Combined Atomic Force Microscope and Volumetric Light Sheet System for Mechanobiology

Kern

Stephens

et al. 2020

Preprint

Self Cite

Nuclei are constantly under external stressbe it during migration through tight constrictions or compressive pressure by the actin capand the mechanical properties of nuclei govern their subsequent deformations. Both altered mechanical properties of nuclei and abnormal nuclear morphologies are hallmarks of a variety of disease states. Little work, however, has been done to link specific changes in nuclear shape to external forces. Here, we utilize a combined atomic force microscope and light sheet microscope (AFM-LS) to show SKOV3 nuclei exhibit a two-regime force response that correlates with changes in nuclear volume and surface area, allowing us to develop an empirical model of nuclear deformation. Our technique further decouples the roles of chromatin and lamin A/C in compression, showing they separately resist changes in nuclear volume and surface area respectively; this insight was not previously accessible by Hertzian analysis. A two-material finite element model supports our conclusions. We also observed that chromatin decompaction leads to lower nuclear curvature under compression, which is important for maintaining nuclear compartmentalization and function. The demonstrated link between specific types of nuclear morphological change and applied force will allow researchers to better understand the stress on nuclei throughout various biological processes.

Section: Combined Atomic Force Microscopy and Side-view Light Sheet Mmentioning

confidence: 99%

Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics

Kern

Stephens

et al. 2020

Preprint

Self Cite

“…2E). The AFM-LS system allows for high-resolution, 3-D imaging of the whole nucleus throughout the compression application (Gavara, 2017;Liu et al, 2019;Nelsen et al, 2020), thereby enabling us to observe the spatio-temporal dynamics of DNA damage (Suppl. Video 4).…”

Section: Nuclear Compression Is Sufficient To Cause Dna Damagementioning

confidence: 99%

“…MDA-MB-231 cells were cultured in DMEM/F12 media without phenol red supplemented with 10% FBS (Sigma-Aldrich), 1× antimycotic (Gibco) and 15mM HEPES. A day before the experiment, 50-70% confluent cultures were trypsinized and plated on polyacrylamide gels (stiffness of 55 kPa) coated with collagen as described before (Nelsen et al, 2020). Briefly, 10 µL of activated gel solution was deposited on APTES-treated 40 mm round coverslips, allowed to dry, and attached to a 10 mm diameter glass cloning cylinder (Corning) using vacuum grease (Dow Corning).…”

Section: Afm-lsmentioning

confidence: 99%

“…Cells were plated such that only 1-3 cells were present per field of view at 60× magnification. To compress individual nuclei, we employed the use of atomic force microscopy (AFM) along with light-sheet (LS) microscopy as described previously Nelsen et al, 2020). Briefly, a beaded cantilever (beaded in house and calibrated using thermal tuning as described in (Gavara, 2017)) was mounted onto the AFM and lowered over a cell of interest.…”

Section: Afm-lsmentioning

confidence: 99%

See 1 more Smart Citation

Nuclear deformation causes DNA damage by increasing replication stress

Shah

Cheng

et al. 2020

Preprint

Cancer metastasis, i.e., the spreading of tumor cells from the primary tumor to distant organs, is responsible for the vast majority of cancer deaths. In the process, cancer cells migrate through narrow interstitial spaces substantially smaller in cross-section than the cell. During such confined migration, cancer cells experience extensive nuclear deformation, nuclear envelope rupture, and DNA damage. The molecular mechanisms responsible for the confined migrationinduced DNA damage remain incompletely understood. While in some cell lines, DNA damage is closely associated with nuclear envelope rupture, we show that in others, mechanical deformation of the nucleus is sufficient to cause DNA damage, even in the absence of nuclear envelope rupture. This deformation-induced DNA damage, unlike nuclear envelope ruptureinduced DNA damage, occurs primarily in S/G2 phase of the cell cycle and is associated with stalled replication forks. Nuclear deformation, resulting from either confined migration or external cell compression, increases replication fork stalling and replication stress, providing a molecular mechanism for the deformation-induced DNA damage. Thus, we have uncovered a new mechanism for mechanically induced DNA damage, linking mechanical deformation of the nucleus to DNA replication stress. This mechanically induced DNA damage could not only increase genomic instability in metastasizing cancer cells, but could also cause DNA damage in nonmigrating cells and tissues that experience mechanical compression during development, thereby contributing to tumorigenesis and DNA damage response activation.

“…In this work, we address some of these open questions regarding the links between mechanics and morphology through the use of our combined atomic force microscope and side-view light sheet microscope (AFM-LS; Nelsen et al. , 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics

Kern

O’Brien

et al. 2020

MBoC