1Brigham and Women's Hospital / Harvard Medical School, Department of Medicine, Cardiovascular Division, 2Weill Institute for Cell and Molecular Biology & Department of Biomedical Engineering, Cornell University
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Lombardi, M. L., Zwerger, M., Lammerding, J. Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation. J. Vis. Exp. (55), e3087, doi:10.3791/3087 (2011).
In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.
1. Substrate strain application
The measurement of normalized nuclear strain includes the preparation of strain dishes with transparent, elastic silicone membranes as cell culture surface, plating cells onto the dishes, and acquiring images of the cells before, during and after (uniaxial or biaxial) strain application.
Preparation of silicone membrane dishes and adherence of cells
Substrate strain experiments
2. Microneedle manipulation assay
Preparation of dishes, adherent cells, and microneedles
Microneedle manipulation experiment
3. Representative results:
Substrate strain application
We acquired images before, during, and after strain application to mouse embryonic fibroblasts from heterozygous and homozygous lamin A/C-deficient (Lmna+/– and Lmna–/–), and wild-type (Lmna+/+) mice and subsequently computed the normalized nuclear strain for each cell. After analysis, the nuclei are validated and cells that become damaged or retract during strain application are excluded from the analysis. Figure 1A depicts nuclei of three cells that are valid, whereas Figure 1B depicts cells that should be excluded from analysis. Normalized nuclear strain data are pooled from at least three independent experiments (each containing measurements from ~5–10 nuclei) and compared with other cell or treatment groups by statistical analysis. Increased normalized nuclear strain indicates reduced nuclear stiffness, as seen in cells with reduced expression of the nuclear envelope proteins lamin A/C (Figure 2).
Microneedle manipulation assay
For the microneedle manipulation assay, we imaged nuclear and cytoskeletal displacements during localized cytoskeletal strain application. Cells that become damaged or detached are excluded from the analysis. For the analysis, we measure the magnitude of the nuclear and cytoskeletal movements towards the force application site in single, adherent cells. For example, in Figure 3, we track mitochondrial (marker for the cytoskeleton) displacements before and after cytoskeletal strain and then plot the displacements as vectors. Each vector represents the displacement computed as the shift between the original location and the newly identified position. Regions with low image intensity or insufficient texture (e.g., regions outside the cell) are excluded from the analysis. The cytoskeletal and nuclear displacements are then quantified in select areas at increasing distances from the strain application site (Figure 4, areas corresponding to the colored boxes in inset). In mouse embryonic fibroblasts with intact nucleo-cytoskeletal coupling, forces are transmitted through the entire cells, resulting in induced nuclear and cytoskeletal deformations that slowly dissipate away from the strain application site (Figure 4). In contrast, fibroblasts with disturbed nucleo-cytoskeletal coupling (or altered cytoskeletal organization) display localized displacements near the application site, as shown in Figure 4 and only little induced deformations further away. Comparable cytoskeletal strain application at the microneedle insertion site (orange box) is observed for both control fibroblasts (mCherry alone) and fibroblasts with a disrupted nucleo-cytoskeletal coupling (DN KASH). However, induced nuclear and cytoskeletal displacements (blue, yellow, and red boxes) at other regions were significantly smaller in the fibroblasts with disrupted nucleo-cytoskeleton coupling (DN KASH) than in control cells (mCherry alone) (Figure 4). Thus, decrease in cytoskeletal and nuclear displacements away from the strain application site, indicates that force transmission between the cytoskeleton and nucleus was disturbed.
Importantly, we have also validated that mitochondria are suitable cytoskeletal marker, by conducting microneedle manipulation on mouse embryonic fibroblasts transfected with GFP- or mCherry actin and GFP-vimentin and fluorescently labeled with Mitotracker Green or Red. Cytoskeletal displacement maps were calculated independently from the fluorescent signal of the mitochondria and the actin or vimentin cytoskeleton. The average absolute displacement was computed for four distinct cytoskeletal regions at increasing distances away from the strain application site. The slope and R-squared values were computed from the linear regression between the measurements obtained from mitochondria and from actin or vimentin, respectively. For actin, the slope was 0.99 and the R2 value was 0.986; for vimentin, the slope was 1.04 and the R2 value was 0.971, confirming that mitochondrial displacements serve as reliable indicators for cytoskeletal deformations.
Figure 1. Substrate strain application on mouse embryonic fibroblasts (MEFs). Mouse embryonic fibroblasts spread over two distinct areas on the silicon membrane were imaged with phase contrast and fluorescence microscopy before, during and after application of 20% uniaxial strain. (A) Example of a successful experiment with valid nuclei from cells that survived the strain application without any damage or detachment and (B) example of cells that retract/partially detached during strain application; results from the cells depicted in (B) are excluded from the analysis. In (B), the cell on the left side shows signs of cytoskeletal damage and nuclear collapse (arrow), while the cell on the right side detaches partly and retracts during strain application. This can be an indication of excessive strain application. For better comparison, in (A) and (B) the border of one of the unstretched cell membranes is outlined in red and superimposed on the same cell during and after strain application. In (A) the border of the unstretched nucleus is outlined in green and superimposed on the same nucleus during and after strain application.
Figure 2. Analysis of normalized nuclear strain in a panel of different MEF cell lines. MEFs of the Lmna–/– and Lmna+/– genetic background ectopically expressing either an empty vector or wild-type lamin A were analyzed. In comparison to MEFs from wild-type littermates (Lmna+/+), loss of lamin A/C expression results in decreased nuclear stiffness that can be fully restored by reintroduction of wild-type lamin A. Notably, reduced nuclear stiffness is reflected by increased values of normalized nuclear strain. The error bars represent standard errors.
Figure 3. Microneedle manipulation assay to measure intracellular force transmission. Phase contrast (A, B) and fluorescence (C, D) images of a fibroblast labeled with nuclear stain (blue) and MitoTracker mitochondrial stain (green). A microneedle was inserted into the cytoskeleton at a defined distance from the nucleus (A and C) and subsequently moved towards the cell periphery (B, D). Cytoskeletal and nuclear displacements were quantified by tracking fluorescently labeled nucleus and mitochondria using a custom-written cross-correlation algorithm. (E) Displacement map of the final cytoskeletal (green) deformations computed from fluorescence image series; arrow length is magnified by 2x for better visibility. Scale bars, 10 μm.
Figure 4. Analysis of intracellular force transmission during microneedle manipulation. Induced cytoskeletal and nuclear displacements during microneedle manipulation, measured in the areas corresponding to the colored boxes (inset in A). The orange box is the strain application site. Despite similar strain application in the cytoskeleton (orange box), induced nuclear and cytoskeletal displacements (blue, yellow, and red boxes) were significantly smaller in the mouse embryonic fibroblasts that with a disrupted nucleo-cytoskeletal coupling (DN KASH) compared to control (mCherry alone) cells.
Substrate strain assay
Strain application has been successfully used by us and other groups to study induced nuclear deformations in cells subjected to mechanical stress and to investigate the contribution of specific nuclear envelope proteins to nuclear stiffness.4-8 The advantage of this technique is that it probes mechanical properties of living nuclei in their normal cellular and cytoskeletal environment and that the substrate strain application resembles physiological load application as found in many tissues such as contracting muscle or blood vessel walls.9 Furthermore, it enables strain application to many cells in parallel, increasing the number of cells that can be analyzed in a single experiment. One limitation of the substrate strain assay is that it does not allow direct measurements of nuclear stiffness. Instead, this method determines the relative stiffness of the nucleus compared to the surrounding cytoskeleton. Detailed analysis of induced nuclear and cytoskeletal strain in cells subjected to uniaxial substrate strain show that in wild-type cells, the cytoskeletal strain is comparable to the applied substrate strain, while the stiffer nucleus deforms significantly less.4 Nonetheless, additional assays, such as the microneedle manipulation assay, may be necessary to assure that observed differences in nuclear deformation between different cell lines are not the results of altered nucleo-cytoskeletal coupling or cytoskeletal structure. Despite these limitations, measuring nuclear mechanics in intact cells rather than in isolated nuclei minimizes the risk of artifacts induced by osmotic effects, damage during the isolation procedure, or other changes associated with nuclear isolation. However, one stringent requirement for the substrate strain assay is that cells firmly adhere to the substrate. To improve cellular adhesion, dishes can be coated with different extracellular matrix proteins such as fibronectin, collagen or laminin at variable concentrations; we recommend determining the optimal coating conditions for each new cell type used in the experiments.
Furthermore, for optimal results, the applied substrate strain must be sufficiently large to detect nuclear deformations while minimizing damage to the cells. For mouse and human fibroblasts, we typically apply 5 % biaxial strain or 20% uniaxial strain without overt cell damage. In general, we find that cells tolerate uniaxial strain application better than biaxial strain application, as smaller changes in membrane area are required. As for the extracellular matrix coating, the optimal conditions of strain application should be determined for each new cell line to be tested.
In addition, we found that the experiments are sensitive to the cell density. Experiments should ideally be conducted on sub-confluent cells to minimize cell-cell interactions; however, cell densities that are too low often result in poor survival of cells in response to strain and in a low yield of cells per experiments. On the other hand, a cell density that is too high makes it difficult to identify the same cells before, during, and after strain application. We recommend to test optimal cell density for individual cell types.
Another important advice for the experiments is that strain application should be limited to 10 minutes or less to avoid cellular adaptation, such as remodeling of cytoskeletal elements. To this end, the use of automated imaging software, e.g. IPlab, in combination with a motorized stage enables automatic re-localization of the cells on the strain dish and faster image acquisition. Furthermore, we use custom-written image analysis software (e.g. MATLAB) for data analysis. The software requires certain marker points on the membrane to calculate the applied substrate strain, such as the applied dot, small fluorescent beads or distinct irregularities on the silicon membrane. Finally, validation of each nucleus is required to exclude those of damaged or retracting cells, as the nuclear deformations measured in these cells are not representative. A possible adaptation of the substrate strain technique is to apply substrate strain to cells cultured in three-dimensional collagen gels, which allows analysis of nuclear mechanical properties under more physiological conditions, as well as to apply the method to more weakly adherent cells. Another variation is to use micropatterned substrates, e.g., round or rectangular patches of fibronectin on the silicon membrane, to achieve consistent cell spreading and alignment during the strain application. Finally, to better understand nuclear mechanics on a more physiological level, cellular strain can be applied on entire tissues instead of isolated cells. We are currently successfully using this technique to measure nuclear stiffness in model organisms such as Drosophila melanogaster or Caenorhabditis elegans.
Microneedle manipulation assay
The microneedle manipulation assay is a single cell-based method to probe intracellular force transmission by quantifying induced nuclear and cytoskeletal displacements after localized cytoskeletal strain application, thereby advancing an earlier approach pioneered by Maniotis and colleagues.10 This technique offers several advantages over other localized force application methods such as magnetic tweezers or optical traps. For instance, strain can be applied directly to the cytoskeleton and can be modified varying the distance of the microneedle. The microneedle can also be positioned in the cell with high accuracy, making the experiment very reproducible, and the force application rate, which is directly related to the microneedle speed, can be precisely controlled by the programmed micromanipulator. In contrast, the magnetic beads used in magnetic tweezer and optical traps are often randomly localized on the apical cellular surface, and both optical and magnetic tweezers generate insufficient forces to result in large scale cytoskeletal deformations in many cell types. Although, the microneedle manipulation assay is an invasive technique and there is limited control over which cytoskeletal structures the microneedle adheres to, we can confirm the results with less invasive approaches (e.g. substrate strain). Thus, microneedle manipulation and substrate strain are two complementary assays that yield information about nuclear mechanics in intact living cells while maintaining normal nuclear and cytoskeletal architecture and preserving the correct chemical composition of the nucleoplasm and cytoplasm.
In the following, we suggest possible modifications or improvements. If cells are too well spread, the microneedle tip may break against the glass bottom dish when trying to position it within the thin (~1-2 μm) cytoskeleton. To avoid these problems, we recommend testing a range of concentrations of extracellular matrix (ECM) molecules, such as fibronectin, collagen, or laminin, to determine conditions that achieve sufficient cell adhesion without allowing the cells to spread too thin. Another modification could be to plate the cells on ECM-coated micropatterned surfaces to assure uniform cell spreading and orientation. An additional or complementary approach could be to plate cells on ECM-coated polyacrylamide gels and insert the microneedle through the cytoskeleton into the gel to provide uniform strain application across the cytoskeleton. The use of an intact and fine-tipped microneedle is absolutely essential for the success of the experiments. We routinely pull our own microneedles, and one can experiment with different shapes and sizes. Alternatively, one can also purchase individually packed microneedles with consistent geometry (e.g., Eppendorf Femtotips). In any case, it is important to assure that the microneedle remains undamaged during the experiments, as a broken microneedle tip, for example after making contact with the glass substrate, will damage cells. During the microneedle manipulation procedure itself, it is important to apply consistent speed and range of the microneedle movement, as the nucleus and cytoskeleton are viscoelastic materials with time-dependent behavior. We recommend using a computer-controlled micromanipulator, which will also assist in the coordination of microneedle movement and image acquisition. From our experience, Mitotracker can by cytotoxic, thus, limit the experiments to 30 minutes or use other fluorescent markers, such as GFP-actin or GFP-vimentin. One modification of the approach described here, particularly when studying nucleo-cytoskeletal coupling, is to insert the microneedle into the nucleus instead of the cytoskeleton and move it towards the cell periphery.
For all studies, due to the typically large cell-to-cell variability, experiments should be performed at least three independent times to obtain measurements from a minimum of 15–25 total valid cells.
No conflicts of interest declared.
This work was supported by the National Institutes of Health (R01 HL082792 and R01 NS059348) and the Brigham and Women's Hospital Cardiovascular Leadership Group Award.
|MitoTracker Red FM and Green FM||Invitrogen||M22425 and M-7514|
|Hank’s Buffered Salt Saline||Invitrogen||14185|
|Phenol free, DMEM||Invitrogen||21063|
|Fetal bovine serum||Aleken Biologicals||FBSS500|
|Borosilicate Glass with filament||Sutter Instrument Co.||BF100-78-10|
|Gloss/Gloss non-reinforced silicone sheeting, 0.005"||Specialty Manufacturing Inc.|
|Dulbecco’s Phosphate Buffered Saline||Invitrogen||14200|
|35 mm glass bottom culture dishes (FluoroDish)||World Precision Instruments, Inc.||FD35-100|
|Braycote 804 Vacuum Grease||SPI Supplies||05133A-AB|