Masatoshi Nishikawa

Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases

We showed that engineered zinc‐finger nucleases (ZFNs), which consist of a zinc‐finger DNA‐binding array and a nuclease domain of the restriction enzyme FokI, can introduce mutations at a specific genomic site in the sea urchin embryo. Using bacterial one‐hybrid screening with zinc‐finger randomized libraries and a single‐strand annealing assay in cultured cells, ZFNs targeting the sea urchin Hemicentrotus pulcherrimus homologue of HesC (HpHesC) were efficiently selected. Consistent with the phenotype observed in embryos injected with an antisense morpholino oligonucleotide against HpHesC, an increase in the primary mesenchyme cell population was observed in embryos injected with a pair of HpHesC ZFN mRNAs. In addition, sequence analysis of the mutations showed that deletions and insertions occurred at the HpHesC target site in the embryos injected with the HpHesC ZFN mRNAs. These results suggest that targeted gene disruption using ZFNs is feasible for the sea urchin embryo.

Excitable signal transduction induces both spontaneous and directional cell asymmetries in the phosphatidylinositol lipid signaling system for eukaryotic chemotaxis.

Intracellular asymmetry in the signaling network works as a compass to navigate eukaryotic chemotaxis in response to guidance cues. Although the compass variable can be derived from a self-organization dynamics, such as excitability, the responsible mechanism remains to be clarified. Here, we analyzed the spatiotemporal dynamics of the phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) pathway, which is crucial for chemotaxis. We show that spontaneous activation of PtdInsP3-enriched domains is generated by an intrinsic excitable system. Formation of the same signal domain could be triggered by various perturbations, such as short impulse perturbations that triggered the activation of intrinsic dynamics to form signal domains. We also observed the refractory behavior exhibited in typical excitable systems. We show that the chemotactic response of PtdInsP3 involves biasing the spontaneous excitation to orient the activation site toward the chemoattractant. Thus, this biased excitability embodies the compass variable that is responsible for both random cell migration and biased random walk. Our finding may explain how cells achieve high sensitivity to and robust coordination of the downstream activation that allows chemotactic behavior in the noisy environment outside and inside the cells.

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B–GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.

Modeling the self-organized phosphatidylinositol lipid signaling system in chemotactic cells using quantitative image analysis

Summary A key signaling event that is responsible for gradient sensing in eukaryotic cell chemotaxis is a phosphatidylinositol (PtdIns) lipid reaction system. The self-organization activity of this PtdIns lipid system induces an inherent polarity, even in the absence of an external chemoattractant gradient, by producing a localized PtdIns (3,4,5)-trisphosphate [PtdIns(3,4,5)P3]-enriched domain on the membrane. Experimentally, we found that such a domain could exhibit two types of behavior: (1) it could be persistent and travel on the membrane, or (2) be stochastic and transient. Taking advantage of the simultaneous visualization of PtdIns(3,4,5)P3 and the enzyme phosphatase and tensin homolog (PTEN), for which PtdIns(3,4,5)P3 is a substrate, we statistically demonstrated the inter-dependence of their spatiotemporal dynamics. On the basis of this statistical analysis, we developed a theoretical model for the self-organization of PtdIns lipid signaling that can accurately reproduce both persistent and transient domain formation; these types of formations can be explained by the oscillatory and excitability properties of the system, respectively.

Intracellular Encoding of Spatiotemporal Guidance Cues in a Self-Organizing Signaling System for Chemotaxis in Dictyostelium Cells

Even in the absence of guidance cues, chemotactic cells are often spontaneously motile, which should accompany a spontaneous symmetry breaking inside the cells. A shallow chemoattractant gradient can induce these cells to move directionally without much change in cell morphology. As the gradient becomes steeper, the accuracy of chemotaxis increases. It is not clear how the steepness is expressed or encoded internally in the signaling network, which in turn coordinately activates the motile apparatus for chemotaxis. In Dictyostelium cells, self-organizing polarization activities in the signaling network have been reported. In this paper, we conducted a theoretical study of the response of this self-organizing system to guidance cues. Our analyses indicate that self-organizing systems respond sharply to a shallow external gradient by increasing the precision of polarity direction and modulating the frequency of self-polarization. We also show how the precision increase and frequency modulation are achieved. Our results indicate that self-organizing activity, independent of external cues, is the basis for the sensitive and robust response to shallow gradients. Finally, we show that the system can sense the direction of space-time waves of a stimulus, for which Dictyostelium cells exhibit chemotaxis in the developmental process.

Fluctuation Analysis of Mechanochemical Coupling Depending on the Type of Biomolecular Motors

Mechanochemical coupling was studied for myosin II and V consistently. The fluctuation in myosin V motility was determined by correlating the stochasticity of the ATPase reaction with regular displacements per one ATP, consistent with a tight mechanochemical coupling. In contrast, myosin II, working in an ensemble, was explained by a loose coupling, generating variable step sizes which depend on [ATP] and realizing a much larger step (200 nm) per one ATP than myosin V through its cooperativity at zero load. These different mechanics are ideal for their physiological functions.

Cooperative actions between myosin heads bring effective functions.

A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1, 1-19]. We have built a model reproducing such a stochastic movement of a myosin molecule incorporated with ATPase reaction cycles and demonstrated that the thermal fluctuation was a key for the function of myosin molecules [Esaki, S., Ishii, Y., Yanagida, T., 2003. Model describing the biased Brownian movement of myosin. Proc. Jpn. Acad. 79 (Ser B), 9-14]. The size of the displacement generated during the hydrolysis of single ATP molecules was limited within a half pitch of an actin filament when a single myosin molecules work separately. However, in muscle the size of the displacement has been reported to be greater than 60 nm [Yanagida, T., Arata, T., Oosawa, F., 1985. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366-369; Higuchi et al., 1991]. The difference suggests cooperative action between myosin heads in muscle. Here we extended the model built for an isolated myosin head to a system in which myosin heads are aligned in muscle arrangement to understand the cooperativity between heads. The simulation showed that the rotation of the actin filament [Takezawa, Y., Sugimoto, Y., Wakabayashi, K., 1998. Extensibility of the actin and myosin filaments in various states of skeletal muscles as studied by X-ray diffraction. Adv. Exp. Med. Biol. 453, 309-317; Wakabayashi, K., Ueno, Y., Takezawa, Y., Sugimoto, Y., 2001. Muscle contraction mechanism: use of X-ray synchrotron radiation. Nat. Enc. Life Sci. 1-11] associated with the release of ATPase products and binding of ATP as well as interaction between myosin heads allowed the myosin filament to move greater than a half pitch of the actin filament while a single ATP molecule is hydrolyzed. Our model demonstrated that the movement is loosely coupled to the ATPase cycle as observed in muscle.

Nonadaptive fluctuation in an adaptive sensory system: bacterial chemoreceptor.

Background Sensory systems often exhibit an adaptation or desensitization after a transient response, making the system ready to receive a new signal over a wide range of backgrounds. Because of the strong influence of thermal stochastic fluctuations on the biomolecules responsible for the adaptation, such as many membrane receptors and channels, their response is inherently noisy, and the adaptive property is achieved as a statistical average. Methodology/Principal Findings Here, we study a simple kinetic model characterizing the essential aspects of these adaptive molecular systems and show theoretically that, while such an adaptive sensory system exhibits a perfect adaptation property on average, its temporal stochastic fluctuations are able to be sensitive to the environmental conditions. Among the adaptive sensory systems, an extensively studied model system is the bacterial receptor responsible for chemotaxis. The model exhibits a nonadaptive fluctuation sensitive to the environmental ligand concentration, while perfect adaptation is achieved on average. Furthermore, we found that such nonadaptive fluctuation makes the bacterial behavior dependent on the environmental chemoattractant concentrations, which enhances the chemotactic performance. Conclusions/Significance This result indicates that adaptive sensory systems can make use of such stochastic fluctuation to carry environmental information, which is not possible by means of the average, while keeping responsive to the changing stimulus.

The Relation of Signal Transduction to the Sensitivity and Dynamic Range of Bacterial Chemotaxis

Complex networks of interacting molecular components of living cells are responsible for many important processes, such as signal processing and transduction. An important challenge is to understand how the individual properties of these molecular interactions and biochemical transformations determine the system-level properties of biological functions. Here, we address the issue of the accuracy of signal transduction performed by a bacterial chemotaxis system. The chemotaxis sensitivity of bacteria to a chemoattractant gradient has been measured experimentally from bacterial aggregation in a chemoattractant-containing capillary. The observed precision of the chemotaxis depended on environmental conditions such as the concentration and molecular makeup of the chemoattractant. In a quantitative model, we derived the chemotactic response function, which is essential to describing the signal transduction process involved in bacterial chemotaxis. In the presence of a gradient, an analytical solution is derived that reveals connections between the chemotaxis sensitivity and the characteristics of the signaling system, such as reaction rates. These biochemical parameters are integrated into two system-level parameters: one characterizes the efficiency of gradient sensing, and the other is related to the dynamic range of chemotaxis. Thus, our approach explains how a particular signal transduction property affects the system-level performance of bacterial chemotaxis. We further show that the two parameters can be derived from published experimental data from a capillary assay, which successfully characterizes the performance of bacterial chemotaxis.

Adaptive Responses Limited by Intrinsic Noise

Sensory systems have mechanisms to respond to the external environment and adapt to them. Such adaptive responses are effective for a wide dynamic range of sensing and perception of temporal change in stimulus. However, noise generated by the adaptation system itself as well as extrinsic noise in sensory inputs may impose a limit on the ability of adaptation systems. The relation between response and noise is well understood for equilibrium systems in the form of fluctuation response relation. However, the relation for nonequilibrium systems, including adaptive systems, are poorly understood. Here, we systematically explore such a relation between response and fluctuation in adaptation systems. We study the two network motifs, incoherent feedforward loops (iFFL) and negative feedback loops (nFBL), that can achieve perfect adaptation. We find that the response magnitude in adaption systems is limited by its intrinsic noise, implying that higher response would have higher noise component as well. Comparing the relation of response and noise in iFFL and nFBL, we show that whereas iFFL exhibits adaptation over a wider parameter range, nFBL offers higher response to noise ratio than iFFL. We also identify the condition that yields the upper limit of response for both network motifs. These results may explain the reason of why nFBL seems to be more abundant in nature for the implementation of adaption systems.