No description available.

Axons are directed along stereotypic pathways to their targets by cues arrayed in the extracellular environment. Identifying the cellular and molecular nature of these signals is of high interest and the developing optic pathway is a useful model system for achieving this. Although previous studies have identified several molecules essential for optic pathway formation, in vivo only subsets of retinal axons rely on them. I focused on the Dscam (Down’s syndrome cell adhesion molecule) and Sidekick (Sdk) cell adhesion molecules for potentially playing crucial roles in this system. In situ hybridisation in the embryonic mouse visual system showed Dscam and Sdk-1 expression in the RGC layer of the retina, along the optic pathway and in the visual targets. Sdk-2 was detected in the glia of the optic nerve and optic chiasm, marking the pathway that RGC axons follow, but not in RGCs. No DscamL1 was detected in RGCs or the optic pathway at the stages investigated and it was discounted from future analysis. In vitro, DSCAM promoted RGC axon outgrowth, whereas SDK 1 was inhibitory. SDK 2 had no effect on RGC axon outgrowth, suggesting it does not play a direct role in their pathfinding. Repeating this assay using retinal explants from the Dscamdel17 mouse mutant, showed that DSCAM enhanced retinal axon outgrowth, at least in part, through homophilic interactions. Analysis of visual system development in Dscam mutants showed DSCAM involvement in RGC axon fasciculation and in enhancing their growth, particularly within the ipsilateral optic tract. Retinal cell counts revealed that DSCAM played diverse roles in controlling cell number. Pre- and postnatal retinas lacking DSCAM contained more RGCs and mitotic cells. Postnatally, Dscam-/- retinas also show decreased cell death. In many cases, defect severity was dose-dependent, with an intermediate phenotype in the heterozygous mice, implicating DSCAM in the neurological defects of Downs’ Syndrome patients.

Cell adhesion is a critical process that allows the organisation and functioning of tissues in three-dimensions. However, the replenishing of cells, via cell division, within tissues is equally important for functioning complex life. Both cell adhesion and division are tightly controlled processes and rely on a complex network of signals that, as yet, are not wholly understood. This Thesis presents a structural analysis of several proteins involved in these processes. In the case of cell adhesion, we have made use of high-throughput (HTP) cloning and expression screening technologies in the Oxford Protein Production Facility (OPPF) for the study of the Kindlin protein family – a recently discovered set of proteins essential for integrin-mediated cell adhesion. As a direct result of the HTP pipeline used we were able to determine the high resolution crystal structure of a single domain, the Pleckstrin Homology Domain, from the isoform Kindlin-1. Deletion of this domain in the full-length protein resulted in impaired integrin activation in vivo. This structure, in combination with molecular dynamics simulation demonstrated that, unlike other well characterised PH domains, the binding of secondary messenger lipids (phosphoinositides) is dictated by a, previously unseen, salt bridge that occludes the putative binding site. Mutation of the salt bridge alters the binding characteristics of this domain in vitro. In addition to the PH domain, we have also studied and biophysically characterised full-length Kindlin-3, a blood cell specific isoform. By optimising baculovirus-infected Sf9 cell expression systems we were able to obtain, for the first time, sufficient quantities of protein for characterisation. Furthermore, by using small-angle X-ray scattering (SAXS) in solution we were able to determine a low resolution solution structure of Kindlin-3, revealing a linear arrangement of its FERM domain - a novel conformation known only otherwise in talin. We characterised the interaction of full-length Kindlin-3 with β-integrin cytoplasmic tails using nuclear magnetic resonance spectroscopy, which confirmed that a direct interaction with a membrane distal NPxY motif occurs, and demonstrated the importance of a preceding Serine/Threonine rich region in peptide binding. In the case of cell division, we have determined the crystal structure of the cell cycle checkpoint control related protein, Cid1, a terminal uridine tranferase from Schizzosaccharomyces pombe, alone and in complex with UTP. Structural and biochemical analysis of Cid1 identified a novel Uridine selection mechanism that is suggested to be conserved in metazoan ZCCHC enzymes involved in let-7 miRNA biogenesis, which are important for proliferation, differentiation and cell fate. We have also demonstrated that Cid1 is an RNA binding protein, a property essential for activity that employs a novel mechanism of RNA binding in the absence of RNA binding motifs. The structural work undertaken in this thesis has focussed on two distinct, but interwoven, aspects of cell biology and has significantly added to both fields of research. Excitingly, this has opened many new avenues of investigation and, in the case of Cid1, has the strong potential to lead to the development of novel anticancer therapies.

No description available.

Tissue cells exhibit varying responses according to the stiffness of their extracellular matrix (ECM). The mechanism of this stiffness sensing is not fully understood; however, it is known that cells probe stiffness by applying intracellular force to the ECM via integrin-mediated focal adhesions. The bonds between integrins and ECM have been described as “catch bonds�, and it is unclear how ECM viscoelasticity affects these bonds. We have observed the effects of ECM stiffness on the binding strength of integrins to ECM ligands by measuring the dissociation force of individual integrin-ligand bonds of 3T3 fibroblasts on collagen-coated polyacrylamide gels using atomic force microscopy. Results show that integrins exhibit higher rates of activation on stiff substrates. Furthermore, increased matrix stiffness results in the occurrence of larger, multi-bond dissociation events, which suggests that substrate stiffness may affect the cellular response by promoting integrin clustering as well as by modulating the maximum possible force between individual integrins and the ECM.

A very important aspect for the functioning of an organism is that cells adapt their behaviour to external stimuli. They continuously interact with their environment, and biochemical and physical cues can activate cellular signalling, which leads to changes in cell behaviour such as proliferation and shape. Understanding cells' interactions with their environment is also important for understanding diseases. For example mechanosensing, which is the sensing of the cell's mechanical environment, has been associated with cancer development. In order for a cell to be able to sense its mechanical environment, it needs to form attachments to the environment. In my thesis, I have worked on three different tasks: the development of a new measurement technique and the study of initial cell adhesion and of cell spreading. When a cell from suspension first comes into contact with a substrate, it forms initial attachment bonds with proteins on the substrate surface. These bonds are mediated through integrins, which are transmembrane heterodimers, binding to the cell's environment on one side and to the cell's cytoskeleton on the other side. I study this initial cell attachment by measuring the force needed to detach cells, called cell adhesion strength. For these experiments I built a detachment device, which allows the detachment of cells from a substrate by vibrating the substrate in liquid. The device combines cell incubation, detachment and imaging. I measured the dependence of initial integrin bond formation on external factors such as incubation temperature and substrate stiffness. Once initial integrin bonds are formed, many different proteins are recruited to the adhesion site in order to form stronger adhesions. Amongst these proteins are signalling proteins, which direct the behaviour of the cell as a whole. One of the first cellular reactions to a substrate after initial integrin binding is cell spreading. This can be seen by the cell changing its shape from spherical to dome-like on the substrate. Because cell spreading is a very early response of a cell to a substrate, the onset time of spreading can be used as a quantitative measure for the time it takes the cell to sense a substrate and signal shape change. In my work, I look at the distribution of the time of initial cell spreading in a population of cells. I measure this distribution under different growth conditions such as pH, change of incubation medium from DMEM to PBS, substrate stiffness and incubation temperature. In my detachment experiments, I observe that vibration accelerates cell spreading in those cells which remain on the substrate. This is a connection between the detachment experiments and the cell spreading experiments and it shows how cells react to external forces. By changing the medium temperature in the cell detachment and cell spreading experiments, I am able to analyse the kinetics of these two processes. I use a signalling network model to analyse the internal cellular signalling path that leads from a spherical to a spread cell.

Metastatic melanoma is among the most aggressive forms of cancer for which there are no effective therapies. Emerging evidence indicates that melanomas can be sensitized to chemotherapy by increasing the function of integrin transmembrane adhesion receptors. Current integrin therapies work by targeting the extracellular domain, resulting in complete gains or losses of integrin function that lead to toxicity.An attractive alternative approach is to target proteins from inside the cell, such as vinculin, that associate with the integrin cytoplasmic domains and regulate its ligand binding properties. The work presented in this thesis describes a novel reagent, denoted vinculin activating peptide or VAP, which increases integrin activity from within the cell as measured by elevated: (1) numbers of active integrins, (2) adhesion of cells to extracellular matrix ligands, (3) numbers of cell-matrix adhesions, and (4) downstream signaling. The effects of VAP are dependent on both integrins and a key regulatory residue A50 in the vinculin head domain. I further show that VAP dramatically increases the sensitivity of melanomas to chemotherapy in clonal growth assays and in vivo mouse models of melanoma. Finally, we demonstrate that the increase in chemosensitivity results from increases in DNA damage-induced apoptosis by a mechanism that requires both p53 and β1 integrin. Collectively these findings demonstrate that integrin function can be manipulated from within the cell and validate integrins as a new therapeutic target for the treatment of chemoresistant melanomas.

PVA hydrogels are potential biomaterials for various tissue-engineering applications. PVA hydrogels are relevant to vascular graft development due to their excellent biocompatibility and the capability to possess a wide range of mechanical properties based on compositional and processing parameters. This thesis aims to characterize some PVA hydrogels mechanically, biologically, and physically. A constitutive formulation is used for mechanical characterization, which allows for analysis of any possible stress-strain configuration applied to the material. A bovine aortic endothelial cell adhesion study under physiologic blood flow conditions comprises the biologic characterization, which gives insight into how human endothelial cells might interact with PVA hydrogels in a vascular graft application. A high-resolution SEM study is used to physically characterize the material, which furthers the understanding of the reactions of this material in vivo. These characterizations of PVA hydrogels will aid in the development of tissue-engineered products, in particular, the potential use as a vascular grafting biomaterial.

Nature encourages diversity in life forms (morphologies). The study of morphogenesis deals with understanding those processes that arise during the embryonic development of an organism. These processes control the organized spatial distribution of cells, which in turn gives rise to the characteristic form for the organism. Morphogenesis is a multi-scale modeling problem that can be studied at the molecular, cellular, and tissue levels. Here, we study the problem of morphogenesis at the cellular level by introducing an integrated biomechanical model of cells embedded in the extracellular matrix. The fundamental aspects of mechanobiology essential for studying morphogenesis at the cellular level are the cytoskeleton, extracellular matrix (ECM), and cell adhesion. Cells are modeled using tensegrity architecture. Our simulations demonstrate cellular events, such as differentiation, migration, and division using an extended tensegrity architecture that supports dynamic polymerization of the micro-filaments of the cell. Thus, our simulations add further support to the cellular tensegrity model. Viscoelastic behavior of extracellular matrix is modeled by extending one-dimensional mechanical models (by Maxwell and by Voigt) to three dimensions using finite element methods. The cell adhesion is modeled as a general Velcro-type model. We integrated the mechanics and dynamics of cell, ECM, and cell adhesion with a geometric model to create an integrated biomechanical model. In addition, the thesis discusses various computational issues, including generating the finite element mesh, mesh refinement, re-meshing, and solution mapping. As is known from a molecular level perspective, the genetic regulatory network of the organism controls this spatial distribution of cells along with some environmental factors modulating the process. The integrated biomechanical model presented here, besides generating interesting morphologies, can serve as a mesoscopic-scale platform upon which future work can correlate with the underlying genetic network.

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, June 2001. / Includes bibliographical references. Also available on the Internet.