The mechanical behaviour of cells is essential in ensuring continued physiological function, and deficiencies therein can result in a variety of diseases. Also, altered mechanical response of cells can in certain cases be an indicator of a diseased state, and even actively promoting progression of pathology. In this thesis, methods to model cell and cytoskeletal mechanics are developed and analysed. In Paper A, a constitutive model for the response of transiently cross-linked actin networks is developed using a continuum framework. A strain energy function is proposed and modified in terms of chemically activated cross-links. In Paper B, a finite element framework was used to assess the influence of numerous geometrical and material parameters on the response of cross-linked actin networks, quantifying the influence of microstructural properties and cross-link compliance. Also, a micromechanically motivated constitutive model for cross-linked networks in a continuum framework was proposed. In Paper C, the discrete model is extended to include the stochastic nature of cross-links. The strain rate dependence observed in experiments is suggested to depend partly on this. In Paper D, the continuum model for cross-linked networks is extended to encompass more composite networks. Favourable comparisons to experiments indicate the interplay between phenomenological evolution laws to predict effects in biopolymer networks. In Paper E, experimental and computational techniques are used to assess influence of the actin cytoskeleton on the mechanical response of fibroblast cells. The influence of cell shape is assessed, and experimental and computational aspects of cell mechanics are discussed. In Paper F, the filament-based cytoskeletal model is extended with an active response to predict active force generation.  Importantly, experimentally observed stiffening of cells with applied stress is predicted. / <p>QC 20151209</p>

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Protein structure and functions are tightly regulated. Studying the integration of multiple modifications in single systems is a novel approach. Hisactophilin protein from Dictyostelium discoideum, is an actin binding protein that serves to induce formation of actin filaments and is regulated by protonation and myristoylation. Utilizing hisactophilin as a model, I determined the effect of pH and myristoyl-switching on actin binding and filament induction using fluorescence spectroscopy, light scattering, and time-course electron microscopy. Results revealed the accessible myristoyl group slows binding and the rate of actin polymerization compared to when the group is sequestered. Hisactophilin induces pH-dependent actin aggregates before reorganizing them into filaments and bundles. Hisactophilin mutants impact initial actin binding and the kinetics of the aggregated state. I determined the cooperativity of myristoylation and protonation as interdependent protein regulatory mechanisms, their impact on actin binding and proposed a novel mechanism for actin polymerization as a result of these integrated regulators. / NSERC

The Actin Related Protein (ARP) 2/3 complex is a major regulator of the actin cytoskeleton that is implicated in cell morphogenesis in plants. However, a similar role is attributed to the endoplasmic reticulum (ER). My research explored the relationship between the two systems by using transgenic plants simultaneously expressing fluorescent proteins highlighting F-actin and ER organization in living cells. A comparison of F-actin organization in cells of wild type Arabidopsis thaliana and mutants with aberrant actin cytoskeleton suggests bundling in the distorted2 mutant but a relatively fine F-actin arrangement in klunker. These differences correlate with ER organization into cisternae, fenestrated sheets and tubules. A model relating ER-organization to the degree of actin bundling in a cell emerges and is supported by drug-induced interference in actin polymerization, altered ionic conditions and temperature. The study adds to the mechanistic understanding of cell morphogenesis in plants.

Understanding how hyphal organisms grow and develop is essential in order to manipulate mycelial colonies for purposes such as disease prevention and food production. One aspect of hyphal development that is not well understood is hyphal branching. Hyphal organisms branch as a way of creating new hyphal tips required for the search for nutrients, the acquisition of these nutrients and for hyphal fusion events that facilitate communication of signals within a mycelial colony. This investigation focused on the branching process occurring in the fungus N. crassa and in the oomycete A. bisexualis. An induction technique was developed to study branching in N. crassa involving local application of amino acids towards hyphae. This induced a branch to form along the hypha within the field of view. The use of this technique will enable the study of underlying events occurring internally prior to the visible branching stages. The role of Ca²⁺ hyphal branching was investigated in N. crassa suggesting Ca²⁺ is involved in apical dominance of the hyphal tip. This is based on a dose dependent response of increased branch frequency, decreased colony radius and decreased distance between the hyphal tip and the first branch, to the Ca²⁺ channel inhibitor verapamil. The stretch-activated Ca²⁺ channel inhibitors also had an effect on mycelial morphology. Gd³⁺ resulted in an increased branch frequency and a decreased colony radius and La³⁺ resulted in a decreased colony radius. The local application of verapamil towards N. crassa showed an increase in the number of multiple branches forming. Cytoplasmic Ca²⁺ was imaged in hyphae of A. bisexualis and N. crassa showing a tip-high Ca²⁺ gradient in A. bisexualis and Ca²⁺ sequestered into organelles in N. crassa. The role of F-actin in the process of hyphal branching was investigated using Lifeact N. crassa where F-actin could dynamically be seen at the site of both growing and non-growing hyphal branches. The involvement of F-actin at sites of septa development and associated with suspected vesicles was also observed.

One of the hallmarks of postprandial glucose homeostasis is the ability of insulin to promote glucose uptake into skeletal muscles. Insulin achieves this feat by enhancing the recruitment of glucose transporter 4 (GLUT4) from an intracellular compartment to the plasma membrane of muscles in order to create a net increase in surface GLUT4, which results in elevated glucose uptake. From a molecular perspective, this insulin-regulated GLUT4 traffic action requires the independent activation of Akt and Rac-1 in muscle cells because perturbation of either molecule results in an impaired response. Although Rac-1 has been validated as key component of insulin response, its downstream signalling capacity contributing to GLUT4 translocation remains unexplored. Studies on Rac-1 have shown that it is responsible for the formation of cortical remodelled actin that facilitates GLUT4 translocation following insulin stimulation. However, the downstream Rac-dependent molecules governing this actin remodelling are undetermined. Here we identified Arp2/3 and cofilin as the Rac-dependent regulators of insulin-induced actin remodelling in muscle cells. While Arp2/3 acts to initiate a burst of actin polymerization, cofilin balances out the actin dynamics through its severing/depolymerizing activity. Inhibition of either molecule’s function leads to defective GLUT4 translocation mediated by insulin in muscle cells, suggesting the requirement of actin dynamics to facilitate GLUT4 traffic to the plasma membrane. Furthermore, given the importance of Rac-1 in insulin-mediate GLUT4 traffic, its application potential to reverse insulin resistance has never been explored. We discovered that providing muscle cells with additional Rac-1 activity produces an insulin-independent gain in surface GLUT4 with magnitude comparable to that normally elicited by insulin. This phenotype is accomplished because of the concomitant cross-activation of Akt pathway when supplying the cells with active Rac-1. Interestingly, this response can bypass signalling defects imposed by cellular insulin resistance conditions, leading to restoration of GLUT4 translocation in muscle cells. Overall, these results not only reinforce the functional impact of Rac-1 on GLUT4 traffic but also identify additional molecules governed by Rac-1 contributing to the integrity of this insulin-mediated response in muscle cells.

It is becoming clear that mechanical stimuli are critical in regulating cell biology; however, the short-term structural response of a cell to mechanical forces remains relatively poorly understood. We mechanically stimulated cells expressing actin-EGFP with controlled forces (0-20nN) in order to investigate the cell’s structural response. Two clear force dependent responses were observed: a short-term local deformation of actin stress fibres and a long-term force-induced remodelling of stress fibres at cell edges, far from the point of contact. We were also able to quantify strain dynamics occurring along stress fibres. The cell exhibits complex heterogeneous negative and positive strain fluctuations along stress fibres, indicating localized dynamic contraction and expansion. A ~50% increase in myosin contractile activity is apparent following application of 20nN force. Directly visualizing force-propagation and stress fibre strain dynamics has revealed new information about the pathways involved in mechanotransduction which ultimately govern the downstream response of a cell.

The microfilament system consists of actin filaments as the major component and is regulated by a number of actin binding proteins. It is juxtaposed to the plasma membrane where it forms a dense cortical weave from where it pervades into the cell interior. This filament system has multiple roles and participates in virtually all motile processes where its dynamic activities depend on receptor mediated signaling leading to constant polymerizations and depolymerizations. These activities are now also known to affect gene regulation. This thesis discusses these dynamic reorganizations of the microfilament system and how components are supplied to support these motile processes. The focus is on profilin/profilin:actin, actin polymerization and the localization of the transcripts of these proteins. The localization of profilin mRNA was examined in relation to the distribution of β-actin mRNA using fluorescent in situ hybridization. It was concluded that both these mRNAs localize to sites of massive actin polymerization called dorsal ruffles albeit the data obtained suggests that this localization must be dependent on distinct mechanisms. Additionally signal transduction and cell motility was studied after depleting the two profilin isoforms 1 and 2. The activity of the transcription factor SRF is known to be coupled to microfilament system dynamics via the cofactor MAL which binds to actin monomers and is released upon receptor mediated actin polymerization. Depletion of profilin was seen to influence SRF dependent signaling, most likely because the lack of profilin enables more MAL to bind actin monomers thereby preventing SRF dependent transcription. Finally, it was also investigated how the synaptic vesicle protein synaptotagmin 1 which is involved in exocytosis, has a role in actin polymerization. This protein has previously been described to cause filopodia formation when ectopically expressed. A polybasic sequence motif was identified as the effector sequence for this activity and it was established that this sequence interacts with anionic lipids. It is also discussed how this sequence could have a role in neurotransmitter release and actin polymerization in the nerve synapse. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Submitted.