Cyclase-associated protein (CAP)2 is an evolutionarily highly conserved actin-binding protein implicated in striated muscle development, carcinogenesis, and wound healing in mammals. To date, the presence as well as the putative role(s) of CAP2 in platelets, however, remain unknown. Therefore, mice constitutively lacking CAP2 (Cap2gt/gt mice) were examined for platelet function. These studies confirmed the presence of both mammalian CAP isoforms, CAP1 and CAP2, in platelets. CAP2-deficient platelets were slightly larger than WT controls and displayed increased GPIIbIIIa activation and P-selectin recruitment in response to the (hem)ITAM-specific agonists collagen-related peptide and rhodocytin. However, spreading of CAP2-deficient platelets on a fibrinogen matrix was unaltered. In conclusion, the functionally redundant CAP1 isoform may compensate for the lack of CAP2 in murine platelets. Moreover, the studies presented in this thesis unveiled a severe macrothrombocytopenia that occurred independently of the targeted Cap2 allele and which was preliminarily termed orphan (orph). Crossing of the respective mice to C57BL/6J wild-type animals revealed an autosomal recessive inheritance. Orph mice were anemic and developed splenomegaly as well as BM fibrosis, suggesting a general hematopoietic defect. Strikingly, BM MKs of orph mice demonstrated an aberrant morphology and appeared to release platelets ectopically into the BM cavity, thus pointing to defective thrombopoiesis as cause for the low platelet counts. Orph platelets exhibited marked activation defects and spread poorly on fibrinogen. The unaltered protein content strongly suggested a defective alpha-granule release to account for the observed hyporesponsiveness. In addition, the cytoskeleton of orph platelets was characterized by disorganized microtubules and accumulations of filamentous actin. However, further experiments are required to elucidate the activation defects and cytoskeletal abnormalities in orph platelets. Above all, the gene mutation responsible for the phenotype of orph mice needs to be determined by next-generation sequencing in order to shed light on the underlying genetic and mechanistic cause. / Cyclase-associated protein 2 (CAP)2 ist ein evolutionär hoch konserviertes Aktin-bindendes Protein, welches in der Entwicklung der quergestreiften Muskulatur, der Krebsentstehung und der Wundheilung von Säugetieren eine Rolle spielt. Bis heute sind jedoch das Vorhandensein sowie die mutmaßliche(n) Funktion(en) von CAP2 in Thrombozyten unbekannt. Aus diesem Grund wurden Mäuse, denen konstitutiv CAP2 fehlt (Cap2gt/gt-Mäuse), im Hinblick auf ihre Thrombozytenfunktion untersucht. Diese Untersuchungen bestätigten die Anwesenheit beider Säugetierisoforme von CAP, CAP1 und CAP2, in Thrombozyten. CAP2-defiziente Thrombozyten waren geringfügig größer als WT-Kontrollen und zeigten eine erhöhte GPIIbIIIa-Aktivierung und P-Selektin-Rekrutierung nach Stimulation durch die (hem)ITAM-spezifischen Agonisten collagen-related peptide und Rhodozytin. Demgegenüber verlief die Adhäsion (sog. spreading) CAP2-defizienter Thrombozyten auf einer Fibrinogen-Matrix unverändert. Dies legt den Schluss nahe, dass die funktionell redundante CAP1-Isoform in der Lage ist, den Mangel an CAP2 in Mäusethrombozyten zu kompensieren. Darüber hinaus offenbarten die in dieser Dissertation präsentierten Untersuchungen eine schwere Makrothrombozytopenie, welche unabhängig von dem veränderten Cap2-Allel auftrat und welche vorläufig als orphan (orph) bezeichnet wurde. Das Kreuzen der entsprechenden Mäuse mit C57BL/6J-Wildtyp-Tieren enthüllte einen autosomal rezessiven Erbgang. Orph-Mäuse waren anämisch und entwickelten eine Milzvergrößerung sowie eine Knochenmarkfibrose, was einen generellen hämatopoetischen Defekt nahelegte. Bemerkenswerterweise waren Knochenmarksmegakaryozten von orph-Mäusen morphologisch auffällig und gaben allem Anschein nach Thrombozyten ektop in das Knochenmarkstroma ab, was auf eine defekte Thrombopoese als Ursache für die niedrigen Thrombozytenzahlen hindeutet. Orph-Thrombozyten zeigten ausgesprochene Aktivierungsdefekte und adhärierten kaum auf Fibrinogen. Der unveränderte Gehalt an Proteinen lenkte den Verdacht auf eine defekte Exozytose von Alpha-Granula als Ursache der Mindererregbarkeit. Des Weiteren war das Zytoskelett von orph-Thrombozyten durch unorganisierte Mikrotubuli und Akkumulationen von filamentösem Aktin charakterisiert. Weitere Experimente sind jedoch notwendig, um die Aktivierungsdefekte und die Zytoskelettveränderungen aufzuklären. Vor allem aber muss die Genmutation, welche für den Phänotyp der orph-Mäuse verantwortlich ist, mittels Sequenziermethoden der nächsten Generation (next-generation sequencing) aufgeklärt werden um Aufschluss über die zugrunde liegende genetische und mechanistische Ursache zu geben.

The cytoskeleton is a remarkable system of filaments that helps in the organisation and functioning of living cells. In plant cells, this cytoskeleton comprises actin microfilaments and microtubules that polymerise from actin and tubulin respectively. While these proteins are highly conserved in eukaryotes, the plant cytoskeleton performs many plant-specific functions. The organisation and functions of the cytoskeleton are determined by a plethora of accessory proteins (actin-binding proteins, microtubule-associated proteins) that link the cytoskeletal filaments to other cell components and to each other. While there is extensive data for the subcellular localisation of actin-binding proteins with actin microfilaments in animal cells, surprisingly few experiments of this type have previously been tried in plants, and the subcellular localisation of most plant actin-binding proteins remains unknown. Such information is important in assessing functions of these proteins to give a better understanding of the actin cytoskeleton. In this study, an attempt was made to visualise the association of actin microfilaments and actin-binding proteins. A range of antibodies raised against various plant and animal actin-binding proteins were screened in two model systems for plant cytoskeleton research, the root of Arabidopsis thaliana and in whole cells of the tobacco BY2 liquid cell culture. Further, because previous data in the localisation of the actin-binding protein tropomyosin have suggested that the localisation of this actin-biding protein with the finer cortical actin microfilaments in Arabidopsis roots might not be discerned due to high cytoplasmic background, immunolabelling experiments were also conducted on plasma membrane ghosts generated from tobacco BY2 from which any non-specific cytoplasmic labelling could be washed away. There experiments gave some preliminary suggestions for the association of the actin-binding proteins to the actin cytoskeleton in plant cells. The most intriguing observations were obtained with antibodies against the β-subunit of capping protein which colocalised with larger microfilament bundles in tobacco BY2 cells. No colocalisation was observed on membrane ghosts on which these bundles are not well retained. However, the previous experiments in which there were suggestions of tropomyosin-related proteins associated with fine cortical microfilaments in Arabidopsis could not be replicated. As no cytoskeletal localisation was observed in either Arabidopsis or tobacco with antibodies raised against known actin-bundling proteins from Arabidopsis such as villin and fimbrin, it is speculated that the labelling protocols, currently optimised for visualising the actin cytoskeleton, might not to be modified to allow visualisation of actin-binding proteins in plant cells.

Konstanz, Univ., Diss., 2006.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23551号 / 生博第462号 / 新制||生||62(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 中野 雄司, 教授 見学 美根子, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Actin cytoskeleton reorganization plays essential roles in many cellular processes such as cell structure maintenance, cell motility, and force generation. Cytoskeletal drugs are small molecules that act on cytoskeletal components by either stabilizing or destabilizing them. Swinholide A is an actin-binding drug derived from the marine sponge. Swinholide A binds actin dimers as well as severs filaments. The main objective of this project is to determine how Swinholide A modulates actin filament assembly dynamics in the presence of macromolecular crowding. We utilize total internal reflection fluorescence (TIRF) microscopy imaging to directly visualize Swinholide A-mediated actin filament disassembly and severing. Filament disassembly and severing are evaluated by calculating actin filament lengths and length distribution controlled by Swinholide A. This study helps us better understand the fundamental mechanism by which Swinholide A affects actin assembly and disassembly dynamics. Further studies will allow for investigating new methods of treatment for a range of different diseases that have pathogenetically high levels of filamentous actin, such as cystic fibrosis, as well as a drug to combat the explosive expansion of cancers.

No description available.

Hyphal organisms are a commonly used model system for studies of polarised growth. While growing hyphal tips offer a good example of polarised growth, little detail of the process of polarisation can be determined from them. Hyphal branching offers a good example of the development of polarity, however to date it has been largely impractical to study hyphal branching, due to the irregular timing and location along the hypha of natural branch formation. Chemical induction of branches circumnavigates this problem, using a localised concentration of nutrients adjacent to the growing hypha to stimulate controlled branching. Using previous studies of hyphal branching combined with the current understanding of hyphal tip growth, a model of the branching process was established (Jackson et al. 2001). Reception of a branching cue leads to the formation of a radial F-actin array at the new branch site. This, by means of either delivery of cell wall softening enzymes or direct mechanical pressure, leads in turn to the emergence of a visible bump in the hyphal wall. This bump enlarges and then progresses into the branch proper. The bump stage of the branching process is perhaps the least understood, with existing studies giving detail of pre- and post-bump events. The research described in this thesis suggests that bump emergence is a two stage process; an early bump stage, where localised cell wall softening leads to turgor pressure in the cell pushing out the bump, and a late bump, where F-actin is arranged into the developing branch. The addition of an F-actin inhibitor to the induction solution confirmed that the early bump stage is relatively independent of the F-actin cytoskeleton, however this experiment was unable to test F-actin's role in full branch development.

Tip growth is a form of cellular expansion characteristic of fungal hyphae and some types of plant cells. Currently there is no unified model that satisfactorily describes this in hyphal species. Traditionally turgor has been considered an essential driving force behind cell expansion. In recent years this hypothesis has been challenged by evidence that in some species tip growth can occur despite the absence of measurable hydrostatic pressure. There are currently two contentious theories of hyphal extension. These are the turgor-driven model and the amoeboid-movement theory. Though the essential mechanism underlying cell growth differs between these theories, the actin cytoskeleton is considered important in both. It has been suggested that both the turgor-driven and amoeboid-like modes of growth could occur depending on the whether the hyphae are growing invasively or non-invasively respectively (Money, 1990). It has also been proposed that both modes may occur within the same mycelium (Garrill, 2000). Two distinct patterns of actin have been identified in the hyphal tips of oomycetes. This has lead to the hypothesis that two different mechanisms of apical extension may be employed by some hyphal organisms. During the course of this thesis, actin deplete zones have been observed in a significantly higher number of invasive compared to non-invasive hyphae of the oomycete Achlya bisexualis. Furthermore the difference between burst pressures was found to be lower in invasive hyphae compared to non-invasive hyphae suggestive of a weaker cell wall. A lack of significant difference in turgor pressures between the invasive and non-invasive hyphae of this organism suggests that the deplete zone and weaker wall plays a functional role in enabling hyphae to penetrate substrate. Fractal analysis of mycelial colonies shows that the variation in agar concentration and therefore substrate solidity has a significant effect on mycelial morphology. This is most likely due to an effect at the cellular level. The results of the experiments carried out during the course of this thesis provide the basis for future work towards elucidating the mechanisms of hyphal extension.

Actin is one of the 2 main proteins in muscle, and is also involved in many non-muscle motile processes. When actin is extracted from non-muscle cells, it is often found as a 1:1 complex with profilin which reduces its tendency to polymerise. The control of this interaction is important and necessary for the production of some labile, filamentous actin containing structures, and for some non-muscle motile events. The profilin:actin complex was originally crystallised as an unknown inhibitor of DNase I and, although actin alone will form a tight 1:1 complex with Dnase I, no physiological explanation has been found for these interactions. The profilin:actin crystals crystallise in the orthorhombic space group P2<SUB>1</SUB>2<SUB>1</SUB>2<SUB>1</SUB>, with cell dimensions of a = 3.85 nm, b = 7.23 nm and c &61 18.74 nm, and have one molecule of each protein per asymmetric unit. The crystals diffract to high resolution, but are susceptible to large changes in the cell dimensions; in particular the c-axis will shrink to as little as 16.5 nm. Interaction of heavy metal salts with the crystals is one of the factors which produce large changes in the cell dimensions, so much work had been done to stabilise the crystals to make isomorphous heavy atom derivatives. At the start of this project, 3 fairly isomorphous heavy atom derivatives had already been discovered and data to high resolution collected on 2 of them. However, the data at high resolution contained large errors due to absorption and the derivatives were not totally isomorphous. Thus I set out to discover some new heavy atom derivatives and also to try to improve the use of the existing data so that the structure of the protein complex could be determined at high resolution. Two new derivatives were discovered, one utilising a modified ATP molecule to introduce a reactive group into the complex and both using the mercury salt p-hydroxy mercuribenzoate. Precession film, diffractometer and oscillation film data were collected on these two derivatives as well as recollecting poor data and missing data for the other derivatives. Some of the previously collected data were reprocessed and all the derivatives, at all resolution limits, from all sources were reexamined to find the best possible set of heavy atom solutions and thus produce the best set of phases and the most reliable electron density map. The result of this work was an electron density map which was much more interpretable in terms of protein structure. Previously, very little regular secondary structure could be seen but in the new map α-helices, β-sheets and the ATP molecule could all be found. Once regular secondary structure can be found, modifications can be made to the phases and the results can be observed to improve, or otherwise, the map. The structure of the profilin:actin complex is now being built and even if the complete atomic coordinates can not be found, there exists a rigorous framework on which to base further work.

Proper contraction of striated muscle requires the assembly of actin filaments with precise spacing, polarity and lengths, however the mechanisms by which the cell accomplishes this remain unclear. In one model, the giant protein nebulin is proposed to function as a "molecular ruler" specifying the final lengths of the actin filaments. This dissertation focuses on determining the mechanisms by which nebulin regulates thin filament assembly. We found that nebulin physically interacts with CapZ, a protein that caps the barbed end of the actin filament within the Z-disc. Reduction of nebulin levels in chick skeletal myocytes via siRNA results in a reduction of assembled CapZ, and a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin-filament barbed ends to the Z-disc via a direct interaction with CapZ. Unexpectedly, the CapZ binding site was mapped to a site on nebulin that was previously predicted to localize outside of the Z-disc. Thus, we also propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres. To determine the mechanism by which nebulin regulates thin filament length and directly test the molecular ruler hypothesis, a unique small nebulin molecule ("mini-nebulin") was constructed. The introduction of mini-nebulin into chick skeletal myocytes, with endogenous nebulin knocked down, does not result in corresponding shorter actin filaments; an observation that is inconsistent with a strict ruler function. Treatment of these cells, however, with the actin depolymerizing agent Latrunculin A produces filaments that match the length of the mini-nebulin molecule, indicating mini-nebulin stabilizes the actin filaments. Furthermore, knockdown of nebulin results in more dynamic populations of the thin filament components actin, tropomyosin and tropomodulin. Strikingly, introduction of mini-nebulin is able to restore the normal stability of the actin filaments. Taken together, these data indicate that nebulin is responsible for proper actin organization within the Z-disc and contributes to actin filament length regulation by stabilizing the filament, preventing actin depolymerization.