This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP).
Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can benefit the scientific community. This paper introduces the complete operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled “Automatic Multi-functional Integration Program (AMFIP),” which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages.
This integrated system is applied to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in CRISPR/Cas9-engineered human normal cells (B2B) and lung cancer cells (PC9). Leveraging this system’s capability of multi-channel control and readout, the result shows: (1) B2B normal cells and PC9 cancer cells show a distinct relationship between YAP expression, traction, and cell dynamics during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-functional imaging and analysis to elucidate YAP mechano-sensitivity. These tools open the possibility for detailed explorations of multifaceted signaling dynamics in the context of cell physiology and pathology.
The overall goal of this method is to enable all-optical multi-functional imaging and analysis of living cells. An all-in-one imaging program that enables the automatic coordination of multi-functional optoelectronic devices will reduce labor-intensive and error-prone manual operations and is essential for researchers to conduct long-term live-cell imaging1,2,3,4. However, most existing public programs in the biomedical research community either only apply to limited optoelectronic devices or require additional hardware for the coordination of different equipment5,6,7,8,9. Recently, an open-source and software-based program has been developed, titled "Automatic Multi-functional Integration Program (AMFIP)," enabling multi-channel and time-lapse imaging. Based on Java language and the Application Programming Interface (API) of µManager11,12, AMFIP was developed as a plugin in µManager that executes customized Java scripts to accomplish software-based communications of multiple optoelectronic hardware and software platforms, including but not limited to those from Nikon. The establishment of AMFIP opens the possibility for programmable and multi-functional interrogation of cell behaviors. An integrated experimental and computational system is developed in this paper and combines AMFIP with digital imaging analysis and cell traction force microscopy. The system enables the elucidation of the distinct YAP mechanobiology in CRISPR/Cas9-engineered human normal B2B (Figure 1) and lung cancer PC9 (Figure 2) cell lines. The system provides the scientific community a comprehensive solution that avoids the demand of purchasing additional controlling devices that may not be available for and/or compatible with every imaging system.
The protocols presented in this paper introduce how to (1) apply AMFIP to conduct automatic long-term imaging for both CRISPR/Cas9-engineered cell lines that express mNEonGreen2-tagged YAP; and (2) combine Fiji ImageJ, MATLAB, and Origin for the quantitative analysis of YAP nuclear/cytoplasm (N/C) ratio based on their fluorescent intensity (Figure 3 and Figure 4), cellular displacement field (Figure 1C and Figure 2C), and cellular traction field (Figure 1D and Figure 2D). The results suggest that (1) during the first 10 h of cell spreading on the substrates that have physiologically relevant mechanical stiffness13,14,15,16,17,18, the YAP N/C ratio of single B2B cells shows more noticeable time-dependent variation and fluctuation compared to that of single PC9 cells (Figure 5 and Figure 6); and (2) PC9 cancer cells generate noticeable traction in their peri-nuclear regions (Figure 7). The integrated system and methodologies described in this protocol transcend the specific types of cells and optogenetic molecules. Researchers can apply the protocols to customize their specific live-cell interrogation experiments and elucidate multifaceted signaling dynamics in the context of cell physiology and pathology.
1. Generation of stable CRISPR/Cas9-edited human lung cancer cell line (PC9) and human bronchial epithelial cell line (Beas2B) that endogenously express mNeonGreen21-10/11-tagged YAP protein
2. Maintenance of PC9 and B2B cells
3. Setup of hardware and software environment
4. Gel preparation
5. Cell culture
NOTE: Perform cell culture using aseptic technique.
6. Cell imaging
NOTE: AMFIP enables automatic, multi-channel, and long-term imaging by coordinating with different hardware and software systems: (1) AMFIP manipulates µManager to automatically move the motorized stage of the Ti2-E microscope to multiple fields-of-view (FOVs) and acquire bright-field images through a monochrome camera (Table of Materials); and (2) AMFIP activates multiple macro files inside Elements with a customized Java script to accomplish automatic operations for confocal z-stack imaging and the switching of different laser channels (405 nm and 488 nm).
7. Measurement of the YAP N/C ratio
8. Measurement of traction field
Distinct YAP distribution and dynamics in CRISPR/Cas9-engineered PC9 cancer and B2B normal cells during cell spreading
Representative fluorescence images of YAP distribution in single B2B and PC9 cells on 2, 5, 40 kPa PAA gels and glass coverslip are shown in Figure 1A and Figure 2A. The nuclear localization of YAP in B2B cells increased with increasing substrate stiffness (Figure 1A), whereas PC9 cells showed similar YAP concentration in the nucleus and cytoplasm on substrates of varying stiffness (Figure 2A). Representative fluorescence images of YAP distribution in single, spreading B2B and PC9 cells on the 5 kPa hydrogel substrate (from the 0th h to the 10th h after the cells attached to the substrates) are shown in Figure 1B and Figure 2B, respectively. The B2B cell monotonically increased the spread area over time along with a decrease in the YAP N/C ratio (Figure 1B), while the PC9 cell maintained a comparatively unchanging cell spread area, orientation, and YAP N/C ratio throughout the 10 h spreading process (Figure 2B). During the 10 h duration of early spreading, the representative B2B cell constitutively deformed the substrate surface and applied time-evolving cell traction across the whole cell area (Figure 1C and Figure 1D).
In contrast, the representative PC9 cell only developed displacement and traction at the two ends of the cell body and its traction diminished after 7.5 h (Figure 2C and Figure 2D). More time-lapse images and traction measurements of B2B and PC9 cells at the early spreading stage are provided in Supplemental Figure S2 and Supplemental Figure S3. Other modes of PC9 cell dynamics were also observed (Figure 6). In parallel to these different spreading characteristics, B2B and PC9 cells showed distinct YAP distribution and dynamics (Figure 3). On a 5 kPa gel, YAP in B2B cells was concentrated in the nucleus at the 0th h and became more homogeneously distributed across the cell body at the 10th h. However, PC9 cells showed a more homogeneous distribution of YAP in the nucleus and the cytoplasm throughout the entire 10 h of the spreading process. To quantitatively analyze the YAP activity and translocation in B2B and PC9 cells, the YAP N/C ratio was calculated using the algorithm described in Figure 4.
To further investigate the distinct YAP dynamics, temporal changes in YAP N/C ratio, cell/nucleus area, and traction of multiple single B2B cells (n = 10) and PC9 cells (n = 5) were compared (Figure 5). It was found that the average YAP N/C ratio of B2B cells decreased from 2.54 ± 0.22 to 1.79 ± 0.21 (n = 10; p = 0.0022**; Figure 5A), while the average YAP N/C ratio of PC9 cells changed from 1.92 ± 0.26 to 1.57 ± 0.07 (n = 5; p = 0.187 (not significant (ns)); Figure 5A). The average dipole traction of B2B cells changed from 256.17 ± 123.69 nN to 287.44 ± 99.79 nN (p = 0.7593 (ns); Figure 5B). The average dipole traction of PC9 cells changed from 141.19 ± 33.62 nN to 168.52 ± 73.01 nN (p = 0.7137 (ns); Figure 5B). The average cell spread area of B2B cells increased from 613.89 ± 102.43 µm2 to 942.51 ± 226.71 µm2 (p = 0.0512 (ns); Figure 5C).
The average cell spread area of PC9 cells changed from 495.78 ± 97.04 µm2 to 563.95 ± 89.92 µm2 (p = 0.5804 (ns); Figure 5C). The average nucleus spread area of B2B cells increased from 181.55 ± 36.18 µm2 to 239.38 ± 43.12 µm2 (p = 0.1217 (ns); Figure 5D) and the average nucleus spread area of PC9 cells changed from 133.31 ± 30.05 µm2 to 151.93 ± 22.49 µm2 (p = 0.5944 (ns); Figure 5D). These results suggest that (1) B2B cells show a constitutively substrate-stiffness-dependent YAP N/C ratio; (2) the traction of B2B cells is higher than that of PC9 cells; and (3) in contrast to B2B cells, PC9 cells show a limited increase in cell area and changes in YAP N/C ratio during the 10 h spreading process.
Correlation of YAP distribution and dynamics to the migration states of B2B cells
YAP N/C ratio and dipole traction of all B2B (n=10) and PC9 (n=5) cells as a function of cell spread area and nucleus spread area were compared. The YAP N/C ratio and dipole traction of PC9 cells did not clearly correlate with their small cell and nucleus spread area ranges (Figure 6). In contrast, the YAP N/C ratio and dipole traction of B2B cells appeared to follow two distinct trends (Figure 6A and Figure 6C), suggesting that there might be two groups of B2B cells that co-exist in this experiment. In the first group, the YAP N/C ratio and dipole traction increase along with the enlargement of the cell spread area and reach their maxima at ~ 1000 µm2 (Figure 6C and Figure 6D, indicated by the yellow dashed line). In the second group, the YAP N/C ratio and dipole traction increase at a slower rate with the enlargement of the cell spread area and maintain nearly constant values when the cell spread area continues to increase (Figure 6C,D, indicated by the green dashed line).
PC9 cancer cells generate tractions in peri-nuclear regions
Single, spreading PC9 cells displace the substrates at the peri-nuclear regions, starting from the 6th h of culture (Figure 7C). To visualize the peri-nuclear displacement caused by cell traction, we overlapped the images of fluorescent beads taken before (red) and after (green) the removal of the cells from the substrates (see the protocol section for details). The beads that do not have any displacement will appear yellow in the overlapped images, i.e., the addition of red and green colors. In contrast, the beads that are displaced from their resting positions due to cell traction will show separated green and red colors.
Notably, in both PC9 (Figure 7C,D) and B2B (Figure 7E) cells, bead displacement was observed in the cytoplasm and within the nucleus, in addition to those at the cell boundary. To highlight the peri-nuclear displacement, the Boussinesq equation from linear-elasticity theory is used to predict the 2D theoretical displacement generated by a hypothetical dipole force at the cell boundary (black dashed line in Figure 7B)24. Comparing this theoretical curve with the real substrate displacement measured along the same axis (white dashed line in Figure 7D), the real displacements within the nucleus were found to be 1.5-8-fold-larger than the theoretical value (Figure 7B), indicating the existence of traction force at the peri-nuclear regions.
Figure 1: Changes in YAP expression/distribution, substrate displacement field, and traction field of a B2B normal cell on substrates of varying stiffness and during early spreading. (A) The YAP expression of a B2B cell seeded on 2, 5, and 40 kPa PAA gels and a glass coverslip after 60 h from initial cell-substrate attachment. (B) The B2B cell was seeded on a 5 kPa PAA gel and imaged over 10 h after initial cell-substrate attachment. YAP expression is represented by green fluorescence intensity. Note: The YAP intensity inside the nucleus gradually decreases but remains higher than that in the cytoplasm over time. The color bars indicate the levels of YAP expression (green = high expression; black = low expression) in (A) and (B). (C) Substrate deformation (overlapped with the bright-field image) at cell location is represented by the displacement field at each time point. Displacement direction and magnitude are shown by the arrow direction and color, respectively. The displacement becomes larger at the ends of the B2B cell body as the cell spread area increases. The color bar indicates displacement magnitude (crimson = high magnitude; black = low magnitude). (D) Traction field (overlapped with the bright-field image) calculated from the displacement field. The traction is concentrated on the boundary of the B2B cells. The white and yellow dotted outlines delineate the boundaries of the cell and nucleus, respectively. The color bar indicates traction magnitude (crimson = high magnitude; black = low magnitude). Scale bars = 20 µm. Abbreviations: YAP = Yes-associated protein; PAA = polyacrylamide. Please click here to view a larger version of this figure.
Figure 2: Changes in YAP expression/distribution, substrate displacement field, and traction field of a PC9 cancer cell on substrates of varying stiffness and during early spreading. (A) The YAP expression of a PC9 cell seeded on 2, 5, and 40 kPa PAA gels and glass coverslip after 65 h from initial cell-substrate attachment. (B) The PC9 cell was seeded on a 5 kPa PAA gel and imaged over 10 h after initial cell-substrate attachment. YAP expression is represented by green fluorescence intensity. Note: The YAP intensity plateaus from 1.5 h onwards. The color bars indicate the levels of YAP expression (green = high expression; black = low expression) in (A) and (B). (C) Substrate deformation (overlapped with the bright-field image) at cell location is represented by fluorescent bead displacement field at each time point. Displacement direction and magnitude are shown by the arrow direction and color, respectively. The displacement field caused by PC9 cells is smaller than that caused by the B2B cell. Throughout the 10 h spreading process, the area of PC9 cells remains nearly constant. The color bar indicates displacement magnitude (crimson = high magnitude; black = low magnitude). (D) Traction field (overlapped with the bright-field image) calculated from displacement field. The traction generated by this representative PC9 cell gradually decreases from the 6th h to the 10th h. The white and yellow dotted outlines delineate the boundaries of the cell and nucleus, respectively. The color bar indicates traction magnitude (crimson = high magnitude; black = low magnitude). Scale bars = 20 µm. Abbreviations: YAP = Yes-associated protein; PAA = polyacrylamide. Please click here to view a larger version of this figure.
Figure 3: YAP distribution in B2B and PC9 cells at the early spreading stage. (A) YAP intensity of the B2B cell is measured along the assigned red axis at the 0th and the 10th h. (B) At the 0th h, YAP intensity shows dramatic concentration differences between the nucleus and the cytoplasm. At the 10th h, YAP intensity becomes more homogenous across the whole cell body. (C) YAP intensity of the PC9 cell is measured along the assigned blue axis at the 0th and the 10th h. (D) At the 0th h, YAP intensity in the nucleus appears higher than that in the cytoplasm, although the difference is not as remarkable as that in B2B cells. At the 10th h, YAP intensity in the nucleus still appears slightly higher than that in the cytoplasm, with a variation trend similar to that at the 0th h. Scale bars = 20 µm (A, C). Abbreviation: YAP = Yes-associated protein. Please click here to view a larger version of this figure.
Figure 4: Measuring the YAP N/C ratio. (1) Apply Fiji ImageJ to draw the outline of the nucleus and measure its 2D projected area Anuc. (2) Measure the fluorescence intensity inside the nucleus Inuc. (3) Draw the outline of the cell body and measure its projected area Acel. (4) Measure the fluorescence intensity inside the cell Icel. (5) Calculate the YAP nucleus density Dnuc, YAP cytoplasm density Dcyto, and their ratio R: Dnuc=Inuc/Anuc; Dcyto=(Icel–Inuc)/(Acel–Anuc); R=Dnuc/Dcyto. The color bar indicates the levels of YAP expression (green = high expression; black = low expression). Scale bar = 20 µm. Abbreviations: YAP = Yes-associated protein; N = nucleus; C = cytoplasm. Please click here to view a larger version of this figure.
Figure 5: Distinct YAP expression, cell/nucleus morphology, and cellular traction in PC9 cancer and B2B normal cells during cell spreading. (A) YAP N/C ratio change during the first 10 h of single-cell spreading. Average YAP N/C ratio of B2B cells (red column; n = 10) changed from 2.54 ± 0.22 to 1.79 ± 0.21 (n = 10; p = 0.0022**) while the average YAP N/C ratio of PC9 cells (blue column; n = 5) changed from 1.92 ± 0.26 to 1.57 ± 0.07 (p = 0.187 (ns)). (B) The average dipole traction as a function of time. The average dipole traction of B2B cells changed from 256.17 ± 123.69 nN to 287.44 ± 99.79 nN (p = 0.7593 (ns)) and the average dipole traction of PC9 cells changed from 141.19 ± 33.62 nN to 168.52 ± 73.01 nN (p = 0.7137 (ns)). (C) The average cell area as a function of time. The average cell spread area of B2B cells increased from 613.89 ± 102.43 µm2 to 942.51 ± 226.71 µm2 (p = 0.0512 (ns)) and the average cell spread area of PC9 cells changed from 495.78 ± 97.04 µm2 to 563.95 ± 89.92 µm2 (p = 0.5804 (ns)). (D) The average nucleus area as a function of time. The average nucleus spread area of B2B cells increased from 181.55 ± 36.18 µm2 to 239.38 ± 43.12 µm2 (p = 0.1217 (ns)) and the average nucleus spread area of PC9 cells changed from 133.31 ± 30.05 µm2 to 151.93 ± 22.49 µm2 (p = 0.5944 (ns)). Abbreviations: YAP = Yes-associated protein; N = nucleus; C = cytoplasm; ns = not significant. Please click here to view a larger version of this figure.
Figure 6: YAP N/C ratio and dipole traction force as a function of spread area of cell and nucleus. YAP N/C ratio and dipole traction of B2B cells (n=10) and PC9 cells (n=5) are calculated from the 6th h to the 10th h after attaching to substrate. (A) YAP N/C ratio as a function of the cell spread area. The YAP N/C ratios of B2B cells vary from 1.16 to 2.53, while the YAP N/C ratios of PC9 cells vary from 1.27 to 1.88. The cell spread area of B2B cells varies from 391.94 µm2 to 1986.40 µm2. The cell spread area of PC9 cells ranges from 284.46 µm2 to 830.12 µm2. (B) YAP N/C ratio as a function of nucleus spread area. The nucleus spread area of B2B cells varies from 107.09 µm2 to 514.28 µm2. The nucleus spread area of PC9 cells ranges from 58.03 µm2 to 259.65 µm2. Dipole traction of B2B cells as a function of the cell spread area (C) and the nucleus spread area (D). Spreading and non-migrating B2B cells show higher traction (from 47.50 nN to 1051.48 nN) with lower cell and nucleus area. While spreading and migrating, B2B cells show lower traction (from 105.80 nN to 310.28 nN) with larger ranges of cell and nucleus area. Abbreviations: YAP = Yes-associated protein; N = nucleus; C = cytoplasm. Please click here to view a larger version of this figure.
Figure 7: Peri-nuclear displacement in normal B2B and cancer PC9 cells. (A) Schematic side-view diagram of peri-nuclear and peri-cell displacement measured from bead displacement in the substrate. (B) Substrate displacement underneath the PC9 cell is measured along the cell axis (white dashed line in 7D). The theoretical displacement generated by the dipole force at the cell boundary is shown by the Boussinesq equation (black dashed curve). (C) and (D) Overlapped fluorescent bead images with (red) and without (green) cells for the PC9 cells at the 6th h after attaching (Top view). Yellow (exact overlap of red and green colors) beads indicate no displacement. The separated green and red beads (pointed by yellow arrows) represent the peri-nuclear displacement. Yellow arrows indicate these contracted peri-nucleus spots located at the periphery of the nucleus. (E) Peri-nuclear displacement generated by the B2B cell at 1.5th h after cell-substrate attachment. Scale bars = 10 µm (C–E). Please click here to view a larger version of this figure.
Supplemental Figure S1: The genomic sequence map of YAP-mNeonGreen21-10/11. Please click here to download this File.
Supplemental Figure S2: Changes in YAP expression/distribution, substrate displacement field, and traction field of B2B normal cells during early spreading. (A, D, G, J, M) The B2B cell was seeded on a 5 kPa PAA gel and imaged over 10 h after the initial cell-substrate attachment. YAP expression is represented by green fluorescence intensity. Note: The YAP intensity inside the nucleus gradually decreases but remains higher than in the cytoplasm over time. The color bars indicate the levels of YAP expression (green = high expression; black = low expression) in (A, D, G, J, M). (B, E, H, K, N) Substrate deformation (overlapped with the bright-field image) at cell location is represented by the displacement field at each time point. Displacement direction and magnitude are shown by the arrow direction and color, respectively. The displacement becomes larger at the periphery of the B2B cell body as the cell spread area increases. The color bars indicate displacement magnitude (crimson = high magnitude; black = low magnitude) in (B, E, H, K, N). (C, F, I, L, O) Traction field (overlapped with the bright-field image) calculated from the displacement field using Traction Force Microscopy. The traction is concentrated at the periphery of B2B cells. The color bars indicate traction magnitude (crimson = high magnitude; black = low magnitude) in (C, F, I, L, O). Scale bars = 20 µm. Abbreviations: YAP = Yes-associated protein; PAA = polyacrylamide. Please click here to download this File.
Supplemental Figure S3: Changes in YAP expression/distribution, substrate displacement field, and traction field of PC9 cancer cells during early spreading. (A, D, G, J) The PC9 cell was seeded on a 5 kPa PAA gel and imaged over 10 h after the initial cell-substrate attachment. YAP expression is represented by green fluorescence intensity. Note: The YAP intensity inside the nucleus gradually decreases but remains similar to or slightly lower than that in the cytoplasm over time. The color bars indicate the levels of YAP expression (green = high expression; black = low expression) in (A, D, G, J). (B, E, H, K) Substrate deformation (overlapped with the bright-field image) at cell location is represented by the displacement field at each time point. Displacement direction and magnitude are shown by the arrow direction and color, respectively. The displacement becomes larger at the periphery of the PC9 cell body as the cell spread area increases. The color bars indicate displacement magnitude (crimson = high magnitude; black = low magnitude) in (B, E, H, K). (C, F, I, L) Traction field (overlapped with the bright-field image) calculated from the displacement field. The traction is concentrated at the periphery of PC9 cells. The color bars indicate traction magnitude (crimson = high magnitude; black = low magnitude) in (C, F, I, L). Scale bars = 20 µm. Abbreviations: YAP = Yes-associated protein; PAA = polyacrylamide. Please click here to download this File.
The imaging process (step 6.3) is critical to ensure that the fluorescence images are of sufficiently good quality to yield valid quantification results. The z-stack images of fluorescent protein or beads should have a z-range that is large enough to include the in-focus images for all the Z-positions that the sample spans. Another critical step is collecting the reference images of fluorescent beads after dissolving the cells (step 6.5). Because the reference images need to be taken at the same positions in step 6.3, no relative displacement should be induced between the Petri dish, environment chamber, and the microscope. The investigators performing the dissolving step must be careful to remove the lid of the Petri dish and ensure that the applied mechanical perturbation is not large enough to alter the location of the dish in the environment chamber.
Solutions are provided below to resolve some errors that might occur during experiments. If no macro is activated after clicking Enter in step 6.4, it is most likely because the left bottom area of the screen is occupied by a non-Element window. In such a case, the left bottom area of the window needs to be cleared so that macros can be activated in Elements. Another common error is that the bright-field images appear black. This issue is caused by an insufficient time interval between the acquisitions of fluorescence and bright-field images. Slight delays in the fluorescence imaging time counting can accumulate over time and cause considerable delays and interfere with the bright-field imaging. One solution is to adjust the duration of one imaging cycle of all the positions to be less than (not equal to) the time interval between the start of consecutive motions. This operation refreshes the time counting and eliminates the cumulative error at the beginning of each imaging cycle.
This all-optical interrogation technology supports (1) a wide range of hardware/software, including but not limited to Nikon, (2) diverse types of validated hydrogel systems, including gelatin, PEG, Matrigel, and collagen I gels, and (3) the programmable customization based on different needs of researchers. However, if any of the bottom-level control functions are not available from a commercial microscope, customization of the functions using AMFIP becomes challenging. Another limitation of this technique is the spatial drift of the sample in both the XY and focus (Z) planes. Although this limitation can be overcome during the post-processing of the images, it is essential to improve the auto-focus function to correct the real-time drift of the samples. This improvement will increase the throughput of the imaging process and reduce the potential error caused by the drift during experiments.
Mechanotransducers, such as YAP, may serve as new therapeutic targets for the development of promising cancer therapies25,26,27. Emerging data suggest that YAP promotes the proliferation and invasion of cancer cells. Mechanics-induced YAP translocation from the cytoplasm to the nucleus activates the transcription of genes related to cell migration, proliferation, invasion, and apoptosis, leading to aberrant cell behaviors28,29,30,31. This work aimed to explore the potential correlation of the YAP N/C ratio and cell mechanics in two typical human lung cancer and normal cell lines. During the 10 h cell spreading period, PC9 cells show similar YAP concentrations in the nucleus and cytoplasm (Figure 3D and Figure 5A). B2B cells show a higher YAP concentration in the nucleus than in the cytoplasm (Figure 3C and Figure 5A). This relationship found during the early spreading stage is different from the majority of published findings that compare YAP concentration in the nucleus between normal and cancer cells. Although not necessarily at the early spreading stage, most published findings show that YAP is more concentrated in the nucleus of cancer cells than in the nucleus of normal cells27,28. Only one study on breast cancer reported an exception32 that shows YAP is more concentrated in the cytoplasm, which agrees with our current observations made in lung cancer PC9 cells. To the best of the authors' knowledge, this work is the first to show a lower YAP N/C ratio in a human lung cancer cell line. The authors hypothesize that the reason for a stable YAP N/C ratio in PC9 cells might be due to the low variation in the cell/nucleus spread area and traction in PC9 cells at the early spreading stage. The dissection of the underpinning molecular mechanisms of low YAP N/C ratio in PC9 and B2B cells is ongoing.
During the first 10 h of spreading, these two cell lines show a distinct relationship between the YAP N/C ratio, cell traction, and spread area (Figure 5). For B2B cells, a higher YAP N/C ratio is correlated with a higher cell and nucleus spread area (Figure 6A,B), which is consistent with the reported data of other normal cells33. Interestingly, although the developmental trend of this relationship is generally found in all B2B cells recorded, two different degrees (high and low) of this relationship are found. B2B cells that spread and migrate simultaneously show lower traction and higher cell and nucleus spread area with a higher YAP N/C ratio (2.05 ± 0.32). For B2B cells that spread and remain at the same location, they show higher traction and lower cell and nucleus spread area with a lower YAP N/C ratio (1.74 ± 0.21). These two degrees of relationships are demonstrated in the bifurcated scattered data groups (Figure 6C,D). As reported in the literature, stationary normal cells, such as embryonic fibroblast NIH 3T3 cells, have higher traction than migratory cells34. The data reported in this paper suggest that the spreading and non-migrating B2B cells applied higher traction than spreading and migrating B2B cells, likely suggesting that high traction is needed for non-migrating cells to stabilize on the substrate.
In addition, these data show that stationary normal B2B cells generate a higher peri-nuclear force, whereas previous research conducted by other researchers reported only higher cell traction generated at the periphery of stationary cells34,35,36,37. The authors think that the difference in the intrinsic tendency of migration in the experiments might cause these contradictory results. In the published experiments, square-shaped micropatterning had been used to confine single cells from spreading and inhibit migration; whether the cells had the tendency to migrate is unknown. As migratory cells often show high traction force at the periphery of the cells38, it is likely that cells with the tendency to migrate will still maintain high periphery traction even though their migration is restricted. In this present study, the stationary cells are not restricted by any micropattern but do not migrate, indicating that the cells tend to maintain their non-migrating state. Another possibility is that the cell shape defined by the micropattern may affect the distribution of focal adhesions and traction forces39. The results in this study were generated without any confining micropatterning and represent the force distribution of stationary cells in their original shape.
To the best of the authors' knowledge, only one publication to date specifically reported the finding of peri-nuclear forces in normal cells (mouse embryonic fibroblasts), potentially caused by the actin cap spanning across the nucleus40. YAP cytoplasm-to-nucleus translocation is correlated with the increase in peri-nuclear force40. A thorough search of the relevant literature did not yield any publicationsthat report a peri-nuclear force or the actin cap in cancer cells. An indirect study on melanoma cancer cells demonstrated that the actin rim (another peri-nuclear actin organization located around but not covering the nucleus) reduces cell migration rates41, indirectly suggesting the existence of a peri-nuclear force. However, no direct experimental data are reported. In this study, the authors found that both PC9 and B2B cells show peri-nuclear displacement and traction. The mechanisms of the generation of the peri-nuclear forces and their effects remain controversial. In normal cells, the actin cap was reported to play a role in regulating nucleus morphology and chromatin organization42, transmitting mechanical signals from focal adhesions into the nucleus through linkers of nucleoskeleton and cytoskeleton (LINC) complex43, and regulating cell migration44. Lamin A/C is related to the formation and disruption of the actin cap40,41,42,43,44. However, the report that claimed that the actin cap generates a peri-nuclear force did not consider the potential role of the actin rim40. In cancer cells, overexpression of Lamin A facilitates the formation of an actin rim and restricts cancer cell migration. Overexpression of Lamin B reduces actin rim formation and promotes migration. The peri-nuclear force might be involved in this process due to the existence of peri-nuclear actin organization and the effect of Lamin A. However, the results of this study did not show any evidence of measured peri-nuclear forces or the behavior of the actin cap. Therefore, the discovery of peri-nuclear forces in PC9 cells in this present study is the first report showing peri-nuclear forces and displacements in lung cancer cells. The authors are currently investigating the molecular mechanisms and functions of peri-nuclear forces in CRISPR/Cas9-engineered PC9 and B2B cells.
Beyond the all-optical mechanobiology interrogation that is demonstrated in this paper, the integrated multi-functional system can be applied to optically probe a myriad of other essential physiological and pathobiological signals in living systems. For example, the authors' laboratory has recently established multiple stably transduced human cancer cell lines that co-express three light-responsive membrane proteins: membrane voltage indicator QuasAr2 (excitation: 640 nm; emission: 660 nm-740 nm), membrane voltage depolarizer CheRiff (excitation: 488 nm), and membrane voltage hyperpolarizer eNpHR3 (excitation: 590 nm). These three functional proteins can be activated by spectrum-orthogonal laser lines in a crosstalk-free manner, enabling all-optical two-way signaling communications (readout and control) of membrane electrophysiology. Using an integrated opto-electronics system and a manual patch-clamp, the authors have validated the all-optical control and readout of the membrane voltage (Vm) in single human cancer cells and multicellular tumor spheroids. The all-optical electrophysiology interrogation opens the possibility for detailed explorations of previously inaccessible bioelectricity in cancer cells, which may help advance tumor biology from a new axis.
The authors have nothing to disclose.
This project is financially supported by the Cancer Pilot Award from UF Health Cancer Center (X. T. and D. S.) and the Gatorade Award Start-up Package (X. T.). The authors sincerely appreciate the intellectual discussions with and the technical supports from Dr. Jonathan Licht (UFHCC), Dr. Rolf Renne (UFHCC), Dr. Ji-Hyun Lee (Biostatistics, UF), Dr. Hugh Fan (MAE, UF), Dr. Warren Dixon (MAE, UF), Dr. Ghatu Subhash (MAE, UF), Dr. Mark Sheplak (MAE & ECE, UF), Dr. Malisa Sarntinoranont (MAE, UF), Dr. Scott Banks (MAE, UF), Dr. Matthew Traum (MAE, UF), Dr. David Hahn (University of Arizona), Dr. Weihong Wang (Oracle Corporation), Dr. Youhua Tan (Hong Kong Polytechnic University), and the Support Team of Nikon (Drs. Jose Serrano-Velez, Larry Kordon, and Jon Ekman). The authors are deeply grateful for the generous and effective support from all members of Tang's, Siemann's, and Guan's research laboratories and all staff members of the MAE & ECE & Physics & Radiation Oncology Departments, UF.
(3-Aminopropyl)triethoxysilane | Sigma-aldrich | 440140 | |
0.05 % Trypsin | Corning | 25-051-CI | |
75 cm2 flask | Corning | 430641U | |
8 Benchtop Centrifuge | Thermo | 75007210 | |
A1R confocal system | Nikon | HD25 | |
Acetic acid | Sigma-aldrich | 695092 | glacial, ACS reagent, ≥99.7% |
BEAS-2B (B2B) cells | Sigma-aldrich | 95102433 | human epithelial cells from lung tissue |
Carboxylate-Modified Microspheres | Invitrogen | F8797 | |
Culture medium (RPMI-1640) | Gibco | 11875093 | |
Desktop Computer | Dell | 2018 | with Windows 10 operating system |
Environmental chamber TIZB | Tokai Hit | TIZB | |
Fetal bovine serum (FBS) | Gibco | 26140 | |
Fibronectin Human Protein, Plasma | Gibco | 33016015 | |
Fiji ImageJ | National Institutes of Health and the Laboratory for Optical and Computational Instrumentation | 1.53k | |
Glass-bottom petri dish | MatTek | P35G-1.5-14-C | |
HEPES buffered saline | Sigma-aldrich | 51558 | |
Hydrazine hydrate solution | Sigma-aldrich | 53847 | |
IntelliJ IDEA | JetBrains | 2020 | Java development platform |
Java Development Kit | Oracle | 14.0 | |
Kimwipe | Kimtech Science | 3066-05 | |
MATLAB | MathWorks | 2020b | |
Monochrome Camera | FLIR | BFS-U3-70S7M-C | |
MycoAlert Mycoplasma Detection Kit | Lonza | LT07-218 | |
N,N′-Methylenebisacrylamide solution | Sigma-aldrich | M1533 | |
NIS-Elements software platform | Nikon | 4.50 | software platform |
Origin | OriginLab | OriginPro 2017 (Learning Edition) | data analysis and graphing software |
Penicillin-streptomycin | Gibco | 15140122 | |
PC9 cells | Sigma-aldrich | 90071810 | human adenocarcinoma cells from lung tissue |
Phosphate buffered saline (PBS) | Gibco | 10010023 | |
Phusion High-Fidelity DNA Polymerase | New England Biolabs | F-553S | high-fidelity DNA polymerase |
Scotch tape | Scotch | adhesive tape | |
Sodium dodecyl sulfate solution | Sigma-aldrich | 05030 | |
Super glue | Gorilla | cyanoacrylate glue | |
Ti2-E inverted microscope | Nikon | MEA54000 | |
TI2-S-SE-E Motorized Stage with Encoder | Nikon | MEC56120 | |
μManager | version 2.0 gamma | open source microscopy software (https://micro-manager.org/) |