Journal: Scientific Reports

Article Title: Epithelial layer unjamming shifts energy metabolism toward glycolysis

doi: 10.1038/s41598-020-74992-z

Figure Lengend Snippet: Cell redox potential decreases upon epithelial layer unjamming. Top panels: prior to lifting the PDMS barrier, cells are confined and jammed. ( a ) The cell migration speed shows that very little migration is occurring in the layer. ( b ) Traction forces are low throughout the layer but show an increased edge-effect near the PDMS barrier. ( c , d ) Cells are uniformly small in area and round in shape. ( e , f ) The cytoplasmic redox potential (NAD + /NADH) measured via the Peredox biosensor is high throughout the cell layer. Middle panels: 4 h after lifting the PDMS barrier, cells near the layer edge begin to migrate into the free space and the layer expands. Cell migration speeds are increased near the advancing edge. Traction forces are elevated throughout the layer and a steep gradient appears from the leading edge into the bulk. Cell area is dramatically increased and cell shapes become elongated near the advancing edge. The cell redox potential decreases at the leading edge. Bottom panels: 24 h after lifting the PDMS barrier, the layer has expanded to nearly twice the extent of the confined layer. Migration speeds at the leading edge continue to increase as the cell layer migrates into the free space. Traction forces are elevated at the migrating front and cells have substantially expanded in area and elongated in shape. The cytoplasmic redox potential in the migrating cells remains low relative to the jammed cells near the center of the layer. Error bars represent the standard deviation of the mean of the fluorescence ratio which are subsequently transformed to a redox potential using the Peredox fluorescence response curve (Supplementary Fig. ). As the calibration is non-linear, the conversion results in asymmetric error bars. Figure generated with MATLAB R2017b, https://www.mathworks.com/products/matlab.html .

Article Snippet: Cell morphology is derived using built-in MATLAB region-property functions of a Voronoi tessellation generated from the nuclei centroid positions.

Techniques: Migration, Standard Deviation, Fluorescence, Transformation Assay, Generated

Journal: Scientific Reports

Article Title: Epithelial layer unjamming shifts energy metabolism toward glycolysis

doi: 10.1038/s41598-020-74992-z

Figure Lengend Snippet: Fluorescence Lifetime Imaging Microscopy (FLIM) reveals that a shift toward glycolytic metabolism occurs in the unjamming cell layer. ( a ) Composite color maps of the mean NADH lifetime in the non-migrating cell layer confined by the PDMS barrier and at timepoints 4 h and 24 h after barrier removal. Prior to barrier lifting, cells have a uniform mean NADH lifetime throughout the layer. At 4 h after barrier lifting, the layer begins to expand and the mean NADH lifetime is reduced. After 24 h, a complex spatial gradient emerges across the expanding cell layer. Cells at the unjammed and advancing front of the cell layer have a drastically reduced mean NADH lifetime relative to cells in the non-migrating layer. Similarly, cells in the jammed bulk show a decreased mean NADH lifetime relative to the pre-barrier and 4 h timepoints. ( b ) We plot traces of the mean NADH lifetime prior to barrier lift and at 4 h and 24 h after barrier lift. The data is averaged along the entire y-axis and into bins along the x-axis. Error bars show layer-to-layer standard deviations of the mean NADH lifetimes. ( c ) Composite colormaps of the ratio of free to bound NADH in the non-migrating cell layer confined by the PDMS barrier and at timepoints 4 h and 24 h after barrier removal. Prior to barrier lift, the free-to-bound NADH ratio is uniform and low throughout the cell layer. At 4 h after barrier lifting, the layer shifts towards a higher fraction of free NADH. After 24 h, cells near the leading edge as well as cells in the bulk have shifted significantly towards a high fraction of free NADH. ( d ) We plot traces of the fraction of free-to-bound NADH prior to barrier lift and at 4 h and 24 h after barrier lift. The data is averaged along the entire y-axis and into bins along the x-axis. Error bars show layer-to-layer standard deviations of the free-to-bound NADH ratio. Figure generated with MATLAB R2017b, https://www.mathworks.com/products/matlab.html .

Article Snippet: Cell morphology is derived using built-in MATLAB region-property functions of a Voronoi tessellation generated from the nuclei centroid positions.

Techniques: Fluorescence, Imaging, Microscopy, Generated

Journal: Scientific Reports

Article Title: Epithelial layer unjamming shifts energy metabolism toward glycolysis

doi: 10.1038/s41598-020-74992-z

Figure Lengend Snippet: Glucose uptake per cell increases everywhere while mitochondrial membrane potential increases only near the advancing edge. To investigate the extent to which cells within the layer shift toward glycolysis, we employ the glucose uptake assay by imaging the epithelial cell layer after incubation with 2-NBDG, a fluorescent non-catabolizable glucose analog that accumulates within cells after uptake. ( a ) Representative fluorescence images of the 2-NBDG signal are shown at timepoints prior to barrier lifting and at 4 h and 24 h after barrier lifting. Cells confined by the PDMS barrier have a uniform and low 2-NBDG intensity throughout the layer. The PDMS barrier appears bright in the image as 2-NBDG tends to non-specifically adhere to the cut PDMS edge. At 4 h and 24 h after barrier lifting, the 2-NBDG intensity progressively increases in the jammed interior of the cell layer and remains low at the advancing edge. ( b ) We normalize the 2-NBDG fluorescence intensity of all three timepoints relative to the 2-NBDG intensity at the center of the confined cell layer of the pre-barrier lift condition (inset). Additionally, the average 2-NDBG fluorescence intensity across the cell layer is normalized by the average cell area measurements from tessellations of the Peredox cell nucleus positions. This reveals that on a per-cell basis, prior to barrier lifting, the 2-NBDG intensity is uniform and low. At timepoints after barrier lifting, 2-NDBG uptake per cell, relative to the confined layer, becomes progressively elevated. The 2-NBDG intensity is dramatically increased near the advancing front. To investigate the mitochondrial membrane potential in cells throughout the epithelial cell layer, we use TMRE, a membrane potential dependent dye, imaged on a fluorescence microscope. ( c ) Representative fluorescence images of the TMRE intensity are shown at timepoints prior to barrier lifting and at 4 h and 24 h after barrier lifting. Prior to barrier lifting, TMRE fluorescence intensity across the layer remains low and uniform. At 4 h after barrier lifting, the TMRE signal remains low in the jammed bulk but becomes more intense near the migrating edge. After 24 h, the TMRE signal remains elevated only in the cells at and immediately behind the leading edge. ( d ) Composite traces of the TMRE image intensity for data prior to barrier lift and at 4 h and 24 h after barrier lift. TMRE intensity is observed to increase after barrier lift near the leading edge indicating an increased mitochondrial membrane potential in migrating cells, but remains low everywhere else. Cell layers treated with FCCP show a significant reduction in TMRE signal, indicating a drop in mitochondrial membrane potential as expected. Interestingly, the drop in the TMRE signal in the confined and jammed layer is significantly larger than the drop in the migrating cell layers. Figure generated with MATLAB R2017b, https://www.mathworks.com/products/matlab.html .

Article Snippet: Cell morphology is derived using built-in MATLAB region-property functions of a Voronoi tessellation generated from the nuclei centroid positions.

Techniques: Membrane, Imaging, Incubation, Fluorescence, Microscopy, Generated