Faculty and Research
Faculty by Name
Ehud Isacoff
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Ehud Isacoff
Professor of Neurobiology*
*And Chair, Graduate Group in Biophysics
Research Interests
- Work in the lab is focused in three intersecting areas: a) The mechanism of ion channel function, b) Synapse development and plasticity, and c) The design of novel probes for the optical detection and manipulation of neuronal signaling. The projects all involve optical spectroscopy and the new tools that we develop are employed for our studies of channels and synapses.
Current Projects
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A) Allosteric control of channel and enzyme gates. Ion channels respond to physiological signals by opening and closing molecular gates that control the flow of ions across the membrane. Our goal is to understand the general principles of operation of voltage and ligand-gated channels. Our approach combines electrophysiology with biochemical probing and spectroscopy to detect conformational changes in the moving parts of the channels during identified functional transitions, and to apply force to those moving parts in order to understand the mechanical coupling of sensor domains to gates or one sensor domain to another, and thus to investigate the mechanism of allosteric control of gating. At the heart of the effort are two method, which we developed. The first is Voltage Clamp Fluorometry (VCF) (Mannuzzu et al., Science 271, 213-16, 1996; Glauner et al., Nature 402, 813-17, 1999). For VCF channel proteins are labeled site-specifically with fluorescent dyes and the structural rearrangements of the labeled segment of the channel are detected, in real-time, as changes in the fluorescence of environmentally-sensitive probes or of FRET between donor-acceptor pairs. This is done in a native cellular environment under voltage clamp, at the ensemble and single molecule level, enabling specific protein motions to be associated with particular functional transitions. We are using the approach to study how protein motions in sensing domains that detect changes in voltage or the present of ligands allosterically control the gates of channels and the activity of catalytic sites. The second method is a single molecule TRIFM imaging of single protein complexes in which subunits are fused to fluorescent proteins and their number is determined by counting their steps of photobleaching (Ulbrich and Isacoff, Nature Methods 4, 319-21, 2007). This is being used to determine the subunit composition of channels and receptors and the stoichiometry and stability of interaction with auxilliary regulatory proteins.
B) Synapse formation and plasticity. The wiring up of the nervous system is accomplished in several steps. Axons extend from cell bodies, follow long-range guidance cues, and, when they arrive at their termination bed, use contact-mediated cues to select specific cells with which to synapse and to induce synapse formation. We are studying the last two of these steps in two model preparations.
i) Molecular adhesion to induction of release sites. One of the big challenges to studying the molecular basis of synapse formation among mammalian neurons is the inability to control the time and place where neurons encounter one another. This problem can be solved by replacing the dendrite of the postsynaptic cell with a surrogate HEK293 cell expressing a postsynaptic adhesion protein, or with a bilayer-coated bead into which purified protein is incorporated in a GPI-linked form. Hippocampal axons contacted by such a surrogate react as if they have contacted a compatible dendrite and form a presynaptic specialization and release transmitter in an activity dependent manner. This enables us to define the time and location of pre-post contact and follow the dynamics of assembly of fluorescent protein (FP) tagged axonal proteins in forming presynaptic terminals, starting with the adhesion proteins, to the scaffolding proteins, active zone proteins to the secretory apparatus. In collaboration with with Jay Groves in Chemistry we have developed a new assay (Pautot et al., Nature Chem. Bio. 1, 283-9, 2005) in which lipid bilayers incorporating the postsynaptic adhesion protein are deposited flat on a coverslip, and cells expressing neurexin are dropped onto them. This makes it possible to study the adhesion process that triggers presynaptic differentiation at the single molecule level using total internal reflection (TIR) microscopy. This approach is the equivalent of placing the TIR objective inside a dendrite. It provides an unparalleled view of the membrane-associated proteins in the presynaptic terminal (ideal also for studying the exocytotic/endocytotic cyle, etc.), and does so at a membrane junction that mimics the real axon-dendrite contact. In addition, we have developed a new method that provides an unprecedented ability to move differentiated neurons growing on beads without shearing off their axons or dendrites (Pautot et al., 2008) to place them in contact with perspective partners at known times and locations.
ii) Synaptic homeostasis. Two mechanisms (one activity-dependent, and the other activity-independent) regulate synaptic size and strength in the Drosophila larval NMJ. They ensure that the motor neuron augments its transmitter release to match the ~100-fold expansion of muscle surface area (and the concomitant drop in input resistance) during development. Both mechanisms depend on retrograde signaling from muscle to motor neuron, and appear to involve postsynaptic bone morphogenic protein (BMP) as a retrograde signaling factor. We developed postsynaptic targeted calcium sensors (Guerrero et al., 2005) and are using them to image synaptic transmission. We have recently achieved quantal resolution with the method, enabling us to determine precisely the release properties of single active zones and define their mechanism of plasticity. Optical manipulation of glutamate receptors (see below) is being used to determine the activity depedence of retrograde transmission.
C) Novel probes for the optical detection and manipulation of neuronal signaling. Fluorescent indicator dyes have revolutionized our understanding of cellular signaling. Chemical indicators have been very powerful, but are limited in scope because they cannot be targeted to specific cell types or locations within a cell. Our solution for this problem has been to make sensors from proteins--the very biological molecules that transduce, transmit and receive cellular signals. This includes genetically encoded optical sensors of membrane potential and of synaptic transmission and new tools for the optical manipulation of neuronal activity. The ability to artificially stimulate and inhibit neurons is important for investigating how they contribute to the function of neural circuits. Our goal is to develop new ways to regulate neuronal activity by creating ion channels that are regulated by light. Because light can be applied rapidly and accurately, this approach offers great promise even for neurons that communicate using sparse temporal codes. The functional switch consists of a tethered ligand containing a photo-isomerizable prosthetic group. Different wavelengths of light either increase or decrease the length of the tether, thus regulating the probability that the ligand will reach its binding site. Several strategies make it possible to change the sign of light-activated neural response (i.e. inhibition vs. excitation) and the location within the cell where the current is induced. We have already made K+ channels and ionotropic glutamate receptors that are gated by light (Banghart et al., 2004; Volgraf et al., 2006; Gorostiza et al., 2007; Numano et al., 2007) and shown that these can be used to control the behavior of flies (unpublished) and zebrafish (Szobota et al., 2007). We are now expanding the toolset to other channels, GPCRs and enzymes involved in synaptic plasticity and employing these remote controls of synaptic signaling to study the development of neural circuits and the circuit basis of sensory integration and locomotion (Wyart et al., 2009).
Selected Publications
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Wyart, C., Bene, F.D., Warp, E., Scott, E.K., Trauner, D., Baier, H. and Isacoff, E.Y. (2009). Optogenetic dissection of a behavioral module in the vertebrate spinal cord. Nature (in press).
Numano, R., Szobota, S., Lau, A.Y., Gorostiza, P., Volgraf, M., Roux, B., Trauner, D. and Isacoff, E.Y. (2009). Nanosculpting a Yin/Yang photoswitch for an ionotropic glutamate receptor. PNAS 106, 6814-9.
Ulbrich, M. and Isacoff, E.Y. (2008). Rules of engagement for NMDA receptor subunits. PNAS 105, 14163-8.
Pautot, S., Wyart, C. and Isacoff, E.Y. (2008). Colloid-guided assembly of oriented 3D neuronal networks. Nature Methods 5, 735-40.
Tombola, F., Ulbrich, M. and Isacoff, E.Y. (2008). The voltage-gated proton channel Hv1 has two pores each controlled by one voltage sensor. Neuron 58, 546-556.
Kohout, S., Ulbrich, M., Bell, S.C. and Isacoff, E.Y. (2008). Subunit organization and functional transitions in Ci-VSP. Nat. Struct. Molec. Bio. 15, 106-8.
Stowers, R.S. and Isacoff, E.Y. (2007). Drosophila HIP14 is a Presynaptic Protein Required for Photoreceptor Synaptic Transmission and Expression of the Palmitoylated Proteins SNAP-25 and CSP. J. Neurosci. 27, 12874-83.
Pathak, M.M., Yarov-Yarovoy, V., Agarwal, G., Roux, B., Barth, P., Kohout, S., Tombola, F. and Isacoff, E.Y. (2007). Closing in on the resting state of the Shaker K+ channel. Neuron 56, 124-40.
Szobota, S., Gorostiza, P., Del Bene, F., Wyart, C., Fortin, D.L., Kolstad, K., Tulyathan, O., Volgraf, M., Numano, R., Aaron, H., Scott, E.K., Kramer, R.H., Flannery, J., Baier, H., Trauner, D. and Isacoff, E.Y. (2007). Remote control of neuronal activity with a light-gated glutamate receptor. Neuron 45, 535-45.
Tombola, F. Pathak, M., Gorostiza, P. and Isacoff, E.Y (2007). The twisted ion-permeation pathway of a resting voltage-sensing domain. Nature 445, 546-9.
Guerrero, G., Agarwal, G., Reiff, D.F., Ball, R.W., Borst, A., Goodman, C.S. and Isacoff, E.Y. (2005). Heterogeneity in synaptic transmission along a Drosophila larval motor axon. Nature Neurosci. 8, 1188-96.
Last Updated 2009-08-02