Cell-Matrix Interactions and Migration

National Institute Of Dental & Craniofacial Research

Investigators

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Abstract

Extracellular matrix molecules, integrin receptors, cadherins, cytoskeletal proteins, and regulators of these molecular systems contribute to cell migration and signaling by complex, integrated mechanisms. We are addressing the following specific questions: 1. What biophysical phenomena and signaling mechanisms are important for efficient cell migration in two-dimensional and three-dimensional environments? 2. How are the functions of integrins, cadherins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce effective cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including live-cell wide-field, confocal, and two-photon time-lapse microscopy with fluorescent protein chimeras, biochemical and signal transduction analyses, as well as methods for evaluating local matrix deformations in response to forces from an individual migrating mammalian cell. We use a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on the functions and regulation of specific integrins and their associated extracellular and intracellular molecules in the mechanisms and spatial governance of cell migration. A series of studies of non-malignant versus cancer cells has been focusing on the biophysical mechanisms of efficient cell migration in 3D collagen matrices. The dynamic interactions of cells as they apply forces to a 3D matrix while migrating was characterized using particle image velocimetry (PIV), which permits quantitative measurements of matrix fibril displacement using confocal microscopy time-lapse movies of normal versus cancer cells. Normal cells undergoing migration apply strong pulling forces to the extracellular matrix originating from the anterior region of the cell, resulting in matrix displacement toward the cell, i.e., an anterior prestrain of the matrix. This local anterior matrix displacement indicates that cells stiffen the matrix in front of them oriented in the direction of ongoing cell migration, which provides pre-tensioning of the extracellular matrix for effective formation and maintenance of cell adhesions. In fact, a laser-mediated cut in the matrix in front of migrating human fibroblasts results in prompt retraction of the leading edge, inhibition of forward migration, and sometimes complete reversal of their direction of migration away from the cut after the matrix slackens and anterior adhesions disassemble. Both fibrosarcoma and metastatic breast cancer cells displayed deficits or defects in this phenomenon of anterior force generation. Anterior prestrain was also strongly diminished after genetic ablation by CRISPR-Cas9 of myosin IIA in primary human fibroblasts, with much less effect of myosin IIB ablation. These studies have established a novel mode of 3D cell migration. In early embryonic development, epithelial cells in organs undergoing branching morphogenesis undergo striking levels of cell migration, which is lost after organs mature. Using 2-photon microscopy, the motility of virtually every cell in a developing mouse embryonic salivary gland was tracked. Cells at the basement membrane had the highest rates of migration, but interior cells also migrated. For cell division, cells moved inward and away from their basement membrane migratory substrate into the cell interior. However, 100% of such dividing cells eventually returned to the cell surface. Most returned within 4 hours, and the remainder returned by 18 hours. This striking but transient motility of embryonic epithelial cells may be important to permit both plasticity for ingression of gland clefts and for cell positional rearrangements to allow cells to sort out and to return to the bud surface. This combined approach involving mechanistic characterization of the regulation of cell migration and phenotypes in various microenvironments should provide novel approaches to understanding, preventing, or ameliorating migratory processes used by cells during abnormal embryonic development, as well as in cancer invasion. An in-depth understanding of the precise manner by which cells move and interact with their extracellular matrix environment, e.g., during embryonic development, should also facilitate tissue engineering studies in future regenerative medicine.