The effect of mechanical stimulation in plants has been studied in depth for more than a century. This type of stress has been shown to trigger alterations in development such as stunting, thickened stems and differential cell wall deposition. These responses are very likely to be initiated at a subcellular level, but the molecular mechanisms transducing mechanical signals into intracellular responses still remain unknown in plants. In this thesis I test the hypothesis that the membrane anchored protein Defective Kernel 1 (DEK1) could act as a plant-specific mechanosensor in plants. Constitutive overexpression of the cytoplasmic CALPAIN domain DEK1 causes a phenotype in Arabidopsis, that that resembles that of mechanically stressed plants. The CALPAIN domain of DEK1 shows a very high homology with animal calpains; a class of calcium-dependent Cysteine proteases which undergo a calciumstimulated CALPAIN domain-releasing autolytic cleavage event during activation. A similar autolytic cleavage event has been observed in DEK1 which, together with the fact that the CALPAIN domain alone can rescue the embryo-lethality associated with loss of DEK1 function, has led to the suggestion that this domain represents an activated form of the protein. I show that like mechanically stressed plants, CALPAIN overexpressing plants show a modified call wall composition. Consistent with this, transcriptional analysis of these plants shows a deregulation of genes encoding cell wall modifying enzymes, amongst others. Other characteristics of mechanically stimulated plants which I have characterized in CALPAIN overexpressing lines include late flowering and thickened stems. Therefore, I proposed a model in which the CALPAIN domain of DEK1 acts as an effector which is normally activated by mechanical stimulation. In this model, the transmembrane domains of DEK1 would regulate activation (cleavage) of the CALPAIN domain, potentially in response to mechanical stress. In order to test this model further, CALPAIN overexpressing lines were generated in a dek1 mutant background. If the model is correct, these plants should not only behave as if responding constitutively to mechanical stimulation, but should also lack appropriate responses to applied mechanical stimuli due to lack of the mechanosensory integral membrane domain of DEK1. My results confirm that the absence of the transmembrane domains of DEK1 is indeed translated into a lack of some, but not all responses to mechanical stimulation compared to wild-type plants. Furthermore, the lack of the transmembrane domains of DEK1 correlates with the absence of a mechanically-triggered calcium flux in the plant. Thus my work suggests that the transmembrane domains of DEK1 are involved in sensing mechanical stimulation, via the regulation activity of a mechano-sensitive calcium flux at the plasma membrane. In summary, my proposal is that Defective Kernel 1 (DEK1) acts both as a key mechanosensory cellular component, and as the first effector of the signalling cascade in response to mechanical stimulation, via an autolytic activation in response to mechanical stress.

Low Intensity Pulsed Ultrasound (LIPUS) is used clinically to promote wound healing. In vivo studies show that LIPUS is effective in a wide range of tissue types, and in vitro experiments show that multiple cell types respond to LIPUS stimulation. Despite this, there is no unifying mechanism of how LIPUS stimulation is sensed by cells, and it is unknown what the early signalling events are. The LIPUS signal is a mechanical one; therefore I hypothesised that mechanosensitive organelles, called focal adhesions, would be essential for the induction of cellular signalling events in response to this type of stimulation. Proteins within these structures (such as vinculin and talin) link the actin cytoskeleton to the extracellular matrix via integrins, and are known to be sensitive to mechanical forces, capable of generating intracellular signalling events in response to mechanical stimulation. The purpose of this work was to identify the early signalling events occurring within minutes of LIPUS stimulation; determine the molecular mechanisms behind such events; and to investigate whether such events require integrin-mediated adhesions. In the first part of the work, I established the use of live-cell imaging together with LIPUS stimulation to directly observe the cellular response. I determined rapid reorganizations of the actin cytoskeleton, which led to increased cell velocity. These effects were found to be Rac dependent, and, using FRET-based probes, I measured rapid increases in Rac activity occurring within minutes of LIPUS stimulation. The second part of this work identified an increase in the number of early endosomes in cells stimulated with LIPUS. This phenotype was also Rac dependent, as well as requiring the early endosomal regulator protein Rab5. In this chapter, I observed an increase in the association between Rac and Rab5 in response to LIPUS stimulation, and this contributes to Rac activation. Using substrates to block integrin-mediated adhesion, I determined that cell-matrix adhesions are required for the effects of LIPUS stimulation. Using vinculin-deficient cells, I determined that this mechanosensitive protein is vital for co-ordinating Rac activation in response to LIPUS. In particular, the actin binding tail is needed for mechanosensing of this LIPUS signal. In the final chapter I established the use of photoactivatable (PA) GFP to assess adhesion protein turnover. This technique was used to show that LIPUS stimulation directly affects the turnover of vinculin. Overall, this work shows that the mechanosensitive protein vinculin is crucial for sensing the mechanical stimulation provided by LIPUS, orchestrating downstream Rab5-mediated Rac activation to enhance cell motility.

The protein p130Cas (human ortholog BCAR1) is a major substrate for phosphorylation by the Src family kinase and plays a central role in oncogenic transformation. Increased level of BCAR1 correlates with primary tumour growth and cancer progression. Localized to focal adhesion, p130Cas serves as a mechanosensor and mediates key interactions with the extracellular environment. The structure of p130Cas is crucial for its function, mainly the anchoring domains SH3 and CCH, together with the substrate domain which is extended when under tension. This Master's thesis presents a newly developer FRET mechanosensor based on the structure of p130Cas. The sensor utilizes the anchoring domains of p130Cas for proper localization to focal adhesions, where it can detect tension in living cells. Key words: p130CAS, FRET, focal adhesions, mechanosensing

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20587号 / 農博第2239号 / 新制||農||1052(附属図書館) / 学位論文||H29||N5076(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 矢﨑 一史, 教授 宮川 恒 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The protein p130Cas (human ortholog BCAR1) is a major substrate for phosphorylation by the Src family kinase and plays a central role in oncogenic transformation. Increased level of BCAR1 correlates with primary tumour growth and cancer progression. Localized to focal adhesion, p130Cas serves as a mechanosensor and mediates key interactions with the extracellular environment. The structure of p130Cas is crucial for its function, mainly the anchoring domains SH3 and CCH, together with the substrate domain which is extended when under tension. This Master's thesis presents a newly developer FRET mechanosensor based on the structure of p130Cas. The sensor utilizes the anchoring domains of p130Cas for proper localization to focal adhesions, where it can detect tension in living cells. Key words: p130CAS, FRET, focal adhesions, mechanosensing

The mechanical properties of the tissue vary widely around the body, from the soft brain to the rigid bone. Tissue cells are able to sense mechanical signals from their environment, which influence many aspects of cell behaviour such as migration, proliferation and differentiation. Focal adhesions (FAs) are large protein complexes that form the bridge between the extracellular matrix (ECM)-binding integrins and the contractile actin cytoskeleton. Here, they sense the rigidity of the local environment and translate this information into a cellular response, a process known as mechanotransduction. However, the FA proteins required for mechanotransduction, and the molecular mechanisms involved in this fundamental process, remain to be elucidated. Talin, vinculin, FAK and paxillin are four core FA-associated proteins that are thought to be involved in mechanotransduction. These proteins associate and dissociate from the complex in a constant state of flux. Using a live-cell imaging approach, I found that the rate of dynamic exchange of an FA protein correlates to its function. The FA appears to have a modular organisation; the slowest proteins have a structural role, such as talin and vinculin, responsible for directly linking integrin to actin and sensing the ECM stiffness. The signalling proteins are turned over more rapidly, including FAK and paxillin, and are responsible for directing the cellular response to force-generated signals from the ECM.The second results chapter focused on the force-dependent interactions between talin, vinculin and actin. The talin domains R2R3 were identified as the key mechanosensitive vinculin-binding sites, which are exposed upon the application of force across the talin rod. Vinculin binding to R2R3 led to actin associating with the central actin-binding site in the talin rod (ABS2), which is required for the transmission of actomyosin tension onto the underlying substrate as cellular traction force. Finally, the protein turnover data were incorporated into two mathematical models, describing talin and vinculin turnover, which were able to simulate the dynamic exchange of various talin and vinculin mutants in response to changing ECM stiffness. Using these models, the talin ABS2-actin and vinculin tail-actin interactions were found to be extremely important for sensing the stiffness of the ECM. These findings significantly increase our knowledge of the molecular mechanisms underpinning cellular mechanotransduction. Increased understanding of how mechanical signals are sensed and interpreted by the cell could lead to a number of novel therapies for a wide range of associated diseases, such as atherosclerosis, muscular dystrophy and cancer.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18657号 / 工博第3966号 / 新制||工||1610(附属図書館) / 31571 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 岩田 博夫, 教授 木村 俊作, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Myxococcus xanthus, referred to as a 'social bacterium', demonstrates unique behaviors such as coordinated motility, cooperative feeding, and multicellular structure formation. Its complex social behaviors and developmental processes make M. xanthus a model organism for studying bacterial social behaviors and their underlying mechanisms. Much of the social behavior of M. xanthus hinges on coordination of cell motility among bacteria in close proximity. M. xanthus moves on moist solid surfaces, using its Adventurous (A)-motility and Social (S)-motility systems. A striking feature of M. xanthus motility is the periodic reversal of its direction of movement. The reversal frequency is influenced by chemical and mechanical cues in the surrounding environment. The modulation of the reversal frequency upon physical contact between cells is believed to be a key factor in the bacterium's social behaviors, especially in the formation of complex patterns and structures within the cell population. Here I utilized mathematical modeling to study the motility regulation in M. xanthus, focusing on contact-dependent reversal control, mechanosensing response and impact of motility regulation in solitary (single-cell) predation. My goal is to provide experiment-guiding theories and hypotheses for M. xanthus motility regulation, which is essential to fully understand the social behaviors in this bacterium. In Chapter 2, I developed a single-cell model based on a hypothesis that the motility regulation in M. xanthus is mediated by the interplay between the cell polarity regulation pathway and the A-motility machinery. The aim of this model is to elucidate the cellular mechanism governing contact-dependent motility coordination among cells and to understand how contact-dependent responses at the single-cell level contribute to population-level patterns. This model suggests that the A-motility machinery of M. xanthus potentially serves as a 'mechanosensor' that transduces mechanical cues in the environment into a reversal modulation signal. Chapter 3 addresses a puzzling observation: cells with A-motility alone (A+S−) show a dependence of reversal frequency on substrate stiffness that is opposite to what is observed in wild-type cells that possess both motility systems. Specifically, A+S− cells reverse less frequently on harder substrates, whereas wild-type cells reverse more frequently. To elucidate this perplexing phenomenon, I refined the single-cell model developed in Chapter 2 to study the mechanosensing behaviors with or without S-motility. The base model was sufficient to explain the mechanosensing response in A+S− cells. I then proposed possible interactions between the A-motility and S-motility systems that could explain the contrasting responses to substrate stiffness when S-motility is present or absent. This provides a testable prediction for future experimental investigations. The model suggests that the A-motility system in M. xanthus functions as a central hub of mechanosensing-based reversal control, modulating cell reversal in response to environmental mechanical cues. In Chapter 4, I constructed an agent-based model to investigate the optimal motility strategies for nutrient consumption by M. xanthus during its solitary predation. For different nutrient source types and their uptake latencies, the model identifies 'explore', 'inch', and 'fast explore' as the three most effective motility strategies. Variability in velocity and cell reversal period changes the optimal strategies from 'explore' mode to 'revisit' mode and to 'speed-controlled explore' mode, respectively, for massive remains of prey nutrient sources with moderate uptake latency. The experimental observation that solitary M. xanthus cells combined the 'revisit' and 'inch' mode—as predicted by the model for nutrient acquisition respectively from prey remains and macromolecules—suggests that some of the dead preys may not release its cellular contents immediately and that release of molecular nutrients may require multiple digestion cycles. This model provides insights into the functional role of complex motility regulation in M. xanthus during solitary predation. / Doctor of Philosophy / A fundamental question in biology is how a cell responds to physical, chemical and biological stimuli. Such responses are usually mediated by complex coupling between multiple cellular processes. Bacterial motility and its regulation present many excellent examples of this kind. This dissertation focuses on Myxococcus xanthus, a model organism for bacterial social behavior due to the highly coordinated motility of cells in M. xanthus colonies and their functional cooperation. In this dissertation, I built theoretical models to study the motility regulation in M. xanthus, which is essential for understanding the social behaviors and survival in this bacterium. The specific focuses are to comprehend how environmental mechanical cues regulate M. xanthus's motility, and how the observed motility regulation in M. xanthus facilitates its predatory behavior at the single-cell level. The key aspect of this work is to construct a modeling framework to provide coherent explanations for the experimental observations. It is anticipated that the hypotheses generated through modeling will guide new experiments in the field of myxobacterial biology. The findings offer general insights into how bacterial cells sense, respond, and adapt to the chemical, physical, and biological cues.

Doctor of Philosophy / Department of Entomology / David C. Margolies and Yoonseong Park / Organisms are capable of sensing environmental conditions through diverse mechanisms. Transient receptor potential channels (TRPs) are a cation channel family that has been found to function in diverse sensing mechanisms. In this dissertation, I identified the function of several TRPs in thermosensing and mechanosensing in the red flour beetle, Tribolium castaneum. Candidate TRPs were chosen based on homology to TRPs found and studied in Drosophila melanogaster. To identify the function of candidate TRPs in T. castaneum, I suppressed the expression of target genes by RNA interference technique and investigated the phenotype of each treated beetle. Temperature is a major limiting environmental factor for organisms. I tested the function of candidate TRPs in thermotaxis (behavior) and thermal acclimation (physiology). Using bioinformatics approaches, I identified three candidate TRPs – painless, pyrexia, and trpA1 – involved in high temperature sensing. To test thermotactic behavior, I investigated beetle movement on a temperature arena with two separate temperature zones. Thermal acclimation was tested by pre-exposing beetles to either 42 °C for 10 min. When treated with double stranded RNA of TRPA1 (dstrpA1), the thermotactic response of beetles at 39 and 42 °C was reduced when compared to control groups. With pre-exposure at 42 °C, survivorship of dstrpA1-treated beetles significantly increased after one minute exposure at 52 °C compared to beetles that were not pre-exposed. With dspainless treatment, beetles showed lower response to thermal acclimation and lower long-term survivorship. Beetles treated with dspyrexia showed lower recovery after heat treatment without pre-exposure at 42 °C. To identify the function of candidate TRPs in mechanosensing, I evaluated dsRNA treated beetles for survival, walking behavior, and tonic immobility. Treatment with dsnompC and dstrpA5 resulted in failure in eclosion, causing 93 % mortality in both treatments. Survivors in dsnompC showed defects in elytra sclerotization. In dsnanchung and dsinactive treatments, adults showed abnormal walking behavior and reduced walking speed that were likely caused by defects of mechanosensing in folding of the joint between the femur and tibia. For tonic immobility, beetles with dsnanchung, dsinactive, dswaterwitch and dsick2 (insect cytokine 2) treatments showed increased sensitivity to mechanical stimulation leading to tonic immobility.

Cell proliferation and contact inhibition play a major role in maintaining epithelial cell homeostasis. A hallmark of epithelial cells is strong cell-cell junctions. These junctions include E-Cadherin, a type of adherens junction that is critical for both barrier function and contact inhibition. Prior experiments by other groups have shown that adherens junctions are subject to mechanical tension. Externally applied forces (e.g. stretch) results in changes in E-Cadherin forces that coordinate proliferation. My current work tests the hypothesis that E-Cadherin forces mediate the spatial regulation of cell proliferation even in the absence of externally applied forces.