Dissecting the kinetics of phosphoinositide metabolism and signaling using FRET, translocation, and ion channels
Bertil Hille, Ph.D.
Professor of Physiology and Biophysics
University of Washington School of Medicine
Dr. Hille and his laboratory have studied biophysical mechanisms of ion permeation and pharmacological modification of ion channels in nerve and muscle. In the last twenty years, his research interests turned to regulation of ion channel function by G-protein-coupled receptors, dynamics of intracellular calcium, and regulation of exocytosis in neurons, endocrine cells, epithelia, and reproductive cells. He showed regulation of inward rectifier K channels by pertussis toxin sensitive G proteins without a second messenger. He defined modulation of Ca channels by a fast and a slow signaling pathway, showed the fast pathway used pertussis toxin-sensitive G proteins and Gbg subunits, showed regulation of K and Ca channels by a slow pathway involving depletion of the phosphoinositide PIP2 by action of PLC, and determined the kinetics of phosphoinositide metabolism by optical and electrophysiological measurements.
Dynamic Visualization of Kinase Activities and Second Messenger Dynamics
Jin Zhang, Ph.D.
Department of Pharmacology and Molecular Sciences
Neuroscience and Oncology
Johns Hopkins University
To achieve a comprehensive understanding of the spatiotemporal regulation of signal transduction, tools that are capable of tracking signaling dynamics in living systems with single-cell resolution are essential. To generate such tools for a broad spectrum of signaling molecules, we have developed several general strategies for engineering fluorescent biosensors to track the activities of second messengers, kinases and phosphatases. We are currently applying these molecular tools, in combination with other cellular and molecular techniques, to investigate the spatiotemporal regulation or dysregulation of several signaling pathways, such as cAMP/PKA, PI3K/Akt and MAPK pathways, in the context of cell migration, energy metabolism and cancer development. Quantitative measurement from live-cell fluorescence imaging is combined with mechanistic computational modeling for systems analyses of signaling networks.
Dynamics of protein access to retinal photoreceptor signaling compartments probed by confocal and multiphoton imaging
Peter Calvert, Ph.D.
Upstate Medical University
Assistant Professor in Ophthalmology
Adjunct Assistant Professor in Biochemistry & Molecular Biology
Adjunct Assistant Professor in Neuroscience & Physiology
Our lab studies signaling dynamics within discrete subcellular compartments. Imaging of signaling in live cells has gained significant interest with the increased availability of expressible probes. To overcome the problem posed by cell signaling compartments below the resolution limit of fluorescence microscopy, we have developed a high-resolution imaging approach using Xenopus photoreceptors as model cells. The major signaling compartments in these large neurons fully accommodate diffraction limited confocal and multiphoton excitation beams, permitting the analysis of spatial as well as temporal signaling dynamics. Moreover, the ultrastructure of photoreceptors is well characterized, allowing the impact of compartment geometry on dynamics to be examined. Our current work has revealed that the geometry of the photoreceptor signaling compartment plays a critical role in determining protein access and thus may be important for the control of light sensitivity.
Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination
Thomas Planchon, Ph.D.
Janelia Farms Research Campus (HHMI)
Thomas received his PhD in Physics from Ecole Polytechnique in Paris, where he developed simulation tools and adaptive optics methods for the next generation of high intensity ultrafast laser. At the University of Michigan in the lab of Gerard Mourou he demonstrated the world's highest laser intensity to date (10^22 W/cm^2). He then joined the groups of Chip Durfee and Jeff Squier where he developed spatio-temporal pulse shaping methods and adaptive optics for the generation of white-light filaments in air and non linear (THG) microscopy. He recently joined the lab of Eric Betzig as a Research Specialist to develop novel bioimaging methods. His recent work is on the development of a new kind of plane illumination microscope, using a shaped laser excitation beam in the form of a Bessel beam. This microscope allows fast isotropic 3D imaging suitable for live cell imaging of single cells and multiorganisms.
Two-photon calcium imaging in drosophila during visually guided behavior
Joahannes Seelig, Ph.D.
Janelia Farms Research Campus (HHMI)
Johannes Seelig studied physics at the University of Basel and received his PhD from ETH Zurich where he worked in the field of single molecule biophysics. In 2007, he joined Vivek Jayaraman's lab, where he has worked on novel techniques to record the activity of identified neurons in head-fixed, behaving fruit flies. He is now using these tools to understand circuit processing underlying visually guided behavior.
Synapses and microglia: dynamic interactions in the healthy brain
Ania Majewska, Ph.D.
Department of Neurobiology and Anatomy
Center for Visual Science
University of Rochester Medical Center
My lab uses two-photon microscopy to track individual cells in the brain during brain plasticity and pathophysiology. We are particularly interested in how glial cells interact with synapses on neurons and aid in synaptic remodeling during visual plasticity. We are actively studying the brain extracellular matrix in order to describe the extracellular mechanisms that may impact plasticity within the visual cortex during the critical period.
A ground plan for networks in the hindbrain
Joe Fetcho, Ph.D.
Department of Neurobiology and Behavior
Our lab studies how movements are produced by the brain and spinal cord of vertebrates. We use zebrafish as a model system because they allow us to combine genetic and optical methods with more conventional physiological approaches to study the neural circuits. In addition to studies of normal function, we examine disruptions of function by genetic mutations, and are developing approaches to cure spinal injuries.
Fluorescence Microscopy of the Retina in the
David R. Williams, Ph.D.
William G. Allyn Professor of Medical Optics
Director, Center for Visual Science
University of Rochester
Since 1991, Williams has served as Director of Rochester's Center for Visual Science, an interdisciplinary research program of 32 faculty interested in the mechanisms of human vision. Williams' research marshals optical technology to address questions about the fundamental limits human vision. His research team demonstrated the first adaptive optics system for the eye, showing that vision can be improved beyond that provided by conventional spectacles. Most recently, his group has been studying the normal and diseased retina using adaptive optics to obtain microscopic images with unprecedented resolution in the living eye.
Functional and structural imaging of a cortical circuit
R. Clay Reid, M.D., Ph.D.
Professor of Neurobiology
Harvard Medical School
My laboratory has always studied the general question: what is the relationship between the functional properties of neurons, as measured in vivo, and the connections between these neurons? We started as an electrophysiology lab that concentrated on functional properties—the receptive fields—of neurons in the mammalian visual system. In the past six years, we have changed approaches by using optical methods, primarily two-photon calcium imaging, to study physiology in vivo. This allows us to study the response properties of thousands of neurons within local cortical circuits to examine the interactions between simple behaviors and sensory processing. We are using the parallel nature of optical recordings to examine the sensory response properties of populations of neurons in multiple cortical areas.