2005 — 2009 |
Suter, Daniel Marcel |
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 Neuronal Growth Cone Guidance @ Purdue University West Lafayette
Understanding the mechanisms of neuronal growth cone guidance and motility is imperative, if we want to develop successful strategies for nerve regeneration after injury and neurodegenerative diseases. Although a large number of axon guidance molecules have been characterized in recent years, there are significant gaps in our understanding of the molecular and cellular mechanisms that the growth cone uses to integrate its sensory, signaling and motile functions. We have recently provided evidence that the immunoglobulin superfamily cell adhesion molecule apCAM mediates growth cone steering by substrate-cytoskeletal coupling. pCAM-actin coupling depends on Src kinase activity and results in actin flow attenuation ollowed by microtubule extension. Recent findings further suggest that microtubules influence Src kinase activity at adhesion sites. The goal of this project is to test the following hypothesis: Src kinase activity and microtubule dynamics regulate apCAM-cytoskeletal coupling in neuronal growth cone steering. Using the well-established high-resolution Aplysia growth cone system, advanced live cell imaging techniques, and a new set of molecular tools for Src tyrosine kinases, we will address three Specific Aims: (1) to determine if microtubules play a role early during apCAM-mediated growth cone steering. We will achieve this goal by combining a novel in vitro growth cone steering assay with microtubule fluorescent speckle microscopy to quantify the dynamic behavior of microtubules early before the major microtubule rearrangement occurs. The second Aim of this study is: (2) to identify Aplysia Src family kinases, an important group of tyrosine kinases implicated in the regulation of axonal growth, and to determine their subcellular localization, activation state and dynamics in growth cones. To achieve this goal, we will prepare antibodies and EGFP-fusion constructs of newly identified Src kinases in Aplysia. The third Aim is: (3) to determine the role of these Src family kinases in apCAM-mediated growth cone steering. Therefore, we will image Src-EGFP protein dynamics during growth cone steering events and test the effect of active and inactive Src mutants on apCAM-actin coupling and growth cone guidance. These studies will not only provide new insights into the role of microtubules and Src kinases in growth cone steering, but also unprecedented information on the dynamic behavior of this key signaling enzyme within a living neuron. Thus, they will have an impact on our understanding of axon guidance and nerve regeneration, as well as of tumor cell metastasis, another motile process, in which Src has been implicated.
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2012 — 2017 |
Suter, Daniel |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Nanomechanics of Src Signaling in Neuronal Growth Cones
During development and regeneration of the nervous system, neurons send out processes guided by the neuronal growth cone, a highly motile sensing structure and an excellent model system to study the dynamic interactions between cell surface, signaling, and cytoskeletal proteins such as actin. Although significant progress has been made in identifying the molecules involved in directional growth cone migration, the underlying mechanisms are not well understood. Particularly, the relationship between mechanical and biochemical signaling in neuronal growth and guidance remains a mystery. To fill this knowledge gap, this project will investigate how Src tyrosine kinase, a key signaling enzyme, regulates actin organization and dynamics as well as adhesion-mediated growth and force production of live neurons. Specifically, this project will determine which aspects of actin dynamics are regulated by Src as well as the relationship between force production and Src signaling in adhesion-mediated growth. To reach this goal, quantitative fluorescent imaging of cytoskeletal dynamics and Src activation will be combined with biophysical approaches involving micropipettes and the Atomic Force Microscope (AFM).
This research will significantly improve the understanding of how cells integrate sensing, signaling, and cytoskeletal dynamics during directional migration. The project involves training of High School, undergraduate, and graduate students in cutting-edge live cell imaging and biophysical techniques. The results will not only be disseminated to the scientific community, but also to the broad public through a web site called "CELLebration" displaying movies, images, and educational material about cell motility, through presentations at local schools, and public outreach events at Purdue University (Spring Fest and NanoDays). In summary, through high-quality digital imaging presentations, this work will increase the public awareness for the need for quantitative microscopy and biophysical approaches to solve key cell biological problems.
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2020 — 2021 |
Suter, Daniel Marcel |
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. |
Nadph Oxidase Regulates Growth Cone Guidance
Reactive oxygen species (ROS) can act as signaling molecules mediating physiological functions in immunity, cell proliferation, differentiation, and migration. Whether ROS have a major signaling function as second messengers in axonal growth and guidance is currently unclear. The neuronal growth cone is a highly motile structure at the tip of neuronal processes, guiding them to appropriate target cells during development and regeneration of the nervous system. The growth cone integrates molecular information from the environment and transduces it via multiple signaling cascades to affect underlying cytoskeletal dynamics. Whereas most major second messenger systems have been implicated in regulating directional growth cone movement, such a role has not been established for ROS. The present study has two major objectives focusing on ROS produced by nicotinamide adenine dinucleotide phosphate-(NADPH) oxidase (Nox): (1) to determine the cellular and molecular mechanism by which ROS control neurite growth; and (2) to determine whether ROS act as second messengers downstream of specific guidance cues to control axonal growth and guidance. The four central hypotheses state that (1) a physiological level of ROS is optimal and required for adhesion-mediated neurite growth; (2) Src tyrosine kinase is a key target of ROS signaling in neuronal growth cones; (3) neuronal Nox2-derived ROS regulate axonal pathfinding; and (4) specific axon guidance cues such as slit2 control axonal pathfinding via Nox2-derived ROS both in vitro and in vivo. This project will take advantage of two excellent model systems to test these hypotheses: large Aplysia growth cones for quantitative live cell imaging of growth cone motility and intracellular ROS in vitro and developing zebrafish embryos for imaging and manipulating axonal development in vivo. In vitro growth cone guidance assays, novel fluorescent dyes and biosensors specific for hydrogen peroxide and Src activity, respectively, advanced imaging techniques, chimeric analysis of Nox2-deficient zebrafish lines as well as retinal ganglion cell-specific Nox2-mutant fish lines will be used to address the following two Specific Aims: (1) The first aim is to determine the cellular and molecular mechanism by which ROS in control neurite growth. (2) The second aim is to determine the role of neuronal Nox2 in axonal pathfinding of retinal ganglion cells. The proposed work is highly innovative because it investigates ROS as a novel group of signaling molecules in axonal growth and guidance and develops several new zebrafish lines suitable for studying Nox function in the nervous system. In summary, these studies have the potential of leading to breakthrough findings in the field of neuronal development and regeneration. Furthermore, since basic mechanisms of axonal growth and guidance are highly conserved across species, these studies will impact the development of antioxidant treatments for neurodegenerative diseases and central nervous system injuries.
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