Analysis of Nuclear Pore Complex Permeability in Mammalian Cells and Isolated Nuclei Using Fluorescent Dextrans.

In eukaryotic cells the nuclear envelope encloses the genome separating it from the rest of the cell. Nuclear pore complexes are large multi protein channels that perforate the nuclear envelope, connecting the nucleus and the cytoplasm. Besides controlling nucleocytoplasmic molecule exchange, nuclear pore complexes create a permeability barrier that defines the maximum size of molecules that can freely diffuse into the nucleus. Accumulating evidence indicate that the permeability barrier of the nucleus can vary in different cellular conditions, during aging and in disease. Here we provide a simple protocol to analyze changes in nuclear permeability in plasma membrane-permeabilized cells and isolated nuclei using fluorescent dextrans of different sizes and confocal microscopy. The methods described herein represent a valuable resource to researchers studying the function of nuclear pore complexes and the dynamics of nuclear permeability in different cell types and processes.

Structure, Maintenance, and Regulation of Nuclear Pore Complexes: The Gatekeepers of the Eukaryotic Genome.

In eukaryotic cells, the genetic material is segregated inside the nucleus. This compartmentalization of the genome requires a transport system that allows cells to move molecules across the nuclear envelope, the membrane-based barrier that surrounds the chromosomes. Nuclear pore complexes (NPCs) are the central component of the nuclear transport machinery. These large protein channels penetrate the nuclear envelope, creating a passage between the nucleus and the cytoplasm through which nucleocytoplasmic molecule exchange occurs. NPCs are one of the largest protein assemblies of eukaryotic cells and, in addition to their critical function in nuclear transport, these structures also play key roles in many cellular processes in a transport-independent manner. Here we will review the current knowledge of the NPC structure, the cellular mechanisms that regulate their formation and maintenance, and we will provide a brief description of a variety of processes that NPCs regulate.

Loss of Nup210 results in muscle repair delays and age-associated alterations in muscle integrity.

Nuclear pore complexes, the channels connecting the nucleus with the cytoplasm, are built by multiple copies of ∼30 proteins called nucleoporins. Recent evidence has exposed that nucleoporins can play cell type-specific functions. Despite novel discoveries into the cellular functions of nucleoporins, their role in the regulation of mammalian tissue physiology remains mostly unexplored because of a limited number of nucleoporin mouse models. Here we show that ablation of Nup210/Gp210, a nucleoporin previously identified to play a role in myoblast differentiation and Zebrafish muscle maturation, is dispensable for skeletal muscle formation and growth in mice. We found that although primary satellite cells from Nup210 knockout mice can differentiate, these animals show delayed muscle repair after injury. Moreover, Nup210 knockout mice display an increased percentage of centrally nucleated fibers and abnormal fiber type distribution as they age. Muscle function experiments also exposed that Nup210 is required for muscle endurance during voluntary running. Our findings indicate that in mammals, Nup210 is important for the maintenance of skeletal muscle integrity and for proper muscle function providing novel insights into the in vivo roles of nuclear pore complex components.

Inhibition of Nuclear Pore Complex Formation Selectively Induces Cancer Cell Death.

Nuclear pore complexes (NPC) are the central mediators of nucleocytoplasmic transport. Increasing evidence shows that many cancer cells have increased numbers of NPCs and become addicted to the nuclear transport machinery. How reducing NPC numbers affects the physiology of normal and cancer cells and whether it could be exploited for cancer therapies has not been investigated. We report that inhibition of NPC formation, a process mostly restricted to proliferating cells, causes selective cancer cell death, prevents tumor growth, and induces tumor regression. Although cancer cells die in response to NPC assembly inhibition, normal cells undergo a reversible cell-cycle arrest that allows them to survive. Mechanistically, reducing NPC numbers results in multiple alterations contributing to cancer cell death, including abnormalities in nuclear transport, catastrophic alterations in gene expression, and the selective accumulation of DNA damage. Our findings uncover the NPC formation process as a novel targetable pathway in cancer cells. SIGNIFICANCE: Reducing NPC numbers in cancer cells induces death, prevents tumor growth, and results in tumor regression. Conversely, normal cells undergo a reversible cell-cycle arrest in response to inhibition of NPC assembly. These findings expose the potential of targeting NPC formation in cancer.This article is highlighted in the In This Issue feature, p. 1.

Nuclear Pore Complexes Are Key Regulators of Oligodendrocyte Differentiation and Function.

Increasing evidence points to nuclear pore complexes as important regulators of cell fate and tissue homeostasis. A recent report by Liu et al. (2019) in this issue of Neuron uncovers that nucleoporin Seh1 is required for the expression of genes critical for oligodendrocyte differentiation and myelination.

Nuclear pore complex-mediated modulation of TCR signaling is required for naïve CD4+ T cell homeostasis.

Nuclear pore complexes (NPCs) are channels connecting the nucleus with the cytoplasm. We report that loss of the tissue-specific NPC component Nup210 causes a severe deficit of naïve CD4+ T cells. Nup210-deficient CD4+ T lymphocytes develop normally but fail to survive in the periphery. The decreased survival results from both an impaired ability to transmit tonic T cell receptor (TCR) signals and increased levels of Fas, which sensitize Nup210-/- naïve CD4+ T cells to Fas-mediated cell death. Mechanistically, Nup210 regulates these processes by modulating the expression of Cav2 (encoding Caveolin-2) and Jun at the nuclear periphery. Whereas the TCR-dependent and CD4+ T cell-specific upregulation of Cav2 is critical for proximal TCR signaling, cJun expression is required for STAT3-dependent repression of Fas. Our results uncover an unexpected role for Nup210 as a cell-intrinsic regulator of TCR signaling and T cell homeostasis and expose NPCs as key players in the adaptive immune system.

Nuclear Pores Regulate Muscle Development and Maintenance by Assembling a Localized Mef2C Complex.

Nuclear pore complexes (NPCs) are multiprotein channels connecting the nucleus with the cytoplasm. NPCs have been shown to have tissue-specific composition, suggesting that their function can be specialized. However, the physiological roles of NPC composition changes and their impacts on cellular processes remain unclear. Here we show that the addition of the Nup210 nucleoporin to NPCs during myoblast differentiation results in assembly of an Mef2C transcriptional complex required for efficient expression of muscle structural genes and microRNAs. We show that this NPC-localized complex is essential for muscle growth, myofiber maturation, and muscle cell survival and that alterations in its activity result in muscle degeneration. Our findings suggest that NPCs regulate the activity of functional gene groups by acting as scaffolds that promote the local assembly of tissue-specific transcription complexes and show how nuclear pore composition changes can be exploited to regulate gene expression at the nuclear periphery.

Nuclear pore complexes and regulation of gene expression.

Nuclear pore complexes (NPCs), are large multiprotein channels that penetrate the nuclear envelope connecting the nucleus to the cytoplasm. Accumulating evidence shows that besides their main role in regulating the exchange of molecules between these two compartments, NPCs and their components also play important transport-independent roles, including gene expression regulation, chromatin organization, DNA repair, RNA processing and quality control, and cell cycle control. Here, we will describe the recent findings about the role of these structures in the regulation of gene expression.

Nuclear pore complex composition: a new regulator of tissue-specific and developmental functions.

Nuclear pore complexes (NPCs) are multiprotein aqueous channels that penetrate the nuclear envelope connecting the nucleus and the cytoplasm. NPCs consist of multiple copies of roughly 30 different proteins known as nucleoporins (NUPs). Due to their essential role in controlling nucleocytoplasmic transport, NPCs have traditionally been considered as structures of ubiquitous composition. The overall structure of the NPC is indeed conserved in all cells, but new evidence suggests that the protein composition of NPCs varies among cell types and tissues. Moreover, mutations in various nucleoporins result in tissue-specific diseases. These findings point towards a heterogeneity in NPC composition and function. This unexpected heterogeneity suggests that cells use a combination of different nucleoporins to assemble NPCs with distinct properties and specialized functions.

Organismal propagation in the absence of a functional telomerase pathway in Caenorhabditis elegans.

To counteract replication-dependent telomere shortening most eukaryotic cells rely on the telomerase pathway, which is crucial for the maintenance of proliferative potential of germ and stem cell populations of multicellular organisms. Likewise, cancer cells usually engage the telomerase pathway for telomere maintenance to gain immortality. However, in ∼10% of human cancers telomeres are maintained through telomerase-independent alternative lengthening of telomeres (ALT) pathways. Here, we describe the generation and characterization of C. elegans survivors in a strain lacking the catalytic subunit of telomerase and the nematode telomere-binding protein CeOB2. These clonal strains, some of which have been propagated for >180 generations, represent the first example of a multicellular organism with canonical telomeres that can survive without a functional telomerase pathway. The animals display the heterogeneous telomere length characteristic for ALT cells, contain single-stranded C-circles, a transcription profile pointing towards an adaptation to chronic stress and are therefore a unique and valuable tool to decipher the ALT mechanism.

Age-dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells.

In dividing cells, nuclear pore complexes (NPCs) disassemble during mitosis and reassemble into the newly forming nuclei. However, the fate of nuclear pores in postmitotic cells is unknown. Here, we show that NPCs, unlike other nuclear structures, do not turn over in differentiated cells. While a subset of NPC components, like Nup153 and Nup50, are continuously exchanged, scaffold nucleoporins, like the Nup107/160 complex, are extremely long-lived and remain incorporated in the nuclear membrane during the entire cellular life span. Besides the lack of nucleoporin expression and NPC turnover, we discovered an age-related deterioration of NPCs, leading to an increase in nuclear permeability and the leaking of cytoplasmic proteins into the nucleus. Our finding that nuclear "leakiness" is dramatically accelerated during aging and that a subset of nucleoporins is oxidatively damaged in old cells suggests that the accumulation of damage at the NPC might be a crucial aging event.

C. elegans telomeres contain G-strand and C-strand overhangs that are bound by distinct proteins.

Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in C. elegans, chromosome termini possess 3' G-strand overhangs as well as 5' C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that C. elegans telomere homeostasis relies on a novel mechanism that involves 5' and 3' single-stranded termini.

A mutant ankyrin protein kinase from Medicago sativa affects Arabidopsis adventitious roots.

A family of plant kinases containing ankyrin-repeats, the Ankyrin-Protein Kinases (APKs), shows structural resemblance to mammalian Integrin-Linked Kinases (ILKs), key regulators of mammalian cell adhesion. MsAPK1 expression is induced by osmotic stress in roots of Medicago sativa (L.) plants. The Escherichia coli-purified MsAPK1 could only phosphorylate tubulin among a variety of substrates and the enzymatic activity was strictly dependent on Mn2+. MsAPK1 is highly related to two APK genes in Arabidopsis thaliana (L.), AtAPK1 and AtAPK2. Promoter-GUS fusions assays revealed that the Arabidopsis APK genes show distinct expression patterns in roots and hypocotyls. Although Medicago truncatula (L.) plants affected in MsAPK1 expression could not be obtained using in vitro regeneration, A. thaliana plants expressing MsAPK1 or a mutant MsAPK1 protein, in which the conserved aspartate 315 of the kinase catalytic domain was replaced by asparagines (DN-lines), developed normally. The DN mutant lines showed increased capacity to develop adventitious roots when compared with control or MsAPK1-expressing plants. APK-mediated signalling may therefore link perception of external abiotic signals and the microtubule cytoskeleton, and influence adventitious root development.

A CDPK isoform participates in the regulation of nodule number in Medicago truncatula.

Medicago spp. are able to develop root nodules via symbiotic interaction with Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in various signalling pathways in plants, and we found that expression of MtCPK3, a CDPK isoform present in roots of the model legume Medicago truncatula, is regulated during the nodulation process. Early inductions were detected 15 min and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers was also present in inoculated M. truncatula dmi mutants and in wild-type roots subjected to salt stress, indicating that this rapid response is probably stress-related. In contrast, the later response was concomitant with cortical cell division and the formation of nodule primordia, and was not observed in wild-type roots inoculated with nod (-) strains. This late induction correlated with a change in the subcellular distribution of CDPK activities. Accordingly, an anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in epidermal onion cells, a localization that depends on myristoylation and palmitoylation sites of the protein, suggesting a dual subcellular localization. MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone linked to the regulation of cortical cell division and other nodulation-related responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium rhizogenes-transformed roots. Although no major phenotype was detected in these roots, when infected with rhizobia, the total number of nodules was, on average, twofold higher than in controls. This correlates with the lack of MtCPK3 induction in the inoculated super-nodulator sunn mutant. Our results suggest that CPK3 participates in the regulation of the symbiotic interaction.

Uncoupling of longevity and telomere length in C. elegans.

The nematode Caenorhabditis elegans, after completing its developmental stages and a brief reproductive period, spends the remainder of its adult life as an organism consisting exclusively of post-mitotic cells. Here we show that telomere length varies considerably in clonal populations of wild-type worms, and that these length differences are conserved over at least ten generations, suggesting a length regulation mechanism in cis. This observation is strengthened by the finding that the bulk telomere length in different worm strains varies considerably. Despite the close correlation of telomere length and clonal cellular senescence in mammalian cells, nematodes with long telomeres were neither long lived, nor did worm populations with comparably short telomeres exhibit a shorter life span. Conversely, long-lived daf-2 and short-lived daf-16 mutant animals can have either long or short telomeres. Telomere length of post-mitotic cells did not change during the aging process, and the response of animals to stress was found independent of telomere length. Collectively, our data indicate that telomere length and life span can be uncoupled in a post-mitotic setting, suggesting separate pathways for replication-dependent and -independent aging.

Regulation of CDPK isoforms during tuber development.

CDPK activities present during tuber development were analysed. A high CDPK activity was detected in the soluble fraction of early stolons and a lower one was detected in soluble and particulate fractions of induced stolons. The early and late CDPK activities displayed diverse specificity for in vitro substrates and different subcellular distribution. Western blot analysis revealed two CDPKs of 55 and 60 kDa that follow a precise spatial and temporal profile of expression. The 55 kDa protein was only detected in early-elongating stolons and the 60 kDa one was induced upon stolon swelling, correlating with early and late CDPK activities. A new member of the potato CDPK family, StCDPK3, was identified from a stolon cDNA library. Gene specific RT-PCR demonstrated that this gene is only expressed in early stolons, while the previously identified StCDPK1 is expressed upon stolon swelling. This expression profile suggests that StCDPK3 could correspond to the 55 kDa isoform while StCDPK1 could encode the 60 kDa isoform present in swelling stolons. StCDPK1 has myristoylation and palmitoylation consensus possibly involved in its dual intracellular localization. Transient expression studies with wild-type and mutated forms of StCDPK1 fused to GFP were used to show that subcellular localization of this isoform is controlled by myristoylation and palmitoylation. Altogether, our data suggest that sequential activation of StCDPK3 and StCDPK1 and the subcellular localisation of StCDPK1 might be critical regulatory steps of calcium signalling during potato tuber development.

StCDPK1 is expressed in potato stolon tips and is induced by high sucrose concentration.

StCDPK1 encodes a calcium-dependent protein kinase (CDPK) from Solanum tuberosum, which is transiently induced upon tuberization in swelling stolons. In situ hybridization determined that StCDPK1 mRNA is localized in the apical dome of tuberizing stolon tips, close to the region where sucrose was reported to accumulate. The expression of StCDPK1, and other tuber-specific genes was enhanced when in vitro-cultured potato plants were transferred to high sucrose or high sorbitol containing media. Glucose, fructose or a mixture of both showed no effect on CDPK expression. Okadaic acid blocked sucrose-inducible gene expression, suggesting that phosphatases from the PP1/PP2A family could also participate in the regulation of StCDPK1 and other tuberization-related genes.

Mitochondrial respiration in muscle and liver from cold-acclimated hypothyroid rats.

The effects of long-term cold exposure on muscle and liver mitochondrial oxygen consumption in hypothyroid and normal rats were examined. Thyroid ablation was performed after 8-wk acclimation to 4 degrees C. Hypothyroid and normal controls remained in the cold for an additional 8 wk. At the end of 16-wk cold exposure, all hypothyroid rats were alive and normothermic and had normal body weight. At ambient temperature (24 degrees C), thyroid ablation induced a 65% fall in muscle mitochondrial oxygen consumption, which was reversed by thyroxine but not by norepinephrine administration. After cold acclimation was reached, suppression of thyroid function reduced muscle mitochondrial respiration by 30%, but the hypothyroid values remained about threefold higher than those in hypothyroid muscle in the warm. Blockade of beta- and alpha1-adrenergic receptors in both hypothyroid and normal rats produced hypothermia in vivo and a fall in muscle, liver, and brown adipose tissue mitochondria respiration in vitro. In normal rats, cold acclimation enhanced muscle respiration by 35%, in liver 18%, and in brown adipose tissue 450% over values in the warm. The results demonstrate that thyroid hormones, in the presence of norepinephrine, are major determinants of thermogenic activity in muscle and liver of cold-acclimated rats. After thyroid ablation, cold-induced nonshivering thermogenesis replaced 3,5,3'-triiodothyronine-induced thermogenesis, and normal body temperature was maintained.

Brown fat thermogenesis in cold-acclimated rats is not abolished by the suppression of thyroid function.

The effects of long-term cold exposure on brown adipose tissue (BAT) thermogenesis in hypothyroid rats have been examined. Thyroid ablation was performed in normal rats after 2 mo of exposure to 4 degrees C, when BAT hypertrophy and thermogenic activity were maximal. After ablation, hypothyroid and normal controls remained in the cold for 2 additional months. At the end of the 4-mo cold exposure, all untreated hypothyroid rats were alive, had normal body temperature, and had gained an average 12.8% more weight than normal controls. Long-term cold exposure of hypothyroid rats markedly increased BAT weight, mitochondrial proteins, uncoupling protein (UCP)-1, mRNA for UCP-1, and oxygen consumption to levels similar to those seen in cold-exposed normal rats. The results indicate that thyroid hormones are required for increased thermogenic capacity to occur as an adaptation to long-term cold exposure. However, cold adaptation can be maintained in the absence of thyroid hormone.

Jasmonic acid affects plant morphology and calcium-dependent protein kinase expression and activity in Solanum tuberosum.

The effect of jasmonic acid (JA) on plant growth and on calcium-dependent protein kinase (CDPK) activity and expression was studied in non-photoperiodic potato plants, Solanum tuberosum L. var. Spunta, grown in vitro. Stem cuttings were grown for 45 days (long treatment, LT) in MS medium with increasing concentrations of JA. For short treatments (ST) adult plants grown in MS were transferred for 1, 4 and 20 h to JA containing media. During the LT, low concentrations of JA promoted cell expansion and shoot elongation while higher concentrations caused growth inhibition. Under these conditions, treated plants showed root shortening and tuber formation was not induced. Morphological and histochemical studies using light microscopy and TEM analysis of leaves from treated plants revealed that JA also affected subcellular organelles of mesophyll cells. Peroxisomes increased in size and number, and an autophagic process was triggered in response to high concentrations of the hormone. CDPK activity, determined in crude extracts of treated plants (LT), was inhibited (up to 80%). Plant growth and CDPK inhibition were reverted upon transfer of the plants to hormone-free medium. Soluble CDPK activity decreased in response to JA short treatment. Concomitantly, a decline in the steady state levels of StCDPK2 mRNA, a potato CDPK isoform that is expressed in leaves, was observed. These data suggest that the phytohormone down-regulated the expression and activity of the kinase.