Raymond Cheong

Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs

Heart tissue possesses complex structural organization on multiple scales, from macro- to nano-, but nanoscale control of cardiac function has not been extensively analyzed. Inspired by ultrastructural analysis of the native tissue, we constructed a scalable, nanotopographically controlled model of myocardium mimicking the in vivo ventricular organization. Guided by nanoscale mechanical cues provided by the underlying hydrogel, the tissue constructs displayed anisotropic action potential propagation and contractility characteristic of the native tissue. Surprisingly, cell geometry, action potential conduction velocity, and the expression of a cell–cell coupling protein were exquisitely sensitive to differences in the substratum nanoscale features of the surrounding extracellular matrix. We propose that controlling cell–material interactions on the nanoscale can stipulate structure and function on the tissue level and yield novel insights into in vivo tissue physiology, while providing materials for tissue repair.

Information transduction capacity of noisy biochemical signaling networks.

Noise limits information transfer through a single signaling pathway in a single cell to just one bit. Molecular noise restricts the ability of an individual cell to resolve input signals of different strengths and gather information about the external environment. Transmitting information through complex signaling networks with redundancies can overcome this limitation. We developed an integrative theoretical and experimental framework, based on the formalism of information theory, to quantitatively predict and measure the amount of information transduced by molecular and cellular networks. Analyzing tumor necrosis factor (TNF) signaling revealed that individual TNF signaling pathways transduce information sufficient for accurate binary decisions, and an upstream bottleneck limits the information gained via multiple integrated pathways. Negative feedback to this bottleneck could both alleviate and enhance its limiting effect, despite decreasing noise. Bottlenecks likewise constrain information attained by networks signaling through multiple genes or cells.

Transient IκB Kinase Activity Mediates Temporal NF-κB Dynamics in Response to a Wide Range of Tumor Necrosis Factor-α Doses

Dynamic properties of signaling pathways control their behavior and function. We undertook an iterative computational and experimental investigation of the dynamic properties of tumor necrosis factor (TNF)α-mediated activation of the transcription factor NF-κB. Surprisingly, we found that the temporal profile of the NF-κB activity is invariant to the TNFα dose. We reverse engineered a computational model of the signaling pathway to identify mechanisms that impart this important response characteristic, thus predicting that the IKK activity profile must transiently peak at all TNFα doses to generate the observed NF-κB dynamics. Experimental confirmation of this prediction emphasizes the importance of mechanisms that rapidly down-regulate IKK following TNFα activation. A refined computational model further revealed signaling characteristics that ensure robust TNFα-mediated cell-cell communication over considerable distances, allowing for fidelity of cellular inflammatory responses in infected tissue.

Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk.

Loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to abnormal activation of the normally silent maternal allele, is a common human epigenetic population variant associated with a 5-fold increased frequency of colorectal neoplasia. Here, we show first that LOI leads specifically to increased expression of proliferation-related genes in mouse intestinal crypts. Surprisingly, LOI(+) mice also have enhanced sensitivity to IGF-II signaling, not simply increased IGF-II levels, because in vivo blockade with NVP-AEW541, a specific inhibitor of the IGF-II signaling receptor, showed reduction of proliferation-related gene expression to levels half that seen in LOI(−) mice. Signal transduction assays in microfluidic chips confirmed this enhanced sensitivity with marked augmentation of Akt/PKB signaling in LOI(+) cells at low doses of IGF-II, which was reduced in the presence of the inhibitor to levels below those found in LOI(−) cells, and was associated with increased expression of the IGF1 and insulin receptor genes. We exploited this increased IGF-II sensitivity to develop an in vivo chemopreventive strategy using the azoxymethane (AOM) mutagenesis model. LOI(+) mice treated with AOM showed a 60% increase in premalignant aberrant crypt foci (ACF) formation over LOI(−) mice. In vivo IGF-II blockade with NVP-AEW541 abrogated this effect, reducing ACF to a level 30% lower even than found in exposed LOI(−) mice. Thus, LOI increases cancer risk in a counterintuitive way, by increasing the sensitivity of the IGF-II signaling pathway itself, providing a previously undescribed epigenetic chemoprevention strategy in which cells with LOI are “IGF-II addicted” and undergo reduced tumorigenesis in the colon upon IGF-II pathway blockade.

Transient IKK activity mediates NF-κB temporal dynamics in response to a wide range of TNFα doses

Dynamic properties of signaling pathways control their behavior and function. We undertook an iterative computational and experimental investigation of the dynamic properties of tumor necrosis factor (TNF)α-mediated activation of the transcription factor NF-κB. Surprisingly, we found that the temporal profile of the NF-κB activity is invariant to the TNFα dose. We reverse engineered a computational model of the signaling pathway to identify mechanisms that impart this important response characteristic, thus predicting that the IKK activity profile must transiently peak at all TNFα doses to generate the observed NF-κB dynamics. Experimental confirmation of this prediction emphasizes the importance of mechanisms that rapidly down-regulate IKK following TNFα activation. A refined computational model further revealed signaling characteristics that ensure robust TNFα-mediated cell-cell communication over considerable distances, allowing for fidelity of cellular inflammatory responses in infected tissue.

Subcellular-resolution delivery of a cytokine through precisely manipulated nanowires

Precise delivery of molecular doses of biologically active chemicals to a pre-specified single cell among many, or a specific sub-cellular location, is still a largely unmet challenge hampering our understanding of cell biology. Overcoming this could allow unprecedented levels of cell manipulation and targeted intervention. Here, we show that gold nanowires conjugated with cytokine, such as tumour necrosis factor-alpha (TNFα), can be transported along any prescribed trajectory or orientation using electrophoretic and dielectrophoretic forces to a specific location with subcellular resolution. The nanowire, 6 μm long and 300 nm in diameter, delivered the cytokine and activated canonical nuclear factor-kappaB signaling in a single cell. Combined computational modeling and experimentation indicated that cell stimulation was highly localized to the nanowire vicinity. This targeted delivery method has profound implications for controlling signaling events on the single cell level.

Engineering neuronal growth cones to promote axon regeneration over inhibitory molecules

Neurons in the central nervous system (CNS) fail to regenerate axons after injuries due to the diminished intrinsic axon growth capacity of mature neurons and the hostile extrinsic environment composed of a milieu of inhibitory factors. Recent studies revealed that targeting a particular group of extracellular inhibitory factors is insufficient to trigger long-distance axon regeneration. Instead of antagonizing the growing list of impediments, tackling a common target that mediates axon growth inhibition offers an alternative strategy to promote axon regeneration. Neuronal growth cone, the machinery that derives axon extension, is the final converging target of most, if not all, growth impediments in the CNS. In this study, we aim to promote axon growth by directly targeting the growth cone. Here we report that pharmacological inhibition or genetic silencing of nonmuscle myosin II (NMII) markedly accelerates axon growth over permissive and nonpermissive substrates, including major CNS inhibitors such as chondroitin sulfate proteoglycans and myelin-associated inhibitors. We find that NMII inhibition leads to the reorganization of both actin and microtubules (MTs) in the growth cone, resulting in MT reorganization that allows rapid axon extension over inhibitory substrates. In addition to enhancing axon extension, we show that local blockade of NMII activity in axons is sufficient to trigger axons to grow across the permissive–inhibitory border. Together, our study proposes NMII and growth cone cytoskeletal components as effective targets for promoting axon regeneration.

Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor

The signaling pathway mediated by JNK transduces different types of signals, such as stress stimuli and cytokines, into functional responses that mediate apoptosis, as well as proliferation, differentiation, and inflammation. To better characterize the dynamic information flow and signal processing of this pathway in the cellular context, a genetically encoded, fluorescent protein-based biosensor was engineered to detect endogenous JNK activity. This biosensor, named JNKAR1 (for JNK activity reporter), specifically detects stress- (ribotoxic and osmotic) and cytokine- (TNF-α) induced JNK activity in living cells with a 15 to 30% increase in the yellow-to-cyan emission ratio because of a phosphorylation-dependent increase in FRET between two fluorescent proteins. JNK activity was detected not only in the cytoplasm, but also in the nucleus, mitochondria, and plasma membrane with similar kinetics after induction of ribotoxic stress by anisomycin, suggesting relatively rapid signal propagation to the nuclear, mitochondrial, and plasma membrane compartments. Furthermore, quantitative single-cell analysis revealed that anisomycin-induced JNK activity exhibited ultrasensitivity, sustainability, and bimodality, features that are consistent with behaviors of bistable systems. The development of JNKAR1, therefore, laid a foundation for evaluating the signaling properties and behaviors of the JNK cascade in single living cells.

High Content Cell Screening in a Microfluidic Device

A comprehensive, systems level understanding of cell signaling networks requires methods to efficiently assay multiple signaling species, at the level of single cells, responding to a variety of stimulation protocols. Here we describe a microfluidic device that enables quantitative interrogation of signaling networks in thousands of individual cells using immunofluorescence-based readouts. The device is especially useful for measuring the signaling activity of kinases, transcription factors, and/or target genes in a high throughput, high content manner. We demonstrate how the device may be used to measure detailed time courses of signaling responses to one or more soluble stimuli and/or chemical inhibitors as well as responses to a complex temporal pattern of multiple stimuli. Furthermore we show how the throughput and resolution of the device may be exploited in investigating the differences, if any, of signaling at the level of a single cell versus at the level of the population. In particular, we show that NF-κB activity dynamics in individual cells are not asynchronous and instead resemble the dynamics of the population average in contrast to studies of cells overexpressing p65-EGFP.

How Information Theory Handles Cell Signaling and Uncertainty

Information theory allows analyses of cell signaling capabilities without necessarily requiring detailed knowledge of the signaling networks. Intracellular biochemical networks have traditionally been studied by stimulating populations of genetically identical cells and measuring the aggregate response. However, such population-based measurements may obscure the idiosyncrasies of individual cells and therefore suggest deceptively precise input-output relationships. Consequently, signaling pathways have been viewed as the finely tuned circuitry that programs the cell to behave in a predefined manner (1). Detailed studies of cellular biochemistry at the single-cell level now show that cells responding en masse may have quite varied behaviors when examined individually (2), raising the question of how precisely signaling pathways can control a cells actions (3).