Menu

Close

Nuclear stiffness decreases with disruption of the extracellular matrix in living tissues

Kaitlin P. McCreery, Xin Xu, Adrienne K. Scott, Apresio K. Fajrial, Sarah Calve, Xiaoyun Ding, Corey P. Neu

Preprint posted on August 31, 2020 https://www.biorxiv.org/content/10.1101/2020.08.28.273052v1

Article now published in Small at http://dx.doi.org/10.1002/smll.202006699

Understanding cell dynamics within tissues.

Selected by Mariana De Niz

Categories: biophysics, cell biology

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.

Figure 1. AFM-based method for probing the cell nucleus of viable cells in tissues. (From Ref.1).

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?
  1. 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?
  1. 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?
  1. 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?
  1. As future directions, do you envisage the AFM needle tip technique to be used in the context of, for instance, cancer and infection biology?
  1. 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.

 

Posted on: 2nd December 2020

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

Read preprint (No Ratings Yet)




Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the cell biology category:

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka, Madhuja Samaddar, Miguel V. Almeida, Sejal Davla, Jennifer Ann Black, Dey Lab

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Preprint missing? Don't hesitate to let us know.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage
Close