Glucagon-like peptide-1 (GLP-1) is secreted from the enteroendocrine L cell following nutrient ingestion. Although GLP-1 regulates several aspects of nutrient homeostasis, one important function is to enhance glucose-dependent insulin secretion. In type 2 diabetes, post-prandial GLP-1 secretion is impaired. Insulin resistance, which is required for the pathogenesis of type 2 diabetes, is also associated with impaired GLP-1 secretion. I, therefore, hypothesized that insulin modulates GLP-1 secretion from the intestinal L cell and, furthermore, insulin resistance directly impairs the function of the endocrine L cell. In well-characterized L cell models, I established that insulin stimulates GLP-1 secretion through the MEK1/2-ERK1/2 pathway, and induction of insulin resistance in vitro attenuated insulin- and heterologous secretagogue-induced GLP-1 release. Furthermore, glucose-stimulated GLP-1 secretion was decreased in hyperinsulinemic-insulin resistant MKR mice, demonstrating that insulin resistance is associated with impaired L cell function. I next examined the role of the actin cytoskeleton in insulin-stimulated GLP-1 secretion. Insulin treatment transiently induced actin depolymerization, and depolymerization of the actin cytoskeleton potentiated insulin-stimulated GLP-1 release from the L cell, demonstrating that the cytoskeleton functions as a permissive barrier. Central to insulin’s effects on actin dynamics is the Rho GTPase, Cdc42, as siRNA-mediated knockdown and over-expression of a dominant-negative mutant, prevented insulin-stimulated actin remodeling and GLP-1 release. Insulin also promoted activation of PAK1, the downstream kinase of Cdc42, and over-expression of a kinase-dead PAK1 mutant attenuated insulin-stimulated GLP-1 release. In cells that expressed dominant-negative Cdc42 or kinase-dead PAK1, activation of ERK1/2 following insulin treatment was attenuated, demonstrating that the Cdc42-PAK1 axis regulates the activity of the canonical ERK1/2 pathway. In summary, this thesis demonstrates, for the first time, that insulin is a GLP-1 secretagogue, and this effect of insulin is mediated through the canonical ERK1/2 pathway and the Cdc42-PAK1 axis. Insulin resistance in the L cell impairs the responsiveness of the L cell to heterologous secretagogues. Collectively, these findings suggest that an alternative approach to treat type 2 diabetes and/or insulin resistance may be to directly improve the function of the L cell, thereby enhancing endogenous GLP-1 release.

Sensory-motile cells fulfill various biological functions ranging from immune activity or wound healing to the formation of the highly complex nervous systems of vertebrates. In the case of neurons, a dynamic structure at the tip of outgrowing processes navigates towards target cells or areas during the generation of neural networks. These fan shaped growth cones are equipped with a highly complex molecular machinery able to detect various external stimuli and to translate them into directed motion. Receptor and adhesion molecules trigger signaling cascades that regulate the dynamics of an internal polymeric scaffold, the cytoskeleton. It plays a crucial role in morphology maintenance as well as in the generation and distribution of growth cone forces. The two major components, actin and microtubules (MTs) connect on multiple levels through interwoven biochemical and mechanical interactions. Actin monomers assemble into semiflexible filaments (F-actin) which in turn are either arranged in entangled networks in the flat outer region of the growth cone (lamellipodium) or in radial bundles termed filopodia. The dynamic network of actin filaments extends through polymerization at the front edge of the lamellipodium and is simultaneously moving towards the center (C-domain) of the growth cone. This retrograde flow (RF) of the actin network is driven by the polymerizing filaments themselves pushing against the cell membrane and the contractile activity of motor proteins (myosins), mainly in the more central transition zone (T-zone). Through transmembrane adhesion molecules, a fraction of the retrograde flow forces is mechanically transmitted to the cellular substrate in a clutch-like mechanism generating traction and moving the GC forward. MTs are tubular polymeric structures assembled from two types of tubulin protein subunits. They are densely bundled in the neurite and at the growth cone “neck” (where the neurite opens out into the growth cone) they splay apart entering the C-domain and more peripheral regions (P-domain). Their advancement is driven by polymerization and dynein motor protein activity. The two subsystems, an extending array of MTs and the centripetal moving actin network are antagonistic players regulating GC morphology and motility. Numerous experimental findings suggest that MTs pushing from the rear interact with actin structures and contribute to GC advancement. Nevertheless, the amount of force generated or transmitted through these rigid structures has not been investigated yet. In the present dissertation, the deformation of MTs under the influence of intracellular load is analyzed with fluorescence microscopy techniques to estimate these forces. RF mechanically couples to MTs in the GC periphery through friction and molecular cross-linkers. This leads to MT buckling which in turn allows the calculation of the underlying force. It turns out that forces of at least act on individual MT filaments in the GC periphery. Compared to the relatively low overall protrusion force of neuronal GCs, this is a substantial contribution. Interestingly, two populations of MTs buckle under different loads suggesting different buckling conditions. These could be ascribed to either the length-dependent flexural rigidity of MTs or local variations in the mechanical properties of the lamellipodial actin network. Furthermore, the relation between MT deformation levels and GC morphology and advancement was investigated. A clear trend evolves that links higher MT deformation in certain areas to their advancement. Interactions between RF and MTs also influence flow velocity and MT deformation. It is shown that transient RF bursts are related to higher MT deformation in the same region. An internal molecular clutch mechanism is proposed that links MT deformation to GC advancement. When focusing on GC dynamics it is often neglected that the retraction of neurites and the controlled collapse of GCs are as important for proper neural network formation as oriented outgrowth. Since erroneous connections can cause equally severe malfunctions as missing ones, the pruning of aberrant processes or the transient stalling of outgrowth at pivotal locations are common events in neuronal growth. To date, mainly short term pausing with minor cytoskeletal rearrangements or the full detachment and retraction of neurite segments were described. It is likely that these two variants do not cover the full range of possible events during neuronal pathfinding and that pausing on intermediate time scales is an appropriate means to avoid the misdetection of faint or ambiguous external signals. In the NG108-15 neuroblastoma cells investigated here, a novel type of collapse was observed. It is characterized by the degradation of actin network structures in the periphery while radial filopodia and the C-domain persist. Actin bundles in filopodia are segmented at one or multiple breaking points and subsequently fold onto the edge of the C-domain where they form an actin-rich barrier blocking MT extension. Due to this characteristic, this type of collapse was termed fold collapse. Possible molecular players responsible for this remarkable process are discussed. Throughout fold collapse, GC C-domain area and position remain stable and only the turnover of peripheral actin structures is abolished. At the same time, MT driven neurite elongation is hindered, causing the GC to stall on a time scale of several to tens of minutes. In many cases, new lamellipodial structures emerge after some time, indicating the transient nature of this collapse variant. From the detailed description of the cytoskeletal dynamics during collapse a working model including substrate contacts and contractile actin-myosin activity is derived. Within this model, the known and newly found types of GC collapse and retraction can be reduced to variations in local adhesion and motor protein activity. Altogether the results of this work indicate a more prominent role of forward directed MT-based forces in neuronal growth than previously assumed. Their regulation and distribution during outgrowth has significant impact on neurite orientation and advancement. The deformation of MT filaments is closely related to retrograde actin flow which in turn is a regulator of edge protrusion. For the stalling of GCs it is not only required that actin dynamics are decoupled from the environment but also that MT pushing is suppressed. In the case of fold collapse, this is achieved through a robust barrier assembled from filopodial actin bundles.

Various genes have evolved to protect the cell against stressor-induced damage or death including the heat shock proteins (HSPs). Stressor-induced HSP gene expression involves the activation of heat shock factor (HSF), which binds to the heat shock element (HSE) found in the promoter region of <em>hsp</em> genes. Previously, our laboratory has examined the expression and function of <em>hsp</em> genes in the South African clawed frog, <em>Xenopus laevis</em>. Amphibians are particularly susceptible to adverse environmental conditions, including high temperatures and toxicants. In contrast to the many known inducers of HSF activation in poikilothermic vertebrates, few inhibitors have been either discovered or described in the literature. The present study has compared for the first time the effect of two heat shock response (HSR) inhibitors, quercetin and KNK437, on <em>hsp</em> gene expression in <em>Xenopus</em> A6 cells, demonstrating their efficacy in poikilotherms. Northern blot and densitometric analysis showed that cells treated with either quercetin or KNK437 decreased the heat shock-induced accumulation of <em>hsp70</em>, <em>hsp47</em>, and <em>hsp30</em> mRNAs. Additionally, constitutive levels of <em>hsp47</em> and <em>hsc70</em> mRNAs were reduced. In comparison, neither quercetin nor KNK437 affected the levels of constitutively expressed <em>ef1&alpha;</em> mRNAs under control or heat shock conditions. Western blot and densitometric analysis in this study showed that under heat shock conditions, exposure to quercetin or KNK437 significantly decreased the accumulation of HSP30, and that KNK437 was more effective in doing so than quercetin. In comparison, levels of actin were not significantly affected by either heat shock or exposure to DMSO, quercetin, or KNK437. These findings suggest that one mechanism by which quercetin and KNK437 inhibits the HSR in <em>Xenopus</em> is through the inhibition of HSF activity. <br /><br /> Results of this study also suggest that KNK437 inhibits the acquisition of thermotolerance in poikilotherms, similar to observations in mammalian systems. In the presence of KNK437, cells given a 2 h heat pretreatment at 33ºC followed by a thermal challenge for 1 h at 37ºC, showed numerous ruffled membrane edges and some aggregates of disrupted stress fibers. In comparison, cells directly challenged for 1 h at 37ºC, showed a marked decrease in HSP30, which was located predominantly at the cellular periphery in conjunction with actin aggregates. These cells showed virtually no intact stress fibers spanning cells and no coherent cell-cell connections. A 3-D analysis of cells given a 1 h thermal challenge at 37ºC (after a prior 2 h heat shock at 33ºC) in the absence of KNK437, showed numerous linear actin bundles transversing the entire cell, even extending into areas of cell-cell contact, and abundant HSP30 concentrated in the perinuclear region surrounding an intact nucleus. However, in the presence of KNK437, there was a significant emergence of membrane ruffles indicating global instability of cellular adhesion. This study has demonstrated that KNK437, which is the more specific and efficient HSR inhibitor, will be an important inhibitor to compare with the well-documented quercetin for future investigations.

Cell motility has been found to play a role in many important body functions as well as the embryogenenis of mulitcellular organisms like vertebrates. From a physics point of view the interesting questions behind every motion are: Why is it moving? Where do the forces come from? Today we know that the motility of many cells is dependent on an active polymer network. Actin, one of the most abundant proteins in the body, is constantly polymerized, being moved around and depolymerized in motile cells. Until now, only forces outside the cell like traction forces could be measured. The direct measurement of the force generated by polymerizing actin filaments has only been measured by our lab and the lab of M. Radmacher. In these measurements fish keratocytes were used. Whereas I did these experiments, for the first time, on mammalian cells. To measure forward forces on neuronal growth cones I stabilized the SFM, as measurement times went up from minutes to hours. Furthermore measurements had to be performed at 37°C instead of room temerature, this induced drifts of the substrate. I incorporated an optical trap into the microscope to track the motion of the substrate. A feedback loop moved the SFM cantilever to minimize relative motion of substrate and cantilever. For keratocytes I directly measured the forces produced by actin polymerization and, to my knowledge for the first time, the forces associated with the retrograde actin flow using a SFM. The result was that both actin and myosin play important but different roles in motility. For actin it turned out that considering just the polymerization was not enough. Actin depolymerization and the resulting entropic forces are a completely new physical effect in actin based cell motility. With this new force in the force balance I can explain all effects observed in my experiments without introducing any new biochemical feedback loops. Finally I showed that neuronal growth cones are very soft and weak structures. They are at least one order of magnitude softer and weaker as for example fibroblasts or cells forming the blood vessel walls. As neurons are usually located in soft environments this does not impede their normal outgrowth. It could even serve as a safety mechanism that prevents cell from growing into wrong areas like breaching the blood-brain-barrier, on a physical level. For a neuron the wall of a blood vessel feels like a brick wall for us.

Various genes have evolved to protect the cell against stressor-induced damage or death including the heat shock proteins (HSPs). Stressor-induced HSP gene expression involves the activation of heat shock factor (HSF), which binds to the heat shock element (HSE) found in the promoter region of <em>hsp</em> genes. Previously, our laboratory has examined the expression and function of <em>hsp</em> genes in the South African clawed frog, <em>Xenopus laevis</em>. Amphibians are particularly susceptible to adverse environmental conditions, including high temperatures and toxicants. In contrast to the many known inducers of HSF activation in poikilothermic vertebrates, few inhibitors have been either discovered or described in the literature. The present study has compared for the first time the effect of two heat shock response (HSR) inhibitors, quercetin and KNK437, on <em>hsp</em> gene expression in <em>Xenopus</em> A6 cells, demonstrating their efficacy in poikilotherms. Northern blot and densitometric analysis showed that cells treated with either quercetin or KNK437 decreased the heat shock-induced accumulation of <em>hsp70</em>, <em>hsp47</em>, and <em>hsp30</em> mRNAs. Additionally, constitutive levels of <em>hsp47</em> and <em>hsc70</em> mRNAs were reduced. In comparison, neither quercetin nor KNK437 affected the levels of constitutively expressed <em>ef1&alpha;</em> mRNAs under control or heat shock conditions. Western blot and densitometric analysis in this study showed that under heat shock conditions, exposure to quercetin or KNK437 significantly decreased the accumulation of HSP30, and that KNK437 was more effective in doing so than quercetin. In comparison, levels of actin were not significantly affected by either heat shock or exposure to DMSO, quercetin, or KNK437. These findings suggest that one mechanism by which quercetin and KNK437 inhibits the HSR in <em>Xenopus</em> is through the inhibition of HSF activity. <br /><br /> Results of this study also suggest that KNK437 inhibits the acquisition of thermotolerance in poikilotherms, similar to observations in mammalian systems. In the presence of KNK437, cells given a 2 h heat pretreatment at 33ºC followed by a thermal challenge for 1 h at 37ºC, showed numerous ruffled membrane edges and some aggregates of disrupted stress fibers. In comparison, cells directly challenged for 1 h at 37ºC, showed a marked decrease in HSP30, which was located predominantly at the cellular periphery in conjunction with actin aggregates. These cells showed virtually no intact stress fibers spanning cells and no coherent cell-cell connections. A 3-D analysis of cells given a 1 h thermal challenge at 37ºC (after a prior 2 h heat shock at 33ºC) in the absence of KNK437, showed numerous linear actin bundles transversing the entire cell, even extending into areas of cell-cell contact, and abundant HSP30 concentrated in the perinuclear region surrounding an intact nucleus. However, in the presence of KNK437, there was a significant emergence of membrane ruffles indicating global instability of cellular adhesion. This study has demonstrated that KNK437, which is the more specific and efficient HSR inhibitor, will be an important inhibitor to compare with the well-documented quercetin for future investigations.

<p>The ability of cells to migrate is of fundamental importance to a diverse array of biological processes, both physiological and pathological, such as development, the immune response and cancer cell metastasis, to name a few. The process of cell movement is a complicated cycle of coordinated steps involving dynamic and precise rearrangement of the actin-myosin cytoskeleton. As a critical component of the migration machinery, the molecular motor protein nonmuscle myosin II (myosin II) has long been a subject of scientific inquiry. It is now generally accepted that the contractile forces generated by myosin II contribute directly or indirectly to every step in migration. Interestingly, three isoforms of myosin II (myosin IIA, IIB and IIC) have been identified, and although each isoform performs the same basic molecular functions, recent findings suggest that the different myosin II isoforms make unique contributions to the motile process. In this dissertation work I used RNA interference technology to specifically deplete cells of myosin IIA and IIB in order to characterize the distinct migration phenotypes associated with loss-of-function of each individual isoform. Surprisingly, I found that the two myosin II isoforms perform not only distinct but opposing functions in cell migration, with myosin IIA and IIB normally inhibiting and facilitating proper cell movement, respectively. Furthermore, using pharmacological and microscopy techniques, I investigated the cellular mechanisms allowing for isoform-specific function. My results provide evidence for at least two isoform-specific regulatory mechanisms, namely selectivity in signaling pathways and subcellular distribution. A particularly significant finding is the identification of the different assembly properties of myosin IIA and IIB as the key element responsible for directing isoform-distinct distribution. Together the data presented herein represent a considerable advance in our understanding of the distinct functions and regulation of myosin II in cell motility.</p> / Dissertation

Atomic force microscopy (AFM) can be used to acquire high-resolution topographical images of surfaces, but has the additional capability of detecting the local nanometer scale mechanical properties. For these reasons, it becomes a standard research tool in the surface science recently. In this paper, we used AFM to measure the several properties of two different human hepatocellular carcinoma cell lines, Hep G2 ( known as well differentiated and more highly carcinomatous hepatoma cell lines ) and SK-HEP-1 ( known as poorly differentiated and more lightly carcinomatous hepatoma cell lines ) cells fixed on the glass substrate, which including the surface morphology and the relationship between the cantilever deflections and loading forces ( force curve ). Considered the heterogeneous characteristics of the cell surface, the preferred experimental method is to make pixel-by-pixel force curves in a designated area ( force map ) , both adhesion forces and elasticity associated with different locations on the cell surfaces will be obtained. Finally, we use Hertzian model to calculate Young's modulus of Hep G2 and SK-HEP-1 respectively. Based on these results, we can understand the surface properties of two human hepatocarcinoma cell lines with different differentiated stage. The results showed the difference of the morphology, height, cell migration, degree of cell aggregation, roughness, elasticity, adhesive force of two cells. SK-HEP-1 cell has the wide distance of the folds, better cell migration, homogeneous properties of elasticity. It can be assumed that the SK-HEP-1 cells have a dense network structure of actin filaments under the cell membrane like branches (branched networks); Hep G2 cell has the narrow distance of the folds, poor cell migration, heterogeneous properties of elasticity. It can be assumed that the Hep G2 cells have the individual actin filaments and cross-linked network structure of actin filaments under the cell membrane. The above results can be speculated that the elastic properties of the membrane surface will be influenced of actin filaments.

No description available.

No description available.

Huntington's disease (HD) is a rare, dominantly inherited neurodegenerative disorder characterized by progressive chorea, emotional and behavioral disturbances, and cognitive decline. The single, causative mutation is an expanded trinucleotide repeat of cytosine, adenosine, and guanine (CAG) of more than 37 residues in the HD gene (currently referred to as HTT). Genetic evidence suggests that the CAG repeat expansion results in a gain of huntingtin function. While huntingtin and its numerous interactors have been implicated in a variety of essential cellular processes, the role of the full-length, endogenous protein remains unclear. Multiple studies have implicated huntingtin in processes related to cytoskeletal structure and dynamics in HD patients and model organisms. However, alterations in cellular dynamics related to the cytoskeleton &mdash; including cell adhesion &mdash; have not been characterized in a comprehensive, rigorous manner. Using Mus musculus genetic models of the HD mutation and/or deficiency and a Dictyostelium discoideum genetic deficiency model, I have undertaken an investigation of evolutionarily conserved huntingtin function in the cytoskeleton and cell adhesion. The results of these studies support a role for huntingtin in cell-cell and cell-substrate adhesion, as well as maintaining actin cytoskeletal structure. Furthermore, my thesis research sets the stage for future work to elucidate the molecular mechanism by which huntingtin is acting and determine the effect of the CAG repeat expansion on huntingtin function. Evolutionary conservation affords an invaluable tool to identify crucial function(s) of the huntingtin molecule and the effect of the pathogenic HD mutation on function, enabling therapeutic development while providing novel insights into cytoskeletal biology and cell adhesion.