Kevin Gillis - US grants

DESCRIPTION: (Applicant's Abstract) The secretion of neurotransmitter and hormones from neurons and neuroendocrine cells is a highly regulated process. It is now widely accepted that a rise in intracellular [Ca2+] rapidly triggers secretion from excitable cells. However, it has recently become clear that Ca2+ also slowly modulates ("primes") release, in part through activation of protein kinase C (PKC), which, in turn, accelerates the rate that secretory vesicles become ready to be released. Therefore it is likely that there are multiple fast (triggering) and slow (modulating) Ca2+ sensors for exocytosis. A long-range goal of the investigator is to understand how Ca2+ triggers exocytosis from excitable cells and how exocytosis is regulated by Ca2+ and other second messengers. The goal of this project is to characterize fast and slow Ca2+ sensing for exocytosis in individual cells using optical and electrophysiological techniques which allow both fine control of [Ca2+] and high-time-resolution measurements of exocytosis. The 3 aims are: Aim I. To determine how the protein SNAP-25 is involved in Ca2+ priming and triggering steps. The effect of mutations of SNAP-25 on exocytosis will be measured to test the hypothesis that the C-terminus of the protein participates in both Ca2+-priming and triggering steps. Aim II. To determine how fast Ca2+ can prime exocytosis. Experiments will elevate [Ca2+]i in 2 steps to sequentially prime and trigger secretion to test the hypothesis that Ca2+ priming occurs in less than 1 second. Aim III. To quantify the ionic selectivity of the Ca2+ trigger for exocytosis. Multivalent cations such as Sr2+, Ba2+, and Pb2+ can act as "Ca2+ surrogates" in triggering exocytosis and other Ca2+-activated cellular responses. The ability of Ca2+ surrogates to rapidly trigger exocytosis will be measured to provide clues about the approximate size, flexibility and accessibility of the Ca2+-binding cavity of the triggering Ca2+ sensor. Achieving these aims will provide new insights into the mechanisms whereby secretion is regulated. Such basic knowledge is essential to understand complex processes such as short-term memory formation in the brain, the modulation of insulin secretion by glucagon in the endocrine pancreas, and the neurotoxicity of Pb2+ in the central nervous system.

0089018
Gillis
The objective of the proposed research is to replace the patch-clamp pipette with an aperture on a microchip and to replace the carbon fiber with electrochemical electrodes fabricated on microdevices. The specific objectives are to: (1) record the current through the cell membrane using microchip technology, (2) measure secretion from individual cells with high time resolution using electrochemical electrodes constructed on a microchip, and (3) automatically target individual cells to electrophysiological probes on the microchip. This award is co-funded by the Division of Engineering Education and Centers, the Division of Chemical and Transport Systems, and the Division of Bioengineering and Environmental Systems.

Here we propose to design a Nanomedicine Center for Molecular Membrane Physiology. This Center will develop and apply nanoscience tools to study and manipulate the protein and lipid nanomachines that precisely regulate the flow of information and material between the cell and its environment. Defects in these nanomachines are responsible for a host of diseases including cystic fibrosis, diabetes, and cardiovascular disease. In addition, membrane proteins are the targets of about 2/3 of clinically relevant drugs and thus represent a tremendous opportunity for therapeutic intervention. The principle reasons for suggesting Molecular Membrane Physiology as a Center theme are that problems in this area are interrelated and a number of powerful nanoscale techniques are uniquely suited to study cellular nanomachinery in a membrane environment. We propose that the Center focus on developing and applying nanoscience tools to understand and manipulate: 1) vesicular transport, exocytosis and endocytosis, 2) the gating of ion channels, and 3) membrane receptor-mediated signaling. The Specific Aim of this proposal is to create a Concept Development Plan for the Nanomedicine Center. This process will be directed by "facilitators" who are nanotechnology-savvy experts in the 3 subthemes who will help focus discussions on the major problems of their field. They will assemble a Panel of Scientific and Technical Experts and lead discussions at a Membrane Nanomediciene Workshop to be held in December, 2004. Emerging nanoscience technology applicabe to questions in the 3 subthemes will be brought forward. Workshop participants will prioritize promising nanoscience approaches according to their potential impact on membrane nanomedicine. The Panel will also make recommendations for the organization of both the specific Membrane Nanomedicine Center and the entire NIH Nanomedicine Center Initiative with input provided from a survey of nationally prominent Center directors and Center Investigators.

DESCRIPTION (provided by applicant): The long-term objective of our research is to develop microdevices for high-throughput measurement of quantal exocytosis from neurons and neuroendocrine cells. Patch-clamp electrophysiological and carbon-fiber electrochemical approaches represent the state-of-the-art for high time resolution and high information content assays of exocytosis but are slow and labor-intensive. Biochemical assays of secretion from cell populations have limited time resolution and can not resolve individual quantal fusion events. We will use microchip technology to develop devices that can assay quantal exocytosis from thousands of cells in a day in order to greatly accelerate the pace of basic neuroscience research. This approach will also enable, for the first time, rapid and high information content screening of drug candidates that affect exocytosis of neurotransmitter. For example, L-DOPA used to treat Parkinson's disease acts by increasing the quantal content of dopamine release. The approach will be interdisciplinary and will bring together investigators with expertise is biomedical, electrical and mechanical engineering, materials science, physics, electrochemistry, physiology and biophysics. The specific aims are: 1) Develop approaches to automatically target individual cells to electrochemical microelectrodes on microfabricated devices. 2) Develop approaches to stimulate exocytosis from cells on microdevices including rapid microfluidic solution exchange, photolysis of caged Ca and electrical stimulation of action potentials. 3) Integrate new electrochemical electrode materials into microdevices to increase sensitivity and performance. 4) Develop electronic instrumentation to allow simultaneous recording of many channels of electrochemical or electrophysiological data. The five-year goal of the project is to have actual devices on the market to serve the exocytosis research community that are at least an order of magnitude faster than current carbon-fiber approaches. Our first-year milestones for each aim are: 1) Position one or more cells at predetermined sites on a microchip. 2) Exchange extracellular solution in <100 ms on a microchip. 3) Characterize the electrochemical properties of a diamond-like carbon microelectrode. 4) Develop inexpensive modular circuitry for basic electrochemical measurements using off-the-shelf components that can be easily scaled for approximately 12 simultaneous channels.

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Professors Timothy Glass and Kevin Gillis of the University of Missouri are studying how neurotransmitters are released into synapses. Neurotransmitters are critical to the regulation of the nervous system and control a number of functions such as learning, memory, sleep, and movement. Understanding the machinery of synaptic release, and the chemical activity of neurotransmitters is vital to understanding both normal and atypical neuronal processes. Furthermore, understanding the basic mechanisms of synaptic vesicle fusion and transmitter release via exocytosis is of broad significance because it will not only aid in the development of therapies for diseases where release of neurotransmitters is compromised, but it will also advance our understanding of FDA-approved treatments that modulate transmitter release, such as botulinum toxin A and B. As part of the broader impacts of this work, a new robotics camp for underserved high school students will be held. It has been demonstrated that activities such as these camps enhances the likelihood that participants pursue STEM (science, technology, engineering and mathematics) coursework during their secondary education.<br/><br/>This project involves the preparation and evaluation of fluorescent chemical sensors for catecholamines with a view toward the fluorescent detection of neurotransmitters. These sensors are related to the NeuroSensor class of probes developed in PI Glass’s lab, which have already been used to detect norepinephrine in isolated chromaffin cells. Sensors will be developed that produce fluorescence enhancements upon exocytosis. The sensors will be used in combination with novel transparent electrodes developed in PI Gillis’ lab to study mechanisms of exocytosis via fluorescence imaging in combination with amperometric measurements. These combination experiments will measure both release and retention of catecholamine from individual vesicles to test the hypothesis that the amount of catecholamine released from a vesicle is modulated by the stimulus intensity. This project is expected to result in the development and application of a new set of important chemical biology tools for the study of neurochemistry with potentially broad scientific impact.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.