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Retinoic acid-inducible gene I (RIG-I) is the most front-line cytoplasmic viral RNA sensor and induces antiviral immune responses. RIG-I recognizes short double-stranded (dsRNA) ( 500 bp) to trigger antiviral signaling. Since RIG-I is capable of binding with dsRNA irrespective of size, length-dependent RIG-I signaling remains elusive. Here, we demonstrated that RIG-I bound to long dsRNA with slow kinetics. Remarkably, RIG-I/short dsRNA complex efficiently dissociated in an ATP hydrolysis-dependent manner, whereas RIG-I/long dsRNA was stable and did not dissociate. Our study suggests that the dissociation of RIG-I from RIG-I/dsRNA complex could be a step for efficient antiviral signaling. Dissociated RIG-I exhibited homo-oligomerization, acquiring ability to physically associate with MAVS, and biological activity upon introduction into living cells. We herein discuss common and unique mechanisms of viral dsRNA recognition by RIG-I and MDA5.

Clathrin-mediated endocytosis is pivotal to signal transduction pathways between the extracellular environment and the intracellular space. Evidence from live-cell imaging and super-resolution microscopy of mammalian cells suggests an asymmetric distribution of actin fibres near the clathrin-coated pit, which induces asymmetric pit-closing rather than radial constriction. However, detailed molecular mechanisms of this ‘asymmetricity’ remain elusive. Herein, we used high-speed atomic force microscopy to demonstrate that CIP4, a multi-domain protein with a classic F-BAR domain and intrinsically disordered regions, is necessary for asymmetric pit-closing. Strong self-assembly of CIP4 via intrinsically disordered regions, together with stereospecific interactions with the curved membrane and actin-regulating proteins, generates a small actin-rich environment near the pit, which deforms the membrane and closes the pit. Our results provide mechanistic insights into how disordered and structured domain collaboration promotes spatio-temporal actin polymerisation near the plasma membrane.

The cystine/glutamate transporter system xc − consists of the light-chain subunit xCT (SLC7A11) and the heavy-chain subunit CD98 (4F2hc or SLC3A2) and exchanges extracellular cystine for intracellular glutamate at the plasma membrane. The imported cystine is reduced to cysteine and used for synthesis of GSH, one of the most important antioxidants in cancer cells. Because cancer cells have increased levels of reactive oxygen species, xCT, responsible for cystine–glutamate exchange, is overexpressed in many cancers, including glioblastoma. However, under glucose-limited conditions, xCT overexpression induces reactive oxygen species accumulation and cell death. Here we report that cell survival under glucose deprivation depends on cell density. We found that high cell density (HD) down-regulates xCT levels and increases cell viability under glucose deprivation. We also found that growth of glioblastoma cells at HD inactivates mTOR and that treatment of cells grown at low density with the mTOR inhibitor Torin 1 down-regulates xCT and inhibits glucose deprivation-induced cell death. The lysosome inhibitor bafilomycin A1 suppressed xCT down-regulation in HD-cultured glioblastoma cells and in Torin 1–treated cells grown at low density. Additionally, bafilomycin A1 exposure or ectopic xCT expression restored glucose deprivation–induced cell death at HD. These results suggest that HD inactivates mTOR and promotes lysosomal degradation of xCT, leading to improved glioblastoma cell viability under glucose-limited conditions. Our findings provide evidence that control of xCT protein expression via lysosomal degradation is an important mechanism for metabolic adaptation in glioblastoma cells.

Despite numerous recent developments in bioimaging techniques, nanoscale and live-cell imaging of the plasma membrane has been challenging because of the insufficient z-resolution of optical microscopes, as well as the lack of fluorescent probes to specifically label small membrane structures. High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualising the dynamics of a specimen surface and is therefore suitable for observing plasma membrane dynamics. Recent developments in HS-AFM for live-cell imaging have enabled the visualisation of the plasma membrane and the network of cortical actin underneath the membrane in a living cell. Furthermore, correlative imaging with fluorescence microscopy allows for the direct visualisation of morphological changes of the plasma membrane together with the dynamic assembly or disassembly of proteins during the entire course of endocytosis in a living cell. Here, we review these recent advances in HS-AFM in order to analyse various cellular events occurring at the cell surface.

Viral proteins interact with different sets of host cell components throughout the viral life cycle and are known to localize to the intracellular membraneless organelles (MLOs) of the host cell, where formation/dissolution is regulated by phase separation of intrinsically disordered proteins and regions (IDPs/IDRs). Viral proteins are rich in IDRs, implying that viruses utilize IDRs to regulate phase separation of the host cell organelles and augment replication by commandeering the functions of the organelles and/or sneaking into the organelles to evade the host immune response. This review aims to integrate current knowledge of the structural properties and intracellular localizations of viral IDPs to understand viral strategies in the host cell. First, the properties of viral IDRs are reviewed and similarities and differences with those of eukaryotes are described. The higher IDR content in viruses with smaller genomes suggests that IDRs are essential characteristics of viral proteins. Then, the interactions of the IDRs of flaviviruses with the MLOs of the host cell are investigated with emphasis on the viral proteins localized in the nucleoli and stress granules. Finally, the possible roles of viral IDRs in regulation of the phase separation of organelles and future possibilities for antiviral drug development are discussed.

The actin cytoskeleton plays critical roles in numerous cellular events and functions, and its spatiotemporal dynamics are maintained and regulated by several actin cofactor proteins. MISP/Caprice is a recently reported actin-bundling protein that is also involved in the progression of mitosis. In this study, we investigated how the actin-regulatory function of MISP is modulated by phosphorylation. A series of mutation studies demonstrated that phosphorylation of S394, S395, and S400 induced stress fiber formation in interphase cells. In vitro studies revealed that these phosphorylation events increased the actin-bundling activity but not the actin-binding activity of MISP. Moreover, actin-binding activity was suppressed by mitotic phosphorylation, including that at S376, S471, and S541. These results indicate that phosphorylation during interphase and mitosis differentially regulates the actin-binding and -bundling activities of MISP, in turn regulating the higher-order architecture of the actin cytoskeleton during cell cycle.

Together with lamellipodia and stress fibers, a dynamic network of actin filaments in the cell cortex plays a major role in the maintenance of cell morphology and motility. In contrast to lamellipodia, which have been well studied in various motile cells, the dynamics of actin filaments in the cell cortex have not yet been clarified due to a lack of proper imaging techniques. Here, we utilized high-speed atomic force microscopy for live-cell imaging and analyzed cortical actin dynamics in living cells. We successfully measured the polymerization rate and the frequency of filament synthesis in living COS-7 cells, and examined the associated effects of various inhibitors and actin-binding proteins. Actin filaments are synthesized beneath the plasma membrane and eventually descend into the cytoplasm. The inhibitors, cytochalasin B inhibited the polymerization, while jasplakinolide, inhibited the turnover of actin filaments as well as descension of the newly synthesized filaments, suggesting that actin polymerization near the membrane drives turnover of the cortical actin meshwork. We also determined how actin turnover is maintained and regulated by the free G-actin pool and G-actin binding proteins such as profilin and thymosin β4, and found that only a small amount of free G-actin was present in the cortex. Finally, we analyzed several different cell types, and found that the mesh size and the orientation of actin filaments were highly divergent, indicating the involvement of various actin-binding proteins in the maintenance and regulation of cortical actin architecture in each cell type.

In this study, we examined how channel-forming subunits of the nuclear pore complex (NPC) are assembled into a selective channel within a highly structured scaffold ring during postmitotic assembly. We focused on non-structured domains of the scaffold Nups and performed in vitro self-assembled particle assays with those derived from channel-forming FG-Nups. We found that non-structured domains of ELYS and Nup35N interacted with channel-forming FG-Nups to form a self-assembled particle. Sequential addition of FG-Nups into the scaffold particle revealed that ELYS, which initiates postmitotic NPC reassembly, interacts with early assembling FG-Nups (Nups98 and 153) but not middle stage-assembling FG-Nups (Nups58 and 62). Nup35, which assembles between the early and middle stages, facilitated the assembly of Nup62 into the early assembling Nups both in vitro and in vivo. These results demonstrate that ELYS and Nup35 have a role of facilitator in the ordered assembly of channel-forming FG-Nups during mitosis.

During the later stages of lung development, two types of pneumocytes, cuboidal type II (AECII) and flattened type I (AECI) alveolar epithelial cells, form distal lung saccules. Here, we highlight how fibroblasts expressing MAP-microtubule affinity regulating kinase 1 (Mark1) are required for the terminal stages of pulmonary development, called lung sacculation. In Mark1-knockout (KO) mice, distal sacculation and AECI flattening are significantly impaired. Fetal epithelial cells generate alveolar organoids and differentiate into pneumocytes when co-cultured with fibroblasts. However, the size of organoids decreased and AECI flattening was impaired in the presence of Mark1 KO fibroblasts. In Mark1 KO fibroblasts themselves, cilia formation and the Hedgehog pathway were suppressed, resulting in the loss of type I collagen expression. The addition of type I collagen restored AECI flattening in organoids co-cultured with Mark1 KO fibroblasts and rescued the decreased size of organoids. Mathematical modeling of distal lung sacculation supports the view that AECI flattening is necessary for the proper formation of saccule-like structures. These results suggest that Mark1-mediated fibroblast activation induces AECI flattening and thereby regulates distal lung sacculation.

The nuclear pore complex forms a highly crowded selective barrier with intrinsically disordered regions at the nuclear membrane to coordinate nucleocytoplasmic molecular communications. Although oxidative stress is known to alter the barrier function, the molecular mechanism underlying this adaptive control of the nuclear pore complex remains unknown. Here we uncover a systematic control of the crowding barrier within the nuclear pore in response to various redox environments. Direct measurements of the crowding states using a crowding-sensitive FRET (Förster resonance energy transfer) probe reveal specific roles of the nuclear pore subunits that adjust the degree of crowding in response to different redox conditions, by adaptively forming or disrupting redox-sensitive disulfide bonds. Relationships between crowding control and the barrier function of the nuclear pore are investigated by single-molecular fluorescence measurements of nuclear transport. Based on these findings, we propose a proximal control model of molecular crowding in vivo that is dynamically regulated at the molecular level., 酸化ストレスが細胞の核膜機能を変える機構を解明 --環境に応じて分子の「混み具合」が変わる仕組み--. 京都大学プレスリリース. 2020-12-16.

Animal cells undergo rapid rounding during mitosis, ensuring proper chromosome segregation, during which an outward rounding force abruptly increases upon prometaphase entry and is maintained at a constant level during metaphase. Initial cortical tension is generated by the actomyosin system to which both myosin motors and actin network architecture contribute. However, how cortical tension is maintained and its physiological significance remain unknown. We demonstrate here that Cdk1-mediated phosphorylation of DIAPH1 stably maintains cortical tension after rounding and inactivates the spindle assembly checkpoint (SAC). Cdk1 phosphorylates DIAPH1, preventing profilin1 binding to maintain cortical tension. Mutation of DIAPH1 phosphorylation sites promotes cortical F-actin accumulation, increases cortical tension, and delays anaphase onset due to SAC activation. Measurement of the intra-kinetochore length suggests that Cdk1-mediated cortex relaxation is indispensable for kinetochore stretching. We thus uncovered a previously unknown mechanism by which Cdk1 coordinates cortical tension maintenance and SAC inactivation at anaphase onset., Cell rounding at mitosis is driven by cortical tension and maintained through metaphase, although the mechanism is unknown. Here, the authors demonstrate that Cdk1 phosphorylation of DIAPH1 is required for both cortical tension maintenance and inactivation of the spindle assembly checkpoint.

The primary cilium functions as an "antenna" for cell signaling, studded with characteristic transmembrane receptors and soluble protein factors, raised above the cell surface. In contrast to the transmembrane proteins, targeting mechanisms of nontransmembrane ciliary proteins are poorly understood. We focused on a pathogenic mutation that abolishes ciliary localization of retinitis pigmentosa 2 protein and revealed a dual acylation-dependent ciliary targeting pathway. Short N-terminal sequences which contain myristoylation and palmitoylation sites are sufficient to target a marker protein into the cilium in a palmitoylation-dependent manner. A Golgi-localized palmitoyltransferase DHHC-21 was identified as the key enzyme controlling this targeting pathway. Rapid turnover of the targeted protein was ensured by cholesterol-dependent membrane fluidity, which balances highly and less-mobile populations of the molecules within the cilium. This targeting signal was found in a set of signal transduction molecules, suggesting a general role of this pathway in proper ciliary organization, and dysfunction in ciliary disorders.

Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open–close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME., Author summary Cells communicate with their environments via the plasma membrane and various membrane proteins. Clathrin-mediated endocytosis (CME) plays a central role in such communication and proceeds with a series of multiprotein assembly, deformation of the plasma membrane, and production of a membrane vesicle that delivers extracellular signaling molecules into the cytoplasm. In this study, we utilized our home-built correlative imaging system comprising high-speed atomic force microscopy (HS-AFM) and confocal fluorescence microscopy to simultaneously image morphological changes of the plasma membrane and protein localization during CME in a living cell. The results revealed a tight correlation between the size of the pit and the amount of clathrin assembled. Actin dynamics play multiple roles in the assembly, maturation, and closing phases of the process, and affects membrane morphology, suggesting a close relationship between endocytosis and dynamic events at the cell cortex. Knock down of dynamin also affected the closing motion of the pit and showed functional correlation with actin.

The central channel of the nuclear pore complex (NPC) is occupied by non-structured polypeptides with a high content of Phe-Gly (FG) motifs. This protein-rich environment functions as an entropic barrier that prevents the passage of molecules, as well as the binding sites for karyopherins, to regulate macromolecular traffic between the nucleoplasm and the cytoplasm. In this study, we expressed individual Nups fused with a crowding-sensitive probe (GimRET) to determine the spatial distribution of protein-rich domains within the central channel in vivo, and characterize the properties of the entropic barrier. Analyses of the probe signal revealed that the central channel contains two protein-rich domains at both the nucleoplasmic and cytoplasmic peripheries, and a less-crowded central cavity. Karyopherins and other soluble proteins are not the constituents of the protein-rich domains. The time-lapse observation of the post-mitotic reassembly process also revealed how individual protein-rich domains are constructed by a sequential assembly of nucleoporins.

Karyopherin β family proteins mediate the nuclear/cytoplasmic transport of various proteins through the nuclear pore complex (NPC), although they are substantially larger than the size limit of the NPC. To elucidate the molecular mechanism underlying this paradoxical function, we focused on the unique structures called HEAT repeats, which consist of repetitive amphiphilic α helices. An in vitro transport assay and FRAP analyses demonstrated that not only karyopherin β family proteins but also other proteins with HEAT repeats could pass through the NPC by themselves, and serve as transport mediators for their binding partners. Biochemical and spectroscopic analyses and molecular dynamics simulations of purified HEAT-rich proteins revealed that they interact with hydrophobic groups, including phenyl and alkyl groups, and undergo reversible conformational changes in tertiary structures, but not in secondary structures. These results show that conformational changes in the flexible amphiphilic motifs play a critical role in translocation through the NPC., タンパク質が核膜孔を通り抜ける際の構造変化を解明 -細胞核内部への遺伝子導入技術への応用に期待-. 京都大学プレスリリース. 2014-11-27.

The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser-66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. Here we show that phosphorylation of Ser-66 induces a conformational change of ezrin that enables its association with syntaxin 3 (Stx3) and provides a spatial cue for H,K-ATPase trafficking. This conformation-dependent association is specific for Stx3, and the binding interface is mapped to the N-terminal region. Biochemical analyses show that inhibition of ezrin phosphorylation at Ser-66 prevents ezrin-Stx3 association and insertion of H,K-ATPase into the apical plasma membrane of parietal cells. Using atomic force microscopic analyses, our study revealed that phosphorylation of Ser-66 induces unfolding of ezrin molecule to allow Stx3 binding to its N terminus. Given the essential role of Stx3 in polarized secretion, our study presents the first evidence in which phosphorylation-induced conformational rearrangement of the ezrin molecule provides a spatial cue for polarized membrane trafficking in epithelial cells.

Cellular proteins do not work in isolation. Instead, they often function as part of large macromolecular complexes, which are transported and concentrated into specific cellular compartments and function in a highly crowded environment. A central theme of modern cell biology is to understand how such macromolecular complexes are assembled efficiently and find their destinations faithfully. In this Opinion article, we will focus on HEAT repeats, flexible arrays of amphiphilic helices found in many eukaryotic proteins, such as karyopherins and condensins, and discuss how these uniquely designed helical repeats might underlie dynamic protein–protein interactions and support cellular functions in crowded environments. We will make bold speculations on functional similarities between the action of HEAT repeats and intrinsically disordered regions (IDRs) in macromolecular phase separation. Potential contributions of HEAT–HEAT interactions, as well as cooperation between HEATs and IDRs, to mesoscale organelle assembly will be discussed.

An active targeting drug delivery system that targets the nucleus could solve the problem of the treatment of genetic disorders through gene delivery, but it has met with limited success. The purpose of this study was to establish an RNA aptamer-modified nucleus-targeting liposomal carrier system referred to as NupApt-liposomes. RNA aptamers against the Nup358 protein are prepared using a newly established Protein SELEX method. After confirming aptamer binding to the recombinant protein, an aptamer-lipid conjugate (Apt-PEG-DSPE) was prepared. Aptamer-modified liposomes and simple polyethylene glycol (PEG) liposomes were prepared to check its ability to bind to isolated nuclei. Confocal studies indicated that the aptamer-modified liposomes had the ability to bind to isolated nuclei, whereas PEG-liposomes showed only weak binding. Confocal laser scanning microscopy studies of inhibition assays also supported the above conclusion. The dissociation constant of the Nucleoporin358-specific aptamer referred to as NupApt01 and NupApt02 were 36 and 70 nM, respectively. Finally, with aptamer-modified liposomes, gene expression studies showed a two times better gene expression in NupApt-liposome-treated nuclei in comparison to that of PEG-liposomes. This represents the first artificial RNA aptamer-modified liposomes promoting the specific binding of a nanocarrier to the nucleus, thus improving gene expression in comparison to PEG-liposomes.

Using fast-scanning atomic force microscopy, we directly visualized the interaction of Escherichia coli RNA polymerase (RNAP) with DNA at the scan rate of 1–2 frames per second. The analyses showed that the RNAP can locate the promoter region not only by sliding but also by hopping and/or segmental transfer. Upon the addition of 0.05mM NTPs to the stalled complex, the RNAP molecule pulled the template DNA uni-directionally at the rates of 15 nucleotides/s on average. The present method is potentially applicable to examine a variety of protein–nucleic acid interactions, especially those involved in the process of gene regulation.

Establishing a nanobiohybrid device largely relies on the availability of various bioconjugation procedures which allow coupling of biomolecules and inorganic materials. Especially, site-specific coupling of a protein to nanomaterials is highly useful and significant, since it can avoid adversely affecting the protein's function. In this study, we demonstrated a covalent coupling of a protein of interest to the end of carbon nanotubes without affecting protein's function. A modified Staudinger-Bertozzi ligation was utilized to couple a carbon nanotube end with an azide group which is site-specifically incorporated into a protein of interest. We demonstrated that Ca(2+)-sensor protein, calmodulin, can be attached to the end of the nanotubes without affecting the ability to bind to the substrate in a calcium-dependent manner. This procedure can be applied not only to nanotubes, but also to other nanomaterials, and therefore provides a fundamental technique for well-controlled protein conjugation.

The nuclear pores located on the nuclear envelope work as a selective barrier to nuclear import. The transport of large cargo molecules (larger than ∼40 kD) into the nucleus generally requires the aid of nuclear transport proteins, such as importins, which bind to nuclear localization signal (NLS) sequences in the cargo molecules and transport these molecules into the nucleus through the nuclear pore. In our previous paper, we showed that maltotriose(Glc3)-displaying quantum dots (QDs) can pass through the nuclear pore in digitonin-treated HeLa cells. The aim of this study is to clarify the mechanism of the nuclear import of maltooligosaccharide-displaying QDs (maltooligo-QDs). We prepared maltopentaose(Glc5) and maltoheptaose(Glc7)-QDs in addition to Glc3-QDs. The effect of the number of glucose units in the maltooligosaccharide on the rate of nuclear import of QDs has been explored by confocal laser scanning microscopy (CLSM). Further, we analyzed that the direct interactions of maltooligo-QDs to an internal protein in the nuclear pore (Nucleoporin62) using a surface plasmon resonance (SPR) system. We found that maltooligo-QDs with more than three glucose units (Glc3, Glc5 and Glc7-QDs) rapidly entered the cellular nucleus, and had a higher affinity to nucleoporin62 than those of PEG and Glc1-QDs. These data indicate that the interaction between nuclear pores and carbohydrates is the driving force behind the nuclear import of maltooligo-QDs.

The elucidation of structure-function relationships of biological samples has become important issue in post-genomic researches. In order to unveil the molecular mechanisms controlling gene regulations, it is essential to understand the interplay between fundamental DNA properties and the dynamics of the entire molecule. The wide range of applicability of atomic force microscopy (AFM) has allowed us to extract physicochemical properties of DNA and DNA-protein complexes, as well as to determine their topographical information. Here, we review how AFM techniques have been utilized to study DNA and DNA-protein complexes and what types of analyses have accelerated the understanding of the DNA dynamics. We begin by illustrating the application of AFM to investigate the fundamental feature of DNA molecules; topological transition of DNA, length dependent properties of DNA molecules, flexibility of double-stranded DNA, and capability of the formation of non-Watson-Crick base pairing. These properties of DNA are critical for the DNA folding and enzymatic reactions. The technical advancement in the time-resolution of AFM and sample preparation methods enabled visual analysis of DNA-protein interactions at sub-second time region. DNA tension-dependent enzymatic reaction and DNA looping dynamics by restriction enzymes were examined at a nanoscale in physiological environments. Contribution of physical properties of DNA to dynamics of nucleosomes and transition of the higher-order structure of reconstituted chromatin are also reviewed.

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.

Nucleosome is a fundamental structural unit of chromatin, and the exposure from or occlusion into chromatin of genomic DNA is closely related to the regulation of gene expression. In this study, we analyzed the molecular dynamics of poly-nucleosomal arrays in solution by fast-scanning atomic force microscopy (AFM) to obtain a visual glimpse of nucleosome dynamics on chromatin fiber at single molecule level. The influence of the high-speed scanning probe on nucleosome dynamics can be neglected since bending elastic energy of DNA molecule showed similar probability distributions at different scan rates. In the sequential images of poly-nucleosomal arrays, the sliding of the nucleosome core particle and the dissociation of histone particle were visualized. The sliding showed limited fluctuation within approximately 50nm along the DNA strand. The histone dissociation occurs by at least two distinct ways: a dissociation of histone octamer or sequential dissociations of tetramers. These observations help us to develop the molecular mechanisms of nucleosome dynamics and also demonstrate the ability of fast-scanning AFM for the analysis of dynamic protein-DNA interaction in sub-seconds time scale.

A systematic search was performed for DNA-binding sequences of YgiP, an uncharacterized transcription factor of Escherichia coli, by using the Genomic SELEX. A total of 688 YgiP-binding loci were identified after genome-wide profiling of SELEX fragments with a high-density microarray (SELEX-chip). Gel shift and DNase-I footprinting assays indicated that YgiP binds to multiple sites along DNA probes with a consensus GTTNATT sequence. Atomic force microscope observation indicated that at low concentrations, YgiP associates at various sites on DNA probes, but at high concentrations, YgiP covers the entire DNA surface supposedly through protein–protein contact. The intracellular concentration of YgiP is very low in growing E. coli cells under aerobic conditions, but increases more than 100-fold to the level as high as the major nucleoid proteins under anaerobic conditions. An E. coli mutant lacking ygiP showed retarded growth under anaerobic conditions. High abundance and large number of binding sites together indicate that YgiP is a nucleoid-associated protein with both architectural and regulatory roles as the nucleoid proteins Fis and IHF. We then propose that YgiP is a novel nucleoid protein of E. coli under anaerobiosis and propose to rename it Dan (DNA-binding protein under anaerobic conditions).

The cell-surface expression and function of multisubunit plasma membrane proteins are regulated via interactions between catalytic subunits and auxiliary subunits. Subunit assembly in the endoplasmic reticulum is required for the cell-surface expression of the enzyme, but little is known about subunit interactions once it reaches the plasma membrane. Here we performed highly quantitative analyses of the catalytic (alpha1) and auxiliary (beta1 and beta3) subunits of Na(+)/K(+)-ATPase in the HeLa cell plasma membrane using isoform-specific antibodies and a cell-surface protein labeling procedure. Our results indicate that although the beta-subunit is required for the cell-surface expression of the alpha-subunit, the plasma membrane contains more alpha-subunits than beta-subunits. Pulse-labeling and chasing of the cell-surface proteins revealed that degradation of the beta-subunits was much faster than that of the alpha1-subunit. Ubiquitylation, as well as endocytosis, was involved in the fast degradation of the beta1-subunit. Double knockdown of the beta1- and beta3-subunits by RNAi resulted in the disappearance of these beta-subunits but not the alpha1-subunit in the plasma membrane. All these results indicate that the alpha- and beta-subunits of Na(+)/K(+)-ATPase are assembled in the endoplasmic reticulum, but are disassembled in the plasma membrane and undergo different degradation processes.

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., Karyopherin-dependent molecular transport through the nuclear pore complex is maintained by constant recycling pathways of karyopherins coupled with the Ran-dependent cargo catch-and-release mechanism. Although many studies have revealed the bidirectional dynamics of karyopherins, the entire kinetics of the steady-state dynamics of karyopherin and cargo is still not fully understood. In this study, we used fluorescence recovery after photobleaching and fluorescence loss in photobleaching on live cells to provide convincing in vivo proof that karyopherin-mediated nucleocytoplasmic transport of cargoes is bidirectional. Continuous photobleaching of the cytoplasm of live cells expressing NLS cargoes led to progressive decrease of nuclear fluorescence signals. In addition, experimentally obtained kinetic parameters of karyopherin complexes were used to establish a kinetic model to explain the entire cargo import and export transport cycles facilitated by importin β. The results strongly indicate that constant shuttling of karyopherins, either free or bound to cargo, ensures proper balancing of nucleocytoplasmic distribution of cargoes and establishes effective regulation of cargo dynamics by RanGTP.

In order to fold non-native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP-dependent reaction cycle. We constructed the real-time three-dimensional-observation system at high resolution using a newly developed fast-scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with a lifetime of 6 s (k=0.17 s(-1)). We also caught ATP/ADP-induced open-closed conformational changes of individual GroEL in the absence of qGroES and substrate proteins. Namely, the ATP/ADP-bound GroEL can change its conformation 'from closed to open' without additional ATP hydrolysis. Furthermore, the lifetime of open conformation in the presence of ADP ( approximately 1.0 s) was apparently lower than those of ATP and ATP-analogs (2-3 s), meaning that ADP-bound open-form is structurally less stable than ATP-bound open-form. These results indicate that GroEL has at least two distinct open-conformations in the presence of nucleotide; ATP-bound prehydrolysis open-form and ADP-bound open-form, and the ATP hydrolysis in open-form destabilizes its open-conformation and induces the 'from open to closed' conformational change of GroEL.

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 approximately 100 pN on average. By using this cantilever, we succeeded in measuring the interaction force between importin alpha and importin beta.

Higher-order architectures of chromosomes play important roles in the regulation of genome functions. To understand the molecular mechanism of genome packing, an in vitro chromatin reconstitution method and a single-molecule imaging technique (atomic force microscopy) were combined. In 50 mM NaCl, well-stretched beads-on-a-string chromatin fiber was observed. However, in 100 mM NaCl, salt-induced interaction between nucleosomes caused partial aggregation. Addition of histone H1 promoted a further folding of the fiber into thicker fibers 20-30 nm in width. Micrococcal nuclease digestion of these thicker fibers produced an approximately 170 bp fragment of nucleosomal DNA, which was approximately 20 bp longer than in the absence of histone H1 ( approximately 150 bp), indicating that H1 is correctly placed at the linker region. The width of the fiber depended on the ionic strength. Widths of 20 nm in 50 mM NaCl became 30 nm as the ionic strength was changed to 100 mM. On the basis of these results, a flexible model of chromatin fiber formation was proposed, where the mode of the fiber compaction changes depending both on salt environment and linker histone H1. The biological significance of this property of the chromatin architecture will be apparent in the closed segments ( approximately 100 kb) between SAR/MAR regions.

The proper function of the genome largely depends on the higher order architecture of the chromosome. Our previous application of nanotechnology to the questions regarding the structural basis for such macromolecular dynamics has shown that the higher order architecture of the Escherichia coli genome (nucleoid) is achieved via several steps of DNA folding (Kim et al., 2004). In this study, the hierarchy of genome organization was compared among E. coli, Staphylococcus aureus and Clostridium perfringens. A one-molecule-imaging technique, atomic force microscopy (AFM), was applied to the E. coli cells on a cover glass that were successively treated with a detergent, and demonstrated that the nucleoids consist of a fundamental fibrous structure with a diameter of 80 nm that was further dissected into a 40-nm fiber. An application of this on-substrate procedure to the S. aureus and the C. perfringens nucleoids revealed that they also possessed the 40- and 80-nm fibers that were sustainable in the mild detergent solution. The E. coli nucleoid dynamically changed its structure during cell growth; the 80-nm fibers releasable from the cell could be transformed into a tightly packed state depending upon the expression of Dps. However, the S. aureus and the C. perfringens nucleoids never underwent such tight compaction when they reached stationary phase. Bioinformatic analysis suggested that this was possibly due to the lack of a nucleoid protein, Dps, in both species. AFM analysis revealed that both the mitotic chromosome and the interphase chromatin of human cells were also composed of 80-nm fibers. Taking all together, we propose a structural model of the bacterial nucleoid in which a fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes.

The cell-surface expression of the Na(+)/K(+)-ATPase is tightly regulated at the ER/Golgi transit level. The assembly of the alpha- and beta-subunits is a prerequisite for the maturation of the enzyme and its exit from the ER. At present, the fate of the functional ATPase, once it reaches the plasma membrane, is obscure, although endocytosis, recycling, and degradation in the lysosome are known to be involved. In this study, we shall demonstrate the differential degradation of the Na(+)/K(+)-ATPase subunits in the plasma membrane: that is, lysosomal degradation of alpha-subunits and proteasomal degradation of beta-subunits, and propose the possibility of the subunit dissociation in the plasma membrane.

Since the inception of atomic force microscopy (AFM) in 1986, the value of this technology for exploring the structure and biophysical properties of a variety of biological samples has been increasingly recognized. AFM provides the opportunity to both image samples at nanometer resolution and also measure the forces on the surface of the sample. Here, we describe a variety of methods for studying nuclear samples including single nucleic acid molecules, higher-order chromatin structures, the nucleolus, and the nucleus. Protocols to prepare nucleic acids, nucleic acid-protein complexes, reconstituted chromatin, the cell nucleus, and the nucleolus are included, as well as protocols describing how to prepare the AFM substrate and the AFM tip. Finally, we describe how to perform conventional imaging, high-speed imaging, recognition imaging, force spectroscopy, and nanoindentation experiments.

Background: The plant vacuole is a multifunctional organelle that has various physiological functions. The vacuole dynamically changes its function and shape, dependent on developmental and physiological conditions. Our current understanding of the dynamic processes of vacuolar morphogenesis has suffered from the lack of a marker for observing these processes in living cells. Results: We have developed transgenic Arabidopsis thaliana expressing a vacuolar syntaxin-related molecule (AtVam3/SYP22) fused with green fluorescent protein (GFP). Observations using confocal laser scanning microscopy demonstrated that the plant vacuole contained a dynamic membrane system that underwent a complex architectural remodelling. Three-dimensional reconstitution and time-lapse analysis of GFP-fluorescence images revealed that cylindrical and sheet-like structures were present in the vacuolar lumen and were moving dynamically. The movement, but not the structure itself, was abolished by cytochalasin D, an inhibitor of actin polymerization. This moving structure, which sometimes penetrated through the vacuolar lumen, possessed a dynamic membrane architecture similar to the previously recognized ‘transvacuolar strand.’ Conclusion: We propose two possible models for the formation of the vacuolar lumenal structure. Membrane structures including protruding tubules and reticular networks have recently been recognized in many other organelles, and may be actively involved in intra- and/or inter-organelle signalling.

Condensin and cohesin are two protein complexes that act as the central mediators of chromosome condensation and sister chromatid cohesion, respectively. The basic underlying mechanism of action of these complexes remained enigmatic. Direct visualization of condensin and cohesin was expected to provide hints to their mechanisms. They are composed of heterodimers of distinct structural maintenance of chromosome (SMC) proteins and other non-SMC subunits. Here, we report the first observation of the architecture of condensin and its interaction with DNA by atomic force microscopy (AFM). The purified condensin SMC heterodimer shows a head-tail structure with a single head composed of globular domains and a tail with the coiled-coil region. Unexpectedly, the condensin non-SMC trimers associate with the head of SMC heterodimers, producing a larger head with the tail. The heteropentamer is bound to DNA in a distributive fashion, whereas condensin SMC heterodimers interact with DNA as aggregates within a large DNA-protein assembly. Thus, non-SMC trimers may regulate the ATPase activity of condensin by directly interacting with the globular domains of SMC heterodimer and alter the mode of DNA interaction. A model for the action of heteropentamer is presented.

The regulation of DNA replication and transcription is achieved by dynamic structural changes of chromatin in which a series of proteins will acquire accessibility to specific regions of the DNA strand. A combination of biochemistry and nano-technology is essential to address questions regarding the structural basis for such macromolecular mechanisms. In the present study, we established an efficient salt-dialysis method of chromatin reconstitution and employed atomic force microscopy (AFM) as a single-molecule-imaging technique, to monitor the efficiency of the reconstitution. At first, the reconstitution efficiency with short DNA molecules of several kilo-base pairs was low, although the salt dialysis yielded a "beads-on-a-string" structure of oligonucleosomes with each nucleosome trapping 158+/-27 bp DNA. However, the efficiency for nucleosome formation became higher when longer DNA molecules with a super-helical constraint were used. A statistical analysis of the obtained AFM images identified a first-order relationship between the efficiency of the reconstitution and the length of the super-coiled DNA used. A high efficiency of approximately 290 bp/nucleosome that is close to the in vivo situation was obtained with a approximately 100 kbp template DNA. This enabled the structure-function studies of long chromatin molecules under well-defined conditions.

Since the inception of atomic force microscopy (AFM) in 1986, the value of this technology for exploring the structure and biophysical properties of a variety of biological samples has been increasingly recognized. AFM provides the opportunity to both image samples at nanometer resolution and also measure the forces on the surface of the sample. Here, we describe a variety of methods for studying nuclear samples including single nucleic acid molecules, higher-order chromatin structures, the nucleolus, and the nucleus. Protocols to prepare nucleic acids, nucleic acid-protein complexes, reconstituted chromatin, the cell nucleus, and the nucleolus are included, as well as protocols describing how to prepare the AFM substrate and the AFM tip. Finally, we describe how to perform conventional imaging, high-speed imaging, recognition imaging, force spectroscopy, and nanoindentation experiments.

DNA is flexible and easily subjected to bending and wrapping via DNA/protein interaction. DNA supercoiling is known to play an important role in a variety of cellular events, such as transcription, replication, and recombination. It is, however, not well understood how the superhelical strain is efficiently redistributed during these reactions. Here we demonstrate a novel property of an initiator protein in DNA relaxation by utilizing a one-molecule-imaging technique, atomic force microscopy, combined with biochemical procedures. A replication initiator protein, RepE54 of bacterial mini-F plasmid (2.5 kb), binds to the specific sequences (iterons) within the replication region (ori2). When RepE54 binds to the iterons of the negatively supercoiled mini-F plasmid, it induces a dynamic structural transition of the plasmid to a relaxed state. This initiator-induced relaxation is mediated neither by the introduction of a DNA strand break nor by a local melting of the DNA double strand. Furthermore, RepE54 is not wrapped by DNA repeatedly. These data indicate that a local strain imposed by initiator binding can induce a drastic shift of the DNA conformation from a supercoiled to a relaxed state.

The DNase I-hyper-sensitive sites (HS2-HS4) in the beta-globin gene enhancer region (locus control region; LCR) have been known as the target of Bach1/MafK heterodimers. We have demonstrated previously by utilizing atomic force microscopy (AFM) that Bach1/MafK mediates the formation of a looped-DNA structure in the LCR fragment. Here we perform further detailed analyses of the loop structure formed between each HSs by AFM, and propose a novel model for the enhancer/protein interaction: the Bach1/MafK heterodimer preferentially binds to HS2 with highest affinity and to HS3 with lower affinity. However, they assemble to each other to form a stable complex of four heterodimers and mediate a DNA-loop formation. Once the DNA loop is formed between HS2 and HS3, the Bach1/MafK complex at the HS3 side leaves the HS3 and starts to slide along the DNA strand towards HS2 with the other side of the complex fixed at the HS2 region. This 'kiss and pull' model will contribute to understand the function of regulatory proteins at enhancer regions in terms of higher-order structure of DNA, e.g. nucleosomes and chromatin.

The endocytosis proceeds with a series of morphological changes of the plasma membrane, which is mediated by a successive assembly of a number of membrane-associated proteins. In addition to a local binding of those proteins, a mechanical tension of the membrane, as well as a dynamics of cortical cytoskeletal network is also involved in this process. Here, we utilized a fast-scanning atomic force microscope (AFM) which is specially designed for live-cell imaging (BIXAM), to directly visualize the dynamic morphological changes of the plasma membrane and cortical actin network, together with localizations of fluorescently-labeled proteins. COS-7 cells expressing EGFP-fused clathrin were observed by BIXAM. Overlaying confocal fluorescence images and AFM images clearly identified clathrin-coated membrane pits on the cell surface. The diameter of the pit was ∼300 nm, which is similar to that observed by an electron microscope, and the life time was 300 seconds. The membrane started to invaginate 10-20 seconds after the initial accumulation of clathrin. The clathrin signal kept slowly increasing, and suddenly disappeared 20-30 seconds after the pit closes. Interestingly, when the pit closes, a brief swelling of the adjacent membrane (∼50 seconds, ∼50 nm in height) appeared and covered the pit. Knock-down of dynamin did not affect this closing motion, indicating that dynamin is not necessary for the pit closure in COS-7 cells. Scanning of cortical actin with increased loading force of AFM, we found that the membrane swelling accompanied with local formation of densed cortical actin network. Inhibitors for actin polymerization/depolymerization (cytochalasin B and Jasplakinolide), as well as inhibitor for Arp2/3 complex (CK-666), inhibited the formation of the membrane swelling and caused reversible open-close motions of the pit, suggesting that actin dynamics is a major driving force for irreversible closing of the clathrin-dependent membrane pit.

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 β 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 Nups358, 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 Nups62 and 155 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.

Histones are highly conserved proteins among eukaryotes. However, yeast histones are more divergent in their sequences. In particular, the histone tail regions of the fission yeast, Schizosaccharomyces pombe, have fewer lysine residues, making their charges less positive than those of higher eukaryotes. In addition, the S. pombe chromatin lacks linker histones. How these factors affected yeast chromatin folding was analysed by biochemical reconstitution in combination with atomic force microscopy. Reconstitution of a nucleosome array showed that S. pombe chromatin has a more open structure similar to reconstituted human acetylated chromatin. The S. pombe nucleosomal array formed thinner fibers than those of the human nucleosomal array in the presence of mammalian linker histone H1. Such S. pombe fibers were more comparable to human acetylated fibers. These findings suggest that the core histone charges would determine the intrinsic characteristics of S. pombe chromatin and affect inter-nucleosomal interactions.

Highly selective nucleocytoplasmic molecular transport is critical to eukaryotic cells, which is illustrated by the size-filtering diffusion and karyopherin-mediated passage mechanisms. However, a considerable number of large proteins without nuclear localization signals are localized to the nucleus. Here, we provide evidence for spontaneous migration of large proteins in a karyopherin-independent manner. Time-lapse observation of nuclear transport assay revealed that several large molecules spontaneously and independently pass through the NPC. The amphiphilic motifs were shown to be sufficient to overcome the selectivity barrier of the NPC. Furthermore, we report that the characteristic amphiphilic property of these proteins enables altered local conformation in hydrophobic solutions, so that elevated surface hydrophobicity facilitates passage through the nuclear pore. The molecular dynamics simulation revealed the conformational change of the amphiphilic structure that exposes the hydrophobic amino acid residues to the outer surface in hydrophobic solution. These results contribute to the understanding of both nucleocytoplasmic molecular sorting and the nature of the permeability barrier.