(E-164) Diversity in Contact Guidance Sensing by Cells of Different Motility Modes

Introduction:: Cell migration is important for many biological processes, including development, immune response, tissue repair and regeneration and cancer metastasis. When an injury occurs, cells in the surrounding tissue are activated to migrate toward the wound site in order to initiate the wound healing process. Cells interpret different directional migrational signals including collagen fibril alignment within the extracellular matrix. Directed migration along aligned fibrils is called contact guidance. There are many ways to generate contact guidance cues including micro-contact printing, creating topographical structures and aligning fibers based on flow or other mechanical processes. While much work has been done to understand how cells sense contact guidance cues, little of this work has been focused on characterizing differences across cells and the mechanisms that cause this difference. In this study, we use different cells with different migration modes to study their response to the contact guidance cues provided by assembling collagen on mica. This is a powerful system to use as it allows for the transfer of these fibrils onto other substrates including glass and flexible substrates like polyacrylamide.

Materials and Methods:: Collagen fibers were assembled on the surface of the mica at two different concentrations: 10 mg/ml or 100 mg/ml collagen in 50 mM Tris-HCl, 200 mM KCl at pH 9.2. to study the cell’s response to the contact guidance. The assembled collagen was tested with AFM to check the pattern of fibers. The cover slides were chemically functionalized for the HFF cells, which were so strong and able to pull off the collagen fibers from the surface of the mica, and then the collagen fibers were transferred to them. The cells were fixed to localize their F-actin and quantify their response to the contact guidance cues. To track the cell’s migration after putting them on the collagen fibers, a chamber was made and filled with media to supply the cell’s needs during the live cell migration.

Results, Conclusions, and Discussions:: Assembling collagen on mica results in aligned collagen fibrils and these fibrils can be successfully transferred to glass substrates (Figure 1A&B). Cells respond to the contact guidance cues and different cells sense the aligned collagen fibril network with different fidelities (Figure 1F). Human foreskin fibroblasts (HFFs) align well after 2 h, but are so contractile that they were able to delaminate the collagen fibrils after 2 h (Figure 1C&D). To overcome this problem, we chemically functionalized glass and transferred the collagen fibrils to these functionalized surfaces. This inhibited the delamination of fibrils, but cells appeared to be less aligned (Figure 1E). Similarly to HFFs, Epidermal keratinocytes (HaCats) also appear to look well aligned with the collagen fibril network (Figure 1G&F). However, there are two populations of HaCats. The first moves perpendicular to the fibrils and the second moves parallel to the fibrils (Figure 1H-J). These results outline the rich and diverse differences in contact guidance among different cells. Understanding this diversity is absolutely critical in designing artificial tissues or advanced wound dressings where directional migration cues are encoded into to biomaterial. Going forward we are examining two important aspects. The first aspect is how cells sense multiple directional cues simultaneously. We will transfer these aligned collagen fibrils onto polyacrylamide substrates with gradients in mechanical properties oriented parallel and perpendicular to the fibrils. The second aspect is how different cytoskeletal mechanisms allow different cells to sense these cues differently. Our hypothesis is that different F-actin networks (branched vs. linear) determine contact guidance behavior. To test this hypothesis, we perturb the system by knocking down or using pharmacological perturbation of formins and Arp2/3, F-actin regulatory proteins that change the balance between linear and branched F-actin networks. Understanding the mechanisms for directed cell migration will allow use to control the migration of a variety of different cells.


Acknowledgements (Optional): : Funding was from the National Institutes of Health under grant number GM143302

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