Eric A. Vitriol, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The small GTPase RhoA causes growth cone collapse and halts axon growth, thereby inhibiting regeneration of injured nerves. In contrast, RhoA has also been shown to promote growth cone advance under specific circumstances, and is important for guidance in growth cone motility. The broad objective of this proposal is to deduce how, through precise control of RhoA activation kinetics and localization, this protein can stimulate opposite cell behaviors. To resolve this conflict, we will utilize a biosensor to directly visualize the activation of RhoA in growing and retracting neurons, and manipulate microtubules to probe underlying mechanisms in RhoA signaling. Specific Aim 1 will determine the spatiotemporal dynamics of RhoA activation in growth cones during both extension and collapse, using novel image processing tools to generate a dynamic map of RhoA activation with subsecond and submicron resolution. The microtubule cytoskeleton is a master regulator of growth cone motility and guidance; it is both a major downstream target and upstream activator of RhoA. Specific Aim 2 will determine the relationship between RhoA activation and microtubule distribution and dynamics in growth cones. [unreadable] PUBLIC HEALTH RELEVANCE: In addition to nervous system regeneration failure, there are a host of severe and debilitating developmental diseases in which axon growth fails. Understanding the basic mechanism behind growth cone motility and axon extension can lead to the development of new treatments for nervous system injury and disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Decades of research have provided numerous insights as to how Cofilin interacts with and alters actin. However, these actions have complex outputs in the cell, including promoting both assembly and breakdown of actin networks. Thus the same activity can lead to opposite outcomes. For example, the local upregulation of Cofilin has been shown to be both associated with attractive and repulsive growth cone guidance. It is likely that that these seemingly conflicting results are mediated by where, when, and how much Cofilin is being activated. Several studies have shown that localization and timing of Cofilin activation is critical in determining which downstream behaviors it invokes. However, the tools to directly test these ideas have been limited. We hypothesize that a methodology which allows us to instantly inactivate Cofilin with subcellular precision will lead to the discovery of new mechanistic information of how Cofilin functions to regulate actin networks and to control growth cone motility. To determine the spatiotemporal role of Cofilin activity during growth cone migration, we propose the following Specific Aims: (1) To develop a methodology for local and instantaneous inactivation of Cofilin;(2) to determine the effects of instantaneous inactivation of Cofilin on actin distribution and dynamics in growth cones;and (3) to determine the effects of local inactivation of Cofilin on growth cone motility. Using a technique called Chromophore Assisted Laser Inactivation (CALI), we will show that we are able to instantly inactivate Cofilin with subcellular precision. We will develop this methodology so that it will be a generally applicable and useful tool for other labs who want to determine the functional consequences of local Cofilin inactivation. By combining CALI with high resolution live cell microscopy, we will monitor actin network changes in real time after instantaneous Cofilin depletion. Finally, we will use CALI to determine how local inactivation of Cofilin effects growth cone migration and guidance. Defects in axon guidance are associated with developmental disorders and nerve regeneration failure. Understanding the fundamental biological processes that underlie axon growth will allow for the design of better, more effective disease treatments. Cofilin has been implicated in growth cone motility, but its complex functional role has yet to be fully elucidated. In this proposal, we will uncover new mechanistic information about how Cofilin functions to regulate axon growth so that its role in guidance related disorders can be better understood.

Project Summary/Abstract Amyotrophic lateral sclerosis (ALS) is a fatal disease involving motor neuron degeneration. Death occurs 3-5 years after diagnosis, there is no cure, and what limited treatments do exist only extend survival by a matter of months. The mechanism of ALS pathogenesis has remained elusive to researchers; scientists are still unsure exactly what causes the motor neurons to become toxic and die. In this proposal, we will investigate the mechanistic role that defects in the regulation of actin dynamics plays in ALS. This course of investigation was spurred by the discovery that mutations in Profilin1 (PFN1), a key regulator of cytoskeletal dynamics, that inhibit its ability to bind actin are responsible for about 1-2% of familial Amyotrophic lateral sclerosis (fALS). Motor neurons overexpressing these mutant PFN1 constructs displayed inhibited axon growth and had abnormal actin cytoskeletons. The identification of PFN1 mutations as causative agents in fALS presents an exciting new hypothesis that actin cytoskeletal dynamics play a fundamental role in maintaining the health of motor neurons and that impairment of actin dynamics could, over time, lead to neurodegeneration. Because Pfn1 is a G-actin binding protein, the processes that spatially localize G-actin to regulate filament polymerization and the G-/F-actin (G/F) ratio are of particular interest. We hypothesize that defects in actin cytoskeletal dynamics downstream of PFN1, such as G-actin localization, play a crucial role in ALS pathogenesis. To determine if impaired actin dynamics are a hallmark of ALS and to investigate the mechanism of how Pfn1 mutations induce fALS, we propose the following Specific Aims: (1) Determine the role that defects in the dynamic regulation of G-actin plays in ALS; (2) Determine the specific mechanism of how ALS-linked PFN1 mutants alter actin dynamics; and (3) Determine the specific cellular mechanism of dynamic G-actin localization and its function in regulating motor neuron growth and maintenance. We will investigate actin dynamics using high-resolution quantitative imaging in motor neurons and nerve- muscle explants from mouse models of ALS. We will also examine actin in functional motor neurons derived from induced pluripotent stem cells from human ALS patients. Recently, we discovered a novel pathway where G-actin was spatiotemporally localized to regulate cell motility and axon guidance. Thus, we have designed a number of unique assays to visualize G-actin localization, calculate the G/F actin ratio, and quantify actin mobility. The questions addressed in this proposal will yield a deeper understanding of the role that actin dynamics play in motor neuron development and maintenance of the presynaptic terminal of the neuromuscular junction, as well as identify ways that defects in actin regulation can cause ALS.

Project Summary To divide, move, and communicate, cells rely on a dynamic actin cytoskeleton that can rapidly assemble and change. This is achieved by the polymerization of actin monomers into filaments, the construction of filament networks, and the disassembly of these networks back into monomers. Cells maintain a large monomer reserve to meet the demands of actin network assembly. Because of its size, the monomer pool was traditionally considered to be homogeneous. However, recent work by us and others has revealed distinct subgroups of monomers that can drive and modify actin dynamics in ways that profoundly influence cell behavior. These discoveries have shown us that the rules of monomer polymerization are much more complex than previously thought. Addressing this severe knowledge gap in one of the most fundamental aspects of cytoskeletal biology is paramount to understanding how actin functions in cells. It may also finally reveal why defects in monomer regulation are associated with neurodegeneration, inflammatory disorders, cardiac disease and cancer. Most of our current knowledge about actin monomer behavior comes from solution biochemistry. However, these types of experiments cannot reproduce the complex monomer-driven actin dynamics seen in cells. Therefore, we are devising strategies that merge biochemical principles with cellular imaging to reveal how monomeric actin regulates the cytoskeleton in living cells. This includes controlling protein levels with micromolar precision, using quantitative image analysis to extract rate constants and concentrations, and employing computational models to reveal how cellular geometry influences actin dynamics. Additionally, we are developing new tools to describe and quantify actin ultrastructure from super-resolution images. We will use these innovative approaches to address the following fundamental knowledge gaps about monomer-driven actin dynamics: 1) How monomers control the dynamics of complex actin networks, 2) How cell geometry contributes to monomer-regulated actin dynamics, and 3) Novel roles for monomers in the regulation of cellular processes.