"Topographical and mechanical properties of adhesive substrates provide important biological cues that affect cell spreading, migration, growth, and differentiation. The phenomenon has led to the increased use of topographically patterned and flexible substrates in studying cultured cells. However, these studies may be complicated by various limitations. For example, the effects of ligand distribution and porosity are affected by topographical features of 3D biological constructs. Similarly, many studies of mechanical cues are compounded with cellular deformation from external forces, or limited by comparative studies of separate cells on different substrates. Furthermore, understanding cell responses to mechanical input is dependent upon reliable measurements of mechanical properties. This work addresses each of these issues. To determine how substrate topography and focal adhesion kinase (FAK) affect cell shape and movement, I studied FAK-null (FAK -/-) and wild type mouse 3T3 fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars, I found that, compared to cells on flat surfaces, those on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability, which were dependent on both myosin-II and FAK. To study the dynamic responses to changes in substrate stiffness without other confounding effects, I developed a UV-modulatable substrate that softens upon UV irradiation. As atomic force microscopy (AFM) proved inadequate to detect microscale changes in stiffness, I first developed and validated a microsphere indentation method that is compatible with fluorescence microscopy. The results obtained with this method were comparable to those obtained with AFM. The UV-modulatable substrates softened by ~20-30% with an intensity of irradiation that has no detectable effect on 3T3 cells on control surfaces. Cells responded to global softening of the substrate with an initial retraction followed by a gradual reduction in spread area. Precise spatial control of softening is also possible - while there was little response to posterior softening, anterior softening elicited a pronounced retraction and either a reversal of cell polarity or a significant decrease in spread area if the cells move into the softened region. In conclusion, these techniques provide advances in gaining mechanistic insight into cellular responses to topographical and mechanical cues. Additionally, there are various other potential applications of the novel UV-softening substrate, particularly in regenerative medicine and tissue engineering. "

Cells respond to their mechanical environment by changing shape and size, migrating, or even differentiating to a more specialized cell type. A better understanding of the response of cells to surrounding cues will allow for more targeted and effected designs for biomedical applications, such as disease treatment or cellular therapy. The spreading behavior of both human mesenchymal stem cells (hMSCs) and 3T3 fibroblasts is a function of substrate stiffness, and can be quantified to describe the most visible response to how a cell senses stiffness. The stiffness of the substrate material can be modulated by altering the substrate thickness, and this has been done with the commonly-used linearly elastic gel, polyacrylamide (PA). Though easy to produce and tune, PA gel does not exhibit strain-stiffening behavior, and thus is not as representative of biological tissue as fibrin or collagen gel. Fibroblasts on soft fibrin gel show spreading similar to much stiffer linear gels, indicating a difference in cell stiffness sensing on these two materials. We hypothesize cells can sense further into fibrin and collagen gels than linear materials due to the strain-stiffening material property. The goal of this work is to compare the material response of linear (PA) and strain-stiffening (fibrin, collagen gel) substrates through modulation of effective stiffness of the materials. The two-step approach is to first develop a finite element model to numerically simulate a cell contracting on substrates of different thicknesses, and then to validate the numerical model by quantifying fibroblast spreading on sloped fibrin and collagen gels. The finite element model shows that the effective stiffness of both linear and nonlinear materials sharply increases once the thickness is reduced below 10µm. Due to the strain-stiffening behavior, the nonlinear material experiences a more drastic increase in effective stiffness at these low thicknesses. Experimentally, the gradual response of cell area of HLF and 3T3 fibroblasts on fibrin and collagen gels is significantly different (p<0.05) from these cell types on PA gel. This gradual increase in area as substrate thickness decreases was not predicted by the finite element model. Therefore, cell spreading response to stiffness is not explained by just the nonlinearity of the material.

Living organisms are made up of a multitude of individual cells that are surrounded by biomolecules and fluids. It is well known that cells are highly regulated by biochemical signals; however it is now becoming clear that cells are also influenced by the mechanical forces and mechanical properties of the local microenvironment. Extracellular forces causing cellular deformation can originate from many sources, such as fluid shear stresses arising from interstitial or blood flow, mechanical stretching during breathing or compression during muscle contraction. Cells are able to sense variations in the mechanical properties (elasticity) of their microenvironment by actively probing their surroundings by utilizing specialized proteins that are involved in sensing and transmitting mechanical information. The actin cytoskeleton and myosin-II motor proteins form a contractile (actomyosin) network inside the cell that is connected to the extracellular microenvironment through focal adhesion and integrin sites. The transmission of internal actomyosin strain to the microenvironment via focal adhesion sites generates mechanical traction forces. Importantly, cells generate traction forces in response to extracellular forces and also to actively probe the elasticity of the microenvironment. Many studies have demonstrated that extracellular forces can lead to rapid cytoskeletal remodeling, focal adhesion regulation, and intracellular signalling which can alter traction force dynamics. As well, cell migration, proliferation and stem cell fate are regulated by the ability of cells to sense the elasticity of their microenvironment through the generation of traction forces. In vitro studies have largely explored the influence of substrate elasticity and extracellular forces in isolation, however, in vivo cells are exposed to both mechanical cues simultaneously and their combined effect remains largely unexplored. Therefore, a series of experiments were performed in which cells were subjected to controlled extracellular forces as on substrates of increasing elasticity. The cellular response was quantified by measuring the resulting traction force magnitude dynamics. Two cell types were shown to increase their traction forces in response to extracellular forces only on substrates of specific elasticities. Therefore, cellular traction forces are regulated by an ability to sense and integrate at least two pieces of mechanical information - elasticity and deformation. Finally, this ability is shown to be dependent on the microtubule network and regulators of myosin-II activity.

No description available.

Living organisms are made up of a multitude of individual cells that are surrounded by biomolecules and fluids. It is well known that cells are highly regulated by biochemical signals; however it is now becoming clear that cells are also influenced by the mechanical forces and mechanical properties of the local microenvironment. Extracellular forces causing cellular deformation can originate from many sources, such as fluid shear stresses arising from interstitial or blood flow, mechanical stretching during breathing or compression during muscle contraction. Cells are able to sense variations in the mechanical properties (elasticity) of their microenvironment by actively probing their surroundings by utilizing specialized proteins that are involved in sensing and transmitting mechanical information. The actin cytoskeleton and myosin-II motor proteins form a contractile (actomyosin) network inside the cell that is connected to the extracellular microenvironment through focal adhesion and integrin sites. The transmission of internal actomyosin strain to the microenvironment via focal adhesion sites generates mechanical traction forces. Importantly, cells generate traction forces in response to extracellular forces and also to actively probe the elasticity of the microenvironment. Many studies have demonstrated that extracellular forces can lead to rapid cytoskeletal remodeling, focal adhesion regulation, and intracellular signalling which can alter traction force dynamics. As well, cell migration, proliferation and stem cell fate are regulated by the ability of cells to sense the elasticity of their microenvironment through the generation of traction forces. In vitro studies have largely explored the influence of substrate elasticity and extracellular forces in isolation, however, in vivo cells are exposed to both mechanical cues simultaneously and their combined effect remains largely unexplored. Therefore, a series of experiments were performed in which cells were subjected to controlled extracellular forces as on substrates of increasing elasticity. The cellular response was quantified by measuring the resulting traction force magnitude dynamics. Two cell types were shown to increase their traction forces in response to extracellular forces only on substrates of specific elasticities. Therefore, cellular traction forces are regulated by an ability to sense and integrate at least two pieces of mechanical information - elasticity and deformation. Finally, this ability is shown to be dependent on the microtubule network and regulators of myosin-II activity.

No description available.

Cytoskeletal tension plays an important role in numerous biological functions of adherent cells, including mechanosensing of the cell’s microenvironment, mechanotransduction, cell spreading and migration, cell shape stability, and in stem cell lineage. It is believed that for normal biological functions the cell must maintain its cytoskeletal tension stable, at a preferred set-point level, under external perturbations. This is known as tensional homeostasis. Any breakdown of tensional homeostasis is closely associated with disease progression, including cancer, atherosclerosis, and thrombosis. The exact mechanism and the relevant environmental conditions for the maintenance of tensional homeostasis are not yet fully understood. This thesis investigates the impacts of substrate stiffness, availability of functional cadherin junctions and steady shear stress on tensional homeostasis of cells and cell clusters. We define tensional homeostasis as the ability of cells to maintain a consistent level of tension with low temporal traction field fluctuations. Traction forces of isolated cells, multicellular clusters, and monolayer are measured using micropattern traction microscopy. Temporal fluctuations of the traction field are calculated from time-lapsed traction measurements. Results demonstrated that substrate stiffness, cadherin cell-cell junctions and shear stress all impact tensional homeostasis. In particular, we found that stiffer substrates promoted tensional homeostasis in endothelial cells, but were detrimental to tensional homeostasis in vascular smooth muscle cells. We also found that E-cadherins were essential for tensional homeostasis of gastric cancer cells and that extracellular and intracellular mutations of E-cadherin had domain-specific effects on tensional homeostasis. Finally, laminar flow-induced shear stress led to increased traction field fluctuations in endothelial cell monolayers, contrary to reports of physiological shear promoting vascular homeostasis. A possible reason for this discrepancy might be the limitation of our approach which could not account for mechanical balance of traction forces in the monolayers. Through the exploration of these environmental factors, we also found that tensional homeostasis was a length scale-dependent and cell type-dependent phenomenon. These insights suggest that future studies need to take a more comprehensive approach and aim to make observations of different cell types on multiple length scales, in order decipher the mechanism of tensional homeostasis and its role in (patho)physiology. / 2021-08-30T00:00:00Z

Cell-substrate interactions are important to study for development of accurate in vitro research platforms.  Recently it has been demonstrated that physical microenvironment of cells directly affects cellular motility and cytoskeletal arrangement.  Specifically, previous studies have explored the role of material stiffness (Young's modulus: N/m2) on cell behavior including attachment, spreading, migration, cytoskeleton arrangement (stress fiber and focal adhesion distribution) and differentiation. In this study using our recently described non-electrospinning fiber manufacturing platform, customized scaffolds of suspended nanofibers are developed to study single cell behavior in a tunable structural stiffness (N/m) environment.  Suspended fibers of three different diameters (400, 700 and 1200 nm) are deposited in aligned configurations in two lengths of 1 and 2 mm using the previously described STEP (Spinneret based Tunable Engineered Parameters) platform.  These fibers present a gradient of structural stiffness to the cells at constant material stiffness.   Single cells attached to fibers are constrained to move along the fiber axis and with increase in structural stiffness are observed to spread to longer lengths, put out longer focal adhesions, have elongated nucleus with decreased migration rates. Furthermore, more than 60% of cell population is observed to migrate from areas of low to high structural stiffness. Additionally dividing cells are observed to round up and daughter cells are observed to migrate away from each other after division. Interestingly, dividing rounded cells are found to be anchored to the fibers through thin protrusions emanating from the focal adhesion sites. These results indicate a substrate stiffness sensing mechanism that goes beyond the traditionally accepted modulus sensing that cells have been shown to respond to previously.  From this work, the importance of structural stiffness in cellular mechanosensing at the single cell-nanofiber scaled warrants consideration of the above factors in accurate design of scaffolds in future. / Master of Science

One out of eight women in the United States will develop breast cancer during their lifetime. Ninety percent of cancer related deaths are due to metastasis. Metastasis is the biological process where individual or aggregate cancerous cells break away from the primary tumor site and colonize distant, non-adjacent locations throughout the body. It is my objectives to study how mechanical, topographical and biochemical cues affect metastatic breast cancer metastasis at an early developmental stage. ECM components have previously been shown to affect cell motility via ligand-receptor interactions, and physical cues, such as matrix stiffness and protein density. The primary tumor site significantly stiffens during tumor progression. The ability cells have to sense and respond to these matrix features influences and facilitates cell invasion. It is now widely accepted that mechanical properties of the ECM can regulate cell migration; however, presently, tissue modulus and stiffness have been used interchangeably. It is unknown if cell responses are sensitive to a bulk tissue modulus or stiffness on the geometric length scale of the cell. It is my objective to create tunable biomaterials from known materials to independently parse the roles of stiffness and modulus upon the migration of breast cancer cells. I have created a variety of tunable biomaterials which I can parse the roles of mechanical properties and observe their affect upon cell mechanosensing. All systems were coated with collagen I, which is the most abundant ECM protein during tumor development. I was able to quantify the migration along with other parameters of the metastatic breast cancer cell line MDA-MB-231. My results show that the highly metastatic MDA-MB-231 is stiffness sensitive among all biomaterial models. Cells maximum cell speeds are at high concentrations of collagen I on the polymer microlenses and show a biphasic response dependent on stiffness. On poly (ethylene glycol)- 2-Methacryloyloxyethyl phosphorylcholine (PEG-PC) hydrogels cells favor intermediate modulus and show stiffness dependency at low protein concentrations. Cells on Cd/Se and polydimethylsiloxane (PDMS) samples are influenced by the topographical cue more so than the stiffness or modulus of the material. By controlling mechanosensing via force transduction signaling pathways, and determining the appropriate length-scale by which mechanical properties regulate cancer metastasis, I hope to eventually uncover novel therapeutics to block cell invasion.

Pseudomonas Aeruginosa is an opportunistic bacterium which is involved in nosocomial infections and which causes increasing concern in healthcare due to its high antibiotic resistance. During an infection by P.aeruginosa, the bacteria proliferate in the host’s organism by leveraging motility abilities. One of them, twitching motility, is a surface-specific translocation system. To power twitching, P.aeruginosa performs cycles of extension, attachment, and retraction of Type IV pili (T4P), which are long and thin extracellular filaments. On top of allowing cellular traction, T4P can transmit a signal induced by a contact with a solid surface leading to specific biological responses. In particular, we suspect that T4P retraction is triggered in very short timescales by the attachment of the tip of the pilus to a surface. However, the nature of the signal generated by surface contact, and how it is sensed by the cell, are unknown. The aim of this master project is to gain knowledge on how this machinery is coordinated by the cell upon solid surface contact, and particularly investigate the signal induced by surface contact.  To study T4P behavior during extension and retraction, we used label-free interferometric scattering microscopy (iSCAT), a high time and spatial resolution microscopy technology. We aimed at bringing out T4P movements by attaching bacteriophages on their body and tracking their relative position over extension and retraction events. To do so, we first fabricated microstructures in which we confined the cells in order to improve image quality and increase the odds of binding bacteriophages to T4P. Then, we were able to visualize bindings of DMS3 bacteriophages on T4P. During extension and retraction, we identified phage lateral movements around the pilus axis, which we were unable to see on non-retracting pili. Our results therefore support the hypothesis of T4P helical movements. The nature of the signal generated by tip contact and how it is sensed by the cell remains to be elucidated. Nevertheless, we elevated iSCAT to a new application by visualizing the interaction between T4P and bacteriophages.