Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and... Show moreEarly recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the mu(s) heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients' respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD. Show less
Mechanical interactions between cells and their environment play an important role in many biological processes. These interactions are often anisotropic in nature, but most mathematical models in... Show moreMechanical interactions between cells and their environment play an important role in many biological processes. These interactions are often anisotropic in nature, but most mathematical models in the field of cell mechanics describe cells as isotropic entities. In this thesis we theoretically study the role of anisotropic forces in cell mechanics, and compare our predictions to experimental data. Show less
Shape and biological function are tightly connected. Physical descriptions are used to connect the shape of a biological system with its function. One system investigated here is the dendritic... Show moreShape and biological function are tightly connected. Physical descriptions are used to connect the shape of a biological system with its function. One system investigated here is the dendritic spine, which is the connection between neurons. The dendritic spine is mimicked in an artificial system. In this way, I was able to show that the shape of the dendritic spine is important in memory and learning. The shape of a cell itself is governed by its actin cytoskeleton. I showed that a simple model can be used to describe the shape of adherent cells. An adherent cell attaches itself at discrete points to the substrate. The edge of the cell in between these points can be described as a part of an ellipse. I show that all edges of a single cell can all be described with a single ellipse. On a small scale, the shape of the membrane of a cell changes with changing lipid content. I used light to change the lipid content, triggering phase unmixing in an artificial lipid membrane. I show that various important parameters change significantly. Show less
In this thesis we present a new method to simulate realistic three-dimensional networks of biopolymers under shear. These biopolymer networks are important for the structural functions of cells and... Show moreIn this thesis we present a new method to simulate realistic three-dimensional networks of biopolymers under shear. These biopolymer networks are important for the structural functions of cells and tissues. We use the method to analyze these networks under shear, and consider the elastic modulus, the non-affinity during deformation, the normal modes and the density of states. We expand our analysis to composite networks consisting of stiff and floppy filaments. In the final chapter of the thesis we incorporate the thermal and viscous interactions of the surrounding medium in the calculations, and present the results of the simulations of the shear frequency dependence of the network response. We find that non-affine reorientations are important for understanding the network response. These non-affine reorientations make the networks relatively soft under shear and delay the onset of stiffnening. In composite networks non-affine reorientations allow for an intricate interplay between the stiff and floppy filaments. The frequency-dependent response shows a transition from a soft, non-affine regime at low frequencies to a stiff, close-to-affine response at high frequencies. Show less
Blood-flow-induced shear stress plays an important role in cardiovascular development and disease. How endothelial cells sense shear stress remains to be elucidated. We postulated that the primary... Show moreBlood-flow-induced shear stress plays an important role in cardiovascular development and disease. How endothelial cells sense shear stress remains to be elucidated. We postulated that the primary cilium is a component of the endothelial shear sensor. This luminal cell protrusion contains microtubules and is connected to the microtubular cytoskeleton. We identified cilia on endothelial cells of the embryonic heart in areas of low or oscillatory shear stress. This shear-related distribution is reminiscent of the distribution of atherosclerotic lesions in the adult arterial system, as lesions develop at sites of low or oscillating shear (athero-prone flow). Ciliated endothelial cells are exclusively present at these atherosclerotic predilection sites in adult mice. Athero-prone (oscillatory) but not athero-protective (steady or pulsatile) flow induces ciliation of cultured endothelial cells. Moreover, the endothelial shear response is dependent on the microtubular cytoskeleton and primary cilia sensitise the endothelium for shear. Taken together, these data demonstrate that primary cilia are induced by athero-prone flow and that ciliated cells are more sensitive to shear stress. We conclude that the endothelial biosensor for shear stress is the microtubular cytoskeleton and that the attached primary cilium functions as a signal amplifier in areas subjected to athero-prone flow. Show less
Our kidneys play a major role in regulating the body__s internal environment, via transportation of water, salt, potassium and waste products. As a result of this transport function, cells within... Show moreOur kidneys play a major role in regulating the body__s internal environment, via transportation of water, salt, potassium and waste products. As a result of this transport function, cells within the kidney are relatively sensitive to injury. This injury can occur when the kidneys are exposed to anticancer drugs, antibiotics, toxic chemicals or as a result of a drop in blood flow during kidney transplantation (ischemia/reperfusion injury). As a consequence, renal function is rapidly lost. The primary targets for injury are epithelial cells lining the proximal tubule. These cells rest on a basement membrane via cell-matrix interactions and are connected to each other via cell-cell interactions. At these adhesion sites, several signalling complexes are located, which are linked to the F-actin cytoskeleton of the cell. When cells are damaged, they alter or may loose their cell-cell and cell-matrix adhesions in association with reorganization of the actin cytoskeleton. This is associated with changes in the activation status of several signal transduction pathways. The research described in this thesis was designed to identify __new__ signalling pathways involved in renal cell injury and understand their role in this process. The changes in protein expression and phosphorylation that occur in association with changes in cell adhesion and cytoskeletal organization prior to or during renal cell injury were analyzed using 2D-Difference In Gel Electrophoresis (DIGE) and 2D-phosphotyrosine blotting.the protein identifications that are described in this thesis point to a more common observation of alterations in the F-actin cytoskeleton that take place during the process of renal cell injury and regeneration. Assessing the precise role of these proteins and their phosphorylation status will increase our understanding of the events that take place during this process. Show less
This thesis addresses the regulation of TRPM7 channels by receptor-mediated signals and the effects of the ensuing ionic signals on the cytoskeleton. Several signaling pathways were investigated... Show moreThis thesis addresses the regulation of TRPM7 channels by receptor-mediated signals and the effects of the ensuing ionic signals on the cytoskeleton. Several signaling pathways were investigated that activate TRPM7 channel opening. Stimulation of PLC-activating receptors opens TRPM7 channels, leading to influx of extracellular Ca2+. The results contrast markedly with a report by Runnels et al. who showed inhibition of TRPM7, rather than activation by PLC. To explain this discrepancy, we explored the differences in PIP2-mediated regulation of TRPM7 measured in perforated-patch and whole-cell configuration after intracellular Mg2+ depletion. This leads us to propose that the effects of PLC activation on TRPM7 currents as detected in whole cells can best be viewed as ‘accelerated rundown’ rather than as bona fide signal transduction. Agonist-induced TRPM7 activation and subsequent Ca2+ influx affects the cytoskeleton by phosphorylation of the myosin II heavy chain to promote cytoskeletal relaxation and the conversion of focal adhesions to podosomes. Ca2+ influx appears crucial for the in vivo function of the TRPM7-kinase by triggering the association between the kinase and its substrate. A second effect of receptor-mediated Ca2+ influx can affect the cytoskeleton by translocating Rac in a PKC-dependent manner to the plasma-membrane, where it induces extensive membrane ruffling. Show less
Ligation of the right lateral vitelline vein in chicken embryos (venous clip) results in changes in the intracardial blood flow patterns, and in functional and morphological cardiovascular defects.... Show moreLigation of the right lateral vitelline vein in chicken embryos (venous clip) results in changes in the intracardial blood flow patterns, and in functional and morphological cardiovascular defects. This demonstrates that blood flow, of which shear stress is a derivative, plays an important role in cardiovascular development. A general mechanism of shear stress sensing by endothelial cells remained to be elucidated. We postulate that the cytoskeleton functions as a central shear stress transducer, which uses a primary cilium for detection of low shear forces. During normal cardiogenesis we demonstrated that shear stress responsive genes lung Kruppel-like factor (KLF2) and endothelial nitric oxide synthase (NOS-3) are expressed in the endothelium and endocardium of structural lumen constrictions, where endothelin-1 (ET-1), a growth factor and vasoconstrictor, is absent and shear stress is high. After vitelline ligation KLF2 and NOS-3 were locally increased in the heart whereas ET-1 was down-regulated. Infusion of ET-1 or antagonists of the endothelin-A (ETA) and/or endothelin-B (ETB) receptor in the chicken embryonic circulation resulted in similar but less severe defects in cardiac function and morphology, demonstrating that components of the ET-1 pathway play a role in the development of cardiovascular defects in the venous clip model. Show less
