1994 — 1996 |
Harkins, Amy B |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Calcium Channels in Excitation/Secretion Coupling |
0.922 |
2002 — 2003 |
Harkins, Amy B |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Homologous Recombination in a Secretory Mouse Cell Line
DESCRIPTION (Provided By Applicant): Disruptions of the molecular events that regulate exocytosis/synaptic transmission give rise to a variety of neuropathologies. Model systems that allow exploration of the underlying molecular machinery that control these processes are problematic. This exploratory/developmental grant (R21) application seeks to develop a mouse cell line that can be genetically modified to study vesicle release at the single-cell level, and to provide pilot data for subsequent grant applications. In the nervous system, synaptic transmission is initiated by the entry of Ca2+ into the presynaptic terminal, which activates the fusion of transmitter-filled vesicles with the presynaptic membrane. Synaptotagmin I (syt I), an integral vesicular protein, has been postulated to act as the Ca2+ sensor for exocytosis. One genetic approach to study the function of syt I as a Ca2+ sensor has been the generation of knockout mice. The syt I homozygous knockout mice are viable until 48 hours after birth, and the heterozygous animals are phenotypically indistinguishable from wild-type. These problems, not uncommon in knockout animals, make it difficult to study the functional role of syt I. 1. The first aim is to introduce a targeting vector to disrupt the syt I gene by homologous recombination in a mouse cell line of non-embryonic origin. We have identified a mouse pheochromocytoma cell line that exhibits Ca2+-dependent exocytosis of membrane-bound vesicles. We have designed specific targeting vectors to the syt I gene of the cells. We will attempt to introduce the specific targetig vectors into the cells, to screen cells with positive and negative selection, and to obtain and test the genomic DNA from the screened cells for targeted interruption of the syt I gene. We believe that this unique cell line will serve as an excellent model system to probe the function of syt I with biophysical techniques. 2. The second aim is to characterize Ca 2+-regulated exocytosis in the knockout cell line. We will use capacitance and amperometric measurements, in conjunction with [Ca2+]i measurements, to measure exocytosis from patch-clamped single cells. Cells will be stimulated to elicit Ca2+-dependent secretion. From the analysis of Ca2+-influx and vesicle release, we will determine the Ca2+ dependency of secretion for the knockout cell line and compare it to secretion in wild-type cells. Our short-term goal is to establish a syt I homozygous knockout cell line that can be used to determine whether syt I is the Ca2+ sensor for rapid Ca2+-dependent secretion in these secretory cells. Our long-term goal is to knock out each of the syt isoforms in the mouse cell line, using different selection vectors, and then add each one back to the cell by stable transfection to study the role that different syt isoforms play in secretion.
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0.958 |
2008 — 2012 |
Harkins, Amy |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Regulation of Differentially Released Transmitter
INTELLECTUAL MERIT: All organisms' ability to successfully live and function within their own habitat depends on cell-to-cell communication that is necessary for the most basic reflexes to the highest order thought processes. This cellular communication by neurons occurs by specialized signals that rapidly allow a chemical transmitter to be released from one neuronal cell and have an effect on an adjacent neuronal cell. It is not understood how these signals can be modulated to cause more or less chemical transmitter to be released, or to release one type of chemical transmitter preferentially over another. Previous work has established that two chemical transmitters, neuropeptide Y (NPY) and catecholamines (CAs), that are packaged into the same small vesicles undergo differential release. That is, NPY requires a longer stimulation duration and/or a higher frequency of stimulation than required to release CAs from the same vesicles. How this differential regulation of chemical transmitter release occurs is not known. The aims of this research will test the hypothesis that different members of a family of proteins, called synaptotagmins (syt), sense and control calcium-dependent release of the chemical transmitters from the vesicles. This project will utilize a combination of electrophysiology, molecular biology and biochemistry techniques in a model secretory cell system to study how these proteins may regulate release of NPY compared to the CAs. The objectives of this study are 1) to determine what types of stimuli are required for differential release, 2) to determine whether a Ca2+-dependent , or 3) a Ca2+-independent syt protein regulates differential release of chemical transmitter. Intellectually, this project will establish whether one protein that functions to sense Ca2+ and trigger secretion of transmitter can also preferentially determine the release of one particular transmitter over another.
BROADER IMPACT: These results will have broad application to understanding how one type of vesicle can preferentially release transmitter from the same population of vesicles, a basic mechanism that underlies neuronal communication. Results from this project will provide the basis for future research directions to understand how transmitter is regulated in release not only from secretory cells and neurons, but also from brain tissues. Education initiatives will involve students at all levels. The PI will continue to a) participate in an outreach science program for middle school inner city girls, b) participate in a highly competitive high school student research program (STARS), c) involve undergraduate students in research from simple lab techniques to Honors research theses, and d) train graduate students pursuing their PhDs to enter the academic arena. Students at all levels will be introduced to the disciplines of biophysics, molecular biology and chemistry as a combined approach to research. Current efforts to recruit female and minority researchers will be continued and expanded. All generated cell lines will be freely distributed to other researchers. To disseminate findings from this project, students will continue to present results at national conferences, and publish their work in peer-reviewed journals. As an outreach program for high school and undergraduate students, as well as a tool for all researchers to use, the students will provide lay descriptions of their projects and their scientific protocols on the laboratory's website. This research program will provide a platform for integrating research with education for students, scientists, and the lay community.
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1 |
2013 — 2014 |
Harkins, Amy B |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Developing An Electrochemical Assay to Detect Nerve Regeneration in 3d Scaffolds
DESCRIPTION (provided by applicant): The research objective of this proposal is to develop a method to detect whether regenerating neurons are communicating appropriately before they regrow to their target tissues. Currently, successful regeneration of nerves in vivo is evaluated by nerve conduction velocity and muscle contraction. The major drawback to currently available methods is that either complete reconnectivity or final reinnervation of target tissues must occur for testing to be possible. The proposed technique will utilize a multi-disciplinary approach to detect synaptic signals as a measure of proper nerve growth in the initial stages of regeneration, rather than waiting until the nerve has reconnected and innervated target tissues. The aims to accomplish this objective are (i) to characterize synaptic signals from neurons regrowing on a 2 dimensional surface, or within a 3 dimensional scaffold, and detected with an x-y multi-channel interdigitated array detector, and (ii) to design a 3 dimensional (x-y-z) interdigitated array detector to measure synaptic signals from regrowing neurons within clinically relevant 3D scaffold materials. The long-term outcome of this project has the potential to meet the global need for a practical and inexpensive method to rapidly assess the efficacy of nerve regeneration.
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0.958 |