Nuclear transport kinetics in microarrays of nuclear envelope patches

Optical Single Transporter Recording (OSTR) is a technique for analyzing membrane transport kinetics at high sensitivity, selectivity, and spatial resolution. Cellular membranes are firmly attached to microarrays of small test compartments (TCs) with diameters between approximately 0.1 and 100 microm and depths between approximately 10 and 100 microm. This permits to generate either "small" membrane patches containing few transporters or "large" patches containing many transporters. Transport of substrates across membrane patches is recorded by confocal microscopy. The present article reviews recent applications of OSTR to the nuclear pore complex (NPC). The results show that the transport functions of the NPC, previously studied almost exclusively in intact and permeabilized cells, are conserved in isolated nuclei and can be fully reconstituted in purified nuclear envelopes by addition of recombinant transport factors. This opens new avenues to the analysis of nuclear transport including the export of nucleic-acid-protein and ribosomal particles.     

Correlation between nucleocytoplasmic transport and caspase-3-dependent dismantling of nuclear pores during apoptosis

During apoptosis (also called programmed cell death), the chromatin condenses and the DNA is cleaved into oligonucleosomal fragments. Caspases are believed to play a major role in nuclear apoptosis. However, the relation between dismantling of nuclear pores, disruption of the nucleocytoplasmic barrier, and nuclear entry of caspases is unclear. We have analyzed nuclear import of the green fluorescent protein fused to a nuclear localization signal (GFP-NLS) in tissue culture cells undergoing apoptosis. Decreased nuclear accumulation of GFP-NLS could be detected at the onset of nuclear apoptosis manifested as dramatic condensation and redistribution of chromatin toward the nuclear periphery. At this step, dismantling of nuclear pores was already evident as indicated by proteolysis of the nuclear pore membrane protein POM121. Thus, disruption of nuclear compartmentalization correlated with early signs of nuclear pore damage. Both these events clearly preceded massive DNA fragmentation, detected by TUNEL assay. Furthermore, we show that in apoptotic cells, POM121 is specifically cleaved at aspartate-531 in its large C-terminal portion by a caspase-3-dependent mechanism. Cleavage of the C-terminal portion of POM121, which is adjoining the nuclear pore complex, is likely to disrupt interactions with other nuclear pore proteins affecting the stability of the pore complex. A temporal correlation of apoptotic events supports a model where caspase-dependent disassembly of nuclear pores and disruption of the nucleocytoplasmic barrier paves the way for nuclear entry of caspases and subsequent activation of CAD-mediated DNA fragmentation.     

Nuclear electrophysiology

Conclusions Patch-clamp, fluorescence microscopy and high-resolution EM have yielded new data which question current concepts of ion transport across the nuclear envelope. The current challenge is to prove that NICs play an important role in nuclear function either through their identity with NPCs or parts thereof. Electrophysiological designs must incorporate cell biology approaches as done for putative protein-conducting channels of the ER (Simon & Blobel, 1991, 1992).Preliminary studies (J.O. Bustamante et al., in preparation), illustrated in Fig. 1, confirm that, as is the case of NPCs, NICs cannot function in an extracellular environment deprived of cytosolic factors. Our current efforts aim at clarifying if the lysate factors required for macromolecular transport through NPCs (e.g., Adam et al., 199la,b) are those required for NIC open-shut gating. Monoclonal antibodies to identified NPC proteins should be helpful in furthering the identification of NICs with NPCs. Our observation of blockade of NIC activity with wheat germ agglutinin, discussed above, supports the idea that NPCs are the structural foundation for NICs. Should NICs be identified with NPCs or otherwise proven essential to nucleocytoplasmic transport, NIC response to cytoplasmic signals would suggest that they are relevant to mediating gene control by transduction and other cytosolic signals (Karin, 1991; Davis, 1992). NIC influence on intranuclear free ion concentrations is potentially important to controlling gene activation, repression, as well as the efficiency and fidelity of gene expression (e.g., Kroeger, 1963; Lezzi & Gilbert, 1970; Leake et al., 1972; Morgan & Curran, 1986; Li & Rokita, 1991; Lippard, 1993). As electrophysiological and cell/molecular biology approaches merge, the prospects improve for the field of nuclear electrophysiology.The author thanks (in alphabetical order) the intellectual contributions of Drs. Christopher W. Akey, Gregory S. Beckler (Promega), Louis J. DeFelice, Colin Dingwall, Alexander Fabiato, Julio M. Fernández, Larry Gerace, John A. Hanover, Bertil Hille, Stuart L. Jacobson, W. Jonathan Lederer, Andrejs Liepins, Gilbert N. Ling, Michele Mazzanti, Ernst Niggli, Sanford M. Simon, Walter Stühmer, and W. Gil Wier. Special thanks are tendered to Drs. Dingwall, Gerace, Hanover and Liepins for their observations on nuclear electrophysiology within the context of cell/molecular biology. Thanks are also extended to Drs. Lederer and Wier for discussions on fluorescence microscopy of Ca²⁺ transients. Dr. Niggli provided the preprint of his paper, with P. Lipp, confirming previous observations that cardiomyocyte nuclei behave as a barrier to intracellular Ca²⁺ waves. Drs. DeFelice and Mazzanti provided a draft of their review on the biophysics of the nuclear envelope. This work is supported by the American Heart Association, Maryland Affiliate. Institutional support and facilities have come through Drs. C. William Balke, Michael R. Gold, W. Gil Wier and W. Jonathan Lederer, to whom the author is deeply grateful. This work is dedicated to my parents for introducing me to scientific curiosity and for their constant incentive and support. A special dedication to my father who recently passed away.     

核孔复合体的结构及其功能

核孔复合体(Nuclear pore complexes, NPCs)镶嵌在核膜上,是细胞核与细胞质之间的唯一通道。冷冻电子X射线断层扫描将环状NPCs分为三个环,分别称为胞质环、内环和核质环,胞质环上附有胞质纤丝,核质环上附有核篮。由于物种不同,NPCs由30～50多种不同的核孔蛋白(nucleoporins, Nups)组成,但结构和功能高度保守。根据其结构、氨基酸序列,NPCs定位和功能,Nups被分为跨膜Nups、屏障Nups、骨架Nups、胞质纤丝Nups和核篮Nups。相互间作用稳定、紧密连接的数个Nups可组成亚复合体。为了应对不同生理需要,NPCs处于高度动态变化中,间期和有丝分裂期均可通过组装和去组装改变核孔数量和功能。NPCs的主要功能是调控核质转运,小分子物质可自由扩散,大分子物质则需在核转位信号和转运载体的介导下以主动运输的方式进行转运。除了核质转运这一主要功能外,Nups还能以一个独立于转运的方式影响基因组功能。通过影响染色质结构和影响转录调控元件对靶基因的访问,Nups促进或抑制转录。在酵母,Nups介导的基因调控主要由位于NPCs中的Nups执行;在多细胞生物,不仅NPCs中的Nups,核质内游离的Nups也具有基因调控功能。此外,Nups还能通过参与形成染色质边界和形成转录记忆对基因进行调控。在增殖细胞, Nups通过与DNA修复机器相互作用,参与DNA损伤修复,保护基因组完整性。有丝分裂时,Nups协助核膜解体和中心体迁移,并通过作用于着丝粒来控制有丝分裂组件的空间定位与活性,稳定它们与微管之间的相互作用,保证纺锤体正常组装和染色体准确分离。总之,NPCs与生物分子的核质转运、基因表达和细胞周期密切相关,它的结构和功能的稳定是真核细胞生长、增殖、分化等生命活动的基本保证。     

The transmembrane nucleoporin NDC1 is required for targeting of ALADIN to nuclear pore complexes

NDC1 is a transmembrane nucleoporin that is required for NPC assembly and nucleocytoplasmic transport. We show here that NDC1 directly interacts with the nucleoporin ALADIN, mutations of which are responsible for triple-A syndrome, and that this interaction is required for targeting of ALADIN to nuclear pore complexes (NPCs). Furthermore, we show that NDC1 is required for selective nuclear import. Our findings suggest that NDC1-mediated localization of ALADIN to NPCs is essential for selective nuclear protein import, and that abrogation of the interaction between ALADIN and NDC1 may be important for the development of triple-A syndrome.     

Landscape of nuclear transport receptor cargo specificity

Nuclear transport receptors (NTRs) recognize localization signals of cargos to facilitate their passage across the central channel of nuclear pore complexes (NPCs). About 30 different NTRs constitute different transport pathways in humans and bind to a multitude of different cargos. The exact cargo spectrum of the majority of NTRs, their specificity and even the extent to which active nucleocytoplasmic transport contributes to protein localization remains understudied because of the transient nature of these interactions and the wide dynamic range of cargo concentrations. To systematically map cargo–NTR relationships in situ, we used proximity ligation coupled to mass spectrometry (BioID). We systematically fused the engineered biotin ligase BirA* to 16 NTRs. We estimate that a considerable fraction of the human proteome is subject to active nuclear transport. We quantified the specificity and redundancy in NTR interactions and identified transport pathways for cargos. We extended the BioID method by the direct identification of biotinylation sites. This approach enabled us to identify interaction interfaces and to discriminate direct versus piggyback transport mechanisms. Data are available via ProteomeXchange with identifier PXD007976.     

基因打靶技术在人类疾病动物模型研究中的应用

人类疾病动物模型在医学研究中起着非常重要的作用,而自然发生的人类疾病已远远不能满足医学研究的需要。利用基因打靶技术开展人类疾病动物模型的研究具有广阔的应用前景。本文就基因打靶技术及其在人类疾病动物模型研究中的应用做一介绍。     

SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.     

Using peptide arrays to define nuclear carrier binding sites on nucleoporins

In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-beta. Crystallization studies by others have indicated that nuclear carriers such as importin-beta bind to phenylalanine-glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-beta identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-beta. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-beta binding to those repeats. In addition to these expected binding sequences for importin-beta, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-beta, indicating that additional Nup sequences may form binding sites for importin-beta.     

Nuclear-pore-complex dynamics and transport in higher eukaryotes

Summary The nuclear-pore complex controls the passage of macromolecules to and from the nucleus. It is a complex structure spanning the double-membrane nuclear envelope, consisting of many proteins and structural components. Structurally it consists of a series of stacked rings and associated filaments and a central cylinder which appears to contain the transport channel. Much of the pore complex appears to be dynamic, altering conformationally during transport. We review what is known about pore complex structure and dynamics and attempt to relate this to recent new information on transport pathways and the interactions of transport factors with each other and with components of the nuclear-pore complex.     

Nuclear transport and nuclear pores in yeast

The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumulation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes. Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an efficient structural and functional analysis of these components. Due to the evolutionary conservation potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus, yeast can be considered as a eukaryotic model system to study nuclear transport.     

Computational and biochemical identification of a nuclear pore complex binding site on the nuclear transport carrier NTF2

Nuclear transport carriers interact with proteins of the nuclear pore complex (NPC) to transport their cargo across the nuclear envelope. One such carrier is nuclear transport factor 2 (NTF2), whose import cargo is the small GTPase Ran. A domain highly homologous to the small NTF2 protein (14kDa) is also found in a number of additional proteins, which together make up the NTF2 domain containing superfamily of proteins. Using structural, computational and biochemical analysis we have identified a functional site that is present throughout this superfamily, and our results indicate that this site functions as an NPC binding site in NTF2. Previously we showed that a D23A mutant of NTF2 exhibits increased affinity for the NPC. The mechanism of this mutation, however, was unknown as this region of NTF2 had not been implicated in binding to NPC proteins. Here we show that the D23A mutation in NTF2 does not result in gross structural changes affecting other known NPC binding sites. Instead, the D23 residue is located in an evolutionarily important region in the NTF2 domain containing superfamily, that in NTF2, is involved in binding to the NPC.     

The yeast nuclear pore complex: composition, architecture, and transport mechanism

An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.     

Cryo-electron tomography provides novel insights into nuclear pore architecture: implications for nucleocytoplasmic transport

To go beyond the current structural consensus model of the nuclear pore complex (NPC), we performed cryo-electron tomography of fully native NPCs from Xenopus oocyte nuclear envelopes (NEs). The cytoplasmic face of the NPC revealed distinct anchoring sites for the cytoplasmic filaments, whereas the nuclear face was topped with a massive distal ring positioned above the central pore with indications of the anchoring sites for the nuclear basket filaments and putative intranuclear filaments. The rather "spongy" central framework of the NPC was perforated by an elaborate channel and void system, and at the membrane pore interface it exhibited distinct "handles" protruding into the lumen of the NE. The most variable structural moiety of the NPC was a rather tenuous central plug partially obstructing the central pore. Its mobile character was documented by time-lapse atomic force microscopy. Taken together, the new insights we gained into NPC structure support the notion that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargoes.     

Gene targeting in mouse embryos mediated by RecA and modified single-stranded oligonucleotides

Gene targeting is a precise manipulation of endogenous gene by introduction of exogenous DNA and has contributed greatly to the elucidation of gene functions. Conventional gene targeting has been achieved through a use of embryonic stem cells. However, such procedure is often long, tedious, and expensive. This study was carried out to develop a simple procedure of gene targeting using E. coli recombinase A (RecA) and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene in mouse embryos. The single-stranded oligonucleotide to target an exon 3 of HPRT was 74 bases in length including phosphorothioate linkages at each terminus to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except a single nucleotide that induces a mismatch between an introduced oligonucleotide and endogenous HPRT gene. Endogenous repairing of such mismatch would give rise to the conversion of TAT to TAG stop codon thereby losing the function of the target gene. Before an introduction into zygotes, single-stranded oligonucleotides were bound to RecA to enhance the homologous recombination. The RecA–oligonucleotide complex was microinjected into the pronucleus of zygote. Individual microinjected embryos developed to the blastocyst stage were analyzed for the expected nucleotide conversion using polymerase chain reaction (PCR) and subsequent sequencing. The conversion of TAT to TAG stop codon was detected in three embryos among 48 tested blastocysts (6.25% in frequency). The result suggests that the gene targeting was feasible by relatively easier and direct method.     

Chromatin organization in relation to the nuclear periphery

In the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery.