Paul Bridgman - US grants

Probably the most important part of the growing axon is the structure at its tip, the growth cone. Nerve growth cones are essential for directed axonal outgrowth. They are complex sensory-motor machines that respond to environmental cues through cell surface receptors or changes in ion channel permeability. They locally process this information using signal transduction pathways that regulate the motility mechanism responsible for driving forward advancement through the environment. Relatively little is known about the specific mechanisms that integrate the information and regulate its affect on motility. Although signal transduction pathways have been implicated, it has been difficult to link these pathways to specific effecter mechanisms of motility that persist over time. One effecter mechanism is the generation of force by myosin. Myosin activity has multiple roles that include the control of growth cone shape changes and the generation of traction force that drives forward advancement. It is an important effecter of growth cone motility that may be essential for changes in direction of advancement. My overall goal is to identify the various myosins responsible for modulating growth cone motility, understand how pathways that are stimulated by specific environmental cues control myosin activity and how the force that is developed is coupled to the extracellular environment to provide traction force. I propose that myosin II is the most important of the myosins identified in growth cones for regulating motility and producing traction force. I will test this proposal by eliminating the activity of myosin II and then measuring key aspects of motility including traction force. I also plan to identify the major pathways involved in growth cone myosin II regulation. Combined these results will provide the framework for understanding the molecular basis of growth cone motility and how environmental cues stimulate specific pathways to regulate this motility. This is essential for understanding the formation of neuronal circuitry during development and the mechanisms required for successful nerve regeneration.

DESCRIPTION (adapted from applicant's abstract): Dilute-lethal mice (i.e., myosin V null mutants) exhibit ataxia, convulsion with clonic link movements, and opisthotonus suggesting that they have defects in central nervous system. function. Death occurs at about 3 weeks of age. a certain rare human genetic disease (Griscelli disease) has been identified which results from mutations in the myosin V gene, and similar to the mouse mutation, can be lethal. The dilute mouse model is particularly attractive because of the distinct phenotype exhibited by the myosin V null mutants, combined with the fact that it is one of few mutations in which the gene product has been identified and the function of the protein extensively studied. Recent morphological data has also identified a specific defect in smooth endoplasmic reticulum localization in cerebellar Purkinje cells of dilute-lethal mice. There is additional evidence that myosin V associates with synaptic vesicles and may contribute to regulate the transport of these organelles or their precursors along actin filaments. Furthermore, myosin V may associate with a complex of proteins responsible for the anchoring of NMDA type glutamate receptors. Taken together these results suggest that the neurological defects in dilute-lethal mice (and humans) may result from a combination of impaired organelle trafficking and protein localization in neurons. Because of the organelles and proteins involved, this could have profound effects on synaptic efficacy. Thus, this system allows the applicants to address both the molecular mechanism of myosin V function and the important consequences of its activity for the nervous system at both cellular and behavioral levels. The applicant's current goals are to: (1) determine the consequences of the dilute-lethal structural defects for synaptic physiology at the synapse between granule cells and Purkinje cells, (2) determine the mechanism through which myosin V targets smooth endoplasmic reticulum to dendritic spines, (3) determine the consequences of myosin V's absence on the localization of NMDA receptors and associate postsynaptic proteins, and (4) determine if presynaptic terminals of dilute-lethal granule cells have abnormal organelle transport of processing. To accomplish these goals, a combination of electrophysiological, live imaging and morphological techniques will be used.

To understand how phosphatidylinositol (PI) 3-dinase (PI-3K) modulates cell structure and behavior we examined the molecular and cellular defects of a Dictyostelium mutant strain (pik 1?2?) missing two (DdPIK1 and 2) of three Pi-3K genes which are redundant homologues of the mammalian p110 subunit. DdPIK2 rescued the defects of pik 1?2?. Levels of phosphatidylinositol trisphosphate (PI93,4,5)P3) were reduced in pik 1?2? which had major defects in macropinocytosis. This was accompanied by dramatic deficits in a subset F-actin-enriched structures such as circular ruffles, actin crowns, filopodia, pseudopodia and cortical actin. Although pik1?2? were still capable of near normal motility and chemotaxis, they failed to aggragate into streams. Therefore, we conclude that PIK1 and 2, possibly through modulation of the levels of PIP3 and PI93,4)P2, regulate the reorganization of actin filaments necessary for invagination and circular ruffling during macropinocytosis, the extensio n of filopodia and pseuodopodia, and the aggregation of cells into streams but not for the regulation of cell movement during chemotaxis. Confocal microscopy studies for this project were carried out at the NCMIR. This work has been published (Zhou, Pandol, Bokoch, and Traynor-Kaplan, J. Cell Sci, 111: 283-94. 1998).

[unreadable] DESCRIPTION (provided by applicant): We have devised a new substrate patterning method that overcomes the limitations associated with random cell binding on pre-patterned substrates. Our approach provides the ability to create patterns that restrict, contain, allow or direct growth after cells have been plated. The ability to pattern substrates after plating allows selection of specific subsets of cells (i.e., transfected or knockout cells) and modification of patterns in response to changes in outgrowth. When combined with pre-patterned substrates comprising more than one material, polarization of neurons can be induced and interactions oriented. Using this approach we are able to create neuronal circuits with varying complexity and known connectivity. Our patterning technique is similar to maskless photolithography and uses a high-energy pulsed IR laser to directly unbind substrate materials. The areas where protein is removed become non-permissive for neuronal growth. We will use the combination of substrate patterning, microfluidics and electrophysiology to create and test functional neuronal circuits of hippocampal neurons. Once we have verified function and optimized the approach, we will focus on addressing specific biological questions. This will include studying the influence and regulation of neural activity on dendritic spine emergence, development and dynamics. We will also study the influence of an applied exogenous factor on spine formation and maturation, as well as alterations in spine dynamics in the absence of myosin IIB using cells from myosin IIB knockout mice. Finally, we will study the role of Rab5 in presynaptic function of hippocampal neurons connected in simple circuits. PUBLIC HEALTH RELEVANCE We have developed a new method of patterning substrates in the presence of living cells that allows creation of neuronal circuits with known connectivity in cell culture. This will advance our understanding of communication between nerve cells and facilitate modeling of brain circuits. This is important for understanding normal brain function and the potential for recovery from brain or spinal cord injury. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our goal is to develop a system based on optical methods for creating and testing neural circuits in vitro. We have developed a multiphoton laser technique (Live Cell Substrate Patterning or LCSP) that allows substrates to be patterned after cells have been plated. The ability to alter substrates dynamically in response to new growth enables us to create well-defined simple (few-cell) circuits. Functional tests of these circuits have been made using conventional electrophysiological methods. We now plan to record changes in activity using calcium imaging. Cells will be loaded with a calcium indicator dye or transfected to express a calcium indicator protein. We will also use light stimulation to activate or silence cells expressing channelrhodopsin-2 or halorhodopsin, respectively. This all-optical approach will allow repeated activation and imaging of cells as will be necessary for studies of slow-changing events during synapse formation. For example, it will be possible to address questions such as when synaptic activity first becomes functional and how synapse formation may be altered in response to changes in activity. It will also provide a means for testing a wide range of cellular and molecular mechanisms that may underlie structure or function of circuits. PUBLIC HEALTH RELEVANCE: New approaches are needed to create and test neural circuits in order to model neural function. Using minimally invasive optical methods we will be able to control circuit formation and also stimulate and record activity at high resolution over time.