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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.

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.

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

The evolution and development of multicellular organisms requires cells to differentiate, interact and "collaborate". Our understanding of the molecular mechanisms is still hazy. In this dissertation mathematical modelling is used to integrate available experimental data and to make testable predictions about such mechanisms. The thesis is split into three parts, each of which addresses one of the three challenges: differentiation, adhesion and collaboration. In the first part, a mathematical model is developed to explain how, in the absence of polarizing cues from the environment, sister cells with identical genomes can follow distinct routes of differentiation. It is shown that difference in cell size, resulting from asymmetric cell division, is sufficient to induce differential cell fate in Bacillus subtilis. The model predicts that this effect depends on the allosteric behaviour of a kinase and the low catalytic rate of the corresponding phosphatase; both properties were subsequently confirmed in experiments. During the development of multicellular organisms, differentiation can arise in response to gradients. By example of dorso-ventral patterning it is shown how a shallow maternal gradient can be converted into a sharp pattern. In the second part, a model for cell adhesion via integrins is developed, and it is shown that, for physiological parameters, binding of a ligand and of a stabilizing factor such as talin are insufficient for ligand-dependent integrin activation, and that a positive signaling feedback is required. In the final part, antibody affinity maturation is studied as an example for division of labour between collaborating cells. A novel B cell selection mechanism, based on competition for T cell help rather than for antigen, is proposed and shown to reconcile heretofore inexplicable experimental observations. Such a mechanism requires B cells to discriminate among different affinities of binding, and it is further shown that this can be achieved if B cell signaling is initiated by antigen-dependent receptor-inhibitor segregation. The predictions of the model match experimental measurements quantitatively.

Thesis (Ph. D.)--University of Virginia, 2000. / Spine title: Palladin & actin organization. Includes bibliographical references (leaves 209-251). Also available online through Digital Dissertations.

Thesis (M.Ch.E.)--University of Delaware, 2006. / Principal faculty advisor: Eric M. Furst, Dept. of Chemical Engineering. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 189-230).

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Thesis (Ph. D. in Biochemistry)--Vanderbilt University, May 2009. / Title from title screen. Includes bibliographical references.