Tetsuya J. Kobayashi

Atypical cadherins Dachsous and Fat control dynamics of noncentrosomal microtubules in planar cell polarity.

How global organ asymmetry and individual cell polarity are connected to each other is a central question in studying planar cell polarity (PCP). In the Drosophila wing, which develops PCP along its proximal-distal (P-D) axis, we previously proposed that the core PCP mediator Frizzled redistributes distally in a microtubule (MT)-dependent manner. Here, we performed organ-wide analysis of MT dynamics by introducing quantitative in vivo imaging. We observed MTs aligning along the P-D axis at the onset of redistribution and a small but significant excess of + ends-distal MTs in the proximal region of the wing. This characteristic alignment and asymmetry of MT growth was controlled by atypical cadherins Dachsous (Ds) and Fat (Ft). Furthermore, the action of Ft was mediated in part by PAR-1. All these data support the idea that the active reorientation of MT growth adjusts cell polarity along the organ axis.

Automatic Extraction of Nuclei Centroids of Mouse Embryonic Cells from Fluorescence Microscopy Images

Accurate identification of cell nuclei and their tracking using three dimensional (3D) microscopic images is a demanding task in many biological studies. Manual identification of nuclei centroids from images is an error-prone task, sometimes impossible to accomplish due to low contrast and the presence of noise. Nonetheless, only a few methods are available for 3D bioimaging applications, which sharply contrast with 2D analysis, where many methods already exist. In addition, most methods essentially adopt segmentation for which a reliable solution is still unknown, especially for 3D bio-images having juxtaposed cells. In this work, we propose a new method that can directly extract nuclei centroids from fluorescence microscopy images. This method involves three steps: (i) Pre-processing, (ii) Local enhancement, and (iii) Centroid extraction. The first step includes two variations: first variation (Variant-1) uses the whole 3D pre-processed image, whereas the second one (Variant-2) modifies the preprocessed image to the candidate regions or the candidate hybrid image for further processing. At the second step, a multiscale cube filtering is employed in order to locally enhance the pre-processed image. Centroid extraction in the third step consists of three stages. In Stage-1, we compute a local characteristic ratio at every voxel and extract local maxima regions as candidate centroids using a ratio threshold. Stage-2 processing removes spurious centroids from Stage-1 results by analyzing shapes of intensity profiles from the enhanced image. An iterative procedure based on the nearest neighborhood principle is then proposed to combine if there are fragmented nuclei. Both qualitative and quantitative analyses on a set of 100 images of 3D mouse embryo are performed. Investigations reveal a promising achievement of the technique presented in terms of average sensitivity and precision (i.e., 88.04% and 91.30% for Variant-1; 86.19% and 95.00% for Variant-2), when compared with an existing method (86.06% and 90.11%), originally developed for analyzing C. elegans images.

Balancing Acts of Two HEAT Subunits of Condensin I Support Dynamic Assembly of Chromosome Axes

Condensin I is a five-subunit protein complex that plays a central role in mitotic chromosome assembly and segregation in eukaryotes. To dissect its mechanism of action, we reconstituted wild-type and mutant complexes from recombinant subunits and tested their abilities to assemble chromosomes in Xenopus egg cell-free extracts depleted of endogenous condensins. We find that ATP binding and hydrolysis by SMC subunits have distinct contributions to the action of condensin I and that continuous ATP hydrolysis is required for structural maintenance of chromosomes. Mutant complexes lacking either one of two HEAT subunits produce abnormal chromosomes with highly characteristic defects and have contrasting structural effects on chromosome axes preassembled with the wild-type complex. We propose that balancing acts of the two HEAT subunits support dynamic assembly of chromosome axes under the control of the SMC ATPase cycle, thereby governing construction of rod-shaped chromosomes in eukaryotic cells.

Single cell visualization of yeast gene expression shows correlation of epigenetic switching between multiple heterochromatic regions through multiple generations.

A single-cell method allows the assessment of relationships between the dynamic epigenetic behavior of yeast heterochromatin boundaries over multiple generations.

Dynamics of intracellular information decoding

A variety of cellular functions are robust even to substantial intrinsic and extrinsic noise in intracellular reactions and the environment that could be strong enough to impair or limit them. In particular, of substantial importance is cellular decision-making in which a cell chooses a fate or behavior on the basis of information conveyed in noisy external signals. For robust decoding, the crucial step is filtering out the noise inevitably added during information transmission. As a minimal and optimal implementation of such an information decoding process, the autocatalytic phosphorylation and autocatalytic dephosphorylation (aPadP) cycle was recently proposed. Here, we analyze the dynamical properties of the aPadP cycle in detail. We describe the dynamical roles of the stationary and short-term responses in determining the efficiency of information decoding and clarify the optimality of the threshold value of the stationary response and its information-theoretical meaning. Furthermore, we investigate the robustness of the aPadP cycle against the receptor inactivation time and intrinsic noise. Finally, we discuss the relationship among information decoding with information-dependent actions, bet-hedging and network modularity.

Discovery of triazolopyridine GS-458967, a late sodium current inhibitor (Late INai) of the cardiac NaV 1.5 channel with improved efficacy and potency relative to ranolazine.

We started with a medium throughput screen of heterocyclic compounds without basic amine groups to avoid hERG and β-blocker activity and identified [1,2,4]triazolo[4,3-a]pyridine as an early lead. Optimization of substituents for Late INa current inhibition and lack of Peak INa inhibition led to the discovery of 4h (GS-458967) with improved anti-arrhythmic activity relative to ranolazine. Unfortunately, 4h demonstrated use dependent block across the sodium isoforms including the central and peripheral nervous system isoforms that is consistent with its low therapeutic index (approximately 5-fold in rat, 3-fold in dog). Compound 4h represents our initial foray into a 2nd generation Late INa inhibitor program and is an important proof-of-concept compound. We will provide additional reports on addressing the CNS challenge in a follow-up communication.

Discovery of Dihydrobenzoxazepinone (GS-6615) Late Sodium Current Inhibitor (Late INai), a Phase II Agent with Demonstrated Preclinical Anti-Ischemic and Antiarrhythmic Properties.

Late sodium current (late INa) is enhanced during ischemia by reactive oxygen species (ROS) modifying the Nav 1.5 channel, resulting in incomplete inactivation. Compound 4 (GS-6615, eleclazine) a novel, potent, and selective inhibitor of late INa, is currently in clinical development for treatment of long QT-3 syndrome (LQT-3), hypertrophic cardiomyopathy (HCM), and ventricular tachycardia-ventricular fibrillation (VT-VF). We will describe structure-activity relationship (SAR) leading to the discovery of 4 that is vastly improved from the first generation late INa inhibitor 1 (ranolazine). Compound 4 was 42 times more potent than 1 in reducing ischemic burden in vivo (S-T segment elevation, 15 min left anteriorior descending, LAD, occlusion in rabbits) with EC50 values of 190 and 8000 nM, respectively. Compound 4 represents a new class of potent late INa inhibitors that will be useful in delineating the role of inhibitors of this current in the treatment of patients.

Positive and Negative Regulatory Mechanisms for Fine-Tuning Cellularity and Functions of Medullary Thymic Epithelial Cells

Self-tolerant T cells and regulatory T cells develop in the thymus. A wide variety of cell–cell interactions in the thymus is required for the differentiation, proliferation, and repertoire selection of T cells. Various secreted and cell surface molecules expressed in thymic epithelial cells (TECs) mediate these processes. Moreover, cytokines expressed by cells of hematopoietic origin regulate the cellularity of TECs. Tumor necrosis factor (TNF) family RANK ligand, lymphotoxin, and CD40 ligand, expressed in T cells and innate lymphoid cells (ILCs), promote the differentiation and proliferation of medullary TECs (mTECs) that play critical roles in the induction of immune tolerance. A recent study suggests that interleukin-22 (IL-22) produced by ILCs promotes regeneration of TECs after irradiation. Intriguingly, tumor growth factor-β and osteoprotegerin limit cellularity of mTECs, thereby attenuating regulatory T cell generation. We will review recent insights into the molecular basis for cell–cell interactions regulating differentiation and proliferation of mTECs and also discuss about a perspective on use of mathematical models for understanding this complicated system.

Fluctuation Relations of Fitness and Information in Population Dynamics.

Phenotype switching with and without sensing environment is a common strategy of organisms to survive in a fluctuating environment. Understanding the evolutionary advantages of switching and sensing requires a quantitative evaluation of their fitness gain and its fluctuation together with the conditions for the switching and sensing strategies being adapted to a given environment. In this work, by using a pathwise formulation of the population dynamics, we show that the optimal switching strategy is characterized by a consistency condition for time-forward and backward path probabilities. The formulation also clarifies the underlying information-theoretic aspect of selection as a passive information compression. The loss of fitness by a suboptimal strategy is also shown to satisfy a fluctuation relation, which provides us with the information on how environmental fluctuation impacts the advantages of the optimal strategy. These results are naturally extended to the situation that organisms can use an environmental signal by actively sensing the environment. The fluctuation relations of the fitness gain by sensing are derived in which the multivariate mutual information among the phenotype, the environment, and the signal plays the role to quantify the relevant information in the signal for the fitness gain.

Experimental verification of laser photocathode RF gun as an injector for a laser plasma accelerator

The feasibility of the laser photocathode RF gun, BNL/GUN-IV, as an injector for a laser plasma accelerator was investigated at the subpicosecond S-band twin linac system of the Nuclear Engineering Research Laboratory, University of Tokyo. Electron beam energy of 16 MeV, emittance of 6/spl pi/ mm mrad, bunch length of 240 fs (FWHM), and charge per bunch of 350 pC were confirmed at 10 Hz. As for diagnosis of the femtosecond electron bunch, the quantitative comparison of performance of the femtosecond streak camera, the coherent transition radiation (CTR) Michelson interferometer, and the far-infrared polychromator was carried out. We concluded that the streak camera is the most reliable up to 200 fs and that the polychromator is the best for the shorter electron bunch. The 3.5-ps (rms) resolved synchronization between the YLF laser driver for the gun and the electron bunch was achieved. Based on the above experiences, we have designed and installed a much better laser-electron synchronization system using the Kerr-lens mode-locked Ti:Sapphire laser with the min harmonics synchrolocker and the stable 15-MW klystron. The timing jitter is expected to be suppressed down to 320 fs (rms).