1990 — 1993 |
Schroer, Trina A |
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 Cytoplasmic Dynein-Based Vesicle Transport @ Johns Hopkins University
Microtubule-based membrane transport is a complicated process that involves the mechanochemical enzymes dynein and kinesin as well as soluble activator proteins. Assays of organelle motility in vitro have allowed the identification and preliminary characterization of three distinct activators of dynein-driven organelle transport in chick embryo brain. The activators are likely to govern motor function in vivo. The goal of the proposed research is to elucidate the mechanism(s) by which two activators, 150/5OkD and C3, regulate motor activity and to initiate studies of regulation of motor activity in vivo. The following questions will be addressed. 1. How is cytoplasmic dynein activity regulated by the 150/5OkD activator? The 150/5OkD activator will be purified further and antibodies will be raised. The effects of the activator on dynein ATPase activity will be determined. Anti-activator antibodies will be used to immunolocalize the activator in cultured cells and in microinjection studies of live cells. 2. Do membranes affect enzyme activity? The membrane binding properties of dynein will be measured and it will be determined if membranes stimulate dynein ATPase. 3. Is cytoplasmic dynein activity regulated by phosphorylation? The C3 activator has protein kinase activity; it will be determined if cytoplasmic dynein is a substrate for phosphorylation. Dynein phosphorylation patterns will be analyzed to determine if they correlate with patterns of activity. 4. Is cytoplasmic dynein activity regulated during the cell cycle? Membrane traffic is known to be inhibited during mitosis; at this time, cytoplasmic dynein may be involved in the mitotic spindle movements. It will be determined whether "mitotic" dynein can serve as an organelle motor and how dynein activity is regulated during the cell cycle. These studies may provide insight into the role of dynein in mitosis.
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1992 — 2002 |
Schroer, Trina A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Microtubule Based Vesicle Transport in Polarized Epithelia @ Johns Hopkins University
In many eukaryotic cells, the subcellular positioning and dynamics of membranous organelles is dependent on microtubules and microtubule- based transport. The arrangement of the microtubule cytoskeleton along the apico-basal axis of simple polarized epithelia suggests that these filaments provide a structural framework that both yields stability to themonolayer and provides a substrate for transport ofmaterials across the cell. It appears that transcellular movement in the basal-to-apical direction, i.e. toward microtubule minus ends, is highly dependent on microtubules, suggesting tht the motor protein, cytoplasmic dynein, andits activator, dynactin are key players in this process. Polarized epithelia are therefore a useful model system for in-depth study of dynein-and dynactin-based intracellular motility. Understanding of mechanisms ofmicrotubule dynamics and microtubule- based motility has been significantly extended by the application of video-enhanced microscopy techniques, as they allow evaulation in real time of complex subcellular behaviors. Studies of intracellular membrane traffic and endomembrane dynamics have benefited equally fromthis sort of analysis. Inthis proposal, a series of experiments designed to elucidate the transcytotic pathway in WIF-B cells and to determine how cytoplasmic dynein and dynactin contribute to transcellular movement in WIF-B and MDCK are proposed. Membrane dynamics will be visualized using a novel surface labeling technique that allow traffic from the basal surface to the apical surface to be observed directly in real time by video-enhanced fluorescence microscopy. A separate line of investigation will explore the mechanism of microtubule nucleationin WIF-B and MDCK cells. Many epithelia are post-mitotic and their microtubule cytoskeletons lack a centralfocus, suggesting that conventional centrosomal nucleating mechanisms may not be involved. Sites for microtubule nucleationwill be identified and it will be determined if their centrosomes have the capacity to nucleate and release microtubules in vitro and in vivo.
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1994 — 2012 |
Schroer, Trina A |
P41Activity Code Description: Undocumented code - click on the grant title for more information. 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. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Regulation of Cytoplasmic Dynein Based Vesicle Transport @ Johns Hopkins University |
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2007 — 2008 |
Schroer, Trina A |
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.) |
Molecular Mechanism of Sif Formation by Salmonella Typhimurium @ Johns Hopkins University
Salmonella enterica serovar Typhimurium (Salmonella TyphimuriumJ is a significant pathogen that causes gastrointestinal disease in humans and other anima|s and a systemic infection that resembles typhoid fever in mice. As part of its infectious cycle, S. Typhimurium enters host epithelial and phagocytic cells and takes up residence in a late endocytic compartment (the Sa/mone//a-containing vacuole, or SCV) that becomes biochemically and structurally modified to support bacterial replication. These modifications involve the action of a set of bacterially produced effector proteins that are delivered into cells via a type three secretion system. A hallmark of infected epithelial cells is the formation of elongated, membranous tubules, known as Sa/mone//a-induced filaments (SIFs), that emanate from the SCV and are aligned with microtubules. SIF formation is required for both systemic disease and localized infection in the intestinal epithelium, highlighting the importance of these unique structures in pathogenesis. A number of bacterial effector proteins have been identified that contribute to SIF formation, but the molecular details of how these proteins impact the architecture of endosome membranes, particularly the microtubules and motors that contribute to endosome movement, are poorly characterized. The experiments described in this proposal are intended to further understanding of the molecular basis of SIF formation. Late endosomes, the host cell compartment that becomes subverted to form the SCV and SIFs, are ordinarily highly motile, so we will begin by visualizing SIF formation in living cells infected with wild type and mutant Salmonella strains using a vital fluorescent probe of the SIF .membrane. We will then explore the roles of two different microtubule-based motors, kinesin 2 and kinesin 1, in SIF formation, using dominant negative inhibitors. The ability of different bacterial effector proteins to bind microtubules will be tested biochemically using copelleting assays, and their impact on microtubule-based motility will be determined in vitro. How different effectors alter microtubule organization and dynamics in host cells will be explored by evaluating the behavior of cells infected with mutant strains. Together, this analysis will provide a clear picture of how Salmonella modifies the activities of the microtubule cytoskeleton during the course of intracellular infection. Relevance: Salmonella infections cause serious human diseases such as food poisoning and typhoid fever. Salmonella invade and take up residence in host cells, exploiting a number of normal cell functions in the process. A comprehensive understanding of the molecular mechanisms that underlie this process is necessary to identify novel targets for therapy and disease prevention.
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