Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly.

The nucleolus is the site of ribosome assembly and formed through liquid-liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm.

Live imaging of transcription sites using an elongating RNA polymerase II-specific probe.

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe.

Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells.

In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here, we demonstrate a new method to see endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps: First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments.

CLIP-170 is essential for MTOC repositioning during T cell activation by regulating dynein localisation on the cell surface.

The microtubule-organizing centre (MTOC) is repositioned to the centre of the contacted cell surface, the immunological synapse, during T cell activation. However, our understanding of its molecular mechanism remains limited. Here, we found that the microtubule plus-end tracking cytoplasmic linker protein 170 (CLIP-170) plays a novel role in MTOC repositioning using fluorescence imaging.

Multi-color single-molecule tracking and subtrajectory analysis for quantification of spatiotemporal dynamics and kinetics upon T cell activation.

The dynamic properties of molecules in living cells are attracting increasing interest. We propose a new method, moving subtrajectory analysis using single-molecule tracking, and demonstrate its utility in the spatiotemporal quantification of not only dynamics but also the kinetics of interactions using single-color images. Combining this technique with three-color simultaneous single-molecule imaging, we quantified the dynamics and kinetics of molecules in spatial relation to T cell receptor (TCR) microclusters, which trigger TCR signaling.

MKRN2 is a novel ubiquitin E3 ligase for the p65 subunit of NF-κB and negatively regulates inflammatory responses.

Activation of NF-κB transcription factor is strictly regulated to prevent excessive inflammatory responses leading to immunopathology. However, it still remains unclear how NF-κB activation is negatively controlled. The PDZ-LIM domain-containing protein PDLIM2 is a nuclear ubiquitin E3 ligase targeting the p65 subunit of NF-κB for degradation, thus terminating NF-κB-mediated inflammation.

Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-xL in living cells.

Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF.

A facile preparation of glass-supported lipid bilayers for analyzing molecular dynamics.

Many research programs focus on the molecular dynamics of living cells. This research requires cells to be adhered to a substrate while retaining the innate motility of their surface molecules. Lipid bilayer-based systems fulfill this requirement, although current methods are complicated and their utility is limited.

Nuclear actin activates human transcription factor genes including the OCT4 gene.

RNA microarray analyses revealed that nuclear actin activated many human transcription factor genes including OCT4, which is required for gene reprogramming. Oct4 is known to be activated by nuclear actin in Xenopus oocytes. Our findings imply that this process of OCT4 activation is conserved in vertebrates and among cell types and could be used for gene reprogramming of human cells.

Regulation of RNA polymerase II activation by histone acetylation in single living cells.

In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time.

Characterization of nuclear pore complex components in fission yeast Schizosaccharomyces pombe.

The nuclear pore complex (NPC) is an enormous proteinaceous complex composed of multiple copies of about 30 different proteins called nucleoporins. In this study, we analyzed the composition of the NPC in the model organism Schizosaccharomyces pombe using strains in which individual nucleoporins were tagged with GFP. We identified 31 proteins as nucleoporins by their localization to the nuclear periphery.

Vesnarinone suppresses TNFα mRNA expression by inhibiting valosin-containing protein.

Vesnarinone is a synthetic quinolinone derivative used in the treatment of cardiac failure and cancer. It is also known to cause agranulocytosis as a side effect, which restricts its use, although the mechanism underlying agranulocytosis is not well understood. Here, we show that vesnarinone binds to valosin-containing protein (VCP), which interacts with polyubiquitinated proteins and is essential for the degradation of IκBα to activate nuclear factor (NF)κB.

Dynein-driven transport of T cell receptor microclusters regulates immune synapse formation and T cell activation.

When T cells recognize a peptide-major histocompatibility complex on antigen-presenting cells (APCs), T cell receptor microclusters (TCR-MCs) are generated and move to the center of the T cell-APC interface to form the central supramolecular activation cluster (cSMAC). cSMAC formation depends on stimulation strength and regulates T cell activation. We demonstrate that the dynein motor complex colocalized and coimmunoprecipitated with the TCR complex and that TCR-MCs moved along microtubules (MTs) toward the center of the immune synapse in a dynein-dependent manner to form cSMAC.

Spatiotemporal basis of CTLA-4 costimulatory molecule-mediated negative regulation of T cell activation.

T cell activation is positively and negatively regulated by a pair of costimulatory receptors, CD28 and CTLA-4, respectively. Because these receptors share common ligands, CD80 and CD86, the expression and behavior of CTLA-4 is critical for T cell costimulation regulation. However, in vivo blocking of CD28-mediated costimulation by CTLA-4 and its mechanisms still remain elusive.

RNG105 deficiency impairs the dendritic localization of mRNAs for Na+/K+ ATPase subunit isoforms and leads to the degeneration of neuronal networks.

mRNA transport and local translation in dendrites play key roles in use-dependent synaptic modification and in higher-order brain functions. RNG105, an RNA-binding protein, has previously been identified as a component of RNA granules that mediate dendritic mRNA localization and local translation. Here, we demonstrate that RNG105 knock-out in mice reduces the dendritic localization of mRNAs for Na+/K+ ATPase (NKA) subunit isoforms (i.

RNA granule protein 140 (RNG140), a paralog of RNG105 localized to distinct RNA granules in neuronal dendrites in the adult vertebrate brain.

RNA granules mediate the transport and local translation of their mRNA cargoes, which regulate cellular processes such as stress response and neuronal synaptic plasticity. RNA granules contain specific RNA-binding proteins, including RNA granule protein 105 (RNG105), which is likely to participate in the transport and translation of mRNAs. In the present report, an RNG105 paralog, RNG140 is described.

Stochastic emergence of multiple intermediates detected by single-molecule quasi-static mechanical unfolding of protein.

Experimental probing of a protein-folding energy landscape can be challenging, and energy landscapes comprising multiple intermediates have not yet been defined. Here, we quasi-statically unfolded single molecules of staphylococcal nuclease by constant-rate mechanical stretching with a feedback positioning system. Multiple discrete transition states were detected as force peaks, and only some of the multiple transition states emerged stochastically in each trial.

Vav links the T cell antigen receptor to the actin cytoskeleton and T cell activation independently of intrinsic Guanine nucleotide exchange activity.

Background: T cell receptor (TCR) engagement leads to formation of signaling microclusters and induction of rapid and dynamic changes in the actin cytoskeleton, although the exact mechanism by which the TCR initiates actin polymerization is incompletely understood. The Vav family of guanine nucleotide exchange factors (GEF) has been implicated in generation of TCR signals and immune synapse formation, however, it is currently not known if Vav's GEF activity is required in T cell activation by the TCR in general, and in actin polymerization downstream of the TCR in particular.

Methodology/principal Findings: Here, we report that Vav1 assembles into signaling microclusters at TCR contact sites and is critical for TCR-initiated actin polymerization.

Direct observation of multiple and stochastic transition states by a feedback-controlled single-molecule force measurement.

To overcome the ensemble-averaging barrier, single-molecule experiments have been performed, but energy landscapes comprising multiple intermediates have not yet been defined. We performed mechanical unfolding of staphylococcal nuclease using intermolecular force microscopy, modified AFM with high resolution and feedback control of the positioning. The force dropped vertically just after its peak, and multiple transition states were detected as force peaks.

Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C theta translocation.

T cell activation is mediated by microclusters (MCs) containing T cell receptors (TCRs), kinases, and adaptors. Although TCR MCs translocate to form a central supramolecular activation cluster (cSMAC) of the immunological synapse at the interface of a T cell and an antigen-presenting cell, the role of MC translocation in T cell signaling remains unclear. Here, we found that the accumulation of MCs at cSMAC was important for T cell costimulation.

Highly inclined thin illumination enables clear single-molecule imaging in cells.

We describe a simple illumination method of fluorescence microscopy for molecular imaging. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. A high ratio yielded clear single-molecule images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify molecular dynamics, interactions and kinetics in cells for molecular systems biology.

Zinc is a novel intracellular second messenger.

Zinc is an essential trace element required for enzymatic activity and for maintaining the conformation of many transcription factors; thus, zinc homeostasis is tightly regulated. Although zinc affects several signaling molecules and may act as a neurotransmitter, it remains unknown whether zinc acts as an intracellular second messenger capable of transducing extracellular stimuli into intracellular signaling events. In this study, we report that the cross-linking of the high affinity immunoglobin E receptor (Fcepsilon receptor I [FcepsilonRI]) induced a release of free zinc from the perinuclear area, including the endoplasmic reticulum in mast cells, a phenomenon we call the zinc wave.

[Molecular imaging and analysis of microclusters responsible for initiating T cell receptor signaling].

T cell receptors (TCR) are activated by a specific antigen and interact with other signaling molecules, such as kinases and adaptors. Aiming at analyzing precisely the dynamic process of T cell signaling, we used a combined system of a planar bilayer and TIRF microscopy. This system allowed us to observe the T cell activation process from the initial cell-bilayer contact (time 0).