Yasuda Lab PKC alpha integrates spatiotemporally distinct signals to facilitate synaptic plasticity
We study Neuronal Signal Transduction
Dr. Yasuda started as Scientific Director of the Max Planck Florida Institute in January 2012. Before he worked as assistant professor at the Neurobiology department at the Duke University Medical Center, Durham, NC. Yasuda has received a number of awards for his research accomplishments including the Career Award at the Scientific Interface from the Burroughs Wellcome Fund, the Alfred P. Sloan Fellowship, the New Investigator Award from the Alzheimer’s Association, Research Award for Innovative Neuroscience from the Society for Neuroscience and the National Institute of Health’s (NIH) Pioneer award.
Yasuda received his Ph.D. in physics in 1998 from Keio University Graduate School of Science and Technology in Yokohama, Japan. In his Ph.D. study, he demonstrated that the enzyme ATP synthase is a rotary motor made of single molecule and that its energy conversion efficiency is close to 100%. From 2000 to 2005, he was a post-doctoral fellow at the Cold Spring Harbor Laboratory, where he built an imaging device to monitor protein interactions in living cells with high sensitivity and resolution. From 2005 to 2012, he was an assistant professor of the Neurobiology department at the Duke University Medical Center, where he developed a number of techniques to visualize signaling activity in single synapses. From 2009 to 2012, he also served as an Early Career Scientist at the Howard Hughes Medical Institute.
Our research focuses on synaptic plasticity, the ability of synapses to change their connection strength. This process is thought to underlie learning and memory. Cascades of biochemical reactions in dendritic spines, tiny (~0.1 femtoliter) postsynaptic compartments emanating from dendritic surfaces, trigger diverse forms of synaptic plasticity. The Yasuda lab aims to elucidate some of the operation principles of such signaling networks in dendritic spines using various optical techniques.
First, we have been developing techniques to image activity of various proteins in single dendritic spines using 2-photon fluorescence lifetime imaging microscopy (2pFLIM) in combination with new biosensors extensively optimized for 2pFLIM. Using this technique, we have succeeded in imaging signaling proteins, CaMKII, HRas, RhoA and Cdc42, in single dendritic spines undergoing synaptic potentiation. Our results indicate that signaling activity is subjected to a complicated spatiotemporal regulation: some signals are compartmentalized in single synapses, while others spread to affect a short stretch (micrometers) of dendritic segments including multiple synapses. This rich spatiotemporal regulation plays an essential role in coordinating cellular events in different dendritic micro-compartments. Further, Ca2+ elevation in a synapse is relayed in several different stages to induce long-term plasticity: First, Ca2+ signals in the synapse are integrated by a signaling protein CaMKII, of which activity decays over ~10 s. Then, Ras, Cdc42 and RhoA are activated by CaMKII, and relay this transient signal into signals lasting 10-30 min.
We have been developing many more sensors to reveal the precise temporal sequences of signaling events in different microcompartments. In addition, we have been developing tools to activate and inactivate signaling proteins in spines optically. Recently we have developed a photo-inducible CaMKII inhibitor, and demonstrated that 60 s of CaMKII activation is required to activate downstream signaling. By monitoring and manipulating activity of signaling proteins with high spatiotemporal resolution, we hopefully disentangle the complicated signaling networks and understand the signaling mechanisms underlying synaptic plasticity and ultimately learning and memory.
Our development of an imaging technique based on two-photon fluorescence lifetime imaging microscopy (2pFLIM) and extensively optimized biosensors has allowed us to monitor signaling activity in single synaptic compartments in brain slices. Using this technique, we have recently succeeded in measuring the activity of signaling proteins important for LTP and structural LTP (sLTP) in single dendritic spines. The new results provided many insights into the spatiotemporal regulation of biochemical signaling during LTP and sLTP.
Besides monitoring each element of the signaling cascade, tools to manipulate signaling activity in a spatially and temporally controlled manner will be extremely useful to understand the information flow in the signaling network. Previously several photo-activatable proteins for activating small GTPase proteins and gene transcription have been developed. However, it is not easy to scale up these strategies to many more proteins. We have been developing photo-inducible inhibitors using a scalable design based on the light oxygen voltage domain 2 (LOV2)-Jα helix domain of Phototropin1. LOV2-Jα changes its conformation from a closed form to an open form upon blue light absorption. We fuse a pseudo-substrate inhibitor peptide to LOV2-Jα so that LOV2-Jα disables the action of the inhibitor due to steric hindrance when it is in the closed conformation, and upon light absorption, the inhibitor peptide action is switched on by the conformational change of LOV2-Jα and reversed spontaneously. Using this strategy, we developed a photo-activatable kinase inhibitor for CaMKII, a kinase important for learning and memory. This tool allows us to measure the timing.
Imaging endogenous proteins with high specificity, resolution, and contrast is critical for defining their subcellular localization, especially in the brain, due to its extremely complicated morphology. However, a scalable and high-throughput approach to imaging endogenous proteins has not been established. Toward this goal, we have developed a new method termed Single-cell Labeling of ENdogenous proteins by endonuclease-mediated homology-Directed Repair (SLENDR), which allows in vivo genome editing in the mammalian brain for single-cell labeling of endogenous proteins by inserting a tag sequence to a gene of interest through CRISPR-Cas9-mediated homology-directed repair (HDR). SLENDR has allowed us to rapidly determine the subcellular localization and dynamics of endogenous proteins with the resolution of micro- to nanometers in various cell types in widespread brain regions. Therefore, SLENDR provides a novel platform for comprehensive, large-scale analyses.
Imaging neuronal activity using genetically-encoded Ca2+ indicators with two-photon microscopy has been invaluable for the analysis of functional circuits and their plasticity with high spatiotemporal resolution in vivo. However, the behaviors and dynamics of each neuron critically depend on their intracellular biochemical states, and thus it is important to analyze the underlying biochemical signaling that regulates neuronal plasticity. To elucidate the missing connection between the biochemical states and circuit plasticity, the goal of this project is to simultaneously image neural circuit activity and signaling dynamics in individual synapses and neurons comprising the network. Thus, our objective is to combine 2-photon Ca2+ imaging with 2-photon fluorescence lifetime imaging microscopy (2pFLIM) of ongoing intracellular signaling and to implement the system in vivo.
Sr Research Assistant