Shotaro Otsuka

Individual binding pockets of importin-β for FG-nucleoporins have different binding properties and different sensitivities to RanGTP

Importin-β mediates protein transport across the nuclear envelope through the nuclear pore complex (NPC) by interacting with components of the NPC, called nucleoporins, and a small G protein, Ran. Although there is accumulated knowledge on the specific interaction between importin-β and the Phe-Gly (FG) motif in the nucleoporins as well as the effect of RanGTP on this interaction, the molecular mechanism by which importin-β shuttles across the nuclear envelope through the NPC is unknown. In this study, we focused on four binding pockets of importin-β for the FG motifs and characterized the interaction using a single-molecule force-measurement technique with atomic-force microscopy. The results from a series of importin-β mutants containing amino acid substitutions within the FG-binding pockets demonstrate that the individual FG-binding pockets have different affinities to FG-Nups (Nup62 and Nup153) and different sensitivities to RanGTP; the binding of RanGTP to the amino-terminal domain of importin-β induces the conformational change of the entire molecule and reduces the affinity of some of the pockets but not others. These heterogeneous characteristics of the multiple FG-binding pockets may play an important role in the behavior of importin-β within the NPC. Single-molecule force measurement using the entire molecule of an NPC from a Xenopus oocyte also implies that the reduction of the affinity by RanGTP really occurs at the nucleoplasmic side of the entire NPC.

Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM. DOI: http://dx.doi.org/10.7554/eLife.19071.001

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.

Nuclear architecture and chromatin dynamics revealed by atomic force microscopy in combination with biochemistry and cell biology

The recent technical development of atomic force microscopy (AFM) has made nano-biology of the nucleus an attractive and promising field. In this paper, we will review our current understanding of nuclear architecture and dynamics from the structural point of view. Especially, special emphases will be given to: (1) How to approach the nuclear architectures by means of new techniques using AFM, (2) the importance of the physical property of DNA in the construction of the higher-order structures, (3) the significance and implication of the linker and core histones and the nuclear matrix/scaffold proteins for the chromatin dynamics, (4) the nuclear proteins that contribute to the formation of the inner nuclear architecture. Spatio-temporal analyses using AFM, in combination with biochemical and cell biological approaches, will play important roles in the nano-biology of the nucleus, as most of nuclear structures and events occur in nanometer, piconewton and millisecond order. The new applications of AFM, such as recognition imaging, fast-scanning imaging, and a variety of modified cantilevers, are expected to be powerful techniques to reveal the nanostructure of the nucleus.

Development of glutathione-coupled cantilever for the single-molecule force measurement by scanning force microscopy

The accuracy and the fidelity of a single‐molecule force measurement largely rely on how the molecule of interest is attached to the solid substrate surface (bead, cantilever, cover glass and etc.). A site‐specific attachment of a protein without affecting its structure and enzymatic function has been a major concern. Here, we established a glutathione‐coupled cantilever to which any glutathione S‐transferase (GST)‐fused proteins can be attached in a desired direction. The rupture force between glutathione and GST was ∼100 pN on average. By using this cantilever, we succeeded in measuring the interaction force between importin α and importin β.

Intermolecular disulfide bonds between nucleoporins regulate karyopherin-dependent nuclear transport.

Summary Disulfide (S–S) bonds play important roles in the regulation of protein function and cellular stress responses. In this study, we demonstrate that distinct sets of nucleoporins (Nups), components of the nuclear pore complex (NPC), form S–S bonds and regulate nuclear transport through the NPC. Kinetic analysis of importin &bgr; demonstrated that the permeability of the NPC was increased by dithiothreitol treatment and reduced by oxidative stress. The permeability of small proteins such as GFP was not affected by either oxidative stress or a reducing reagent. Immunoblot analysis revealed that the oxidative stress significantly induced S–S bond formation in Nups 358, 155, 153 and 62 but not 88 and 160. The direct involvement of cysteine residues in the formation of S–S bonds was confirmed by mutating conserved cysteine residues in Nup62, which abolished the formation of S–S bonds and enhanced the permeability of the NPC. Knocking down Nup62 reduced the stress-inducible S–S bonds of Nup155, suggesting that Nup62 and Nup155 are covalently coupled via S–S bonds. From these results, we propose that the inner channel of the NPC is somehow insulated from the cytoplasm and is more sensitive than the cytoplasm to the intracellular redox state.

Imaging the assembly, structure, and function of the nuclear pore inside cells.

The nuclear pore complex (NPC) mediates selective transport across the nuclear envelope (NE) and plays crucial roles in several additional cellular functions. In higher eukaryotes, the NPC and the NE disassemble and reassemble during cell division and live-cell imaging has been a powerful tool to analyze these dynamic processes. Here, we present a method for the kinetic analysis of postmitotic NPC assembly and reestablishment of transport competence in intact cells by multicolor 4D imaging and photoswitching. By applying the methods we have established previously using normal rat kidney to HeLa cells, we demonstrate the conservation of NPC assembly in different mammalian cells. We recently showed that the molecular organization of the NPC can be studied by combining stochastic super-resolution microscopy with single-particle averaging and present this method here in detail.

Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink. NPC precursors are found in small membrane holes and dilate radially during assembly of the inner ring complex, forming thousands of transport channels within minutes. This mechanism is fundamentally different from that of interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport competent.Ultrastructural analysis of nuclear membrane topology and assembling NPCs reveals how mitotic cells can rapidly establish a closed nuclear compartment while at the same time making it transport competent.

Dissecting in vivo steady-state dynamics of karyopherin-dependent nuclear transport

The steady-state dynamics of karyopherin-dependent nuclear transport in a living cell is examined. The kinetic model established by a number of experimentally obtained parameters reveals how each step of the transport system contributes to maintaining steady-state cargo gradient and fluxes across the nuclear envelope.

Mechanisms of nuclear pore complex assembly – two different ways of building one molecular machine

The nuclear pore complex (NPC) mediates all macromolecular transport across the nuclear envelope. In higher eukaryotes that have an open mitosis, NPCs assemble at two points in the cell cycle: during nuclear assembly in late mitosis and during nuclear growth in interphase. How the NPC, the largest nonpolymeric protein complex in eukaryotic cells, self‐assembles inside cells remained unclear. Recent studies have started to uncover the assembly process, and evidence has been accumulating that postmitotic and interphase NPC assembly use fundamentally different mechanisms; the duration, structural intermediates, and regulation by molecular players are different and different types of membrane deformation are involved. In this Review, we summarize the current understanding of these two modes of NPC assembly and discuss the structural and regulatory steps that might drive the assembly processes. We furthermore integrate understanding of NPC assembly with the mechanisms for rapid nuclear growth in embryos and, finally, speculate on the evolutionary origin of the NPC implied by the presence of two distinct assembly mechanisms.