Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. While intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.

Axonal chemotaxis is believed to be important in wiring up the developing and regenerating nervous system, but little is known about how axons actually respond to molecular gradients. We report a new quantitative assay that allows the long-term response of axons to gradients of known and controllable shape to be examined in a three-dimensional gel. Using this assay, we show that axons may be nature's most-sensitive gradient detectors, but this sensitivity exists only within a narrow range of ligand concentrations. This assay should also be applicable to other biological processes that are controlled by molecular gradients, such as cell migration and morphogenesis.

The fruitfly, Drosophila melanogaster, has become a critical model system for investigating sleep functions. Most studies use duration of inactivity to measure sleep. However, a defining criterion for sleep is decreased behavioral responsiveness to stimuli. Here we introduce the Drosophila ARousal Tracking system (DART), an integrated platform for efficiently tracking and probing arousal levels in animals. This video-based platform delivers positional and locomotion data, behavioral responsiveness to stimuli, sleep intensity measures, and homeostatic regulation effects – all in one combined system. We show how insight into dynamically changing arousal thresholds is crucial for any sleep study in flies. We first find that arousal probing uncovers different sleep intensity profiles among related genetic background strains previously assumed to have equivalent sleep patterns. We then show how sleep duration and sleep intensity can be uncoupled, with distinct manipulations of dopamine function producing opposite effects on sleep duration but similar sleep intensity defects. We conclude by providing a multi-dimensional assessment of combined arousal and locomotion metrics in the mutant and background strains. Our approach opens the door for deeper insights into mechanisms of sleep regulation and provides a new method for investigating the role of different genetic manipulations in controlling sleep and arousal.

Axon guidance by molecular gradients plays a crucial role in wiring up the nervous system. However, the mechanisms axons use to detect gradients are largely unknown. We first develop a Bayesian ''ideal observer'' analysis of gradient detection by axons, based on the hypothesis that a principal constraint on gradient detection is intrinsic receptor binding noise. Second, from this model, we derive an equation predicting how the degree of response of an axon to a gradient should vary with gradient steepness and absolute concentration. Third, we confirm this prediction quantitatively by performing the first systematic experimental analysis of how axonal response varies with both these quantities. These experiments demonstrate a degree of sensitivity much higher than previously reported for any chemotacting system. Together, these results reveal both the quantitative constraints that must be satisfied for effective axonal guidance and the computational principles that may be used by the underlying signal transduction pathways, and allow predictions for the degree of response of axons to gradients in a wide variety of in vivo and in vitro settings.axon guidance ͉ chemotaxis ͉ growth cone ͉ nerve growth factor ͉ nerve regeneration E ndogenous chemical gradients are a key source of information used by developing axons when wiring up the nervous system. Furthermore, artificially generated gradients are a potential therapy for restoring connectivity after neural injury. Many of the molecular gradients that direct axons in the developing nervous system have recently been identified, together with some of the signaling pathways through which they operate (1-8). However, our understanding of the mechanisms by which axons actually detect gradients remains qualitative. This limits our ability to predict both the response of axons when gradients are perturbed and the optimal parameters for promoting regrowth after injury.To be guided by a gradient, axons must be able to detect small spatial variations in receptor binding. This requires integrating signals from spatially distributed receptors to make a decision as to the direction of the gradient. Resources within the growth cone can then be marshaled appropriately by this information, for instance, via the production of steep gradients of intracellular signaling molecules (8). Although there is evidence for a role for gradients of molecules such as Ca 2ϩ in this latter step (9, 10), very little is known about the computations required to accurately make the initial decision.What constrains the ability of an axon to make a decision regarding gradient direction? Both experimental and computational work addressing chemotaxis in related systems such as bacteria, leukocytes, and Dictyostelium has identified the fundamental role of noise in limiting gradient perception. Noise can arise from low numbers of ligand molecules, from the stochastic nature of receptor binding, and from intracellular signaling pathways (11-16). Such constraints must also apply to axonal gradient sensing (17...