In collective cell migration, the motion results from forces produced by each cell and transmitted to the neighboring cells and to the substrate. Because inertia is negligible and the migration occurs over long time scales, the cell layer exhibits viscous behavior, where force and motion are connected by an apparent friction that results from the breaking and forming of adhesive bonds at the cell–cell and cell–substrate interfaces. Most theoretical models for collective migration include an apparent friction to connect force and motion, with many models making predictions that depend on the ratio of cell–cell and cell–substrate friction. However, little is known about factors that affect friction, leaving predictions of many theoretical models untested. Here, we considered how substrate stiffness and the number of adhesions affected friction at the cell–substrate interface. The experimental data were interpreted through prior theoretical models, which led to the same conclusion, that increased substrate stiffness increased the number of cell–substrate adhesions and caused increased cell–substrate friction. In turn, the friction affected the collective migration by altering the curvature at the edge of the cell layer. By revealing underlying factors affecting friction and demonstrating how friction perturbs the collective migration, this work provides experimental evidence supporting prior theoretical models and motivates the study of other ways to alter the collective migration by changing friction.
Vimentin is a Type III intermediate filament (VIF) cytoskeletal protein that regulates the mechanical and migratory behavior of cells. Its expression is considered to be a marker for the epithelial to mesenchymal transition (EMT) that takes place in tumor metastasis. However, the molecular mechanisms regulated by the expression of vimentin in the EMT remain largely unexplored. We created MCF7 epithelial cell lines expressing vimentin from a cumate-inducible promoter to address this question. When vimentin expression was induced in these cells, extensive cytoplasmic VIF networks were assembled accompanied by changes in the organization of the endogenous keratin intermediate filament networks and disruption of desmosomes. Significant reductions in intercellular forces by the cells expressing VIFs were measured by quantitative monolayer traction force and stress microscopy. In contrast, laser trapping micro-rheology revealed that the cytoplasm of MCF7 cells expressing VIFs was stiffer than the uninduced cells. Vimentin expression activated transcription of genes involved in pathways responsible for cell migration and locomotion. Importantly, the EMT related transcription factor TWIST1 was upregulated only in wild type vimentin expressing cells and not in cells expressing a mutant non-polymerized form of vimentin, which only formed unit length filaments (ULF). Taken together, our results suggest that vimentin expression induces a hybrid EMT correlated with the upregulation of genes involved in cell migration.
It is well-established that cells respond to mechanical cues via cytoskeletal remodeling. However, it is not known whether the mechanical environment influences β-cell maturity and function. To examine how mechanical cues influence the β-cell, we used biocompatible substrates with tunable mechanical properties synthesized with elastic moduli ranging from ~3kPa-33 kPa. The surface was coated with extracellular matrix and MIN6 β-like cells were seeded. To independently examine a link between β-cell function and maturity, we examined islets from mice in which β-cells express CaMPARI, a photoconvertible fluorescent protein that in the presence of high Ca2+ activity changes from green to red. For all conditions in MIN6 cells and CAMPARI-expressing islets, we imaged Ca2+ dynamics via Fluo4 at low (2mM) and high glucose (11, 20mM). qPCR was conducted on MIN6 cells (6, 22 kPa) and flow-sorted CaMPARI-expressing β-cells (green, red) to examine expression of genes linked to β-cell function and maturity. With increased substrate stiffness, MIN6 cell Ca2+ oscillations were more robust, including a significant increase in duty cycle (p<0.01) and were more coordinated, consistent with increased excitability. With increasing stiffness, there was significantly decreased expression of Sur1, Kir6.2, Ins2, Pdx1, Fltp, and Ecad (p<0.01). Thus, the dynamics and coordination of Ca2+ activity is mechanoresponsive and may result from altered β-cell maturity. In CaMPARI islets, Ca2+ oscillations were more robust and coordinated for photo-converted red cells which marks cells with higher Ca2+, compared to green cells. In these red cells that show higher Ca2+, qPCR showed significantly increased expression of cFos, but decreased expression of Pdx1 and Ins2, again showing more excitable cells showed altered maturity. In conclusion, our results suggest there may be an inverse relationship between β-cell maturity and function. Further, that the maturity and function of β-cells is responsive to the mechanical environment. Disclosure K.Vazquez: None. R.K.Benninger: None. Funding National Institutes of Health (R01DK102950, R01DK106412)
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