptures, leading to intermixing of nuclear and cytoplasmic contents. Spontaneous nuclear membrane ruptures (i.e., ruptures occurring in the absence of mechanical stress) have been observed in cultured cells, but they are more frequent in the setting of defects or deficiencies in nuclear lamins and when cells are subjected to mechanical stress. Nuclear membrane ruptures in cultured cells have been linked to DNA damage, but the relevance of ruptures to developmental or physiologic processes in vivo has received little attention. Recently, we addressed that issue by examining neuronal migration in the cerebral cortex, a developmental process that subjects the cell nucleus to mechanical stress. In the setting of lamin B1 deficiency, we observed frequent nuclear membrane ruptures in migrating neurons in the developing cerebral cortex and showed that those ruptures are likely the cause of observed DNA damage, neuronal cell death, and profound neuropathology. In this review, we discuss the physiologic relevance of nuclear membrane ruptures, with a focus on migrating neurons in cell culture and in the cerebral cortex of genetically modified mice.

The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ∼40-nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes, which are mediated by protein complexes within this subcellular compartment. Although fluorescence fluctuation spectroscopy is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional fluorescence fluctuation spectroscopy methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted mean-segmented Q (tsMSQ) analysis and applied it to successfully characterize protein homo-oligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE heteroprotein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies heteroprotein complexes and their stoichiometry in the NE by accounting for spectral cross talk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the linker of the nucleoskeleton and cytoskeleton complex, a heteroprotein complex composed of Klarsicht/ANC-1/SYNE homology and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the Klarsicht/ANC-1/SYNE homology protein nesprin-2 to form a heterocomplex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE heteroprotein complexes.

In S. cerevisiae prophase meiotic chromosomes move by forces generated in the cytoplasm and transduced to the telomere via a protein complex located in the nuclear membrane. We know that chromosome movements require actin cytoskeleton [13,31] and the proteins Ndj1, Mps3, and Csm4. Until recently, the identity of the protein connecting Ndj1-Mps3 with the cytoskeleton components was missing. It was also not known the identity of a cytoplasmic motor responsible for interacting with the actin cytoskeleton and a protein at the outer nuclear envelope. Our recent work [36] identified Mps2 as the protein connecting Ndj1-Mps3 with cytoskeleton components; Myo2 as the cytoplasmic motor that interacts with Mps2; and Cms4 as a regulator of Mps2 and Myo2 interaction and activities (Figure 1). Below we present a model for how Mps2, Csm4, and Myo2 promote chromosome movements by providing the primary connections joining telomeres to the actin cytoskeleton through the LINC complex. Fil: Lee, C. Y.. Oklahoma Medical Research Foundation; Estados Unidos Fil: Bisig, Carlos Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina Fil: Conrad, M. N.. Oklahoma Medical Research Foundation; Estados Unidos Fil: Ditamo, Yanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina Fil: Almeida, L. Previato de. Oklahoma Medical Research Foundation; Estados Unidos Fil: Dresser, M.E.. Oklahoma Medical Research Foundation; Estados Unidos Fil: Pezza, Roberto. Oklahoma Medical Research Foundation; Estados Unidos

Xenopus egg extracts are a powerful in vitro tool for studying complex biological processes, including nuclear reconstitution, nuclear membrane and pore assembly, and spindle assembly. Extracts have been further used to demonstrate a moonlighting regulatory role for nuclear import receptors or importins on these cell cycle assembly events. Here we show that exportins can also play a role in these events. Addition of Crm1, Exportin-t, or Exportin-5 decreased nuclear pore assembly in vitro. RanQ69L-GTP, a constitutively active form of RanGTP, ameliorated inhibition. Both Crm1 and Exportin-t inhibited fusion of nuclear membranes, again counteracted by RanQ69L-GTP. In mitotic extracts, Crm1 and Exportin-t negatively impacted spindle assembly. Pulldowns from the extracts using Crm1- or Exportin-t-beads revealed nucleoporins known to be essential for both nuclear pore and spindle assembly, with RanQ69L-GTP decreasing a subset of these target interactions. This study suggests a model where exportins, like importins, can regulate major mitotic assembly events.

Herpes simplex virus capsids are assembled and packaged in the nucleus and move by diffusion through the nucleoplasm to the nuclear envelope for egress. Analyzing their motion provides conclusions not only on capsid transport but also on the properties of the nuclear environment during infection. We utilized live-cell imaging and single-particle tracking to characterize capsid motion relative to the host chromatin. The data indicate that as the chromatin was marginalized toward the nuclear envelope it presented a restrictive barrier to the capsids. However, later in infection this barrier became more permissive and the probability of capsids to enter the chromatin increased. Thus, although chromatin marginalization initially restricted capsid transport to the nuclear envelope, a structural reorganization of the chromatin counteracted that to promote capsid transport later. Analyses of capsid motion revealed that it was subdiffusive, and that the diffusion coefficients were lower in the chromatin than in regions lacking chromatin. In addition, the diffusion coefficient in both regions increased during infection. Throughout the infection, the capsids were never enriched at the nuclear envelope, which suggests that instead of nuclear export the transport through the chromatin is the rate-limiting step for the nuclear egress of capsids. This provides motivation for further studies by validating the importance of intranuclear transport to the life cycle of HSV-1., Author summary Herpes simplex virus type 1 is a DNA virus that is studied intensively due to its high prevalence in humans, its ability to cause many diseases upon lytic replication or to evade the immune system in a latent state, and its potential use in oncolytic and immunotherapeutic applications. The nuclear replication of herpes simplex virus type 1 leads to the emergence of viral replication compartments together with relocalization and condensation of the host cell chromatin to the nuclear periphery. The reorganization of the host chromatin potentially hampers the intranuclear transport and nuclear egress of newly assembled progeny capsids. Nuclear capsids move by diffusion prior to their viral nuclear egress complex-mediated exit through the nuclear envelope. However, it is not understood how the capsids travel through the chromatin network. Our results show that infection-induced changes in chromatin architecture enhance nuclear diffusion of capsids at late infection, and that this transport is the rate-limiting step in the nuclear egress of the virus. Understanding the kinetics of capsid translocation in the nucleus is of fundamental interest in the field of DNA virus research because it explains how viruses are able to move in the chromatin before their nuclear egress.

Chromatin tethers to the nuclear envelope are lost during mitosis to facilitate chromosome segregation. How these connections are reestablished to ensure functional genome organization in interphase is unclear. Ptak et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202103036) identify a phosphorylation and SUMOylation-dependent cascade that links chromatin to the nuclear membrane during late mitosis.

The nuclear lamina protein, Lamin A and inner nuclear membrane protein, emerin participate in maintaining nuclear morphology. However, their correlations with the nuclear shape in the four representative ovarian epithelial cancer subtypes, high-grade serous carcinoma (HGSCa), clear cell carcinoma (CCCa), endometrioid carcinoma (EMCa) and mucinous carcinoma (MUCa), remains unclear. The present study aimed to investigate the association between nuclear morphology and nuclear membrane protein expression in four histological subtypes of ovarian epithelial cancer. A total of 140 surgically resected ovarian cancer specimens were subjected to Feulgen staining to evaluate nuclear morphology, and immunohistochemistry analysis to assess Lamin A and emerin expression. The histological images were analyzed via computer-assisted image analysis (CAIA). The results demonstrated that the mean nuclear area of EMCa was significantly smaller compared with CCCa (P=0.0009). The standard deviation of the mean nuclear area was used to assess nuclear size variation, and the results indicated that EMCa lesions were significantly smaller than CCCa lesions (P=0.0006). Regarding the correlation between the Lamin A-positive rate and nuclear morphological factors, positive correlations were observed with nuclear area in CCCa and EMCa (R=0.2855 and R=0.2858, respectively) and nuclear perimeter in CCCa, EMCa and MUCa (R=0.2409, R=0.4054 and R=0.2370, respectively); however, a negative correlation with nuclear shape factor was observed in HGSCa and EMCa (R=−0.2079 and R=−0.3707, respectively). With regards to the correlation between emerin positivity and nuclear morphological factors, positive correlations were observed with nuclear shape factor in HGSCa (R=0.2673) and nuclear area in CCCa (R=0.3310). It is well-known that HGSCa and CCCa have conspicuous nuclear size variation, and EMCa has small nuclei without strong atypia. These findings were verified in the present study via CAIA. Taken together, the results of the present study suggest that Lamin A strongly contributes to the maintenance of nuclear morphology in ovarian epithelial cancer compared with emerin, although their contributions differ based on tumor subtype.

Precise control of gene expression during differentiation relies on the interplay of chromatin and nuclear structure. Despite an established contribution of nuclear membrane proteins to developmental gene regulation, little is known regarding the role of inner nuclear proteins. Here we demonstrate that loss of the nuclear scaffolding protein Matrin-3 (Matr3) in erythroid cells leads to morphological and gene expression changes characteristic of accelerated maturation, as well as broad alterations in chromatin organization similar to those accompanying differentiation. Matr3 protein interacts with CTCF and the cohesin complex, and its loss perturbs their occupancy at a subset of sites. Destabilization of CTCF and cohesin binding correlates with altered transcription and accelerated differentiation. This association is conserved in embryonic stem cells. Our findings indicate Matr3 negatively affects cell fate transitions and demonstrate that a critical inner nuclear protein impacts occupancy of architectural factors, culminating in broad effects on chromatin organization and cell differentiation., Interactions between chromatin and nuclear components and whether these interactions affect development is not well understood. Here the authors show inner nuclear protein Matrin-3 (Matr3) loss leads to accelerated erythroid maturation, and that Matr3 is involved in chromosomal structure organization and compartmentalization to negatively regulate cell differentiation.

A high-throughput screen uncovered a role for the uncharacterized protein, Ybr063c (Cnm1 [contact nucleus mitochondria 1]), as a molecular tether of the nucleus–mitochondria contact in yeast. Cnm1 on the nucleus mediates contact by interacting with Tom70 on mitochondria. Regulation of Cnm1 abundance by phosphatidylcholine enables coupling of phospholipid levels with contact extent., Mitochondrial functions are tightly regulated by nuclear activity, requiring extensive communication between these organelles. One way by which organelles can communicate is through contact sites, areas of close apposition held together by tethering molecules. While many contacts have been characterized in yeast, the contact between the nucleus and mitochondria was not previously identified. Using fluorescence and electron microscopy in S. cerevisiae, we demonstrate specific areas of contact between the two organelles. Using a high-throughput screen, we uncover a role for the uncharacterized protein Ybr063c, which we have named Cnm1 (contact nucleus mitochondria 1), as a molecular tether on the nuclear membrane. We show that Cnm1 mediates contact by interacting with Tom70 on mitochondria. Moreover, Cnm1 abundance is regulated by phosphatidylcholine, enabling the coupling of phospholipid homeostasis with contact extent. The discovery of a molecular mechanism that allows mitochondrial crosstalk with the nucleus sets the ground for better understanding of mitochondrial functions in health and disease.

Nuclear rupture has long been associated with deficits or defects in lamins, with recent results also indicating a role for actomyosin stress, but key physical determinants of rupture remain unclear. Here, lamin-B stably interacts with the nuclear membrane at sites of low Gaussian curvature yet dilutes at high-curvature to favor rupture, whereas lamin-A depletes similarly but only at high strain-rates. Live cell imaging of lamin-B1 gene-edited cancer cells is complemented by fixed-cell imaging of ruptured nuclei in: iPS-derived cells from progeria patients, cells within beating chick embryo hearts, and cancer cells that develop multiple ruptures in migrating through small pores. Dilution and curvature-dependent rupture fit a parsimonious model of a stiff filament that detaches from a curved surface, suggesting an elastic-type response of lamin-B, but rupture is also modestly suppressed by inhibiting myosin-II and by hypotonic stress, which slow the strain rates. Lamin-A dilution and nuclear rupture likelihood indeed increase above a threshold rate of pulling into small pipettes, suggesting a viscoplastic coupling to the envelope for protection against nuclear rupture.Summary statementHigh nuclear curvature drives lamina dilution and nuclear envelope rupture even when myosin stress is inhibited. Stiff filaments generally dilute from sites of high Gaussian curvature, providing mathematical fits of experiments.

Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes (IACs), play key roles in cancer progression and metastasis. We investigated systems-level changes in the integrin adhesome during metastatic progression of a patient-derived cutaneous squamous cell carcinoma (cSCC), and found that the actin regulatory protein Mena is enriched in IACs in metastatic cSCC cells. Mena is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2, with which it interacts and co-localises at the nuclear envelope of metastatic cells. Moreover, Mena potentiates the interactions of nesprin-2 with the actin cytoskeleton and the nuclear lamina. CRISPR-mediated Mena depletion causes altered nuclear morphology, reduces tyrosine phosphorylation of the nuclear membrane protein emerin and downregulates expression of the immunomodulatory gene PTX3 via the recruitment of its enhancer to the nuclear periphery. We have uncovered an unexpected novel role for Mena at the nuclear membrane, where it controls the LINC complex, nuclear architecture, chromatin repositioning and cancer gene expression. This is the first description of an adhesion protein regulating gene transcription via direct signalling across the nuclear envelope.Abstract Figure

The three-dimensional morphology of the Golgi apparatus in osteoclasts was investigated by computer-aided reconstruction. Rat femora were treated for nicotinamide adenine dinucleotide phosphatase (NADPase) cytochemistry, and light microscopy was used to select several osteoclasts in serial semi-thin sections to investigate the Golgi apparatus by backscattered electron-mode scanning electron microscopy. Lace-like structures with strong backscattered electron signals were observed around the nuclei. These structures, observed within the Golgi apparatus, were attributed to the reaction products (i.e., lead precipitates) of NADPase cytochemistry. Features on the images corresponding to the Golgi apparatus, nuclei, and ruffled border were manually traced and three-dimensionally reconstructed using ImageJ/Fiji (an open-source image processing package). In the reconstructed model, the Golgi apparatus formed an almost-continuous structure with a basket-like configuration, which surrounded all the nuclei and also partitioned them. This peculiar three-dimensional morphology of the Golgi apparatus was discovered for the first time in this study. On the basis of the location of the cis- and trans-sides of the Golgi apparatus and the reported results of previous studies, we postulated that the nuclear membrane synthesized specific proteins in the osteoclasts and, accordingly, the Golgi apparatus accumulated around the nuclei as a receptacle.

Numerous membrane-less organelles composed of a combination of RNA and proteins, referred to as RNP granules, are observed in the nucleus and cytoplasm of eukaryotic cells, including stress granules, processing bodies, Cajal bodies, and nuclear speckles. An unresolved issue is how frequently RNA molecules are required for the maintenance of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. Strikingly, we find the majority of RNP granules, including stress granules, processing bodies, Cajal bodies, nuclear speckles and the nucleolus are altered by the degradation of their RNA components. In contrast, super-enhancer complexes and TIS granules were largely unaffected by widespread intracellular RNA degradation. This highlights a critical and widespread role of RNA in the organization of many, but not all, RNP granules.SUMMARYDecker et al. examine the role of RNA in maintaining membrane-less organelles composed of RNA and protein by activating RNase L in the cytoplasm or nucleus. Degradation of RNA alters the majority of membrane-less organelles indicating that RNA plays a widespread role in their organization.

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here we show that the nuclear-localized LINC complex protein SUN1 regulates vascular sprouting and barrier function via effects on endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted barrier function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and barrier function.SUMMARYThe nuclear membrane protein SUN1 promotes blood vessel formation and barrier function by stabilizing endothelial cell-cell junctions. Communication between SUN1 and endothelial cell junctions relies upon proper microtubule dynamics and Rho signaling far from the nucleus, revealing long-range cellular communication from the nucleus to the cell periphery that is important for vascular development and function.

During anaphase B, molecular motors slide interpolar microtubules to elongate the mitotic spindle, contributing to the separation of chromosomes. However, sliding of antiparallel microtubules reduces their overlap, which may lead to spindle breakage, unless microtubules grow to compensate sliding. How sliding and growth are coordinated is still poorly understood. In this study, we have used the fission yeastS. pombeto measure microtubule dynamics during anaphase B. We report that the coordination of microtubule growth and sliding relies on promoting rescues at the midzone edges. This makes microtubules stable from pole to midzone, while their distal parts including the plus ends alternate between assembly and disassembly. Consequently, the midzone keeps a constant length throughout anaphase, enabling sustained sliding without the need for a precise regulation of microtubule growth speed. Additionally, we found that inS. pombe, which undergoes closed mitosis, microtubule growth speed decreases when the nuclear membrane wraps around the spindle midzone.

Background. Hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) are the common diagnostic/prognostic markers in breast cancer. Few articles have recently reported the correlation between cytology and molecular subtypes. We combined nuclear morphological characteristics with HR and HER2 status to observe the relationship and provide ideas for machine learning. Methods. We reanalyzed fine-needle aspiration cytology samples and core-needle puncture histological specimens from 142 patients with invasive breast cancer between March 2019 and December 2019, and the findings were compared with the two groups (HR+/HER2- and HR-/HER2+) following nuclear cytomorphological features: nuclear/cytoplasmic ratio, difference of nuclear size, nuclear pleomorphism, chromatin feature, nuclear membrane and nucleoli, and Nottingham grading. Results. Two groups were significantly associated with the difference of nuclear size, nuclear pleomorphism, and nucleoli ( P < 0.001 ) and consistent with histological grading ( P < 0.001 ). Moreover, nucleolar characteristics of size and number had obviously statistical significance ( P < 0.001 ). Multiple micro-nucleoli were frequently seen in the HR+/HER2- group compared with the HR-/HER2+ group which mostly were observed centered medium-large nucleoli. We described four interesting nuclear morphologies in the experiment. Conclusions. There were significant differences in nuclear characteristics between two groups. HR and HER2 status not only might be predicted in cytological samples, but some specific nuclear morphological features might have potential value to help us understand molecular function and predict more information.

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.

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.

The condensation and compartmentalization of biomacromolecules in the cell are driven by the process of phase separation. The main effectors of phase separation are intrinsically disordered proteins, which include proteins with a phenylalanine-glycine (FG) repeat domain. Our understanding of the biological function of FG repeat proteins during phase separation has been mainly derived from recent research on a member of the nuclear pore complex proteins, nucleoporins containing FG repeat domain (FG-NUPs). FG-NUPs form meshwork structures by inter- and intra-molecular FG domain interactions, which confine the nucleo-cytoplasmic exchange. Whereas FG-NUPs localize in the nuclear membrane, other FG repeat proteins reside in the cytoplasm and the nucleoplasm, and the biological function of the FG repeat domain of these proteins is not well described. In the present review, we list the FG repeat proteins that are known to phase separate in the cell, and review their biological functions. We extract the unraveled features of FG repeat proteins as an activator of barrier formation and homotypic cell-cell interactions. Understanding the regulatory mechanisms of FG repeat proteins will provide a potential delivery tool for therapeutic reagents.