Chemomechanical regulation of integrin activation and cellular processes in acidic extracellular pH
Author(s)Paradise, Ranjani Krishnan
Massachusetts Institute of Technology. Dept. of Biological Engineering.
Krystyn J. Van Vliet and Douglas A. Lauffenburger.
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It is well established that extracellular pH (pHe) becomes acidic in several important physiological and pathological contexts, including the tumor and wound microenvironments. Although it is known that acidic pHe can have profound effects on cell adhesion and migration processes integral to tumor progression and wound healing, the molecular mechanisms underlying the cellular responses to acidic pHe are largely unknown. Transmembrane integrin receptors form a physical linkage between cells and the extracellular matrix, and are thus capable of modulating cell adhesion and migration in response to extracellular conditions. In this thesis, computational and experimental approaches are used to investigate the role of acidic extracellular pH in regulating activation and binding of integrin [alpha]v[beta]3, and to characterize the consequences for downstream subcellular- and cellular-scale processes. Molecular dynamics simulations demonstrate that opening of the integrin [alpha]v[beta]3 headpiece occurs more frequently in acidic pHe than in normal pHe, and that this increased headpiece opening can be partially attributed to protonation of ASP[beta]127 in acidic pHe. These computational data indicate that acidic pHe can promote activation of integrin [alpha]v[beta]3. This is consistent with flow cytometry and atomic force microscope-enabled molecular force spectroscopy experiments, which demonstrate that there are more activated [alpha]v[beta]3 receptors on live [alpha]v[beta]3 CHO-B2 cell surfaces at acidic pHe than at normal pHe 7.4. Put together, these atomistic- and molecular-level data suggest a novel mechanism of outside-in integrin activation regulation by acidic extracellular pH. Next, the consequences of acid-induced integrin activation for subcellular- and cellular-scale processes are investigated. Kymography experiments show that [alpha]v[beta]3 CHO-B2 cell membrane protrusion lifetime is increased and protrusion velocity is decreased for cells in pHe 6.5, compared to cells in pHe 7.4. Furthermore, [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 form more actin-integrin adhesion complexes than cells in pHe 7.4, and acidic extracellular pH results in increased cell area and decreased cell circularity. Cell migration measurements demonstrate that [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 migrate slower than cells in pHe 7.4, and that the fibronectin ligand density required for peak migration speed is lower for cells in pHe 6.5. Together, these data show that acidic pHe affects subcellular- and cellular-scale processes in a manner that is consistent with increased integrin activation in this condition. Finally, the migration behavior of [alpha]v[beta]3 CHO-B2 cells, bovine retinal microvascular endothelial cells, and NIH-3T3 fibroblasts in an extracellular pH gradient is investigated. Results demonstrate that NIH-3T3 fibroblasts do not exhibit directional preferences in the pHe gradient, but that [alpha]v[beta]3 CHO-B2 cells and bovine retinal microvascular endothelial cells migrate preferentially toward the acidic end of the gradient. These data suggest that acidic extracellular pH may serve as a cue that directs migration of angiogenic endothelial cells to poorly vascularized regions of tumors and wounds. Overall, this thesis research results in multiscale, in-depth understanding of extracellular pH as a critical regulator of cell function, with associated implications for tumor growth, wound healing, and the role of proton pumps in cell migration.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student submitted PDF version of thesis.Includes bibliographical references (p. 162-176).
DepartmentMassachusetts Institute of Technology. Dept. of Biological Engineering.
Massachusetts Institute of Technology