Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (≈10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca²⁺ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)–tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) to study the effect of Ca²⁺ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M₁ receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid–acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca²⁺ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.     

Nuclear calcium and the regulation of the nuclear pore complex

In eukaryotic cells the nucleus and its contents are separated from the cytoplasm by the nuclear envelope. Macromolecules, as well as smaller molecules and ions, can cross the nuclear envelope through the nuclear pore complex. Molecules greater than approx. 60 kDa and containing a nuclear localization signal are actively transported across the nuclear membranes, but there has been little evidence for regulatory mechanisms for smaller molecules and ions. Recently, diffusion across the nuclear envelope has been observed to be regulated by nuclear cisternal Ca²⁺ concentrations. Following depletion of Ca²⁺ from the nuclear store by inositol 1,4,5-trisphosphate or Ca²⁺ chelators, a fluorescent 10 kDa marker molecule was no longer able to enter the nucleus. Distinct conformational states of the nuclear pore complexes depended on the Ca²⁺ filling state of the nuclear envelope, supporting the assumption that a switch in the conformation of the nuclear pore complex may control the transport of intermediate-sized molecules across the nuclear envelope. Thus nuclear Ca²⁺ stores may regulate the conformational state of the nuclear pore complex, and thereby passive diffusion of molecules between the cytosol and the nucleoplasm. The physiological significance of this finding is currently unknown.     

Mediators of Nuclear Protein Import Target Karyophilic Proteins to Pore Complexes of Cytoplasmic Annulate Lamellae

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes ofXenopus laevis.In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin β and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central “transporter” and closely resemble those of “transport intermediates” found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.     

Calcium regulation of nucleocytoplasmic transport

Bidirectional trafficking of macromolecules between the cytoplasm and the nucleus is mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) of eukaryotic cell. The NPC functions as the sole pathway to allow for the passive diffusion of small molecules and the facilitated translocation of larger molecules. Evidence shows that these two transport modes and the conformation of NPC can be regulated by calcium stored in the lumen of nuclear envelope and endoplasmic reticulum. However, the mechanism of calcium regulation remains poorly understood. In this review, we integrate data on the observations of calcium-regulated structure and function of the NPC over the past years. Furthermore, we highlight challenges in the measurements of dynamic conformational changes and transient transport kinetics in the NPC. Finally, an innovative imaging approach, single-molecule super-resolution fluorescence microscopy, is introduced and expected to provide more insights into the mechanism of calcium-regulated nucleocytoplasmic transport.     

Calcium-dependent facilitation and graded deactivation of store-operated calcium entry in fetal skeletal muscle

Activation of store-operated Ca²⁺ entry (SOCE) into the cytoplasm requires retrograde signaling from the intracellular Ca²⁺ release machinery, a process that involves an intimate interaction between protein components on the intracellular and cell surface membranes. The cellular machinery that governs the Ca²⁺ movement in muscle cells is developmentally regulated, reflecting maturation of the junctional membrane structure as well as coordinated expression of related Ca²⁺ signaling molecules. Here we demonstrate the existence of SOCE in freshly isolated skeletal muscle cells obtained from embryonic days 15 and 16 of the mouse embryo, a critical stage of muscle development. SOCE in the fetal muscle deactivates incrementally with the uptake of Ca²⁺ into the sarcoplasmic reticulum (SR). A novel Ca²⁺-dependent facilitation of SOCE is observed in cells transiently exposed to high cytosolic Ca²⁺. Our data suggest that cytosolic Ca²⁺ can facilitate SOCE whereas SR luminal Ca²⁺ can deactivate SOCE in the fetal skeletal muscle. This cooperative mechanism of SOCE regulation by Ca²⁺ ions not only enables tight control of SOCE by the SR membrane, but also provides an efficient mechanism of extracellular Ca²⁺ entry in response to physiological demand. Such Ca²⁺ signaling mechanism would likely contribute to contraction and development of the fetal skeletal muscle.     

蛋白质入核转运的机制和研究进展

细胞核膜是由外膜和内膜组成的磷脂双分子层结构,同时镶嵌一些核孔复合体（NPC）.核孔复合体是胞浆和胞核之间主动和被动转运的生理屏障.核内功能蛋白在胞浆内合成后通过核孔复合体进入胞核,这个过程除了需要NPC上核孔蛋白、胞浆内核转运受体和RanGTP等蛋白的参与外, 货物蛋白本身的结构特征在其入核转运过程中亦发挥重要作用.本文着重就蛋白入核转运的机制及近年来取得的相关进展进行综述.     

Peripheral Hot Spots for Local Ca2+ Release after Single Action Potentials in Sympathetic Ganglion Neurons

Ca²⁺ release from the endoplasmic reticulum (ER) contributes to Ca²⁺ transients in frog sympathetic ganglion neurons. Here we use video-rate confocal fluo-4 fluorescence imaging to show that single action potentials reproducibly trigger rapidly rising Ca²⁺ transients at 1–3 local hot spots within the peripheral ER-rich layer in intact neurons in fresh ganglia and in the majority (74%) of cultured neurons. Hot spots were located near the nucleus or the axon hillock region. Other regions exhibited either slower and smaller signals or no response. Ca²⁺ signals spread into the cell at constant velocity across the ER in nonnuclear regions, indicating active propagation, but spread with a (time)^1/2 dependence within the nucleus, consistent with diffusion. 26% of cultured cells exhibited uniform Ca²⁺ signals around the periphery, but hot spots were produced by loading the cytosol with EGTA or by bathing such cells in low-Ca²⁺ Ringer's solution. Peripheral hot spots for Ca²⁺ release within the perinuclear and axon hillock regions provide a mechanism for preferential initiation of nuclear and axonal Ca²⁺ signals by single action potentials in sympathetic ganglion neurons.     

The nucleoporin Nup50 activates the Ran guanine nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport‐competent nucleus. Here, we show that Nup50 plays a crucial role in NPC assembly independent of its well‐established function in nuclear transport. RNAi‐mediated downregulation in cells or immunodepletion of Nup50 protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and for NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α/β, the GTPase Ran, or chromatin is crucial for its function in the assembly process. Instead, an N‐terminal fragment of Nup50 can stimulate the Ran GTPase guanine nucleotide exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in NPC reformation at the end of mitosis. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild‐type protein in in vitro NPC assembly assays, whereas excess RCC1 can compensate the loss of Nup50.     

FRAP法对内源性GFP在活细胞中动态分布的共焦显微镜成像

各种分子在核质问的动态分布与它们的跨膜转运密切相关。离子、r证矾A和多数小分子量蛋白可以通过核孔复合物（NPG,nuclear pore complexes）在核质问自由扩散,而分子量大于70kDa的分子需要ATP和核定位序列才能实现跨膜转运。本实验利用荧光漂白后恢复（FRAP,fluorescence recovery after photobleaching）法观测人肺腺癌肿瘤细胞（ASTC-a-1）中表达的27 kDa EGFP在核质问的被动扩散,并以激光共焦显微镜进行实时成像。转染EGFP外源基因的肿瘤细胞系在经过半年的传代培养后仍能稳定而高效的表达其荧光标记。实验表明,EGFP分子可以通过核孔在核质间被动扩散,但扩散速度远低于在核内或质内的速度,没有证据表明EGFP可以在细胞问扩散。     

Torsin and NEP1R1‐CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid‐dependent and lipid‐independent mechanisms

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1‐CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1‐CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1‐CTDNEP1 downregulation also restores nuclear pore membrane fusion in post‐mitotic dTorsin^KO fat body cells. However, dTorsin‐associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel‐like structures in place of mature nuclear pores. We conclude that the NEP1R1‐CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid‐dependent and lipid‐independent mechanisms, explaining some of the pleiotropic effects of Torsins.     

Structural Analysis of a Metazoan Nuclear Pore Complex Reveals a Fused Concentric Ring Architecture

The sole gateway for molecular exchange between the cytoplasm and the nucleus is the nuclear pore complex (NPC). This large supramolecular assembly mediates transport of cargo into and out of the nucleus and fuse the inner and outer nuclear membranes to form an aqueous translocation channel. The NPC is composed of eight proteinaceous asymmetric units forming a pseudo-8-fold symmetric passage. Due to its shear size, complexity, and plastic nature, dissecting the high-resolution three-dimensional structure of the NPC in its hydrated state is a formidable challenge. Toward this goal, we applied cryo-electron tomography to spread nuclear envelopes from Xenopus oocytes. To compensate for perturbations of the 8-fold symmetry of individual NPCs, we performed symmetry-independent asymmetric unit averaging of three-dimensional tomographic NPC volumes to eventually yield a refined model at 6.4 nm resolution. This approach revealed novel structural features, particularly in the spoke-ring complex and luminal domains. Fused concentric ring architecture of the spoke-ring complex was found along the translocation channel. Additionally, a comparison of the refined Xenopus model to that of its Dictyostelium homologue yielded similar pore diameters at the level of the three canonical rings, although the Xenopus NPC was found to be 30% taller than the Dictyostelium pore. This discrepancy is attributed primarily to the relatively low homology and different organization of some nucleoporins in the Dictyostelium genome as compared to that of vertebrates. Nevertheless, the experimental conditions impose a preferred axial orientation of the NPCs within spread Xenopus oocyte nuclear envelopes. This may at least in part explain the increased height of the reconstructed vertebrate NPCs compared to those obtained from tomographic reconstruction of intact Dictyostelium nuclei.     

Orai1 mutations alter ion permeation and Ca2+-dependent fast inactivation of CRAC channels: evidence for coupling of permeation and gating

Ca²⁺ entry through store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels is an essential trigger for lymphocyte activation and proliferation. The recent identification of Orai1 as a key CRAC channel pore subunit paves the way for understanding the molecular basis of Ca²⁺ selectivity, ion permeation, and regulation of CRAC channels. Previous Orai1 mutagenesis studies have indicated that a set of conserved acidic amino acids in trans membrane domains I and III and in the I–II loop (E106, E190, D110, D112, D114) are essential for the CRAC channel's high Ca²⁺ selectivity. To further dissect the contribution of Orai1 domains important for ion permeation and channel gating, we examined the role of these conserved acidic residues on pore geometry, properties of Ca²⁺ block, and channel regulation by Ca²⁺. We find that alteration of the acidic residues lowers Ca²⁺ selectivity and results in striking increases in Cs⁺ permeation. This is likely the result of enlargement of the unusually narrow pore of the CRAC channel, thus relieving steric hindrance for Cs⁺ permeation. Ca²⁺ binding to the selectivity filter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca²⁺ binding. Unexpectedly, the mutations diminish Ca²⁺-mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca²⁺ selectivity, increase in Cs⁺ permeability, and enlargement of the pore. We propose that the structural elements involved in ion permeation overlap with those involved in the gating of CRAC channels.     

Structure and function of the nuclear pore complex: new perspectives.

The double membrane of the nuclear envelope is a formidable barrier separating the nucleus and cytoplasm of eukaryotic cells. However, movement of specific macromolecules across the nuclear envelope is critical for embryonic development, cell growth and differentiation. Transfer of molecules between the nucleus and cytoplasm occurs through the aqueous channel formed by the nuclear pore complex (NPC)

1 Abbreviations: NPC, nuclear pore complex; GlcNac, N-acetylglucosamine; WGA, wheat germ agglutinin

. Although small molecules may simply diffuse across the NPC, transport of large proteins and RNA requires specific transport signals and is energy dependent. A family of pore glycoproteins modified by O-linked N-acetylglucosamine moieties are essential for transport through the NPC. Recent evidence suggests that the regulation of nuclear transport may also involve the inteaction of RNA and nuclear proteins with specific binding proteins that recognize these transport signals. Are these nuclear pore glycoproteins and signal binding proteins the ‘gatekeepers’ that control access to the genetic material? Recent evidence obtained from a combination of biochemical and genetic approaches suggests – perhaps.     

Nuclear translocation of proteins and the effect of phosphatidic acid

Transport of proteins containing a nuclear localization signal (NLS) into the nucleus is mediated by nuclear transport receptors called importins, typically dimmers of a cargo-binding α-subunit and a β-subunit that mediates translocation through the nuclear pore complexes (NPCs). However, how proteins without canonical NLS move into the nucleus is not well understood. Recent results indicate that phospholipids, such as phosphatidic acid, play important roles in the intracellular translocation of proteins between the nucleus and cytoplasm.     

Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells

Rotavirus infection modifies Ca²⁺ homeostasis, provoking an increase in Ca²⁺ permeation, the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto), and total Ca²⁺ pools and a decrease in Ca²⁺ response to agonists. A glycosylated viral protein(s), NSP4 and/or VP7, may be responsible for these effects. HT29 or Cos-7 cells were infected by the SA11 clone 28 strain, in which VP7 is not glycosylated, or transiently transfected with plasmids coding for NSP4-enhanced green fluorescent protein (EGFP) or NSP4. The permeability of the plasma membrane to Ca²⁺ and the amount of Ca²⁺ sequestered in the endoplasmic reticulum released by carbachol or ATP were measured in fura-2-loaded cells at the single-cell level under a fluorescence microscope or in cell suspensions in a fluorimeter. Total cell Ca²⁺ pools were evaluated as ⁴⁵Ca²⁺ uptake. Infection with SA11 clone 28 induced an increase in Ca²⁺ permeability and ⁴⁵Ca²⁺ uptake similar to that found with the normally glycosylated SA11 strain. These effects were inhibited by tunicamycin, indicating that inhibition of glycosylation of a viral protein other than VP7 affects the changes of Ca²⁺ homeostasis induced by infection. Expression of NSP4-EGFP or NSP4 in transfected cells induced the same changes observed with rotavirus infection, whereas the expression of EGFP or EGFP-VP4 showed the behavior of uninfected and untransfected cells. Increased ⁴⁵Ca²⁺ uptake was also observed in cells expressing NSP4-EGFP or NSP4, as evidenced in rotavirus infection. These results indicate that glycosylated NSP4 is primarily responsible for altering the Ca²⁺ homeostasis of infected cells through an initial increase of cell membrane permeability to Ca²⁺.     

Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles

The initial rate of Ca²⁺ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca²⁺-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca²⁺-selective minielectrodes to determine pCa values. The initial rate of Ca²⁺ influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca²⁺ movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K⁺. In addition, Ca²⁺ was shown to move across the membrane vesicles in the presence of a K⁺ diffusion potential gradient. The potential-stimulated rate of Ca²⁺ transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl₃, verapamil, and nifedipine had little or no effect. These results indicate that Ca²⁺ transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.     

Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study

All molecular traffic between nucleus and cytoplasm occurs via the nuclear pore complex (NPC) within the nuclear envelope. In this study we analyzed the interactions of the nuclear transport receptors kapα2, kapβ1, kapβ1ΔN44, and kapβ2, and the model transport substrate, BSA-NLS, with NPCs to determine binding sites and kinetics using single-molecule microscopy in living cells. Recombinant transport receptors and BSA-NLS were fluorescently labeled by AlexaFluor 488, and microinjected into the cytoplasm of living HeLa cells expressing POM121-GFP as a nuclear pore marker. After bleaching the dominant GFP fluorescence the interactions of the microinjected molecules could be studied using video microscopy with a time resolution of 5 ms, achieving a colocalization precision of 30 nm. These measurements allowed defining the interaction sites with the NPCs with an unprecedented precision, and the comparison of the interaction kinetics with previous in vitro measurements revealed new insights into the translocation mechanism.     

A short perinuclear amphipathic α-helix in Apq12 promotes nuclear pore complex biogenesis

The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (ER) modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here, we identified a short amphipathic α-helix (AαH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an APQ12 (apq12-ah) version in which AαH is disrupted show NPC biogenesis and NE integrity defects, without impacting Apq12-ah topology or NE/ER localization. Overexpression of APQ12 but not apq12-ah triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AαH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE possibly during NPC biogenesis.     

Mitochondrial Participation in the Intracellular Ca2+ Network

Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca²⁺ uptake and extrusion modulate free cytosolic [Ca²⁺] (Ca_c) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca²⁺ entry, and to monitor Ca²⁺-activated currents that report near-surface [Ca²⁺]. We show that rapid recovery from elevations of Ca_c requires both the mitochondrial Ca²⁺ uniporter and the mitochondrial energization that drives Ca²⁺ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca²⁺] (Ca_m). The indicated resting Ca_m of 100–200 nM is similar to the resting Ca_c reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Ca_m and Ca_c at high temporal resolution shows that, although Ca_m increases less than Ca_c, mitochondrial sequestration of Ca²⁺ is fast and has high capacity. We find that mitochondrial Ca²⁺ uptake limits the rise and underlies the rapid decay of Ca_c excursions produced by Ca²⁺ entry or by mobilization of reticular stores. We also find that subsequent export of Ca²⁺ from mitochondria, seen as declining Ca_m, prolongs complete Ca_c recovery and that suppressing export of Ca²⁺, by inhibition of the mitochondrial Na⁺/ Ca²⁺ exchanger, reversibly hastens final recovery of Ca_c. We conclude that mitochondria are active participants in cellular Ca²⁺ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca²⁺.