Background

Reciprocal interactions between the cell nucleus and the extracellular matrix lead to macroscale tissue phenotype changes. Changes in cell gene expression lead to alterations in signalling that regulate cell communication, timing of cellular activities and tissue structure. The extracellular matrix dictates another layer of cellular regulation, as biochemical cues, physical forces, and changes in stiffness of the extracellular matrix microenvironment guide cell migration, proliferation, differentiation, and changes in gene expression. The extracellular environment is physically linked to the nuclear envelope and provides cues to maintain nuclear structure and cellular homeostasis regulated, partly, by mechano-transduction mechanisms. Cells are physically linked to their local matrix via focal adhesions, and this allows cells to respond to their physical environment. Within the cell, the cytoskeleton is connected to the nucleus through the LINC (linker of nucleoskeleton and cytoskeleton) complex. This, variations in extracellular matrix mechanics are propagated to the cell nucleus affecting cellular processes such as protein conformation, localization of transcription factors and chromosome organization. The nucleus regulates homeostasis and cell phenotype partly through mechano-transduction mechanisms. The nuclear envelope acts as a shock absorber to maintain nuclear architecture when the cell environment changes. Altogether, studying the mechanical properties of the nucleus can ultimately provide insight into changes in gene regulation responsible for changes in cell phenotype and cell pathology.  Although in vitro assays exist to study nuclear mechanics, these do not allow studying the complex interplay between extracellular matrix, cell and nuclear stiffness in the context of a tissue. To address this gap of knowledge, in their work McCreery et al (1) present a method that combines atomic force (AFM) and fluorescence confocal microscopy, and enables investigating the impact of extracellular matrix degrading enzymes on nuclear mechanics while cells are embedded in their native tissue environment.

Key findings and developments

The authors begin by reporting a method to directly measure nuclear membrane stiffness while maintaining cell-matrix interactions. They go on to investigate whether biomechanical disruption of tissues is transmitted to the cell and nucleus, and how the responses of the nucleus to the ECM can be measured by AFM. The experiments showed that enzymatic treatment of cartilage tissue explants causes softening of nuclei within embedded cells, and that the AFM needle-tip technique allows distinguishing local extracellular matrix, cell membrane, and nuclear membrane stiffness.  Fluorescence microscopy is used to visualize cell and nuclear structures and to align the needle tip over the desired structures. The authors were able to report stiffness values of the cell membrane and nuclear envelope by fitting the force-displacement data before relaxation of a corresponding needle puncture to a linear model. To validate needle penetration into the cell and nuclear structures, the authors mounted the AFM system onto an inverted laser scanning confocal microscope to observe the force spectroscopy curve, and to image fluorescence of isolated cells. In order to distinguish membrane deformation and membrane puncture, the authors used reporter HeLa cells stably expressing a charged multivesicular body protein 4B (CHMP4B-GFP). They then report differences between HeLa cell and chondrocyte puncture in terms of membrane stiffness, and attribute this to the small distance between the cell and nuclear membranes in HeLa cells causing the membrane to deform and evenly contact the nuclear envelope before puncturing both. They suggest this finding shows a limitation of the AFM needle-tip technique because the nuclear membrane stiffness may not be resolved in cells that spread on culture plates or have plasma membranes that are easily deformable. The authors then used a sphere-tip probe to indent cell membrane structures, and showed little change of fluorescence intensity at the site of measurements, confirming that membrane integrity is not disrupted by a rounded tip. Altogether, the results show that cell functionality and viability are preserved during and immediately after needle penetration, indicating that the AFM needle-tip technique is compatible with live cells and useful for nuclear probing.

            The authors then investigated the impact of biochemical degradation of bovine cartilage tissue on ECM, cell, and nuclear stiffness when treated over time with cartilage degrading enzymes (ADAMTS4-responsible for the pathological cleavage of aggrecan, and MMP13-which cleaves a range of type II collagen peptides). They measured these effects using the AFM needle tip technique combined with fluorescence. They used tissue sections parallel to the cartilage surface to expose middle zone chondrocytes. They then probed the cell structures with AFM, measuring extracellular matrix stiffness outside of the chondron (i.e. the local extracellular matrix to the cell). Since chondrocytes regulate extracellular enzymes, and enzyme activity occurs in the extracellular space, the authors suggest that cell and nuclear stiffness values obtained are probably reflective of extracellular matrix mechanics and not a direct result of cellular exposure to MMP13 and ADAMTS4. Disruption of the cartilage extracellular matrix by ADAMTS4 results in decreased stiffness of cartilage, cell membrane and nuclear envelope of embedded chondrocytes. Equally, biochemical disruption of the matrix by MMP13 reduces the stiffness of the cartilage tissue, local matrix, cell membrane and nuclear envelope. For both enzymes, as they disrupt extracellular matrix molecules and reduce its foundational structure, a cellular response is reduction of nuclear stiffness. An important difference between both enzymes is the timing and extent by which nuclear stiffness variation is observed. The authors suggest that these discrepancies provide insight into a) the propagation of matrix signals to the cell nucleus and b) the impact of disrupting different functional components of cartilage. Altogether, outcomes with both enzymes demonstrate that disruption of the cartilage matrix destabilizes cartilage structure and the transfer of intrinsic forces within the tissue, which may be transmitted to the cell surface. Moreover, disruption of links between the extracellular matrix to the cell nucleus contributes to pathology by triggering chromatin rearrangement and having an effect on gene expression. The authors finalize by suggesting that studied on nuclear stiffness should not be limited to quantification of nuclear mechanics alone or nuclear mechanics in living cells, but should include interactions of the nuclear envelope with the extracellular prestress among tissue types. Moreover they propose the method hereby presented as the basis for future studies of nuclear elastography of cells within living tissues, and indicate that specific challenges will arise and should be addressed, depending on the tissue type studied.

What I like about this preprint

I particularly like studies that explore gaps of knowledge, and generate methods that make it possible for the scientific community to study biological phenomena in a context as close to the true environment as possible. I also like that the authors address a topic in an interdisciplinary manner, which I think also has led to important discoveries and methods widely used by the scientific community.

 

Open questions

1. You mention that several challenges remain, specific to certain tissue types, and briefly mention a limitation of your method under specific conditions. Can you expand more on what other potential challenges different tissues might pose, and how/if your method can circumvent those challenges?

2. Specific to cells, you discussed the differences you observed between HeLa cells and chondrocytes. For experimental setups, have you found whether primary cells are very different from cell lines? Are you considering generating a “map” of multiple cells representative of various possible tissues?

3. Did you investigate the changes in gene expression of the various cells after being submitted to changes in the extracellular matrix caused by the degrading enzymes you used in your study? And further, how and if those gene expression changes impact cell morphology or pathology?

4. Following from the question above, is there a threshold stress level in the nuclear envelope whereby no changes to chromatin organization occur in the nucleus?

5. As future directions, do you envisage the AFM needle tip technique to be used in the context of, for instance, cancer and infection biology?

6. How compatible is the method with multiple other imaging setups- that is, how many hybrid measurements (considering multiple scales) could be done in an integrated setup?

References

1. McCreery et al , Nuclear stiffness decreases with disruption of the extracellular matrix in living tissues, bioRxiv, 2020.

 

doi: https://doi.org/10.1242/prelights.26107

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