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Browsing Theses and Dissertations by Subject "actin"

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    Actin dynamics in silico, in waves, and in rods
    (Colorado State University. Libraries, 2009) Pak, Chi W., author; Bamburg, James, advisor
    The current paradigm of actin dynamics and superorganization has advanced in the past decade from emerging technologies and perspectives, which include the discovery of actin nucleators, real time imaging of the dynamics of single filaments in vitro, and single molecule imaging of actin superstructures in vivo. These advances have influenced each of our studies on multiple levels, sometimes directly. A novel analysis of single actin filament dynamics revealed faster than expected dynamics during treadmilling but not during bulk polymerization. Using an exact stochastic simulation, we investigated whether filament-annealing and -fragmentation might account for faster than expected dynamics; their influence on actin dynamics had not been investigated before in a comprehensive model. Results from our work demonstrated that filament-annealing and -fragmentation alone cannot account for faster than expected dynamics during treadmilling. Thus, strictly through computational modeling, we are able to investigate various hypothetical models and offer insights into a process that cannot be achieved by experimentation. A concept that has also gained support during the past decade has been the self-organizing nature of actin, which was demonstrated by the Listeria actin-comet-tail reconstitution assay. We have proposed that this is a fundamental property of all actin superstructures, whether they are assembled in vitro or in vivo or whether they are involved in development or disease. The concept of actin's self-organization has influenced our study of neuronal waves, which are growth cone-like structures that travel along neurites and which were hypothesized to transport actin to growth cones and support neuritogenesis. Using diffusional analysis, we were able to demonstrate that neuronal waves transport actin. Neuronal waves provide a unique mechanism for transporting actin in that the delivery of actin is dependent upon actin itself and its dynamics. In disease states, the self-organization of actin is often changed but not disrupted, sometimes resulting in the formation of orderly-structured aggregates of cofilin and actin known as cofilin-actin rods (or rods). Using glutamate excitotoxicity as a model system for the cofilin pathology observed in Alzheimer disease (AD), we have determined signaling mechanisms for cofilin-actin rod induction, which in young rat hippocampal neurons require AMPA receptors and are calcium-independent. In addition, cofilin-actin rod interactions with microtubule associated proteins, and associated changes to the microtubule cytoskeleton were studied for its potential relevance to the pathology of AD. Our results suggest that disruptions to the normal organization of actin and microtubules might underlie several pathological hallmarks of early AD.
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    The role of Cdc42, ADF/cofilin, myosin II and waves during the establishment of neuronal polarity
    (Colorado State University. Libraries, 2008) Flynn, Kevin Carl, author; Bamburg, James R., advisor
    The establishment of neuronal polarity is an essential developmental process, underlying the unidirectional flow of information in neurons and the overall function of the nervous system. In cultured hippocampal neurons, the first signs of polarity occur as one of several undifferentiated processes begins to elongate rapidly to form the axon (axonogenesis). The regulation of the cytoskeleton and intracellular trafficking are crucial to the proper development of neuronal polarity. This dissertation explores both of these polarity-developing mechanisms, identifying actin-regulating components in the signaling pathways as well as characterizing growth-cone like "waves" that correlate with axonogenesis. This study begins by analyzing the functional consequences of the loss of the Rho GTPase, cdc42, on the polarization of hippocampal neurons. Neurons from the cdc42 knock-out (cdc42KO) mouse have severe deficiencies in their ability to extend axons in vivo and in culture which is exerted through the regulation of actin dynamics. The actin regulating protein, cofilin is normally asymmetrically enriched in its active form in axonal growth cones but in cdc42 KO neurons there is an increase in the phosphorylation (inactivation) of cofilin. Cofilin expression promotes axon growth, whereas cofilin knockdown results in polarity defects analogous to those seen upon cdc42 ablation. Taken together, these data suggest that cdc42 is a key regulator of axon specification and that cofilin is a downstream effector of during this process. Though these studies suggest the involvement of cofilin downstream of cdc42; active cofilin cannot rescue polarity deficits in cdc42KO neurons. This suggests that other actin regulating proteins may also be required for axon formation. The actin motor protein, myosin-II also shows an increased activation in the cdc42KO brain. Inhibition of myosin II activity promotes axon formation and acts in synergy with cofilin on inducing supernumerary axons. Furthermore, the combined inhibition of myosin and activation of cofilin rescues axon formation in cdc42KO neurons while either treatment individually does not. Thus, the concurrent inhibition of myosin and activation of cofilin can contribute to the regulation of actin dynamics during axon specification. Axon specification is dependent on the transport of materials to the developing axonal growth cone. "Waves"-growth cone-like structures, propagate down neurites and correlate with neurite extension; thus, waves have been suggested as a mechanism for transporting materials that support this growth. Waves occur in all processes during early neuronal development, but are more frequent in the developing axon. Proteins enriched in axonal growth cones are also localized to waves and proteins such as cofilin and actin appears to be transported via waves to the growth cone, suggesting that waves represent a transport mechanism. Wave arrival at neurite tips was also coincident with an increase in growth cone size and dynamics. In addition, waves can promote neurite branching, either by supporting the growth of existing branches or by facilitating the growth of nascent branches. Waves are observed in neurons in organotypic hippocampal slices, a 3-dimensional growth environment reflecting the in vivo environment. Together, these data indicate that waves contribute to axon differentiation and growth both through the transport of actin and by increasing growth cone dynamics.