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PubMed1994

Isolation of a protein that is essential for the first step of nuclear protein import.

We have purified a cytosolic protein from Xenopus eggs that is essential for selective protein import into the cell nucleus. The purified protein, named importin, promotes signal-dependent binding of karyophilic proteins to the nuclear envelope. We have cloned, sequenced, and expressed a corresponding cDNA. Importin shows 44% sequence identity with SRP1p, a protein associated with the yeast nuclear pore complex. Complete, signal-dependent import into HeLa nuclei can be reconstituted by combining importin purified from Xenopus eggs or expressed in E. coli with Ran/TC4. Evidence for additional stimulatory factors is provided.

Adenosine Triphosphate↗
PubMed2005

Coupling of the nucleus and cytoplasm: role of the LINC complex.

The nuclear envelope defines the barrier between the nucleus and cytoplasm and features inner and outer membranes separated by a perinuclear space (PNS). The inner nuclear membrane contains specific integral proteins that include Sun1 and Sun2. Although the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum, it is nevertheless enriched in several integral membrane proteins, including nesprin 2 Giant (nesp2G), an 800-kD protein featuring an NH(2)-terminal actin-binding domain. A recent study (Padmakumar, V.C., T. Libotte, W. Lu, H. Zaim, S. Abraham, A.A. Noegel, J. Gotzmann, R. Foisner, and I. Karakesisoglou. 2005. J. Cell Sci. 118:3419-3430) has shown that localization of nesp2G to the ONM is dependent upon an interaction with Sun1. In this study, we confirm and extend these results by demonstrating that both Sun1 and Sun2 contribute to nesp2G localization. Codepletion of both of these proteins in HeLa cells leads to the loss of ONM-associated nesp2G, as does overexpression of the Sun1 lumenal domain. Both treatments result in the expansion of the PNS. These data, together with those of Padmakumar et al. (2005), support a model in which Sun proteins tether nesprins in the ONM via interactions spanning the PNS. In this way, Sun proteins and nesprins form a complex that links the nucleoskeleton and cytoskeleton (the LINC complex).

Amino Acid Sequence↗
PubMed1993

NUP2, a novel yeast nucleoporin, has functional overlap with other proteins of the nuclear pore complex.

We have isolated a new gene, NUP2, that encodes a constituent of the yeast-nuclear pore complex (NPC). The NUP2 protein sequence shares a central repetitive domain with NSP1 and NUP1, the two previously characterized yeast nucleoporins. Like NUP1 and NSP1, NUP2 localizes to discrete spots in the nuclear envelope, as determined by indirect immunofluorescence. Although the sequence similarity among these three nucleoporins suggests that they have a similar role in the nuclear pore complex, NUP2, in contrast to NSP1 and NUP1, is not required for growth. Some combinations of mutant alleles of NUP1, NSP1, and NUP2 display "synthetic lethal" relationships that provide evidence for functional interaction between these NPC components. This genetic evidence of overlapping function suggests that the nucleoporins act in concert, perhaps participating in the same step of the recognition or transit of macromolecules through the NPC.

Amino Acid Sequence↗
PubMed1978

Mitotic structure of aortic intimal cells induced by mechanical injury in swine.

Repair processes of the aortic wall have been studied electron microscopically after removal of the endothelium by an inflated balloon procedure in the thoracic aortae of swine. Initial responses after injury included a thrombotic reaction, the appearance of intimal cells over the denuded surface, and increased mitotic activity of medial cells adjacent to the dead cells by day 3. Cells which engaged in intimal thickening were classified as modified smooth muscle cells throughout the course of this investigation. Mitosis of intimal cells, which was initially observed at day 3, substantially increased at day 7 and decreased by day 14. Mitotic intimal cells contained the same cytoplasmic organelles as interphase modified smooth muscle cells. A detailed description of the paired cisternae as an ultrastructural feature of cell division of modified smooth muscle cells was provided. The paired cisternae were initially observed among the remnants of the nuclear envelope in late prophase; they remained at the periphery of the mitotic apparatus in meta- and anaphase, and finally attached themselves to the nuclear envelope of the daughter cells in late telophase.

Animals↗
PubMed1976

Electron-microscope observations on cell nuclei in various tissues of a teleost fish: the nucleolus-associated monolayer of chromatin structural units.

Observations on stain uptake by thin sections through condensed interphase chromosomes in cells from epithelial and muscle tissue in kidney and intestine, and also in fibroblasts, show a distribution into DNA-rich and DNA-poor phases similar to that already described in cells from the connective tissue blood. In all the nuclei the nucleolus, when adjacent to the nuclear envelope, is separated from the inner membrane by a monolayer of chromatin structural units, similar to the monolayer enclosed on both sides by nuclear envelope, previously described in a wide variety of organisms. The data provide further support for the hypothesis that the condensed interphase chromosomes in eukaryotes are characterized by essentially similar structural units folded to form similar patterns. This hypothesis, regarding the higher order units, is consistent with data of others which show that histones and DNA fold to form similar repeating subunits in chromatin, irrespective of the base sequence in the DNA and the origin of the histones.

Animals↗
PubMed2000

Nup50, a nucleoplasmically oriented nucleoporin with a role in nuclear protein export.

We present here a detailed analysis of a rat polypeptide termed Nup50 (formerly NPAP60) that was previously found to be associated with the nuclear pore complex (F. Fan et al., Genomics 40:444-453, 1997). We have found that Nup50 (and/or a related 70-kDa polypeptide) is present in numerous rat cells and tissues. By immunofluorescence microscopy, Nup50 was found to be highly concentrated at the nuclear envelope of rat liver nuclei, whereas in cultured NRK cells it also is abundant in intranuclear regions. On the basis of immunogold electron microscopy of both rat liver nuclear envelopes and NRK cells, we determined that Nup50 is specifically localized in the nucleoplasmic fibrils of the pore complex. Microinjection of anti-Nup50 antibodies into the nucleus of NRK cells resulted in strong inhibition of nuclear export of a protein containing a leucine-rich nuclear export sequence, whereas nuclear import of a protein containing a classical nuclear localization sequence was unaffected. Correspondingly, CRM1, the export receptor for leucine-rich export sequences, directly bound to a fragment of Nup50 in vitro, whereas several other import and export receptors did not significantly interact with this fragment. Taken together, our data indicate that Nup50 has a direct role in nuclear protein export and probably serves as a binding site on the nuclear side of the pore complex for export receptor-cargo complexes.

Animals↗
PubMed1999

Sequence requirements for plasmid nuclear import.

The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells.

Animals↗
PubMed1992

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.

Biological Evolution↗
PubMed2000

Dendrimer-assisted patch-clamp sizing of nuclear pores.

Macromolecular translocation (MMT) across the nuclear envelope (NE) occurs exclusively through the nuclear pore complex (NPC). Therefore, the diameter of the NPC aqueous/electrolytic channel (NPCC) is important for cellular structure and function. The NPCC diameter was previously determined to be approximately equal to 10 nm with electron microscopy (EM) using the translocation of colloidal gold particles. Here we present patch-clamp and fluorescence microscopy data from adult cardiomyocyte nuclei that demonstrate the use of patch-clamp for assessing NPCC diameter. Fluorescence microscopy with B-phycoerythrin (BPE, 240 kDa) conjugated to a nuclear localization signal (NLS) demonstrated that these nuclei were competent for NPC-mediated MMT (NPC-MMT). Furthermore, when exposed to an appropriate cell lysate, the nuclei expressed enhanced green fluorescence protein (EGFP) after 5-10 h of incubation with the plasmid for this protein (pEGFP, 3.1 MDa). Nucleus-attached patch-clamp showed that colloidal gold particles were not useful probes; they modified NPCC gating. As a result of this finding, we searched for an inert class of particles that could be used without irreversibly affecting NPCC gating and found that fluorescently labeled Starburst dendrimers, a distinct class of polymers, were useful. Our patch-clamp and fluorescence microscopy data with calibrated dendrimers indicate that the cardiomyocyte NPCC diameter varies between 8 and 9 nm. These studies open a new direction in the investigation of live, continuous NPC dynamics under physiological conditions.

Animals↗
PubMed1996

Single nuclear pores visualized by confocal microscopy and image processing.

How nuclear pore complexes, mediating the transport of nucleic acids, proteins, and metabolites between cell nucleus and cytoplasm, are arranged in the nuclear envelope is essentially unknown. Here we describe a method combining high-resolution confocal imaging with image processing and pattern recognition to visualize single nuclear pore complexes (120 nm diameter), determine their relative positions with nanometer accuracy, and analyze their distribution in situ. The method was tested by means of a model system in which the very same sample areas could be imaged by confocal and electron microscopy. It was thus found that single fluorescent beads of 105 nm nominal diameter could be localized with a lateral accuracy of <20 nm and an axial accuracy of approximately 20 nm. The method was applied to digitonin-permeabilized 3T3 cells, whose nuclear pore complexes were fluorescently labeled with the anti-nucleoporin antibody mAb414. Stacks of optical sections were generated by confocal imaging at high resolution. Herein the nuclear pore complexes appeared as bright diffraction-limited spots whose centers were localized by fitting them by three-dimensional gaussians. The nearest-neighbor distribution function and the pair correlation function were calculated and found to agree well with those of randomly distributed hard cylinders of 138 +/- 17 nm diameter, but not with those of randomly distributed points or nonrandomly distributed cylinders. This was supported by a cluster analysis. Implications for the direct observation of the transport of single particles and molecules through individual nuclear pore complexes are discussed.

3T3 Cells↗
PubMed1985

Foreign protein can be carried into the nucleus of mammalian cell by conjugation with nucleoplasmin.

In studies on the specific migration of macromolecules across the nuclear envelope, a karyophilic protein was injected into the cytoplasm of cultured cells and its subsequent location in the cell was examined. Nucleoplasmin of frog nuclear protein was used for this experiment. When [125I]nucleoplasmin was introduced into the cytoplasm of mammalian cells (human and mouse) by red blood cell-mediated microinjection, it rapidly accumulated in the nucleus. When nucleoplasmin conjugated with [125I]IgG against chromosomal protein was introduced similarly, it also accumulated rapidly in the nucleus, and reacted with its antigen inside the nucleus. On the contrary, when IgG alone or IgG conjugated with BSA were introduced, they did not migrate from the cytoplasm into the nucleus. These findings imply that the migration of macromolecules from the cytoplasm to the nucleus does not depend only on their molecular size but also on a specific transport mechanism, and that karyophilic proteins may act as useful carriers in the transfer of exogenous proteins into the nucleus.

Animals↗
PubMed2003

Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance liquid chromatography-thermospray mass spectrometry.

I have developed a simple method which enabled simultaneous analysis of ceramides in the subcellular fractions from cultured cells by HPLC-thermospray mass spectrometry. The HPLC-thermospray mass spectra from ceramide standards were characterized by the high intensity of the MNa(+) and MH(+)-H(2)O ions. As the other minor ions, MK(+), MH(+) and m/z 282 ions were detected. Although the preponderance of MNa(+) ions compared with the MH(+)-H(2)O ions was detected in non-hydroxy fatty acid-ceramides, the preponderance of MH(+)-H(2)O ions based on the elimination of the hydroxyl group introduced at the alpha-position of acyl-portion compared with the MNa(+) ions was detected in alpha-hydroxy fatty acid-ceramides. In calibrations for authentic ceramides using N-octanoylsphingosine as an internal standard, an approximately linear relationship existed between the ratios of peak-areas of each ceramide to that of the internal standard and the known amounts of each ceramide. The factor (f) of each ceramide was calculated as follows; N-oleoyl-D-sphingosine (f=0.45), N-palmitoyl-D-sphingosine (f=0.40), N-stearoyl-D-sphingosine (f=0.39), N-nervonoyl-D-sphingosine (f=0.39) and N-lignoceroyl-D-sphingosine (f=0.35). In subcellular fractions from A549 and HepG2 cells, although ceramide species content per mg protein was high in the nuclear envelope fractions, the 7000 g pellet fractions and the 100000 g pellet fractions, a large portion of the ceramide species was concentrated in the nuclear envelope fraction. In addition, this method was applied to a mild alkaline hydrolyzate of total ceramides from pig stratum corneum, and MNa(+)/MH(+)-H(2)O ions corresponding to several omega-hydroxyacyl-ceramides were detected.

Animals↗
PubMed1995

Disruption of the nucleoporin gene NUP133 results in clustering of nuclear pore complexes.

We have characterized a protein with an estimated molecular mass of 130 kDa that is contained in a highly enriched yeast nuclear pore complex (NPC) fraction. Partial amino acid sequence from this protein has led us to a previously identified open reading frame on chromosome XI of Saccharomyces cerevisiae encoding a protein of 133 kDa. Due to its coenrichment with NPCs during cell fractionation and the phenotype observed in the disrupted strain, we propose to term the gene encoding this protein NUP133. Cells carrying a disrupted copy of NUP133 were temperature sensitive for growth. In addition, abnormal clustering of NPCs was observed. This phenotype is similar to that previously observed in the disruption of another nucleoporin gene, NUP145. We speculate that the gene product of NUP133, Nup133p, may functionally overlap with the NUP145 gene product, Nup145p, and that these proteins may be involved in maintaining the position of the NPC within the nuclear envelope.

Fluorescent Antibody Technique↗
PubMed1978

Mitosis in Barbulanympha. I. Spindle structure, formation, and kinetochore engagement.

Successful culture of the obligatorily anaerobic symbionts residing in the hindgut of the wood-eating cockroach Cryptocercus punctulatus now permits continuous observation of mitosis in individual Barbulanympha cells. In Part I of this two-part paper, we report methods for culture of the protozoa, preparation of microscope slide cultures in which Barbulanympha survived and divided for up to 3 days, and an optical arrangement which permits observation and through-focus photographic recording of dividing cells, sequentially in differential interference contrast and rectified polarized light microscopy. We describe the following prophase events and structures: development of the astral rays and large extranuclear central spindle from the tips of the elongate-centrioles; the fine structure of spindle fibers and astral rays which were deduced in vivo from polarized light microscopy and seen as a particular array of microtubules in thin-section electron micrographs; formation of chromosomal spindle fibers by dynamic engagement of astral rays to the kinetochores embedded in the persistent nuclear envelope; and repetitive shortening of chromosomal spindle fibers which appear to hoist the nucleus to the spindle surface, cyclically jostle the kinetochores within the nuclear envelope, and churn the prophase chromosomes. The observations described here and in Part II have implications both for the evolution of mitosis and for understanding the mitotic process generally.

Anaerobiosis↗
PubMed2006

Parvoviral nuclear import: bypassing the host nuclear-transport machinery.

The parvovirus Minute virus of mice (MVM) is a small DNA virus that replicates in the nucleus of its host cells. However, very little is known about the mechanisms underlying parvovirus' nuclear import. Recently, it was found that microinjection of MVM into the cytoplasm of Xenopus oocytes causes damage to the nuclear envelope (NE), suggesting that the nuclear-import mechanism of MVM involves disruption of the NE and import through the resulting breaks. Here, fluorescence microscopy and electron microscopy were used to examine the effect of MVM on host-cell nuclear structure during infection of mouse fibroblast cells. It was found that MVM caused dramatic changes in nuclear shape and morphology, alterations of nuclear lamin immunostaining and breaks in the NE of infected cells. Thus, it seems that the unusual nuclear-import mechanism observed in Xenopus oocytes is in fact used by MVM during infection of host cells.

Animals↗
PubMed1998

A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells.

Eukaryotes respond to the presence of unfolded protein in the endoplasmic reticulum (ER) by up-regulating the transcription of genes encoding ER protein chaperones, such as BiP. We have isolated a novel human cDNA encoding a homolog to Saccharomyces cerevisiae Ire1p, a proximal sensor for this signal transduction pathway in yeast. The gene product hIre1p is a type 1 transmembrane protein containing a cytoplasmic domain that is highly conserved to the yeast counterpart having a Ser/Thr protein kinase domain and a domain homologous to RNase L. However, the luminal domain has extensively diverged from the yeast gene product. hIre1p expressed in mammalian cells displayed intrinsic autophosphorylation activity and an endoribonuclease activity that cleaved the 5' splice site of yeast HAC1 mRNA, a substrate for the endoribonuclease activity of yeast Ire1p. Overexpressed hIre1p was localized to the ER with particular concentration around the nuclear envelope and some colocalization with the nuclear pore complex. Expression of Ire1p mRNA was autoregulated through a process that required a functional hIre1p kinase activity. Finally, overexpression of wild-type hIre1p constitutively activated a reporter gene under transcriptional control of the rat BiP promoter, whereas expression of a catalytically inactive hIre1p acted in a trans-dominant-negative manner to prevent transcriptional activation of the BiP promoter in response to ER stress induced by inhibition of N-linked glycosylation. These results demonstrate that hIre1p is an essential proximal sensor of the unfolded protein response pathway in mammalian cells.

Alanine↗
PubMed1997

Nuclear assembly.

We review old and new insights into the structure of the nuclear envelope and the components responsible for its dynamic reassembly during mitosis. New information is coming to light about several of the proteins that mediate nuclear reassembly. These proteins include the lamins and their emerging relationship with proteins such as otefin and the MAN antigens: peripheral proteins that might participate in lamina structure. There are four identified proteins localized to the inner nuclear membrane: the lamina-associated proteins LAP1 and LAP2, emerin, and the lamin B receptor (LBR). LBR can interact independently with lamin B and a chromodomain protein, Hp1, and appears to be a central player in targeting nuclear membranes to chromatin. Intermediates in the assembly of nuclear pore complexes (NPCs) can now be studied biochemically and visualized by high resolution scanning electron microscopy. We discuss the possibility that the filament-forming proteins Tpr/p270, NuMA, and perhaps actin may have roles in nuclear assembly.

Animals↗
PubMed1999

Structural plasticity of the cardiac nuclear pore complex in response to regulators of nuclear import.

Communication between the cytoplasm and nucleoplasm of cardiac cells occurs by molecular transport through nuclear pores. In lower eukaryotes, nuclear transport requires the maintenance of cellular energetics and ion homeostasis. Although heart muscle is particularly sensitive to metabolic stress, the regulation of nuclear transport through nuclear pores in cardiomyocytes has not yet been characterized. With the use of laser confocal and atomic force microscopy, we observed nuclear transport in cardiomyocytes and the structure of individual nuclear pores under different cellular conditions. In response to the depletion of Ca2+ stores or ATP/GTP pools, the cardiac nuclear pore complex adopted 2 distinct conformations that led to different patterns of nuclear import regulation. Depletion of Ca2+ indiscriminately prevented the nuclear import of macromolecules through closure of the nuclear pore opening. Depletion of ATP/GTP only blocked facilitated transport through a simultaneous closure of the pore and relaxation of the entire complex, which allowed other molecules to pass into the nucleus through peripheral routes. The current study of the structural plasticity of the cardiac nuclear pore complex, which was observed in response to changes in cellular conditions, identifies a gating mechanism for molecular translocation across the nuclear envelope of cardiac cells. The cardiac nuclear pore complex serves as a conduit that differentially regulates nuclear transport of macromolecules and provides a mechanism for the control of nucleocytoplasmic communication in cardiac cells, in particular under stress conditions associated with disturbances in cellular bioenergetics and Ca2+ homeostasis.

Adaptation, Physiological↗