Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 97 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 87-88).

Thesis (M.S.)--Montana State University--Bozeman, 2005. / Typescript. Chairperson, Graduate Committee: Algirdas J. Jesaitis. Includes bibliographical references (leaves 75-80).

Köln, Universiẗat, Diss., 2004.

Thesis (Ph. D.)--University of Canterbury, 2008. / Typescript (photocopy). Two offprints bound in the back. Includes bibliographical references (leaves 142-162). Also available via the World Wide Web.

Axons are up-to-a-meter-long cable-like cellular processes of neurons. The proper function of nervous systems requires that axons grow and wire up correctly during development or regeneration. The uniquely challenging architecture of axons has to be sustained for an organism's lifetime, and renders them key lesion sites during healthy ageing, in injury and neurodegenerative diseases. Notably, axon degeneration is considered as the cause rather than consequence for neuron decay in the context of various neurodegenerative diseases. The structural backbones of axons are formed by parallel bundles of microtubules (MTs) which also provide the highways for life-sustaining long-distance transport between cell bodies and the growth cones or synaptic endings. To better understand axon development, regeneration, maintenance and degeneration during ageing, my PhD project has focused on mechanisms underpinning the regulation of MT bundles in axons. For this, I have capitalised on fast and genetically and experimentally amenable research possible in Drosophila neurons, both in primary culture and in vivo. I have used systematic combinatorial genetics and pharmacological approaches to unravel mechanisms and roles of actin as well as the cortical collapse factor Efa6 in MT regulation during axon formation and maintenance. I was able to gain a number of novel mechanisms contributing to the de novo alignment and maintenance of ordered MT bundles. First, it has been proposed that Spectraplakins (large actin-microtubule linkers) guide the extension of polymerising MTs along cortical F-actin, thus directly laying axonal MTs out into parallel bundles. Here, I have used manipulations of actin networks as well as hybrid constructs of Shot where the actin binding domain was replaced by actin associating domains of other molecules. My data strongly suggest that Shot's ABD domain has unique properties that can sense specific properties of F-actin networks, and this is important for its ability to appropriately regulate MT behaviours. Second, using combinations of actin and Shot manipulations, I found that Shot displays not only these actin-dependent guidance functions, but it displays novel actin-independent function in MT bundle maintenance for which I present a working hypothesis. Third, I found a novel and Shot-independent role of axonal actin in maintaining MTs and promoting axon growth, and my results suggest that these functions involve promotion of MT polymerisation. MT maintenance is therefore mediated through two complementary mechanisms involving Shot on the one hand and actin on the other, and simultaneous removal of Shot and actin leads to entire loss of axons. Finally, I have unravelled novel axonal functions of the cortical collapse factor Efa6 which serves as a check point in MT bundle maintenance by eliminating "off track" MTs that have escaped the axonal bundle organisation. In the absence of this factor, a gradual increase of disorganised, criss-crossed MTs occurs as a matter of days. These new mechanisms strongly suggest that different MT-regulatory mechanisms act in parallel in axons and complement each other in one common mechanism of MT bundle formation and maintenance. I propose here a local homeostasis model of axonal MT bundle maintenance which provides new ways to think about problems of ageing as well as a range of different neurodegenerative diseases.

Branched actin networks play an important role in cellular processes ranging from cell motility, endocytosis, and adhesion. The Actin-related protein 2/3 (Arp2/3) complex nucleates actin branches from the sides of existing actin filaments. Arp2/3 complex is highly regulated and requires association with ATP, actin monomers, actin filaments and a class of proteins called nucleation promoting factors (NPFs) to undergo an activating conformational change where the actin-related subunits, Arp2 and Arp3, arrange into a filament-like conformation that templates a new actin branch. While some progress has been made, the individual roles of each of these factors on the activating conformational change is poorly understood. In addition, it is still unclear how Arp2/3 complex is held in its inactive state, which is vital for understanding how activation occurs. In this dissertation, we dissect key interfaces in Arp2/3 complex that are responsible for holding it in an inactive state, and specifically evaluate the roles of ATP and WASP, the canonical NPF, in the activating conformational change of Arp2/3 complex. In chapter II, we investigated the contacts made between the Arp2 and Arp3 subunits in their inactive state, and the role of ATP in stimulating the active conformation. We found that two key interfaces, the αE/αF loop in Arp2 and the C-terminus of Arp3, a conserved extension not present in actin, are vital for holding Arp2/3 complex in its autoinhibited state. Evaluation of the role of ATP demonstrated that binding of ATP is required for the activating conformational change and displaces the Arp3 C-terminus, an important step in destabilization of the inactive state. In chapter III, we investigated the mechanism of WASP-induced conformational changes using an engineered crosslinking assay that only forms crosslinks when Arp2/3 is in its active conformation. We discovered that many WASP-related proteins are capable of stimulating this conformational change through a mechanism that involves displacement of the Arp3 C-terminus. Interestingly, purified Arp2/3 complex crosslinked in the active conformation was hyperactive compared to WASP-mediated activation, demonstrating that WASP activation limits nucleation and that actin monomer delivery is not required for nucleation. This dissertation contains unpublished co-authored material.

Procedures for the isolation and characterization of Ascaris glyceraldehyde-3-phosphate dehydrogenase and actin are described. The properties of these proteins, including molecular weights, isoelectric points, kinetics, peptide maps, and amino acid compositions, are strikingly similar to the respective proteins from rabbit muscle.

The actin cortex is an active biopolymer network that lines the inner side of the plasma membrane in most animal cells. It can both resist detrimental cell deformations and drive necessary cell shape changes. It is a highly dynamic system where actin filaments constantly polymerize and depolymerize and specific proteins form transient cross-links with lifetimes of a few tens of seconds at most. In addition, a particular type of cross-linkers, myosins, can also generate active stresses in the cortex. This dynamic nature allows for variations in the rheological properties of the cortex, which are very sensitive to its organization and in particular to the density and lifetime of cross-links. In vivo, the cortex is subject to variable levels of passive and active contractile stresses. At the molecular scale, these stresses result in tensile forces that can likely be sensed by the bonds between actin filaments and cross-linkers, and potentially other actin-bound cellular components. In this work, we investigated the mechanosensitive regulation of the organization of the cortex, focusing on the dynamics of the binding of cross-linkers to actin and on the variations in the amount of cortical actin filaments. To this aim, we carried out experiments on single HeLa cells, where we simultaneously measured their cortical tension using an atomic force microscope confinement method and assessed the spatial distribution of cross-linkers and actin filaments by confocal microscopy. On the one hand, we assessed the dependence of the actin-binding dynamics of filamins A and B, alpha-actinin-1 and nonmuscle myosin IIA to cortical tension. We measured how their residence time at the cortex and their contribution to the cortical connectivity depend on tension at steady state. Moreover, we also measured their response to peaks in passive cortical tension. The results of these experiments show that the two filamins and alpha-actinin-1 are catch-binding cross-linkers: the lifetime of their bonds to actin filaments increases under increasing tensile load. This leads to an increase in the connectivity of the cortex when the cortical tension is raised. On the other hand, we also probed the cortical response to peaks in active tension. We found that such tension peaks trigger a permanent increase in the amount of actin filaments at the cortex. In turn, this increase in cortical actin leads to a permanent increase in the cortical recruitment of all cross-linkers, in addition to the expected transient increase in the cortical recruitment of the catch-binding ones. We showed that this mechanosensitive process requires signaling by the cytosolic phospholipase A2 and the activity of the actin nucleator Arp2/3 complex. Together, our results show that two types of mechanosensitivity enable the reinforcement of the cortex in response to high mechanical stresses: a direct mechanosensitivity that relies on the catch-binding of cross-linkers, and an indirect mechanosensitivity mediated by biochemical signaling. / Der Aktinkortex ist ein aktives Biopolymer-Netzwerk, das in den meisten tierischen Zellen die Innenseite der Plasmamembran auskleidet. Er kann sowohl möglicherweise schädliche Zellverformungen abpuffern als auch notwendige Änderungen der Zellform vorantreiben. Es handelt sich um ein hochdynamisches System, in dem Aktinfilamente ständig polymerisieren und depolymerisieren und spezifische Proteine vorübergehende Quervernetzungen mit einer Lebensdauer von höchstens ein paar zehn Sekunden bilden. Darüber hinaus kann eine bestimmte Art von Quervernetzern, die Myosine, auch aktive Spannungen im Kortex erzeugen. Diese dynamische Natur ermöglicht Veränderungen der rheologischen Eigenschaften des Kortex, die sehr empfindlich auf seine Organisation und insbesondere auf die Dichte und Lebensdauer der Vernetzungen reagieren. In vivo ist der Kortex unterschiedlich starken passiven und aktiven kontraktilen mechanischen Spannungen ausgesetzt. Auf molekularer Ebene führen diese Spannungen zu Zugkräften, die wahrscheinlich von den Bindungen zwischen Aktinfilamenten und Quervernetzern und möglicherweise anderen aktingebundenen zellulären Komponenten wahrgenommen werden können. In dieser Arbeit haben wir die mechanosensitive Regulierung der Organisation des Kortex untersucht, wobei wir uns auf die Dynamik der Bindung von Quervernetzern an Aktin und die Schwankungen in der Menge der kortikalen Aktinfilamente konzentriert haben. Zu diesem Zweck haben wir Experimente an einzelnen HeLa-Zellen durchgeführt, bei denen wir gleichzeitig die kortikale Oberflächenspannung mit Hilfe einer rasterkraftmikroskopischen Confinement-Methode gemessen und die räumliche Verteilung von Quervernetzern und Aktinfilamenten mittels konfokaler Mikroskopie untersucht haben. Einerseits haben wir die Abhängigkeit der Aktinbindungsdynamik der Filamine A und B, des alpha-Actinin-1 und des nicht-muskulären Myosin IIA von der kortikale Spannung untersucht. Wir haben gemessen, wie ihre Verweildauer im Kortex und ihr Beitrag zur kortikalen Konnektivität von der Spannung im Gleichgewichtszustand abhängen. Wir haben auch ihre Reaktion auf Spitzen in der passiven kortikalen Spannung gemessen. Unsere Ergebnisse zeigen, dass die beiden Filamine und alpha-Actinin-1 'catch'-bindende Vernetzer sind: Die Lebensdauer ihrer Bindungen an Aktinfilamente nimmt mit zunehmender Zugbelastung zu. Dies führt zu einer Zunahme der Konnektivität des Kortex, wenn die kortikale Spannung erhöht wird. Andererseits haben wir auch die kortikale Reaktion auf aktive Spannungsspitzen untersucht. Wir fanden heraus, dass solche Spannungsspitzen einen permanenten Anstieg der Menge an Aktinfilamenten am Kortex auslösen. Dieser Anstieg des kortikalen Aktins wiederum führt zu einem permanenten Anstieg der kortikalen Rekrutierung aller Quervernetzer, zusätzlich zu dem erwarteten vorübergehenden Anstieg der kortikalen Rekrutierung der catch-bindenden Quervernetzer. Wir konnten zeigen, dass dieser mechanosensitive Prozess eine Signalisierung durch die zytosolische Phospholipase A2 und die Aktivität des Aktinnukleators Arp2/3-Komplexes erfordert. Zusammengenommen zeigen unsere Ergebnisse, dass zwei Arten von Mechanosensitivität die Verstärkung des Kortex als Reaktion auf hohe mechanische Belastungen ermöglichen: eine direkte Mechanosensitivität, die auf der Catch-Bindung von Quervernetzern beruht, und eine indirekte Mechanosensitivität, die durch biochemische Signalübertragung vermittelt wird.

Stomata are specialized plant structures required for gaseous exchange with the outer environment. During stomata formation, the cytoskeleton plays an important role in controlling the division of the individual cells leading to the generation of the stomata complex. Two mutants that affect microfilament and microtubule organization in subsidiary mother cells include brk1 and dcd1. While only 20% of the subsidiary cells in the brk1 and dcd1 single mutants are abnormally shaped, it was reported that there is a synergistic effect between the brk1 and dcd1 mutations in the brk1; dcd1 double mutant since 100% of the subsidiary cells are abnormal. The focus of this research is to try to understand this synergistic effect by investigating the actin cytoskeleton and nuclear position in the single and double mutants. The reported results include the observation that the size of actin patch was largest in the wild-type subsidiary mother cells (SMCs) and smallest in dcd1 and brk1; dcd1 SMCs and that brk1 and brk1; dcd1 double mutants had fewer actin patches than wild-type and dcd1 SMCs. Additionally, we observed that some SMCs that did not have actin patches still underwent nuclear migration suggesting that nuclear migration may not be solely dependent on actin patch formation. Finally, during SMC cytokinesis, a large percentage of double mutant (brk1; dcd1) cells showed an off-track development of the phragmoplast as compared to the single mutants and the wild-type plant explaining the large number of abnormally shaped subsidiary cells in the double mutants.

Actin's ability to engage in a wide range of physiological functions requires that it be subject to complex spatial and temporal regulation. This regulation is achieved internally through monomer-monomer contacts and externally through interactions with actin binding proteins. The first part of my thesis focused on better understanding the role of inter-monomeric ionic interactions proposed between subdomains 2 and 3 of opposing monomers in F-actin stabilization. I studied several yeast actin mutants: A167R to disrupt a proposed ionic attraction with R39, A167E to mimic a proposed ionic attraction in muscle actin, and D275R to disrupt a proposed ionic attraction with R39. I investigated the effects of mutations in vivo, effects on filament polymerization characteristics and appearance in vitro, as well as interaction of the mutants with the filament severing protein cofilin. While both in vivo and in vitro data demonstrated the importance of the R39-D275 interaction for yeast actin and the interaction of the filament with cofilin, disruption of this interaction alone did not cause filament fragmentation. Conversely, results with A167 do demonstrate the in vivo and in vitro importance of another potential R39 ionic interaction for filament stabilization. In the second part of my work I used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry as a tool to gain structural insight into yeast and muscle actin and profilin isoform differences and the actin-profilin interaction. The yeast and muscle actin HD analysis showed greater exchange for yeast G-actin compared to muscle actin in the barbed end pivot region and areas in subdomains 1 and 2, and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared to muscle actin. For yeast-muscle hybrid G-actins, the muscle-like and yeast-like parts of the molecule generally showed exchange characteristics resembling their parent actins. There were a few exceptions to this rule, however: a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3. These exhibited muscle actin-like exchange characteristics even though the areas were yeast-like, suggesting that there is crosstalk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared to both yeast and muscle actins are consistent with mismatched yeast-muscle actin interfaces resulting in decreased stability of the hybrid filament contacts. Actin-profilin HD exchange results demonstrated a possible differential interaction of specific profilin isoforms with specific actin isoforms. While profilin binding mostly caused a decreased exchange for yeast actin peptides, it caused an increase in exchange for muscle actin peptides. Many of the changes observed were in peptides that line or contact the nucleotide cleft, consistent with profilin's ability to alter the kinetics of nucleotide exchange.