1989 — 1991 |
Schnapp, Bruce Jeffrey |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Basis of Axonal Transport @ Boston University Medical Campus
The general aim of this research project is to develop a more complete understanding of the molecular interactions which determine how organelles are transported along microtubules in axons. Our proposed investigations are particularly concerned with how kinesin and dynein two soluble, force-generating ATPases that promote movement in opposite directions along microtubules, interact with specific populations of organelles, programmed to move either toward or away from the cell body. In recent experiments, the movement of purified organelles depended on the presence of an axoplasmic cytosol fraction containing many proteins in addition to kinesin and dynein. The specific aim of this proposal is to identify, more precisely, the particular cytosolic factors required for organelle movement. We will isolate, by AMP-PNP induced microtubule affinity, followed by salt or nucleotide induced release, a subset of cytosolic proteins form axoplasm, sufficient to promote organelle movement. Our proposed experiments will determine whether purified dynein and kinesin from axoplasm can alone drive organelle movement, or whether soluble "accessory factors" are also required, as suggested by previous studies. We will also explore the possibility, suggested by recent experiments, that both dynein and kinesin are required for retrograde organelle movement. We will do so in part by recombining the purified components and testing their ability to promote directed movement of organelles; in addition, electron microscopic studies of organelles will localize kinesin and dynein, to determine whether they are bound together on the organelle surface. Finally, nm-scale motion analysis, which previously indicated that kinesin-coated beads track along single protofilaments, while dynein-coated beads "wander" over the microtubule surface, will be applied to organelle movement to test if these same features of kinesin and dynein driven movement are apparent. Tracking of organelle movement at the nm level will provide an assay for molecular interactions between particular proteins in the reconstituted system.
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0.937 |
1992 — 1993 |
Schnapp, Bruce Jeffrey |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Basis of Microtubule-Based Vesicle Transport @ Harvard University (Medical School)
The general aim of this proposal is to develop a more fundamental understanding of the molecular interactions which serve to direct organelle traffic along microtubules in eukaryotic cells. Our studies will continue to focus on axoplasm extruded from the squid giant axon because this preparation is highly enriched for vesicular organelles that are transported along microtubules. One long-term aim is to elucidate the biochemical mechanisms that target kinesin and dynein, two motor proteins for movement in opposite directions on microtubules, to specific populations of organelles programmed to move toward either the plus- or minus-ends of microtubules. In pursuit of this question, we propose two approaches to identify vesicular membrane proteins that interact with these motor proteins. In one approach, biotinylated organelles, attached by rigor to microtubules, will be detergent extracted, leaving the motor- receptor complex attached to the microtubule. The motor-receptor complex will be eluted with ATP and the components further purified; biotinylated proteins will be analyzed by an avidin-chemiluminescent procedure after gel electrophoresis. In a second approach toward identifying a kinesin receptor, the C-terminal tail domain of the kinesin heavy chain, which is believed to interact with organelles, will be expressed as a glutathione S- transferase fusion protein, coupled to glutathione sepharose, and used to screen detergent extracts of highly purified vesicles for proteins that interact with the tail domain. We also propose experiments aimed at identifying mechanisms that regulate the direction of organelle transport. In these studies, we will use an in vitro organelle motility assay and optical tweezers to screen for factors that cause individual organelles to change their direction of movement. A second long-term aim of our research is to develop a molecular model for kinesin and dynein-driven movement. In these studies, optical tweezers will be used to manipulate beads carrying single motor proteins onto microtubules; the motion of these beads will be tracked at nm-scale resolution with an image processing technique, with the aim of imaging the molecular mechanical events underlying motility. These studies will be executed with recombinant kinesin motor domains, expressed as fusion proteins with sites for specific coupling to beads, in an effort to relate elements of the primary structure of kinesin to the process of mechanochemical transduction. In collaborative studies, we will employ instrumentation for tracking the motion of beads with significantly higher temporal resolution than is possible with video. The aim of these studies is to characterize motion during the power stroke in increasing detail.
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0.937 |
1994 — 1997 |
Schnapp, Bruce Jeffrey |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Basis of Microtubule Based Vesicle Transport @ Harvard University (Medical School)
The general aim of this proposal is to develop a more fundamental understanding of the molecular interactions which serve to direct organelle traffic along microtubules in eukaryotic cells. Our studies will continue to focus on axoplasm extruded from the squid giant axon because this preparation is highly enriched for vesicular organelles that are transported along microtubules. One long-term aim is to elucidate the biochemical mechanisms that target kinesin and dynein, two motor proteins for movement in opposite directions on microtubules, to specific populations of organelles programmed to move toward either the plus- or minus-ends of microtubules. In pursuit of this question, we propose two approaches to identify vesicular membrane proteins that interact with these motor proteins. In one approach, biotinylated organelles, attached by rigor to microtubules, will be detergent extracted, leaving the motor- receptor complex attached to the microtubule. The motor-receptor complex will be eluted with ATP and the components further purified; biotinylated proteins will be analyzed by an avidin-chemiluminescent procedure after gel electrophoresis. In a second approach toward identifying a kinesin receptor, the C-terminal tail domain of the kinesin heavy chain, which is believed to interact with organelles, will be expressed as a glutathione S- transferase fusion protein, coupled to glutathione sepharose, and used to screen detergent extracts of highly purified vesicles for proteins that interact with the tail domain. We also propose experiments aimed at identifying mechanisms that regulate the direction of organelle transport. In these studies, we will use an in vitro organelle motility assay and optical tweezers to screen for factors that cause individual organelles to change their direction of movement. A second long-term aim of our research is to develop a molecular model for kinesin and dynein-driven movement. In these studies, optical tweezers will be used to manipulate beads carrying single motor proteins onto microtubules; the motion of these beads will be tracked at nm-scale resolution with an image processing technique, with the aim of imaging the molecular mechanical events underlying motility. These studies will be executed with recombinant kinesin motor domains, expressed as fusion proteins with sites for specific coupling to beads, in an effort to relate elements of the primary structure of kinesin to the process of mechanochemical transduction. In collaborative studies, we will employ instrumentation for tracking the motion of beads with significantly higher temporal resolution than is possible with video. The aim of these studies is to characterize motion during the power stroke in increasing detail.
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0.937 |
1998 — 2001 |
Schnapp, Bruce Jeffrey |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cytoplasmic Mrna Transport in Xenopus Oocytes @ Harvard University (Medical School)
DESCRIPTION: The goal of this proposal is to understand the mechanisms that govern localization of Vg1 RNA on the Xenopus oocyte. Previous experiments in Schnapp's lab have shown that this localization requires the presence of at least one copy each of four different repeats present in the 3'UTR of the mRNA. A five nucleotide repeat UUCAC (E2) appears to play a major role and is the focus of much of this grant. Schnapp has identified a protein, vera, that binds to the VgLE, most likely by direct physical association with the E2 repeat. Because Vera is an ER associated protein, Schnapp proposes that it plays a role in localizing Vg1RNA to the ER. Schnapp has also succeeded in isolating a RNP particle containing the Vg1 localization element (VgLE). All four VgLE repeat types are required for the VgLE's association in the particle. This contrasts with the earlier observation that only three (E1,E2,E4) are required for association with vera protein, suggesting in turn that the latter is not sufficient for particle assembly.
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1 |
2000 — 2003 |
Schnapp, Bruce Jeffrey |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Kinesin Motors @ Harvard University (Medical School)
The broad aim of this project is to elucidate how cells transport organelles and molecular complexes along microtubules (Mts) to specific destinations. To address this problem, we must explain the molecular basis and the overall logic for how different kinesin motors are mobilized to carry specific cargoes. To his end, we will substantiate our hypothesis that processive kinesin motors, which are primarily soluble in the cell, are in an inhibited ground state until they are bound and activated by their cargoes. How the non-motor regions of kinesins direct motor activity and cargo binding is the research plan's principle concern. We will identify, within the non-motor regions of a few kinesins, the domains required for binding cargo and regulating motor activity. Such domain-mapping studies will bring to light plausible mechanisms for cargo binding and motor regulation, and most importantly, pinpoint candidate-binding sites for proteins that implement these activities. Preliminary studies along these lines on conventional kinesin validate this strategy - our domain-mapping studies indicate that the inhibited state of conventional kinesin depends on kinesin light chain (KLC) and involves a folded conformation, in which the C-terminus of kinesin heavy chain (KHC) interacts with its own motor domain. Moreover, these studies have identified the KLC tandem tetratricopeptide repeats (KLC TPRs) as prime candidates to bind factors that either link kinesin to its cargo or activate kinesin's interaction with Mts. In the first specific aim, we will now isolate these KLC TPR partner proteins, using biochemical and genetic approaches, and characterize their functions. In a similar fashion, the second and third specific aims propose to investigate the non-motor regions of a number of other kinesins (heterotrimeric kinesin II and monomeric KIF1A,B, & C) whose cargoes are known. By identifying, within the non- motor regions of these kinesins, sites involved in cargo binding and motor activation, we will again develop a rationale for isolating the protein factors that implement these activities. It is the identification of these interacting proteins that is this plan's ultimate goal. To ensure that we isolate proteins that are functionally relevant, we will introduce, into the nonmotor regions of these kinesins, mutations that disrupt motor regulation or cargo binding. Such kinesin mutants are the keystone of our proposal - proteins that bind specifically to wild-type, but not mutant, sites are likely to be the bona fide partners in vivo. The generation and use of such negative controls fundamentally distinguishes this proposal from previous attempts to identify proteins that interact with kinesins.
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1 |
2003 |
Schnapp, Bruce Jeffrey |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Nipkow-Disk Confocal Microscope For Live-Cell Imaging @ Oregon Health and Science University
DESCRIPTION (provided by applicant): This application supports the purchase of a Perkin-Elmer Ultraview LCI confocal microscope. This newly designed microscope, based on a recent enhancement in Nipkow disk technology, represents a major advance in confocal imaging of living cells. Compared with microscopes that use laser scanning, the Ultraview enables far faster image acquisition, real-time confocal viewing, and greatly reduced phototoxicity (compared with single-photon confocals). The designated user group consists of 13 scientists who serve as PIs on 27 research projects funded by 8 different institutes of the NIH. Key projects demand capturing time-series of confocal images over large regions of interest in multiple focal planes labeled with two or more fluorophores in order to visualize movements at m/sec speeds. These capabilities are integral to the research programs of several of the participating investigators and will greatly benefit the research efforts of all designated users. Projects include studies of RNA transport in Drosophila embryos, kinesin-mediated movements in cultured cells, post-Golgi sorting and axonal transport in cultured neurons, pH changes and protein movements in hair cells, calcium dynamics in brain slices, trafficking of iron transport proteins in epithelial monolayers, lipid raft-associated ion channels in fibroblasts, and potassium channels in pancreatic islet cells. Laser scanning microscopes cannot begin to meet these demands. The requested instrument will be equipped with accessories to permit flexible use by a diverse group of investigators. It will be housed in OHSU's Live-Cell Imaging Facility, which currently serves a group of 35 registered users drawn from among the >200 research laboratories located on the main campus of OHSU. Use of the instrument will be overseen by Bruce Schnapp (Professor, Cell and Developmental Biology), Gary Banker (Senior Scientist, CROET), and a steering committee drawn from other departments. Together, Schnapp and Banker have more than 30 years of expertise with advanced imaging methods. User training and day-to-day operation of the instrument will be the responsibility of Dr. Stefanie Kaech, who has 10 years experience with confocal imaging and participated in beta-testing of the Ultraview while on the staff at the Friedrich Miescher Institute (Basel).
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