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Impact of ECM stiffness on mitochondrial calcium influx. (A, C) Live imaging time-lapse series capturing the intensity of calcium-specific biosensors within HeLa cells: pCMV CEPIA2 mt for mitochondrial calcium (green) and pCMV R-CEPIA1er for endoplasmic reticulum (ER) calcium (red), post-treatment with 1 µM ionomycin on <t>substrates</t> with 1 and 40 kPa stiffness. Ionomycin was administered 5 min after initiating the imaging process, with only the media and cells present in the confocal dish during the initial 5 min. The accompanying color scale bar represents calcium levels, with red indicating high and blue denoting low calcium concentrations (scale bar = 10 µm). (B, D) Quantitative analysis of the time courses of normalized emission intensity from the pCMV CEPIA2 mt and pCMV R-CEPIA1er biosensors in HeLa cells treated with 1 µM ionomycin on substrates with 1 and 40 kPa stiffness. Each line represents the mean value of normalized emission intensity for a group of 10 cells (n = 10), and the error bars reflect the standard error of the mean (SEM), illustrating the dynamic calcium changes in response to ECM stiffness and ionomycin treatment.
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Impact of ECM stiffness on mitochondrial calcium influx. (A, C) Live imaging time-lapse series capturing the intensity of calcium-specific biosensors within HeLa cells: pCMV CEPIA2 mt for mitochondrial calcium (green) and pCMV R-CEPIA1er for endoplasmic reticulum (ER) calcium (red), post-treatment with 1 µM ionomycin on <t>substrates</t> with 1 and 40 kPa stiffness. Ionomycin was administered 5 min after initiating the imaging process, with only the media and cells present in the confocal dish during the initial 5 min. The accompanying color scale bar represents calcium levels, with red indicating high and blue denoting low calcium concentrations (scale bar = 10 µm). (B, D) Quantitative analysis of the time courses of normalized emission intensity from the pCMV CEPIA2 mt and pCMV R-CEPIA1er biosensors in HeLa cells treated with 1 µM ionomycin on substrates with 1 and 40 kPa stiffness. Each line represents the mean value of normalized emission intensity for a group of 10 cells (n = 10), and the error bars reflect the standard error of the mean (SEM), illustrating the dynamic calcium changes in response to ECM stiffness and ionomycin treatment.
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Impact of ECM stiffness on mitochondrial calcium influx. (A, C) Live imaging time-lapse series capturing the intensity of calcium-specific biosensors within HeLa cells: pCMV CEPIA2 mt for mitochondrial calcium (green) and pCMV R-CEPIA1er for endoplasmic reticulum (ER) calcium (red), post-treatment with 1 µM ionomycin on substrates with 1 and 40 kPa stiffness. Ionomycin was administered 5 min after initiating the imaging process, with only the media and cells present in the confocal dish during the initial 5 min. The accompanying color scale bar represents calcium levels, with red indicating high and blue denoting low calcium concentrations (scale bar = 10 µm). (B, D) Quantitative analysis of the time courses of normalized emission intensity from the pCMV CEPIA2 mt and pCMV R-CEPIA1er biosensors in HeLa cells treated with 1 µM ionomycin on substrates with 1 and 40 kPa stiffness. Each line represents the mean value of normalized emission intensity for a group of 10 cells (n = 10), and the error bars reflect the standard error of the mean (SEM), illustrating the dynamic calcium changes in response to ECM stiffness and ionomycin treatment.

Journal: Animal Cells and Systems

Article Title: ECM stiffness regulates calcium influx into mitochondria via tubulin and VDAC1 activity

doi: 10.1080/19768354.2024.2393811

Figure Lengend Snippet: Impact of ECM stiffness on mitochondrial calcium influx. (A, C) Live imaging time-lapse series capturing the intensity of calcium-specific biosensors within HeLa cells: pCMV CEPIA2 mt for mitochondrial calcium (green) and pCMV R-CEPIA1er for endoplasmic reticulum (ER) calcium (red), post-treatment with 1 µM ionomycin on substrates with 1 and 40 kPa stiffness. Ionomycin was administered 5 min after initiating the imaging process, with only the media and cells present in the confocal dish during the initial 5 min. The accompanying color scale bar represents calcium levels, with red indicating high and blue denoting low calcium concentrations (scale bar = 10 µm). (B, D) Quantitative analysis of the time courses of normalized emission intensity from the pCMV CEPIA2 mt and pCMV R-CEPIA1er biosensors in HeLa cells treated with 1 µM ionomycin on substrates with 1 and 40 kPa stiffness. Each line represents the mean value of normalized emission intensity for a group of 10 cells (n = 10), and the error bars reflect the standard error of the mean (SEM), illustrating the dynamic calcium changes in response to ECM stiffness and ionomycin treatment.

Article Snippet: To determine the stiffness differences between the 1 and 40 kPa substrates, we initially employed Cyto-FAK (Addgene #78300) to observe focal adhesion kinase (FAK) activity across the two stiffness conditions (Supplementary Figure S1), as described by Seong et al. ( ).

Techniques: Imaging