Browsing by Subject "Actin"
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Item Actin Isoforms in neuronal structure and function(2011-07) Cheever, Thomas R.The actin cytoskeleton plays critical roles in nearly every aspect of neuronal development and function. During these processes, the localized polymerization of actin is one mechanism employed to carryout crucial tasks for normal neuronal function. While the activity of actin binding proteins is generally thought to be the primary mediator of spatially restricted actin polymerization, another prominent mechanism involves the local translation of β-actin, one of two actin isoforms expressed in neurons. The localized translation of β-actin has been shown previously to be essential for growth cone guidance in cultured neurons. Additionally, defects in the localization of β-actin have been implicated in the motor neuron disease Spinal Muscular Atrophy (SMA). However, no study to date has directly examined the role of β-actin in a mammalian in vivo system. Although the functions of β-actin were thought to be critical for all neurons, the work described in this thesis indicates that specific functions of β-actin are surprisingly confined to select populations in the central nervous system (CNS). β-actin is not required for motor axon regeneration or motor neuron function, but is required for the proper structure of the hippocampus, cerebellum, and corpus callosum, as well as hippocampal-associated behaviors. Thus, the work described here provides the first direct demonstration of specific roles for β-actin in vivo and presents a model to translate provocative findings in cell culture to the mammalian CNS.Item The role of the actin-binding protein Arp2/3 in growth cone actin dynamics and guidance(2014-05) San Miguel-Ruiz, Jose EnriqueNeuronal growth cones are responsible for wiring the immature nervous system. They rely on guidance cues present in the extracellular environment to guide axons to their targets. Guidance cues achieve effective changes in directionality by modulating the growth cone cytoskeleton. Particularly important for this process are the changes in actin dynamics at the leading edge of the growth cone. However, which and how actin-binding protein (ABP) regulate actin dynamics to achieve correct guidance is not clear. This thesis shows evidence on the role of two ABP's during growth cone guidance.The first chapter, The role of the Arp2/3 complex in actin dynamics and growth cone guidance is substrate dependent, characterizes the role of the actin nucleator Arp2/3 in growth cone actin dynamics and guidance. Arp2/3 has been shown to be important in leading edge actin dynamics in non-neuronal cells, but little was known about its role in neuronal growth cones. Because during development growth cones migrate in association with diverse adhesive substrates during development, we probed the hypothesis that the functional significance of Arp2/3 is substrate dependent. We report that Arp2/3 inhibition led to a reduction in the number of filopodia and F-actin content on laminin and L1. However, we found substrate-dependent differences in growth cone motility, actin retrograde flow, and guidance after Arp2/3 inhibition, suggesting that its role, and perhaps that of other ABP's, in growth cone motility is substrate-dependent. The next chapter, Ezrin/radixin/moesin family proteins mediate actin filament dynamics in attractive growth cone guidance to nerve growth factor, describes the role that the ezrin/radixin/moesin (ERM) family of membrane-cytoskeletal linker proteins have during attractive growth cone guidance. This family of proteins have been shown to link actin filaments to the cell membrane when activated. We found that endogenous guidance cues for dorsal root ganglion and retinal ganglion cell neurons can activate ERM proteins. Moreover, the increase in filamentous actin normally triggered by the stimulation of these attractive guidance cues was abolished when ERM protein function was disrupted. Additionally, we found that ERM activity is closely associated with correct placement of substrate adhesions in growth cones. Finally, we found that disruption of ERM protein function abolishes attractive growth cone guidance and causes mislocalization of ADF/cofilin, an active binding protein previously shown by our group to be required for correct guidance. These results suggest that the correct organization of filamentous actin and adhesions in growth cones by ERM proteins are necessary for correct growth cone guidance.The final chapter, The role of Arp2/3 during in-vivo axon guidance in the chick embryo, deals with the role of the Arp2/3 complex during growth cone guidance in-vivo. For this purpose studied the role of Arp2/3 during the development of chick retinotectal projections and that of the sensory-motor innervation of the hindlimb. We found that Arp2/3-inhibited neurons had no deficiencies during the development of the retinotectal projections. However, we did find that Arp2/3 was required for normal sensory-motor innervation of the hindlimb during development. Thus, suggesting that Arp2/3 is required during developmental axon guidance in some tissues.Item Site-directed modifications of myosin(2013-06) Moen, Rebecca JaneMyosins are a diverse class of molecular motors responsible for movement in all eukaryotic cells. The conversion of chemical energy from ATP hydrolysis into mechanical force produces movement along an actin filament. The mechanism of movement is involved in muscle contraction as well as various cellular processes including cytokinesis, adhesion, and vesicle transport. All myosins contain three functionally important domains: the catalytic head domain (CD), the light chain or lever arm domain (LCD), and the tail. The catalytic head domain is very similar between all classes of myosins, containing the site of ATP binding and hydrolysis and the actin-binding interface. The tail domain of myosins are highly divergent, containing either coiled-coil domains, individual subdomains, or both, that confer each myosin's specific function and cellular localization. The biochemical steps of ATP hydrolysis in myosins are accompanied by a sequence of structural transitions. A large-scale conformational change within the myosin molecule occurs where the LCD, functioning as a lever arm, rotates relative to CD. In muscle myosin, this large-scale conformation change is associated with a transition of the actomyosin complex from a state of disordered, weak actin binding to a state of ordered, strong actin binding. This research focuses on two functionally important domains within the myosin molecule: the catalytic domain and the tail domain. First, the structural transitions that occur within the myosin II catalytic domain during the actomyosin ATPase cycle are investigated using a combination of biochemical and spectroscopic approaches, specifically studying how various chemical modifications (chemical crosslinking, oxidative modifications including methionine oxidation and glutathionylation) produce functional and structural changes. Chemical crosslinking is used to capture a dynamic intermediate in the myosin ATPase cycle, resembling a weak binding state, which is defective in actomyosin functional interaction and is dynamically disordered when bound to oriented actin. In vitro oxidative modification of the myosin catalytic domain, as a model for aging and oxidative stress in muscle shows chemical, functional, and structural perturbations are predominantly caused by a specific methionine residue in the actomyosin binding interface. These combined results illustrate a crucial role in proper actin binding cleft structural dynamics in myosin function. Modification of dynamics in this region, either by crosslinking or oxidation at critical residues in cleft, affect muscle function by interfering with the critical structural transitions necessary for actomyosin functional interaction. The focus then shifts to the tail domain of myosin VII, again using biochemical and spectroscopic approaches to elucidate the functional and structural properties of a myosin tail subdomain, the MyTH/FERM domain.Item An unconventional myosin is necessary for chemotaxis in Dictyostellium discoideum.(2009-08) Breshears, Laura MarieDirected cell migration (chemotaxis) is a fundamental biological process necessary for embryonic development, wound healing, and proper function of the immune system. Chemotaxis also plays a significant role in many developmental disorders and post-embryonic diseases in humans, such as cancer. Chemotaxis is driven by extracellular cues that act, in large part, to induce changes in the actin cytoskeleton, such as actin polymerization, that facilitate directed cell migration. Myosins are actin-associated motors that have a variety of functions in different cellular contexts. Myosins can effect cortical tension, pseudopod and filopodia formation, phagocytosis, the function of sensory structures, and the basic mechanics of cell motility. Members of the MyTH/FERM family of unconventional myosins all have roles in actin-based processes and one member, vertebrate myosin X, has recently been shown to play a role in actin dynamics in response to extracellular migration cues. The social amoeba Dictyostelium discoideum is a powerful model system for dissecting chemoattractant signaling pathways and identifying the cytoskeletal components necessary for directed cell migration. MyoG is a novel unconventional myosin characterized by two MyTH/FERM domains in its tail region. The potential role of this myosin in Dictyostelium cell migration was investigated by analyzing the phenotype of three independent myoG null mutants. The initial stages of Dictyostelium development, induced by starvation, depend on chemotaxis to cAMP, resulting in the formation of a multi-cellular aggregate. Upon starvation myoG — cells fail to aggregate, arresting as a smooth monolayer of cells. The myoG — cells neither polarize in a cAMP gradient nor do they chemotax toward the cAMP source. Analysis of the ability of myoG — cells to polymerize actin in response to cAMP revealed that the response is dampened in the mutants. myoG — cells are also defective in signaling to PI3K in response to cAMP. These data show that while the mutant cells retain some ability to respond to the gradient, the major pathways regulating polarity and chemotaxis are not functional. The mutant phenotype suggests that MyoG acts in transducing the chemotactic signal from the cAMP receptor to PI3K and the actin cytoskeleton, facilitating the morphological changes that lead to polarization and directional migration. The role of MyoG in chemotactic signaling represents a novel function for an unconventional myosin. The work presented here clearly demonstrates that MyoG is necessary for signaling from the cAMP receptor to both PI3K and the actin cytoskeleton. Sequence analysis shows that there is no direct homologue of MyoG in other organisms, but the high degree of conservation of the chemotactic signaling pathways indicates that there are likely to be functional homologues in higher eukaryotic cells, such as neutrophils, that rely on chemotaxis for cellular function.