Cellular mechanisms for synaptic function and plasticity
A major focus in the lab concerns how neuronal proteins make it to the correct subcellular location, in the appropriate quantitites, at the right time, and how synaptic activity coordinates this process. For example, the number of ion channels displayed on the cell surface has a profound effect on cellular excitability. Likewise, the number of surface neurotransmitter receptors at synapses governs how a cell will respond to neurotransmitter released from connected neurons. In other words, the identity and levels of cell surface proteins largely define how a neuron will respond to incoming synaptic signals, and whether it will integrate and propagate the signal. Thus, the cellular machinery governing the surface expression of important neural proteins is extremely important for defining the intrinsic properties of neurons. Synaptic activity influences membrane trafficking pathways important for delivering cargoes such as neurotransmitter receptors (e.g. AMPA-type glutamate receptors) to the cell surface. Several projects in the lab are focused on how synaptic activity couples to dendritic membrane trafficking pathways to tune the surface levels of important factors, the spatiotemporal dynamics of the process, the identity of cargoes mobilized to the dendrite surface by synaptic activity and processing of disease-related proteins through dendritic secretory organelles.
Figure 1. A hippocampal pyramidal neuron expressing transferrin receptor (red) and GFP (green). Transferrin receptor is a marker for cellular organelles called recycling endosomes, which are localized throughout neuronal dendrites and harbor important synaptic receptors and presumably other as yet unidentified factors important for neuronal function.
Movie 1. Recycling endosomes fuse with the plasma membrane of dendritic spines upon synaptic stimulation. Transferrin receptor linked to both mCherry and superecliptic pHluorin (pH-sensitive GFP) was visualized in hippocampal dendrites in response to a stimulation designed to activate synaptic NMDA receptors. For details, see Kennedy et al., 2010 Cell. Firefox and Chrome users may have to "right click" after trying to play the movie and select "view video" (Firefox) or "open video in new tab" (Chrome).
Optogenetic tool development
We are actively developing new optogenetic tools for studying neural function. The development and implementation of light-activated channels and pumps (e.g. channelrhodopsin and halorhodopsin) has transformed how we study the brain by allowing remote control of neural firing within genetically-defined populations of neurons. However, there remains an unmet need for controlling more subtle, but fundamentally important aspects of neural function. We are developing tools to control basic cellular processes and signaling pathways important for neuronal function with light.
UVR8-based tools: In 2013 we published the first strategy for controlling protein trafficking through the cellular secretory pathway using light (Chen et al., 2013 Journal of Cell Biology). This system uses a plant photoreceptor called UVR8, which was recently shown to homodimerize in the dark, and monomerize with light to control UV-responses in plants. Fusing this photoreceptor to target cargo molecules leads to retention in the endoplasmic reticulum. Light frees the cargo from the ER, allowing it to progress through the secretory pathway, ultimately reaching the surface of the cell. Using fluorescently tagged cargo molecules, the trafficking itinerary through all of the cellular secretory organelles can be visualized (see movies 2 and 3 below). In neurons, we used this tool to visualize unique, decentralized secretory organelles in dendrites that receive cargo locally released from dendritic ER (Fig. 2).
Movie 2. Light controlled secretory trafficking. Secretory cargo (green in left panel and greyscale in right panel) can be trapped in the endoplasmic reticulum and released with a pulse of UV-B light (timing of light pulse is designated by small circle in upper left corner). Note the massive redistribution of cargo in response to light as it traffics to the Golgi, and is subsequently packaged into small, mobile carriers destined for the plasma membrane. Total time of movie is 1 hour. For details, see Chen et al., 2013 Journal of Cell Biology. Firefox and Chrome users may have to "right click" after trying to play the movie and select "view video" (Firefox) or "open video in new tab" (Chrome).
Movie 3. Total internal reflection fluorescence (TIRF) imaging of UV-B released cargo molecules as they are inserted into the plasma membrane. The cell in the right panel was briefly exposed to UV-B illumination 30 minutes prior to imaging. The cell in the left panel was not exposed to UV-B. For details, see Chen et al., 2013 Journal of Cell Biology. Firefox and Chrome users may have to "right click" after trying to play the movie and select "view video" (Firefox) or "open video in new tab" (Chrome).
Figure 2. Photoswitching strategy for visualizing local secretory trafficking. Secretory cargo retained in the endoplasmic reticulum was photoconverted from green to red at various locations in dendrites. Cargo photoconverted near dendritic branchpoints accumulated at nearby intermediate secretory organelles, providing evidence for local secretion in neural dendrites.
Cryptochrome-based tools: We also have an ongoing collaboration with Chandra Tucker's lab, who pioneered the use of the plant photoreceptor cryptochrome 2 as an optogenetic tool (Kennedy et al., 2010 Nature Methods). Upon blue light exposure, CRY2 rapidly binds to another protein, CIB1. These modules can be fused to target proteins to control their activity with light. We are engineering and implementing new tools for controlling enzyme activity and various cellular processes. Examples include reconstituting split enzymes (e.g. transcription factors, DNA recombinases) and manipulating the subcellular localization of proteins with light.