Increases in the intracellular concentration of calcium ([Ca2+]i) activate various signaling pathways that lead to the expression of genes that are essential for dendritic development, neuronal survival, and synaptic plasticity. The mode of Ca2+ entry into a neuron plays a key role in determining which signaling pathways are activated and thus specifies the cellular response to Ca2+. Ca2+ influx through L-type voltage-activated channels (LTCs) is particularly effective at activating transcription factors such as CREB and MEF-2. We developed a functional knock-in technique to investigate the features of LTCs that specifically couple them to the signaling pathways that regulate gene expression. We found that an isoleucine-glutamine ("IQ") motif in the carboxyl terminus of the LTC that binds Ca2+-calmodulin (CaM) is critical for conveying the Ca2+ signal to the nucleus. Ca2+-CaM binding to the LTC was necessary for activation of the Ras/mitogen-activated protein kinase (MAPK) pathway, which conveys local Ca2+ signals from the mouth of the LTC to the nucleus. CaM functions as a local Ca2+ sensor at the mouth of the LTC that activates the MAPK pathway and leads to the stimulation of genes that are essential for neuronal survival and plasticity.

We describe a microfluidic immunoassay device that permits sensitive and quantitative multiplexed protein measurements on nanoliter-scale samples. The device exploits the combined power of integrated microfluidics and optically encoded microspheres to create an array of approximately 100 μm 2 sensors functionalized with capture antibodies directed against distinct targets. This strategy overcomes the need for performing biochemical coupling of affinity reagents to the device substrate, permits multiple proteins to be detected in a nanoliter-scale sample, is scalable to large numbers of samples, and has the required sensitivity to measure the abundance of proteins derived from single mammalian cells. The sensitivity of the device is sufficient to detect 1000 copies of TNF in a volume of 4.7 nL.

The ability to make sensitive measurements of protein-protein interaction kinetics in single neurons is critical for understanding the molecular and cellular basis of neuronal function. We have developed a reporter technology based on the differential induction of Escherichia coli TEM-1 beta-lactamase (Bla) enzymatic activity that can function as a sensor of the interaction state of two target proteins within single neurons in vivo. To modulate Bla enzymatic activity, we first split the enzyme into two separate, complementary protein fragments that we identified by using a functional screening approach based on circular permutation of the Bla enzyme. The split enzyme was then brought together by the phosphorylation-dependent association of the kinase inducible domain of the cAMP response element binding protein (CREB) and the KIX domain of the CREB binding protein. Using an intracellular substrate whose fluorescence spectrum changes after hydrolysis by Bla, we performed time-lapse ratiometric imaging measurements of Bla enzymatic induction after association of the CREB and CREB binding protein interaction domains. This approach permits direct imaging of protein-protein interactions in single cells with high signal discrimination.