München, Techn. Univ., Diss., 2001. / Computerdatei im Fernzugriff.

München, Techn. Univ., Diss., 2001. / Computerdatei im Fernzugriff.

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.

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.

The Actin Related Protein (ARP) 2/3 complex is a major regulator of the actin cytoskeleton that is implicated in cell morphogenesis in plants. However, a similar role is attributed to the endoplasmic reticulum (ER). My research explored the relationship between the two systems by using transgenic plants simultaneously expressing fluorescent proteins highlighting F-actin and ER organization in living cells. A comparison of F-actin organization in cells of wild type Arabidopsis thaliana and mutants with aberrant actin cytoskeleton suggests bundling in the distorted2 mutant but a relatively fine F-actin arrangement in klunker. These differences correlate with ER organization into cisternae, fenestrated sheets and tubules. A model relating ER-organization to the degree of actin bundling in a cell emerges and is supported by drug-induced interference in actin polymerization, altered ionic conditions and temperature. The study adds to the mechanistic understanding of cell morphogenesis in plants.

Tip growth is the primary form of growth in hyphal organisms and some plant cells. Tip growth in hyphae is highly dependent on F-actin, which acts to regulate and support growth. One of the models suggested for tip growth, the amebae model of tip growth, suggests that F-actin may also be the primary source of protrusive force for tip growth in some conditions, and that proteins with a similar function to animal integrins would be present an involved in tip growth (Heath and Steinberg 1999). In this thesis we examine the role of F-actin in the growth of the oomycete Phytophthora cinnamomi and the effects on growth of the F-actin disrupting compound Latrunculin B. We demonstrate that F-actin plays a critical role in the tip growth of Phytophthora cinnamomi with it's disruption causing rapid cessation in directional growth, followed by significant subapical swelling. Further more we examine Phytophthora cinnamomi for the presence of an B4 integrin like protein that has been previously reported in the oomycete Achlya bisexualis (Chitcholtan & Garrill 2005) and show that the B4 integrin like protein is not present in Phytophthora cinnamomi. These experiments help further our understanding of tip growth in Phytophthora cinnamomi an economically important plant pathogen.

<p>Lipopolysaccharide (LPS) is a structure of the gram-negative bacteria that protect from chemicals and works as a stabilization component for the membrane. Studies show that LPS also may have a function to avoid immune defense. In this project we investigate two <em>Salmonella enterica</em> variants with different LPS conformation. The wild-type Salmonella got an originally LPS structure and the mutant form had a defect one. The bacteria were transfected with a green fluorescent protein (GFP) to allow measuring of phagocytosis. Monocytes were isolated from human blood and were incubated for several days with cytokines to give dendritic cells. The cells were exposed to each type of <em>Salmonella</em> and incubated for different times. After labeling with phalloidin and studies with fluorescent microscopy, phagocytosis and F-actin were measured. The results show that it is a difference in phagocytosis and F-actin depending on LPS conformation. That means that LPS may have a decisive role for the pathogenicity of <em>Salmonella</em>.</p>

Lipopolysaccharide (LPS) is a structure of the gram-negative bacteria that protect from chemicals and works as a stabilization component for the membrane. Studies show that LPS also may have a function to avoid immune defense. In this project we investigate two Salmonella enterica variants with different LPS conformation. The wild-type Salmonella got an originally LPS structure and the mutant form had a defect one. The bacteria were transfected with a green fluorescent protein (GFP) to allow measuring of phagocytosis. Monocytes were isolated from human blood and were incubated for several days with cytokines to give dendritic cells. The cells were exposed to each type of Salmonella and incubated for different times. After labeling with phalloidin and studies with fluorescent microscopy, phagocytosis and F-actin were measured. The results show that it is a difference in phagocytosis and F-actin depending on LPS conformation. That means that LPS may have a decisive role for the pathogenicity of Salmonella.

Tip growth is the primary form of growth in hyphal organisms and some plant cells. Tip growth in hyphae is highly dependent on F-actin, which acts to regulate and support growth. One of the models suggested for tip growth, the amebae model of tip growth, suggests that F-actin may also be the primary source of protrusive force for tip growth in some conditions, and that proteins with a similar function to animal integrins would be present an involved in tip growth (Heath and Steinberg 1999). In this thesis we examine the role of F-actin in the growth of the oomycete Phytophthora cinnamomi and the effects on growth of the F-actin disrupting compound Latrunculin B. We demonstrate that F-actin plays a critical role in the tip growth of Phytophthora cinnamomi with it's disruption causing rapid cessation in directional growth, followed by significant subapical swelling. Further more we examine Phytophthora cinnamomi for the presence of an B4 integrin like protein that has been previously reported in the oomycete Achlya bisexualis (Chitcholtan & Garrill 2005) and show that the B4 integrin like protein is not present in Phytophthora cinnamomi. These experiments help further our understanding of tip growth in Phytophthora cinnamomi an economically important plant pathogen.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23110号 / 医科博第121号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 濵﨑 洋子, 教授 竹内 理, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM