Spatiotemporally resolved subcellular phosphoproteomics

Proteome-wide profiling of protein phosphorylation has been widely used to reveal the underlying mechanism of diverse cellular signaling events. Yet, characterizing subcellular phosphoproteome with high spatial–temporal resolution has remained challenging. Herein, we developed a subcellular-specific uncaging-assisted biotinylation and mapping of phosphoproteome (SubMAPP) strategy to monitor the phosphorylation dynamics of subcellular proteome in living cells and animals. Our method capitalizes on the genetically encoded bioorthogonal decaging strategy, which enables the rapid activation of subcellular localized proximity labeling biotin ligase through either light illumination or small-molecule triggers. By further adopting an integrated orthogonal pull-down strategy with quantitative mass spectrometry, SubMAPP allowed for the investigation of subcellular phosphoproteome dynamics, revealing the altered phosphorylation patterns of endoplasmic reticulum (ER) luminal proteins under ER stress. Finally, we further expanded the scope of the SubMAPP strategy to primary neuron culture and living mice.

Efficient mesoscale phenotypic screening in cultured primary neuron culture

A longstanding question in neuroscience is how the activity of ion channels shapes neuronal activity and, as a result, computation in circuits and networks. Optogenetic reagents are tools to answer this question by enabling precise and dynamic perturbation of cellular states. However, development of these reagents can be hampered by low-throughput assays in non-physiological contexts. Here, we develop an all optical phenotypic screen in cultured primary hippocampal neurons that enables the functional assessment of large libraries of genetically encoded optogenetic actuators. Combining real-time analysis and data reduction methods allows for continuous observation of several thousand neurons for several days without onerous data storage overhead. This screening system may be useful in a diversity of research questions that can be coupled to optical perturbation and sensing.

Toward a Merged Microfluidic/Microelectrode Array Device for Culture of Unidirectional Neural Network

Traditional neural cultures are formed from disassociating primary neurons that are sourced from animals. However by disassociating them, any larger organizational structure between the neurons is lost. In an effort to regain some of the lost organization a device that is capable of recreating and monitoring a unidirectional connection between two populations is proposed. Initial validation toward a fully functional device is presented. Using soft lithography techniques, a microfluidic chip has been designed and produced with a design conducive for facilitating such a neuron culture. Using a specialized adhesive method, this chip can be attached and removed from a commercially available microelectrode array. Finally, cell viability is demonstrated using the PC12 neuron-like cell line after exploring several device configurations. Further efforts will be focused on primary neuron culture and establishment of a unidirectional network.