Professor (Molecular & Cell Biology)
Research areas: , , Cellular and Molecular Neuroscience
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.
A) Allosteric control of channel 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-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 is our method, of Voltage Clamp Fluorometry (VCF) (Mannuzzu et al., Science 271, 213-16, 1996; Glauner et al., Nature 402, 813-17, 1999). Channel proteins are labeled site-specifically with fluorescent dyes and the structural rearrangements of the labeled segment of the channel can be detected 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 and making it possible to animate structures derived from crystallography. 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 channel gates.
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., 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.
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. Part of this signaling pathway uses postsynaptic bone morphogenic protein (BMP), presynaptic BMP receptors, and at least two classes of downstream neuronal signaling schemes: a MAD/SMAD transcriptional switch and a kinase signaling system. Upstream of the retrograde signal that controls synaptic strength is a calcium dependent kinase in the muscle. We are using postsynaptic targeted calcium sensors that we developed (Guerrero et al., 2005) to study the Ca++ dynamics involved in this process and to determine how the retrograde signaling pathway controls transmitter release.
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.
In a new effort in the lab we are collaborating with the labs of Dirk Trauner in Chemistry and Rich Kramer in MCB to develop new tools for the optical manipulation of neuronal activity. The ability to artificially stimulate and inhibit neurons in intact animals is important for investigating how they contribute to the function of neural circuits and behavior. 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). We are now working to expand the toolset to other channels, GPCRs and enzymes involved in synaptic plasticity.
Gandhi, C.S., Loots, E. and Isacoff, E.Y. 2000. Reconstructing voltage sensor - pore interaction from a fluorescence scan of a voltage-gated K+ channel Neuron 27: 585-95.
Glauner, K.S., Mannuzzu, L.M., Gandhi, C.S. and Isacoff, E.Y. 1999. Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel Nature 402: 813-17.
Sonnleitner, A., Mannuzzu, L.M., Terakawa, S. and Isacoff, E.Y. 2002. Structural rearrangements in single ion channels detected optically in living cells PNAS 99: 12759-64.
Guerrero, G., Siegel, M.S., Roska, B., Loots, E. and Isacoff, E.Y. 2002. Tuning FlaSh: Redesign of the dynamics, voltage range and color of the genetically-encoded optical sensor of membrane potential Biophys. J. 83: 3607-18.
Dean, C. Scholl, F.G., Choih, J., Berger, J., Isacoff, E.Y., and Scheiffele, P. 2003. Oligomeric Complexes of Neuroligin Mediate Synaptic Adhesion with Neurexin and Induce the Formation of Presynaptic Terminals Nature Neuroscience 6: 708-16.
Gandhi, C.S., Clark, E., Loots, E., Pralle, A. and Isacoff, E.Y. 2003. The orientation and molecular movement of a K+ channel voltage-sensing domain Neuron 40: 515-525.
Banghart, M., Borges, K., Isacoff, E.Y., Trauner, D. and Kramer, R.H. 2004. Light-activated ion channels for remote control of neuronal firing Nature Neuroscience 7: 1381-86.
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 Neuroscience 8: 1188-96.
Pautot, S., Lee, H., Isacoff, E.Y. and Groves, J.T. 2005. Molecular adhesion of the neuronal synapse reconstituted between live cells and supported lipid bilayer Nature Chem. Bio. 1: 283-9.
Tombola, F. Pathak, M., and Isacoff, E.Y. 2005. Voltage-sensing arginines in a potassium channel permeate and occlude cation-selective pores Neuron 45: 379-88.
Gorostiza, P., Tombola, F., Verdaguer, A., Smith, S.B., Bustamante, C. and Isacoff, E.Y. 2005. Molecular Handles for the Mechanical Manipulation of Single-Membrane Proteins in Living Cells IEEE Transactions on Nanobioscience 4: 269-76.
Volgraf, M., Gorostiza. P, Numano, R, Kramer, R.H., Isacoff, E.Y. and Trauner, D. 2006. Allosteric Control of an Ionotropic Glutamate Receptor With an Optical Switch Nature Chem. Bio. 2: 47-52.