Lorene Lanier, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Understanding how and why the growth cone moves will shed light on the fundamental processes of neurodevelopment and may also suggest strategies to induce growth cone motility and regeneration in damaged neurons. While much progress has been made in identifying growth cone guidance signals and their receptors, relatively little is known about how these signals are transduced into the changes in cytoskeletal dynamics that are required for directed growth cone motility. It is clear, however, that multiple guidance signals converge on the cytoskeleton and must ultimately be integrated into a coordinated response. Proteins that bind to and directly regulate the cytoskeleton may thus serve as the ultimate interpreters of guidance signals. The overall goal of this proposal is to identify key growth cone cytoskeletal regulatory proteins and determine their role in growth cone motility and guidance. Experiments will focus on the Arp2/3 complex, which nucleates the formation of branched actin filaments and plays an essential role in many types of actin-based cell motility. Recent findings indicate that Arp2/3 is a negative regulator of growth cone translocation and that Arp2/3-dependent actin structures play an important role in coordinating actin and microtubule dynamics in the growth cone. We will test the hypothesis that Arp2/3 regulates cytoskeletal dynamics in response to guidance signals. A combination of molecular and biochemical techniques, live cell imaging and correlative electron microscopy will be used to deduce the mechanism of Arp2/3 function in growth cone motility and pathfinding. Tissue specific inhibition of Arp2/3 will be used to characterize the role of Arp2/3 in the developing nervous system. Finally, identification of the proteins that activate Arp2/3 in growth cones will provide insight into how Arp2/3 is regulated by upstream signaling pathways. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): All biological functions are dependent on molecular interactions. Therefore, by studying when, where and how molecules interact, we can elucidate the fundamental principles of biology. The Principle Investigators on this grant come from three different departments at the University of Minnesota and study a wide variety of topics, ranging from the dynamics of phospholipid receptor activation to the mechanisms that control amoeboid cell motility. These research projects each use different model systems and require their own specialized reagents. Yet, these projects are unified by the fact that each would benefit immensely from the use of Total Internal Reflection Fluorescence Microscopy (TIRFM). The long term goal of this proposal is to enhance basic and translational research at the University of Minnesota by providing state-of-the art, shared TIRFM equipment facilities with technical and scientific support. TIRFM is a specialized type of fluorescent microscopy in which only fluorescent molecule(s) within approximately 100 nm of the glass coverslip are excited (i.e. activated to fluoresce). With conventional epifluorescence microscopy, fluorescent excitation occurs though the entire depth of the sample;this generates out of focus background fluorescence, creating a poor signal-to-noise ratio and obscuring visualization. In addition, the sample is exposed to a considerable amount of fluorescent light, which can cause photobleaching (which leads to loss of imaging capacity) and, in the case of live cell imaging, phototoxicity and cell death. With laser confocal microcopy, the excitation beam is focused on a considerably narrower region (diameter: 250 -800 nm and depth: 500-1500 nm), improving the signal to noise ratio and reducing phototoxicity;however, with continuous imaging photobleaching and phototoxicity can still be a problem. All of the projects described in this proposal involve live cell, high speed or time-lapse imaging of fluorescently tagged proteins in order to follow the transport, assembly and/or processing of individual proteins within the cell. These experiments require the extremely narrow region of fluorescent excitation made possible by TIRFM;in each case, the investigators have attempted standard epifluorescence or confocal microscopy and found these techniques insufficient because they failed to provide the necessary resolution and/or sample preservation. Data from these experiments will elucidate the mechanisms that govern cell differentiation, motility and signaling, processes that are fundamental to all living organisms and, when mis-regulated, can deleteriously affect the health and lifespan of the individual. The specific aims of this proposal are: Aim 1) to provide access to specialized and cutting-edge resources not available to individual investigators. Having access to these recourses will extend the research capabilities and expertise of NIH-funded investigators. Aim 2) to foster new collaborations, particularly those that expand the research directions and impact of NIH-funded investigators.