Search

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Various nuclear functional complexes contain cytoskeletal proteins as regulatory subunits; for example, nuclear actin participates in transcriptional complexes, and actin-related proteins are integral to chromatin remodeling complexes. Nuclear complexes such as these are involved in both basal and adaptive nuclear functions. In addition to nuclear import via classical nuclear transport pathways or passive diffusion, some large cytoskeletal proteins spontaneously migrate into the nucleus in a karyopherinindependent manner. The balance of nucleocytoplasmic distribution of such proteins can be altered by several factors, such as import versus export, or capture and release by complexes. The resulting accumulation or depletion of the nuclear populations thereby enhances or attenuates their nuclear functions. We propose that such molecular dynamics constitute a form of cytoskeleton-modulated regulation of nuclear functions which is mediated by the translocation of cytoskeletal components in and out of the nucleus. [ABSTRACT FROM AUTHOR]

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. WIREs Nanomed Nanobiotechnol 2011 3 574-588 DOI: 10.1002/wnan.150 For further resources related to this article, please visit the . [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Ezrin–radixin–moesin protein family provides a regulated link between the cortical actin cytoskeleton and the plasma membrane. Phosphorylation of ezrin has been functionally linked to membrane dynamics and plasticity. Our recent study demonstrated that phosphorylation of the conserved T567 residue of ezrin alters the physiology of gastric parietal cells. However, the molecular mechanism of phosphorylation-induced ezrin activation has remained elusive. Here we use atomic force microscopy (AFM) to probe phosphorylation-mediated activation of ezrin in single molecules. The phospho-mimicking and non-phosphorylatable mutant ezrin proteins were generated and purified to homogeneity. Comparative analyses of two ezrin mutants by AFM demonstrate the unfolding of the N- and C-terminal domains upon the phospho-activation. To measure the physical force underlying the inter-domain contact during mechanical unfolding, we probed the defined region of ezrin using the N-terminal ezrin coated onto the AFM tip. Comparative force measurements indicate that T567 phosphorylation-induced unfolding of ezrin favors the inter-molecular association. Taken together, these results provide molecular illustration of phosphorylation elicited functional activation of ERM proteins and indicate that stimulus-induced protein conformational change can be used as a signaling mechanism orchestrating cellular dynamics. [Copyright &y& Elsevier]

The Obg subfamily protein is one of the P-loop small G proteins and is highly conserved in many organisms from bacteria to human. Two obg genes, obgH1 and obgH2, exist in the human genome. Both ObgH1 and ObgH2 showed similar GTPase activities (0.014 ± 0.005 and 0.010 ± 0.002/min for ObgH1 and ObgH2, respectively) to those of the bacterial Obg proteins and complemented the Obg function in Escherichia coli ribosome maturation, suggesting that the functions of Obg proteins are well conserved through evolution. Immunofluorescence microscopy of HeLa cells revealed that ObgH1 localizes in mitochondria, and ObgH2 in the dense fibrillar compartment region of the nucleolus. Knock-down of ObgH1 by RNAi induced mitochondria elongation, whereas knock-down of ObgH2 resulted in the disorganization of the nucleolar architecture. In conclusion, the two human Obg proteins have similar enzymatic activities that can complement bacterial Obg function, but show different cellular function(s) with different intracellular localizations. [ABSTRACT FROM AUTHOR]

Abstract: 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 ∼100pN on average. By using this cantilever, we succeeded in measuring the interaction force between importin α and importin β. [Copyright &y& Elsevier]

In the telomere region of human chromosomes, the (TTAGGG)n sequence stretches over several kilobases and forms a distinct higher-order structure with various proteins. Telomere repeat binding factors (TRFs) bind specifically to this sequence and play critical roles in the maintenance of telomere structure and function. Here, we prepared a series of linear DNA carrying a stretch of telomeric sequence ((TTAGGG)n, ∼1.8 (kb) with different end-structures and observed their higher-order complexes with TRFs by atomic force microscopy. TRF2 molecules exclusively bound to the telomeric DNA region at several different places simultaneously mainly as a dimer, and often mediated DNA loop formation by forming a tetramer at the root. These multiple-binding, multimerization and DNA loop formation by TRF2 were observed regardless of the DNA-end structure (blunt, 3′-overhanging, telomeric, non-telomeric). However, when the DNA end carried the telomeric-3′-overhanging region, the loop was frequently formed at the end of the DNA. Namely, the TRF2-mediated DNA loop formation is independent of the end-structure and the 3′-overhanging TTAGGG sequence is responsible for the stabilization of the loop. TRF1 also bound to the telomeric DNA as a dimer, but did not mediate DNA loop formation by itself. These results provide a new insight into the molecular mechanism of DNA end-loop formation by TRFs. [ABSTRACT FROM AUTHOR]

Palytoxin (PTX) induces a cation channel through interaction with Na+,K+-ATPase. It is unclear how this action relates to the enzyme catalytic activity. We examined whether the action of PTX depends on the catalytic domain specific for Na+,K+-ATPase. Wild-type Na+,K+-ATPase α-subunit (NNN) or its chimera (NCN), in which the catalytic domain was replaced with that of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, was co-expressed with β-subunit in the yeast Saccharomyces cerevisiae. PTX (0.1–100 nM) increased K+ efflux in NNN- or NCN-transfected cells to a similar degree but not in non-transfected cells. When ouabain-resistant NNN and NCN were expressed, PTX also increased K+ efflux. Ouabain inhibited the effect of PTX in NNN or NCN cells but not in ouabain-resistant cells. These data suggest that the channel-forming action of PTX does not depend on the catalytic domain species. [Copyright &y& Elsevier]

Abstract 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. [ABSTRACT FROM AUTHOR]