(D-149) Chromatin adaptation to confinement during microchannel-based cell migration

Introduction:: In the past decade, the importance of the tissue microenvironment on the determination of cell fate has been more and more appreciated. Single cell migration is a fundamental process in numerous pathological and physiological situations. Current understanding suggests that during directed cell migration, a large number of factors could contribute to this orchestrated signaling including biophysical and biochemical properties such as matrix stiffness¹, fluid viscosity ², biochemical ligand presentation³ and topography⁴. During this process, cells move through tight interstitial spaces, aligned extracellular matrix, and narrow capillaries where they are exposed to direct physical compression⁵. The diverse nature of tissue microenvironments in vivo present various levels of confinement, constriction, and compression to migrating cells in both homeostasis and disease.

How such diverse microenvironments precisely dictate cell fate is a fascinating topic that still remains mysterious. As the largest and stiffest organelle, the nucleus is directly subjected to external forces during confined migration, and also actively deformed via the force-generating cytoskeleton during cellular adaptation to confinement. Previous studies identified the nucleus as an active mechanosensor and a number of engineered systems have been developed to directly control nuclear confinement and analyse resultant cell behaviour⁶. However, much remains unclear about the processes governing the active nuclear reorganization, specifically, the difference between nuclear response to long, sustained confinement recapitulating continuous interstitial spaces and short, rapid, pinch point confinements mimicking small openings in the endothelium.


Materials and Methods:: To address this gap in knowledge, we fabricated PDMS microchannel-based devices as a platform to monitor cell migration with high temporal resolution and reduced spatial complexity. Linear channels with widths of 3 and 10 µm, and 10 µm height were fabricated with a two-step photolithography. The PDMS replica mold generated was attached to glass coverslips and coated with collagen I. Our microchannel system recreated the sustained micro-scale confining environment through which cells voluntarily migrate without the presence of chemoattractant or flow.


Results, Conclusions, and Discussions:: Results

In 3 µm channels, dramatic deformation of cell nuclei is required for successful migration through such extreme confinement. With confocal reconstruction, we showed that invasive MDA-MB-231 cells experienced nuclear elongation as well as a 30% volume reduction under extreme confinement with partial recovery post confinement. Moreover, live tracking of the nuclear trajectory quantitatively demonstrated that cells spent a longer time at the channel entrance, with the nuclei asymmetrically positioned to the back under extreme confinement. Additionally, with DNA double strand break marker gamma-H2AX, we found a downregulation of nuclear damage level under sustained confinement. Together, these observations hint at a potential reorganization stage of the nucleus before entering confinement.

Next, we investigated expression patterns of multiple histone modification markers and altered chromatin architecture during confined migration. Our results demonstrated rapid local chromatin condensation occurs as cell adapt to confinement (Figure 1A). Total nuclear H3K9Ac staining intensity decreased when cells were under both moderate and extreme confinement (Figure 1B-C). Moreover, colocalization analysis showed that H3K9Ac labelled euchromatin regions polarize to nuclear edges under 3 µm sustained channels (Figure 1D-F). Finally, we demonstrated mechanically induced translocation of histone acetyltransferase and deacetylase contribute to the adaptive chromatin condensation.

Conclusions and Discussions

With our microchannel system, we focus on the question of precisely when and how the nucleus is reorganized during exposure to spatial confinement. To understand the mechanistic details of this process, we investigated how the dynamics of nuclear elements change during this process. One of the most profound effects of such rearrangement is the change on nuclear chromosome architecture. Specifically, we demonstrated rapid local chromatin condensation during nuclear morphological reorganization into extreme confinement. Future work will focus on the crosstalk between mechanically altered biochemical enzyme activity as well as changes in nuclear mechanics and gene expression profiles. Together, this will provide novel insight into the determination of cell fate via confined migration.


Acknowledgements (Optional): : The authors would like to acknowledge the support of the Mechanobiology Institute, the National Research Foundation of Singapore, and the Joseph Needham Foundation for Science and Civilisation (Hong Kong).


References (Optional): : 1 Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 2006;126:677–89.

2 Bera K, Kiepas A, Godet I, Li Y, Mehta P, Ifemembi B, et al. Extracellular fluid viscosity enhances cell migration and cancer dissemination. Nature 2022;611:365–73.

3 Young JL, Holle AW, Spatz JP. Nanoscale and mechanical properties of the physiological cell-ECM microenvironment. Exp Cell Res 2016;343:3–6.

4 Reversat A, Gaertner F, Merrin J, Stopp J, Tasciyan S, Aguilera J, et al. Cellular locomotion using environmental topography. Nature 2020;582:582–5.

5 Yamada KM, Sixt M. Mechanisms of 3D cell migration. Nat Rev Mol Cell Biol 2019;20:738–52.

6 Hsia C-R, McAllister J, Hasan O, Judd J, Lee S, Agrawal R, et al. Confined migration induces heterochromatin formation and alters chromatin accessibility. IScience 2022;25:104978.