Masahiro Oka

The role of the nuclear transport system in cell differentiation

The eukaryotic cell nuclear transport system selectively mediates molecular trafficking to facilitate the regulation of cellular processes. The components of this system include diverse transport factors such as importins and nuclear pore components that are precisely organized to coordinate cellular events. A number of studies have demonstrated that the nuclear transport system is indispensible in many types of cellular responses. In particular, the nuclear transport machinery has been shown to be an important regulator of development, organogenesis, and tissue formation, wherein altered nuclear transport of key transcription factors can lead to disease. Importantly, precise switching between distinct forms of importin alpha is central to neural lineage specification, consistent with the hypothesis that importin expression can be a key mediator of cell differentiation.

The Mobile FG Nucleoporin Nup98 Is a Cofactor for Crm1-dependent Protein Export

Nup98 is a mobile nucleoporin that forms distinct dots in the nucleus. Our results show that Nup98 functions as a novel shuttling cofactor in Crm1-mediated nuclear export.

Effective culture conditions for the induction of pluripotent stem cells.

BACKGROUNDnInduced pluripotent stem (iPS) cells, which are functionally comparable to embryonic stem (ES) cells, can be generated from mouse fibroblasts by expression of a defined set of transcription factors Oct4, Sox2, Klf4, and c-Myc. Since iPS cells are generated from somatic cells, they provide an invaluable source of pluripotent stem cells for cell transplantation therapy that does not present ethical problems. However, the reprogramming efficiency is extremely low, and optimal culture conditions for iPS cell derivation have not been clearly defined.nnnMETHODSnTo generate iPS cells efficiently, we tested 10 different culture conditions: DMEM supplemented with 15% fetal bovine serum (FBS), Knockout DMEM with 15% FBS from Invitrogen, Equitech, or HyClone, DMEM with 15% Knockout Serum Replacement (KSR), and Knockout DMEM with 10%, 15%, 20%, 25%, or 35% KSR. These media all contain 2 mM L-glutamine, 100 μM nonessential amino acids, 100 μM beta-mercaptoethanol, 1000 units ml⁻¹ leukemia inhibitory factor (LIF), 50 units ml⁻¹ penicillin, and 50 μg ml⁻¹ streptomycin.nnnRESULTSnMedium containing Knockout DMEM with 20% KSR permits efficient induction of iPS cells from both mouse embryonic fibroblasts (MEFs) and adult tail tip fibroblasts (TTFs). Mouse iPS cells generated in the condition express ES cell marker genes such as Oct4, Sox2, Rex1, and Nanog at levels comparable to those of ES cells. Furthermore, iPS cells derived form MEFs and adult TTFs can contribute to adult chimeras.nnnCONCLUSIONnOur iPS cell induction efficiency is greater than that described in other reports.nnnGENERAL SIGNIFICANCEnThese findings provide an important catalyst for examining different culture environments for the generation of iPS cells.

Nup358, a nucleoporin, functions as a key determinant of the nuclear pore complex structure remodeling during skeletal myogenesis

The nuclear pore complex (NPC) is the only gateway for molecular trafficking across the nuclear envelope. The NPC is not merely a static nuclear‐cytoplasmic transport gate; the functional analysis of nucleoporins has revealed dynamic features of the NPC in various cellular functions, such as mitotic spindle formation and protein modification. However, it is not known whether the NPC undergoes dynamic changes during biological processes such as cell differentiation. In the present study, we evaluate changes in the expression levels of several nucleoporins and show that the amount of Nup358/RanBP2 within individual NPCs increases during muscle differentiation in C2C12 cells. Using atomic force microscopy, we demonstrate structural differences between the cytoplasmic surfaces of myoblast and myotube NPCs and a correlation between the copy number of Nup358 and the NPC structure. Furthermore, small interfering RNA‐mediated depletion of Nup358 in myoblasts suppresses myotube formation without affecting cell viability, suggesting that NUP358 plays a role in myogenesis. These findings indicate that the NPC undergoes dynamic remodeling during muscle cell differentiation and that Nup358 is prominently involved in the remodeling process.

Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. Our results demonstrate that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α.

Differential Role for Transcription Factor Oct4 Nucleocytoplasmic Dynamics in Somatic Cell Reprogramming and Self-renewal of Embryonic Stem Cells

Background: The spatiotemporal dynamic behavior of Oct4 remains largely unknown. Results: Oct4 is a nucleocytoplasmic shuttling protein, and Oct4 mutants with biased nucleocytoplasmic localization show limited potential for cellular reprogramming. Conclusion: An appropriate nuclear retention of Oct4 is critical for cellular reprogramming but not for the self-renewal of ES cells. Significance: Our findings will provide novel insight into the role of Oct4 during cellular reprogramming. Oct4 is a member of the POU family of transcription factors and plays a critical role in both maintenance of the undifferentiated state of embryonic stem (ES) cells and in the reprogramming of somatic cells to induced pluripotent stem cells. Oct4 is imported into the nucleus where it functions as a transcription factor; however, the spatiotemporal dynamic behavior of Oct4 remains largely unknown. In the present study we show that Oct4 is a nucleocytoplasmic shuttling protein. Furthermore, although Oct4 mutants with altered nuclear import/export activity were able to maintain the self-renewal of ES cells, they displayed limited potential for cellular reprogramming. These results indicate that the intracellular localization of Oct4, which is dependent on nucleocytoplasmic shuttling, must be more strictly regulated for cellular reprogramming, suggesting that Oct4 plays differential roles in the self-renewal of ES cells and in somatic cell reprogramming.

The Interaction Between Importin-α and Nup153 Promotes Importin-α/β-Mediated Nuclear Import

Nuclear transport is mediated by transport factors, including the importin β family members. The directionality of nuclear transport is governed by the asymmetrical distribution of the small GTPase Ran. Of note, importin α/β‐mediated import of classical nuclear localization signal (cNLS) – containing cargo is more efficient than other Ran‐dependent import pathways that do not require importin α. In this study, we characterized the role of importin α in nuclear transport by examining import efficiencies of cNLS‐cargo/importin α/β complexes. We first depleted digitonin‐permeabilized semi‐intact cells of endogenous importin α and used the cells to show that the interaction between importin α and Nup153 – a component of the nuclear pore complex (NPC) – is essential for efficient import of importin β‐binding domain containing substrates, but not other cargoes that directly bind to importin β. Moreover, we found that the binding of importin α to Nup153 facilitates cNLS‐mediated import, and demonstrated that importin α in import complexes and cargo‐free importin α prebound to Nup153 promote efficient import of cNLS‐containing proteins. This is the first in vitro study showing that in conjunction with Nup153, importin α contributes to directionally biased exit of cNLS‐containing cargo to the nuclear side of NPCs.

Cytochrome c-551 of the thermophilic bacterium PS3, DNA sequence and analysis of the mature cytochrome

The structural gene for cytochrome c-551 was isolated from genomic DNA of the thermophilic bacterium PS3. The amino acid sequence of cytochrome c-551 as deduced from the DNA sequence consists of 111 amino acid residues and contains one heme c-binding site (-CASCH-) located approximately in the middle of the polypeptide. The N-terminus of isolated cytochrome c-551 was blocked, but treatment with Rhizopus lipase and molecular weight measurement of the mature and lipase-treated forms by ion spray mass spectroscopy suggest that the mature c-551 may have 93 or 94 amino acid residues with a diacylated glycerol-cysteine at the N-terminal region. The first 17 or 18 amino acid residues in the N-terminal region of the nascent polypeptide, rich in hydrophobic and basic amino acid residues, may be a signal peptide to translocate the major portion of cytochrome c-551 to the extracellular surface and to be processed. Similarity of amino acid sequence of this protein is discussed in relation to other c-type cytochromes of bacilli as well as bacterial small cytochromes c such as Pseudomonas aeruginosa cytochrome c-551 and cytochrome c6 of cyanobacteria.

Quantitative regulation of nuclear pore complex proteins by O-GlcNAcylation

The nuclear pore complex (NPC) is a macromolecular assembly consisting of approximately 30 different proteins called nucleoporins. Several nucleoporins are O-GlcNAcylated, which is a post-translational modification in which the monosaccharide β-N-acetylglucosamine (GlcNAc) is attached to serine or threonine residues within proteins. However, the biological significance of this modification on nucleoporins remains obscure. Here we found that Nup62 and Nup88 protein levels were significantly decreased upon knockdown of O-GlcNAc transferase (OGT), which catalyzes the O-GlcNAcylation of intracellular proteins. Although Nup88, unlike Nup62, was not recognized by an anti-O-GlcNAc antibody or WGA-HRP, knockdown of Nup62 caused a reduction in Nup88 protein levels, suggesting that the observed decrease in Nup88 in OGT knocked-down cells is due to a decrease in Nup62. Furthermore, we found that Nup88 was preferentially associated with O-GlcNAcylated Nup62 compared with non-O-GlcNAcylated Nup62. These results indicate that Nup62 protein levels are primarily maintained by O-GlcNAcylation and that Nup88 is quantitatively regulated through its interaction with O-GlcNAcylated Nup62.

Specific Monoclonal Antibody Against the Nuclear Pore Complex Protein, Nup96

Nup96 is a component of the Nup107-160 complex, the largest subunit of the nuclear pore complex. Nup96 is generated as a precursor protein with Nup98. However, the mechanism by which Nup96 contributes to cell function is not clear. We report here on the preparation of a monoclonal antibody (MAb) directed against mouse Nup96. The antibody was produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The antibody, MAb 4H5, specifically recognized Nup96, as evidenced by immunoblotting using the whole cell lysates. In immunostaining using MAb 4H5, a nuclear rim staining pattern was observed. This antibody will be useful in immunoblotting and immunolocalization experiments, as well as further analyses of the biological function and cellular dynamics of this protein.