Asymmetrical localization of Nup107-160 subcomplex components within the nuclear pore complex in fission yeast.

The nuclear pore complex (NPC) forms a gateway for nucleocytoplasmic transport. The outer ring protein complex of the NPC (the Nup107-160 subcomplex in humans) is a key component for building the NPC. Nup107-160 subcomplexes are believed to be symmetrically localized on the nuclear and cytoplasmic sides of the NPC. However, in S. pombe immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast.

Roles of Nup133, Nup153 and membrane fenestrations in assembly of the nuclear pore complex at the end of mitosis.

Reassembly of the nuclear pore complex (NPC) at the end of mitosis is an important event for eukaryotic nuclear function. In this study, we examined the dynamic behaviors of the endoplasmic reticulum (ER) by "Live CLEM" imaging. In metaphase, numerous fenestrations on the ER membrane were observed around chromosomes. In telophase, these fenestrations became filled at the region attached to chromosomes, whereas they remained open at the region unattached to chromosomes, suggesting that NPC assembly takes place at fenestrations on the membrane. To determine the roles of nucleoporins in postmitotic NPC formation, we used artificial beads conjugated with anti-GFP antibody, which captures GFP-fused proteins on the beads when incorporated into cells. Live CLEM imaging of telophase cells containing Nup133-coated beads or Nup153-coated beads showed that Nup133 and Nup153, as the sole effector molecules, assembled the NPC-like structure on the membrane fenestrations. Indirect immunofluorescence staining of the Nup133-coated beads showed that Nup133 effectively assembled Nup107 and ELYS, whereas minimal assembly of Nup98 and Nup62 was observed; the Nup153-coated bead effectively assembled Nup98, Nup62 and Pom121, but assembled neither Nup107 nor ELYS. Our results suggest that Nup133 and Nup153 play different roles in assembling the NPC on membrane fenestrations.

BAF is a cytosolic DNA sensor that leads to exogenous DNA avoiding autophagy.

Knowledge of the mechanisms by which a cell detects exogenous DNA is important for controlling pathogen infection, because most pathogens entail the presence of exogenous DNA in the cytosol, as well as for understanding the cell's response to artificially transfected DNA. The cellular response to pathogen invasion has been well studied. However, spatiotemporal information of the cellular response immediately after exogenous double-stranded DNA (dsDNA) appears in the cytosol is lacking, in part because of difficulties in monitoring when exogenous dsDNA enters the cytosol of the cell. We have recently developed a method to monitor endosome breakdown around exogenous materials using transfection reagent-coated polystyrene beads incorporated into living human cells as the objective for microscopic observations. In the present study, using dsDNA-coated polystyrene beads (DNA-beads) incorporated into living cells, we show that barrier-to-autointegration factor (BAF) bound to exogenous dsDNA immediately after its appearance in the cytosol at endosome breakdown. The BAF(+) DNA-beads then assembled a nuclear envelope (NE)-like membrane and avoided autophagy that targeted the remnants of the endosome membranes. Knockdown of BAF caused a significant decrease in the assembly of NE-like membranes and increased the formation of autophagic membranes around the DNA-beads, suggesting that BAF-mediated assembly of NE-like membranes was required for the DNA-beads to evade autophagy. Importantly, BAF-bound beads without dsDNA also assembled NE-like membranes and avoided autophagy. We propose a new role for BAF: remodeling intracellular membranes upon detection of dsDNA in mammalian cells.

Biased assembly of the nuclear pore complex is required for somatic and germline nuclear differentiation in Tetrahymena.

Ciliates have two functionally distinct nuclei, a somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Understanding this nuclear dimorphism is a central issue in ciliate biology. We show, by live-cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior-posterior polarized nuclei: MAC-specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC-specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Correlative light-electron microscopy shows that addition of new nuclear envelope (NE) precursors occurs through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD results in assembly of MAC-specific NPCs in the division-failed zygotic nuclei, leading to failure of MIC differentiation. Our findings demonstrate that NPC type switching has a crucial role in the establishment of nuclear differentiation in ciliates.

Inner nuclear membrane protein Lem2 augments heterochromatin formation in response to nutritional conditions.

Inner nuclear membrane proteins interact with chromosomes in the nucleus and are important for chromosome activity. Lem2 and Man1 are conserved members of the LEM-domain nuclear membrane protein family. Mutations of LEM-domain proteins are associated with laminopathy, but their cellular functions remain unclear. Here, we report that Lem2 maintains genome stability in the fission yeast Schizosaccharomyces pombe. S. pombe cells disrupted for the lem2(+) gene (lem2∆) showed slow growth and increased rate of the minichromosome loss. These phenotypes were prominent in the rich culture medium, but not in the minimum medium. Centromeric heterochromatin formation was augmented upon transfer to the rich medium in wild-type cells. This augmentation of heterochromatin formation was impaired in lem2∆ cells. Notably, lem2∆ cells occasionally exhibited spontaneous duplication of genome sequences flanked by the long-terminal repeats of retrotransposons. The resulting duplication of the lnp1(+) gene, which encodes an endoplasmic reticulum membrane protein, suppressed lem2∆ phenotypes, whereas the lem2∆ lnp1∆ double mutant showed a severe growth defect. A combination of mutations in Lem2 and Bqt4, which encodes a nuclear membrane protein that anchors telomeres to the nuclear membrane, caused synthetic lethality. These genetic interactions imply that Lem2 cooperates with the nuclear membrane protein network to regulate genome stability.

Inner nuclear membrane protein Ima1 is dispensable for intranuclear positioning of centromeres.

Inner nuclear membrane (INM) proteins play a role in spatial organization of chromosomes within the nucleus. In the fission yeast Schizosaccharomyces pombe, Sad1, an INM protein of the conserved SUN-domain family, plays an active role in moving chromosomes along the nuclear membranes during meiotic prophase. Ima1 is another conserved INM protein recently identified. A previous study claimed that Ima1 is essential for mitotic cell growth, linking centromeric heterochromatin to the spindle-pole body. However, we obtained results contradictory to the previously proposed role for Ima1: Ima1 was dispensable for mitotic cell growth or centromere positioning. This discrepancy was attributed to incorrect ima1 deletion mutants used in the previous study. Our results show that Ima1 collaborates with two other conserved INM proteins of the LEM-domain family that are homologous to human Man1 and Lem2. Loss of any one of three INM proteins has no effect on mitotic cell growth; however, loss of all these proteins causes severe defects in mitotic cell growth and nuclear membrane morphology. Considering that all three INM proteins interact with Sad1, these results suggest that Ima1, Lem2 and Man1 play at least partially redundant roles for nuclear membrane organization.

Visualization of secretory cargo transport within the Golgi apparatus.

To describe trafficking of secretory cargo within the Golgi apparatus, the cisternal maturation model predicts that Golgi cisternae change their properties from cis to trans while cargo remains in the cisternae. Cisternal change has been demonstrated in living yeast Saccharomyces cerevisiae; however, the behavior of cargo has yet to be examined directly. In this study, we conducted simultaneous three-color and four-dimensional visualization of secretory transmembrane cargo together with early and late Golgi resident proteins. We show that cargo stays in a Golgi cisterna during maturation from cis-Golgi to trans-Golgi and further to the trans-Golgi network (TGN), which involves dynamic mixing and segregation of two zones of the earlier and later Golgi resident proteins. The location of cargo changes from the early to the late zone within the cisterna during the progression of maturation. In addition, cargo shows an interesting behavior during the maturation to the TGN. After most cargo has reached the TGN zone, a small amount of cargo frequently reappears in the earlier zone.

Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore.

Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe, the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the γ-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs.

Peroxisomes are formed from complex membrane structures in PEX6-deficient CHO cells upon genetic complementation.

Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti-70-kDa peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double-membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double-membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that 1) Peroxisomal ghosts are complex membrane structures; and 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.

Lipid droplet dynamics during Schizosaccharomyces pombe sporulation and their role in spore survival.

Upon nitrogen starvation, the fission yeast Schizosaccharomyces pombe forms dormant spores; however, the mechanisms by which a spore sustains life without access to exogenous nutrients remain unclear. Lipid droplets are reservoirs of neutral lipids that act as important cellular energy resources. Using live-cell imaging analysis, we found that the lipid droplets of mother cells redistribute to their nascent spores. Notably, this process was actin polymerization-dependent and facilitated by the leading edge proteins of the forespore membrane. Spores lacking triacylglycerol synthesis, which is essential for lipid droplet formation, failed to germinate. Our results suggest that the lipid droplets are important for the sustenance of life in spores.

Aberrant peroxisome morphology in peroxisomal beta-oxidation enzyme deficiencies.

Peroxisomes are ubiquitous organelles in eukaryotic cells and surrounded by a single membrane, and undergo considerable changes in size, shape and number. Peroxisomal disorders are classified into two categories: peroxisome biogenesis disorders (PBDs) and single-enzyme deficiencies (SEDs). Morphologically aberrant peroxisomes called 'peroxisomal ghosts' in PBDs are well known, however, a morphological approach to the study of peroxisomes in SEDs has been rarely reported. Here, we investigated the morphology of peroxisomes in cultured fibroblasts from patients lacking peroxisomal beta-oxidation enzymes, including acyl-CoA oxidase (AOX) or D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-BP). Morphological analysis by immunofluorescence examination using an antibody against catalase revealed a smaller number of large peroxisomes in fibroblasts from these patients. Moreover, immunoelectron microscopy using an antibody against the 70-kDa peroxisomal membrane protein (PMP70) showed large peroxisomes with various horseshoe-shaped membrane structures. These results give an important clue to elucidating the division of peroxisomes and how peroxisomes change in size, shape, number and position within cells, which are subjects for future study.

Interphase adhesion geometry is transmitted to an internal regulator for spindle orientation via caveolin-1.

Despite theoretical and physical studies implying that cell-extracellular matrix adhesion geometry governs the orientation of the cell division axis, the molecular mechanisms that translate interphase adhesion geometry to the mitotic spindle orientation remain elusive. Here, we show that the cellular edge retraction during mitotic cell rounding correlates with the spindle axis. At the onset of mitotic cell rounding, caveolin-1 is targeted to the retracting cortical region at the proximal end of retraction fibres, where ganglioside GM1-enriched membrane domains with clusters of caveola-like structures are formed in an integrin and RhoA-dependent manner. Furthermore, Gαi1-LGN-NuMA, a well-known regulatory complex of spindle orientation, is targeted to the caveolin-1-enriched cortical region to guide the spindle axis towards the cellular edge retraction. We propose that retraction-induced cortical heterogeneity of caveolin-1 during mitotic cell rounding sets the spindle orientation in the context of adhesion geometry.

Deficiency of a lipid droplet protein, perilipin 5, suppresses myocardial lipid accumulation, thereby preventing type 1 diabetes-induced heart malfunction.

Lipid droplet (LD) is a ubiquitous organelle that stores triacylglycerol and other neutral lipids. Perilipin 5 (Plin5), a member of the perilipin protein family that is abundantly expressed in the heart, is essential to protect LDs from attack by lipases, including adipose triglyceride lipase. Plin5 controls heart metabolism and performance by maintaining LDs under physiological conditions. Aberrant lipid accumulation in the heart leads to organ malfunction, or cardiomyopathy. To elucidate the role of Plin5 in a metabolically disordered state and the mechanism of lipid-induced cardiomyopathy, we studied the effects of streptozotocin-induced type 1 diabetes in Plin5-knockout (KO) mice. In contrast to diabetic wild-type mice, diabetic Plin5-KO mice lacked detectable LDs in the heart and did not exhibit aberrant lipid accumulation, excessive reactive oxygen species (ROS) generation, or heart malfunction. Moreover, diabetic Plin5-KO mice exhibited lower heart levels of lipotoxic molecules, such as diacylglycerol and ceramide, than wild-type mice. Membrane translocation of protein kinase C and the assembly of NADPH oxidase 2 complex on the membrane were also suppressed. The results suggest that diabetic Plin5-KO mice are resistant to type 1 diabetes-induced heart malfunction due to the suppression of the diacylglycerol/ceramide-protein kinase C pathway and of excessive ROS generation by NADPH oxidase.

In vivo evidence for the fibrillar structures of Sup35 prions in yeast cells.

Yeast prion [PSI(+)] is caused by aggregated structures of the Sup35 protein. Although Sup35 forms typical amyloid fibrils in vitro, there is no direct evidence for the fibrillar structures of Sup35 in vivo. We analyzed [PSI(+)] cells in which Sup35 fused with green fluorescent protein (GFP) formed aggregates visible by fluorescence microscopy using thin-section electron microscopy (EM). Rapid-freeze EM combined with an immunogold-labeling technique as well as correlative light EM, which allows high-resolution imaging by EM of the same structure observed by light (fluorescence) microscopy, shows that the aggregates contain bundled fibrillar structures of Sup35-GFP. Additional biochemical and fluorescent correlation spectroscopy results suggest that the Sup35 oligomers diffused in the [PSI(+)] lysates adopt fibril-like shapes. Our findings demonstrate that [PSI(+)] cells contain Sup35 fibrillar structures closely related to those formed in vitro and provide insight into the molecular mechanism by which Sup35 aggregates are assembled and remodeled in [PSI(+)] cells.

Virtual breakdown of the nuclear envelope in fission yeast meiosis.

Asymmetric localization of Ran regulators (RanGAP1 and RanGEF/RCC1) produces a gradient of RanGTP across the nuclear envelope. In higher eukaryotes, the nuclear envelope breaks down as the cell enters mitosis (designated "open" mitosis). This nuclear envelope breakdown (NEBD) leads to collapse of the RanGTP gradient and the diffusion of nuclear and cytoplasmic macromolecules in the cell, resulting in irreversible progression of the cell cycle. On the other hand, in many fungi, chromosome segregation takes place without NEBD (designated "closed" mitosis). Here we report that in the fission yeast Schizosaccharomyces pombe, despite the nuclear envelope and the nuclear pore complex remaining intact throughout both the meiotic and mitotic cell cycles, nuclear proteins diffuse into the cytoplasm transiently for a few minutes at the onset of anaphase of meiosis II. We also found that nuclear protein diffusion into the cytoplasm occurred coincidently with nuclear localization of Rna1, an S. pombe RanGAP1 homolog that is usually localized in the cytoplasm. These results suggest that nuclear localization of RanGAP1 and depression of RanGTP activity in the nucleus may be mechanistically tied to meiosis-specific diffusion of nuclear proteins into the cytoplasm. This nucleocytoplasmic shuffling of RanGAP1 and nuclear proteins represents virtual breakdown of the nuclear envelope.

Artificial induction of autophagy around polystyrene beads in nonphagocytic cells.

Autophagy is an intracellular event that acts as an innate cellular defense mechanism to kill invading bacteria such as group A Streptococcus in nonphagocytic epithelial-like cells. The cellular events underlying autophagosome formation upon bacterial invasion remain unclear due to the biochemical complexity associated with uncharacterized bacterial components, and the difficulty of predicting the location as well as the timing of where/when autophagosome formation will take place. To overcome these problems, we monitored autophagosome formation in living nonphagocytic cells by inducing autophagy around artificial micrometer-sized beads instead of bacteria. Beads conjugated with bio-reactive molecules provide a powerful tool for examining biochemical properties in vitro. However, this technique has not been applied to living cells, except for phagocytes, because the beads cannot be easily incorporated into nonphagocytic cells. Here we report that micrometer-sized polystyrene beads coated with transfection reagents containing cationic lipids can be incorporated into nonphagocytic cells, and that autophagy can be efficiently induced around the beads in these cells. Monitoring the process of autophagosome formation for pH-sensitive fluorescent dye (pHrodo)-conjugated beads by fluorescence live cell imaging combined with correlative light and electron microscopy, we found that autophagosomes are formed around the beads after partial breakdown of the endosomal membrane. In addition, the beads were subsequently transferred to lysosomes within a couple of hours. Our findings demonstrate the cellular responses that lead to autophagy in response to pathogen invasion.

Two distinct repeat sequences of Nup98 nucleoporins characterize dual nuclei in the binucleated ciliate tetrahymena.

Ciliated protozoa have two functionally distinct nuclei, a micronucleus (MIC) and a macronucleus (MAC) [1]. These two nuclei are distinct in size, transcriptional activity, and division cycle control, proceeding with cycles of DNA replication and nuclear division at different times within the same cell [2, 3]. The structural basis generating functionally distinct nuclei remains unknown. Here, we show that, in Tetrahymena thermophila, the nuclear pore complexes (NPCs) of MIC and MAC are composed of different sets of nucleoporins. Among the 13 nucleoporins identified, Nup98 homologs were of interest because two out of the four homologs were localized exclusively in the MAC and the other two were localized exclusively in the MIC. The two MAC-localizing Nup98s contain repeats of GLFG [4]. In contrast, the two MIC-localizing Nup98s lack the GLFG repeats and instead contain a novel repeat signature of NIFN. Ectopic expression of a chimeric MIC-localizing Nup98 homolog bearing GLFG repeats obstructed the nuclear accumulation of MIC-specific nuclear proteins, and expression of a chimeric MAC-localizing Nup98 homolog bearing NIFN repeats obstructed the nuclear accumulation of MAC-specific nuclear proteins. These results suggest that Nup98s act as a barrier to misdirected localization of nucleus-specific proteins. Our findings provide the first evidence that the NPC contributes to nucleus-selective transport in ciliates.

Membrane proteins Bqt3 and -4 anchor telomeres to the nuclear envelope to ensure chromosomal bouquet formation.

In many organisms, telomeres cluster to form a bouquet arrangement of chromosomes during meiotic prophase. Previously, we reported that two meiotic proteins, Bqt1 and -2, are required for tethering telomeres to the spindle pole body (SPB) during meiotic prophase in fission yeast. This study has further identified two novel, ubiquitously expressed inner nuclear membrane (INM) proteins, Bqt3 and -4, which are required for bouquet formation. We found that in the absence of Bqt4, telomeres failed to associate with the nuclear membranes in vegetative cells and consequently failed to cluster to the SPB in meiotic prophase. In the absence of Bqt3, Bqt4 protein was degraded during meiosis, leading to a phenotype similar to that of the bqt4-null mutant. Collectively, these results show that Bqt4 anchors telomeres to the INM and that Bqt3 protects Bqt4 from protein degradation. Interestingly, the functional integrity of telomeres is maintained even when they are separated from the nuclear envelope in vegetative cells.

Live cell imaging and electron microscopy reveal dynamic processes of BAF-directed nuclear envelope assembly.

Assembly of the nuclear envelope (NE) in telophase is essential for higher eukaryotic cells to re-establish a functional nucleus. Time-lapse, FRAP and FRET analyses in human cells showed that barrier-to-autointegration factor (BAF), a DNA-binding protein, assembled first at the distinct ;core' region of the telophase chromosome and formed an immobile complex by directly binding with other core-localizing NE proteins, such as lamin A and emerin. Correlative light and electron microscopy after live cell imaging, further showed that BAF formed an electron-dense structure on the chromosome surface of the core, close to spindle microtubules (MTs) prior to the attachment of precursor NE membranes, suggesting that MTs may mediate core assembly of BAF. Disruption of the spindle MTs consistently abolished BAF accumulation at the core. In addition, RNAi of BAF eliminated the core assembly of lamin A and emerin, caused abnormal cytoplasmic accumulation of precursor nuclear membranes and resulted in a significant delay of NE assembly. These results suggest that the MT-mediated BAF accumulation at the core facilitates NE assembly at the end of mitosis.

Nuclear localization of barrier-to-autointegration factor is correlated with progression of S phase in human cells.

Barrier-to-autointegration factor (BAF) is a conserved metazoan protein that plays a critical role in retrovirus infection. To elucidate its role in uninfected cells, we first examined the localization of BAF in both mortal and immortal or cancerous human cell lines. In mortal cell lines (e.g. TIG-1, WI-38 and IMR-90 cells) BAF localization depended on the age of the cell, localizing primarily in the nucleus of >90% of young proliferating cells but only 20-25% of aged senescent cells. In immortal cell lines (e.g. HeLa, SiHa and HT1080 cells) BAF showed heterogeneous localization between the nucleus and cytoplasm. This heterogeneity was lost when the cells were synchronized in S phase. In S-phase-synchronized populations, the percentage of cells with predominantly nuclear BAF increased from 30% (asynchronous controls) to approximately 80%. In HeLa cells, RNAi-induced downregulation of BAF significantly increased the proportion of early S-phase cells that retained high levels of cyclin D3 and cyclin E expression and slowed progression through early S phase. BAF downregulation also caused lamin A to mislocalize away from the nuclear envelope. These results indicate that BAF is required for the integrity of the nuclear lamina and normal progression of S phase in human cells.