Selective Desensitization of Growth Factor Signaling by Cell Adhesion to Fibronectin

Cell adhesion to the extracellular matrix is required to execute growth factor (GF)-mediated cell behaviors, such as proliferation. A major underlying mechanism is that cell adhesion enhances GF-mediated intracellular signals, such as extracellular signal-regulated kinase (Erk). However, because GFs use distinct mechanisms to activate Ras-Erk signaling, it is unclear whether adhesion-mediated enhancement of Erk signaling is universal to all GFs. We examined this issue by quantifying the dynamics of Erk signaling induced by epidermal growth factor, basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) in NIH-3T3 fibroblasts. Adhesion to fibronectin-coated surfaces enhances Erk signaling elicited by epidermal growth factor but not by bFGF or PDGF. Unexpectedly, adhesion is not always a positive influence on GF-mediated signaling. At critical subsaturating doses of PDGF or bFGF, cell adhesion ablates Erk signaling; that is, adhesion desensitizes the cell to GF stimulation, rendering the signaling pathway unresponsive to GF. Interestingly, the timing of growth factor stimulation proved critical to the desensitization process. Erk activation significantly improved only when pre-exposure to adhesion was completely eliminated; thus, concurrent stimulation by GF and adhesion was able to partially rescue adhesion-mediated desensitization of PDGF- and bFGF-mediated Erk and Akt signaling. These findings suggest that adhesion-mediated desensitization occurs with rapid kinetics and targets a regulatory point upstream of Ras and proximal to GF receptor activation. Thus, adhesion-dependent Erk signaling is not universal to all GFs but, rather, is GF-specific with quantitative features that depend strongly on the dose and timing of GF exposure

DIRECTING CELLULAR TRAFFIC USING GEOMETRIC AND BIOMOLECULAR CUES

continued support and guidance throughout my graduate studies. His boundless knowledge, sharp insights and pursuit for excellence have contributed tremendously in the making of the exciting projects discussed in this thesis. It was a pleasure to be able to work with him on the forefront of science and to be given the opportunity to push the envelope in a new field of research. I would also like to thank other great faculty and staff, whose support were invaluable in making my life at Caltech professionally rewarding. I would like to start by thanking my thesis committee members, Prof. David Tirrell, Prof. Paul Sternberg and Prof. Marianne Bronner-Fraser, for their friendliness, encouragement and useful advice. I would also like to thank Prof. Chin-lin Guo for his friendship and expertise. In addition, I would like to thank Guy DeRose, Alireza Ghaffari, Christina Morales and Saurabh Vyawahare for facilitating my experience at Kavli Nanoscience Institute (KNI) at Caltech. Lastly, I would like to show my appreciation to the wonderful ladies, Kathy Bubash, Laura King, Martha Hepworth and Alison Ross, for taking care of various administrative issues and fixing problems in the lab

Directing Cellular Traffic Using Geometric and Biomolecular Signal Alterations

Directed cell migration plays a principal role in various aspects of important cellular phenomena such as wound healing, development and cancer metastasis. Although the mechanism of gradient stimulus leading to directed cell migration is well understood and exploited, the geometrical and topographical cues that cause directed migration has been largely unexplored. With the advent of accessible microfabrication techniques to precisely control the topography of the extracellular matrix (ECM) on substrates, researchers are just starting to study the complex mechanical signals that can alter directed cell motility. A key challenge now is to parse out the precise factors that affect directional movement of cells on certain micropatterns, use that understanding to design strategies to enhance the motility and bias of directed cell migration, and further apply these concepts to multiple cell types and higher-order cell systems. Here, we investigate the tunability of directional bias through various geometrical manipulations using quantitative analysis of cell movement on micropatterns. We observe that MCF-10A epithelial cells in general jump with an unnaturally high bias between teardrop-based islands with specific gap distance, asymmetry and positional placement. Throughout the studies, we observe that lamellipodial protrusions and unilamellar morphology play a crucial role in dictating not only the directional bias of epithelial cells, but also their speed and persistence, and find that moderate alteration of Rac1 signal leads to an unexpected flip of bias. We further extend the concept of directional bias to design patterns to successfully control cell flux and effectively partition cell population, as well as induce unilamellar morphology in different cell types to promote directed cell motility. We also investigate the combinatorial effect of hybrid micropatterns in enhancing motility and unravel the unique properties and possible mechanisms behind directed cell motility on teardrop-based micropatterns. Our results demonstrate a new type of directed cell motility using a micropattern that involves the use of physical constraints to stabilize the unilamellar morphology and guidance of the unilamella in the correct direction through purely geometrical cues. These studies offer multiple design strategies to modulate the cell motility and directional bias on micropatterns for various applications, such as tissue engineering.</p

Reprogramming Directional Cell Motility by Tuning Micropattern Features and Cellular Signals

Simple geometrical constraints of micropatterned substrates can be used to manipulate the direction and directional persistence of motile cells (scale bar: 20 μm). Modulation of the directional bias is demonstrated by altering pattern parameters and signal pathways

Modular Design of Micropattern Geometry Achieves Combinatorial Enhancements in Cell Motility

Basic micropattern shapes, such as stripes and teardrops, affect individual facets of cell motility, such as migration speed and directional bias, respectively. Here, we test the idea that these individual effects on cell motility can be brought together to achieve multidimensional improvements in cell behavior through the modular reconstruction of the simpler “building block” micropatterns. While a modular design strategy is conceptually appealing, current evidence suggests that combining environmental cues, especially molecular cues, such as growth factors and matrix proteins, elicits a highly nonlinear, synergistic cell response. Here, we show that, unlike molecular cues, combining stripe and teardrop geometric cues into a hybrid, spear-shaped micropattern yields combinatorial benefits in cell speed, persistence, and directional bias. Furthermore, cell migration speed and persistence are enhanced in a predictable, additive manner on the modular spear-shaped design. Meanwhile, the spear micropattern also improved the directional bias of cell movement compared to the standard teardrop geometry, revealing that combining geometric features can also lead to unexpected synergistic effects in certain aspects of cell motility. Our findings demonstrate that the modular design of hybrid micropatterns from simpler building block shapes achieves combinatorial improvements in cell motility. These findings have implications for engineering biomaterials that effectively mix and match micropatterns to modulate and direct cell motility in applications, such as tissue engineering and lab-on-a-chip devices

Slope-Dependent Cell Motility Enhancements at the Walls of PEG-Hydrogel Microgroove Structures

In recent years, research utilizing micro- and nanoscale geometries and structures on biomaterials to manipulate cellular behaviors, such as differentiation, proliferation, survival, and motility, have gained much popularity; however, how the surface microtopography of 3D objects, such as implantable devices, can affect these various cell behaviors still remains largely unknown. In this study, we discuss how the walls of microgroove topography can influence the morphology and the motility of unrestrained cells, in a different fashion from 2D line micropatterns. Here adhesive substrates made of tetra­(polyethylene glycol) (tetra-PEG) hydrogels with microgroove structures or 2D line micropatterns were fabricated, and cell motility on these substrates was evaluated. Interestingly, despite being unconstrained, the cells exhibited drastically different migration behaviors at the edges of the 2D micropatterns and the walls of microgroove structures. In addition to acquiring a unilamellar morphology, the cells increased their motility by roughly 3-fold on the microgroove structures, compared with the 2D counterpart or the nonpatterned surface. Immunostaining revealed that this behavior was dependent on the alignment and the aggregation of the actin filaments, and by varying the slope of the microgroove walls, it was found that relatively upright walls are necessary for this cell morphology alterations. Further progress in this research will not only deepen our understanding of topography-assisted biological phenomena like cancer metastasis but also enable precise, topography-guided manipulation of cell motility for applications such as cancer diagnosis and cell sorting

Lectin-Tagged Fluorescent Polymeric Nanoparticles for Targeting of Sialic Acid on Living Cells

In this study, we fabricated lectin-tagged fluorescent polymeric nanoparticles approximately 35 nm in diameter using biocompatible polymers conjugated with lectins for the purpose of detecting sialic acid on a living cell surface, which is one of the most important biomarkers for cancer diagnosis. Through cellular experiments, we successfully detected sialic acid overexpression on cancerous cells with high specificity. These fluorescent polymeric nanoparticles can be useful as a potential bioimaging probe for detecting diseased cells