Hutchinson–Gilford progeria syndrome alters nuclear shape and reduces cell motility in three dimensional model substrates

Abstract

Cell migration through tight interstitial spaces in three dimensional (3D) environments impacts development, wound healing and cancer metastasis and is altered by the aging process. The stiffness of the extracellular matrix (ECM) increases with aging and affects the cells and cytoskeletal processes involved in cell migration. However, the nucleus, which is the largest and densest organelle, has not been widely studied during cell migration through the ECM. Additionally, the nucleus is stiffened during the aging process through the accumulation of a mutant nucleoskeleton protein lamin A, progerin. By using microfabricated substrates to mimic the confined environment of surrounding tissues, we characterized nuclear movements and deformation during cell migration into micropillars where interspacing can be tuned to vary nuclear confinement. Cell motility decreased with decreased micropillar (μP) spacing and correlated with increased dysmorphic shapes of nuclei. We examined the effects of increased nuclear stiffness which correlates with cellular aging by studying Hutchinson-Gilford progeria syndrome cells which are known to accumulate progerin. With the expression of progerin, cells showed a threshold response to decreased μP spacing. Cells became trapped in the close spacing, possibly from visible micro-defects in the nucleoskeleton induced by cell crawling through the μP and from reduced force generation, measured independently. We suggest that ECM changes during aging could be compounded by the increasing stiffness of the nucleus and thus changes in cell migration through 3D tissues.

On-chip assay of the effect of topographical microenvironment on cell growth and cell-cell interactions during wound healing

“…42,48 Nuclei deformation is most likely the result of a balance between the rigidity of the nucleus and the force that the cytoskeleton is able to exert on it. 23 All together, these results confirmed that cells could interact with the micropillar substrates and respond dynamically and spatiotemporally to the surrounding extracellular matrix.…”

“…The specific geometric dimensions of micropillars in the array were chosen to expect to deform the cell morphology because they are close to cells sizes. 12,13,23 For clarity, the diameter of the micropillars and spacing between neighbor micropillars in the different arrays were denoted as d15, d18, and d18 lm and s15, s18, and s21 lm, respectively, in the next description. SEM image showed that the micropillars were well arranged on the substrate (Figures 1(e) and 1(f)).…”

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

“…However, traditionally the scratch assay has lacked a standardized set-up and quantification protocol, which has led to issues with reproducibility 7,10 . Many of the modifications and optimizations for improving the scratch assay have reduced these issues, including individual live-cell imaging, standardized stopper-based assays, and three dimensional micro-pillar assays 4,8,10 . However, these assays can be difficult to customize or adapt 10 and for some applications, these techniques can also be cost prohibitive.…”

“…Importantly, such an assay must be sufficiently sensitive to detect relatively subtle changes in cellular migratory capacity after damage. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness 1,4,5 . The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.…”

“…Polydimethlysiloxane (PDMS) elastomers cured over a precise mold and treated with ozone and fibronectin produces a homogenous and non-degradable microenvironment. Micro-pillar spacing can also be varied as needed to gauge the ability of cells to enter the array 4 . The mold is created through deep reactive ion etching of silicon wafers to create a negative version of the high aspect-ratio array 13 .…”