Research in my lab examines protein trafficking mechanisms at synapses, and seeks to understand how these mechanisms are used to regulate synaptic transmission.
The dendrites of neurons receive and integrate inputs from hundreds or thousands of partner cells, and most of this input arrives at highly specialized sites called synapses that are distributed over the dendritic tree. Improper regulation of synaptic transmission is implicated in an enormous variety of psychiatric disorders: an imbalance of glutamatergic neurotransmission in particular has been identified in the pathophysiology of diseases ranging from schizophrenia and autism to epilepsy and addiction, and increasing evidence suggests that excitatory synapses are among the earliest targets of Alzheimer's Disease (AD) pathogenesis. A key means of synaptic regulation is the control of the number and subsynaptic position of postsynaptic neurotransmitter receptors, and so understanding the mechanisms involved has broad implications not only for understanding the etiology of many diseases but more generally for defining the cellular basis of nervous system function and disorder.
We focus on processes that operate rapidly and very locally to regulate synaptic function on a moment-to-moment basis. These mechanisms are of particular interest because they likely underlie the initial stages of memory formation, and their perturbation produces severe and long-lasting damage to synaptic and neuronal morphology. Mechanisms we are studying currently include the trans-synaptic molecular bridges that align presynaptic nerve terminals with postsynaptic specializations, the continuous internalization and recycling of synaptic receptors, and the postsynaptic actin cytoskeleton that controls postsynaptic morphology and regulates receptor trafficking in many ways.
Understanding the complex set of molecular reactions that underlie endocytosis and other trafficking events requires examining these events in live cells. Thus, the lab has turned to high-resolution, high-speed, multi-color confocal imaging of proteins in cultured neurons as a way of examining the kinetics and spatial features of the molecular events at individual living synapses. We utilize a number of novel fluorescence assays to examine protein positioning and movement at the synapse in living cells at the highest resolution possible. These tools provide the perfect complement to whole-cell and single-channel electrophysiological assays, which naturally track receptor function at high speed but typically with little or no spatial resolution. The lab aims to combine these approaches with molecular perturbation of protein function to provide unprecedented spatial, temporal, and molecular understanding of the protein trafficking events in spines.
Lab Techniques and Equipment
Most research in the lab revolves around the ability to measure cellular events in real time and in living neurons using fluorescence microscopy and electrophysiology. Recent, enormous advances in microscopy have opened amazing new opportunities to measure and to manipulate molecular events as they happen in the cell. To take advantage of this revolution, the lab will continue developing techniques and analysis to understand how actions at the level of molecules and organelles regulate synaptic transmission and other neuronal functions.
Students and postdocs in the lab can expect to utilize state-of-the-art time-lapse microscopy techniques to visualize receptor trafficking and other forms of protein dynamics, along with simultaneous electrophysiological assays of cell surface receptors and channels. Two super-resolution microscopes in the lab are heavily used for single-molecule-based imagine (PALM and STORM). Confocal microscopy is used to perform photobleaching (FRAP), photoactivation, and energy transfer (FRET) analyses. A variety of molecular techniques are used to tag and alter proteins of interest for live-cell imaging, and lab members will be encouraged to develop biochemical assays to test mechanistic hypotheses outside the cell.
Current lab members
- Harold MacGillavry, PhD, Postdoctoral Fellow
- Emily Lu, PhD student in Molecular Medicine
- Tamar Davis, Research Assistant
- Haiwen Chen, MD/PhD student in the Program in Neuroscience
- Sarah Ransom, PhD student in the Program in Neuroscience
- Tuo Peter Li, MD/PhD student in the Program in Neuroscience
- Nicholas Frost, MD/PhD, moved to residency at UCSF
- Justin Kerr, PhD, moved to Postdoctoral Fellow at NIH
- Mustafa Chowdhury, Postdoctoral Fellow
- Minerva Contreras, Research Assistant
- Ella Kong, Research Assistant
Postdoctoral fellows: We are always seeking talented individuals interested in using super-resolution cellular imaging (PALM) and single-molecule tracking to study cell biological mechanisms of synapse function. Ongoing projects include dynamics of the postsynaptic density and cytoskeletal organization in spines. These are exciting, new lines of work using cutting-edge techniques that require creative development to reach their full potential, and we are looking for motivated and independent scientists who will push the projects forward. Applicants should have a Ph.D. in Neuroscience, Cell Biology, or a related field and have experience and a strong interest in imaging, biophysics, or synaptic transmission. Programming experience or a strong quantitative orientation is a plus. Opportunities for integrating imaging with patch-clamp electrophysiology are available. Postdocs will additionally be expected to apply for further support via individual NRSAs, local training grants, and other sources.
Frost, N.A., Lu, H.E., Blanpied, T.A. (2012) Optimization of cell morphology measurement via single-molecule tracking PALM. PLoS One 7(5):e36751. PMCID: PMC3343014
MacGillavry, H.D., Kerr, J.M., Blanpied, T.A. (2011) Lateral organization of the postsynaptic density. Mol Cell Neurosci., 48: 321-31. PMCID: PMC3216044
Lieberman, J.A., Frost, N.A., Hoppert, M., Fernandes, P.J., Vogt, S.L., Raivio, T.L., Blanpied, T.A., Donnenberg, M.S. (2012) Outer membrane targeting, ultrastructure, and single molecule localization of the enteropathogenic Escherichia coli type IV pilus secretin BfpB. J Bacteriol. 194(7): 1646-58. PMCID: PMC3302462
Weinman, E.J., Steplock, D., Shenolikar, S., Blanpied, T.A. (2011) Dynamics of PTH-induced disassembly of Npt2a/NHERF-1 complexes in living OK cells. Am J Physiol Renal Physiol. 300(1): F231-5. PMCID: PMC3023216
Frost, N.A., Shroff, H., Kong, H., Betzig, E., Blanpied, T.A. (2010) Local dynamics of actin polymerization resolved within dendritic spines by single-molecule tracking PALM. Neuron, 67: 86-99.
Lu, J., Helton, T.D., Blanpied, T.A., Racz, B., Newpher, T.M., Weinberg, R.J., and Ehlers, M.D. (2007) Postsynaptic positioning of endocytic zones and AMPA receptor cycling by physical coupling of dynamin-3 to homer. Neuron 55: 874-889
Racz, B.L., Blanpied, T.A., Ehlers, M.D., and Weinberg, R.J. (2004) Lateral organization of endocytic machinery in dendritic spines. Nat. Neurosci. 7:917-8.