Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemical and Biomolecular Engineering

First Advisor

Dennis E. Discher

Abstract

‘Nuclear mechanosensing’ encompasses a wide range of biophysical pathways that are emerging as key processes in the regulation of cell function and fate. Many of these mechanisms involve the main structural protein of the nucleus, lamin-A, which is abundant in stiff and mechanically stressed tissues such as striated muscle, but is comparatively low in soft tissues such as the brain. Lamin-A’s increase with tissue stiffness correlates strongly with elevated levels of collagen-I fibers in the extracellular matrix (ECM), but mechanisms and functional consequences of any matrix-nucleus interplay remain unclear. Here, in the first set of studies, we show that lamin-A and collagen-I exhibit tightly coupled mechano-sensitivity in the first functional vertebrate organ, the beating embryonic heart, following a mechanism for tension-suppressed turnover that confers mechano-protection against DNA damage. Lamin-A and collagen-I increase together as the heart stiffens daily in embryogenesis, but their levels are found here to be modulated within 1-2 hours by rapid and reversible perturbations of actomyosin contractility or ECM mechanics. In both intact hearts and in isolated cardiomyocytes, suppression of lamin-A – combined with high contractile stress – results in i) increased nuclear envelope rupture, ii) cytoplasmic mis-localization of DNA repair factors, and iii) accumulation of DNA damage, which ultimately causes arrythmia. Embryonic cardiomyocytes on stiff collagen-coated gels show increased lamin-A levels compared to those on soft gels, suggesting a cell-intrinsic protective mechanism against DNA damage. Interphase phosphorylation of lamin-A emerges as a key posttranslational modification that gives rise to such mechano-sensitivity, as phosphorylation and subsequent degradation of lamin-A are suppressed with myosin-II-dependent cell spreading. This mechanism of tension-suppressed turnover is further examined in a second set of studies, which focuses on the aging-associated lamin-A mutant, ‘progerin’. Using a novel mass spectrometry-based workflow, we find that progerin phosphorylation in patient iPS-derived cells is lower and less mechanosensitive compared to normal lamin-A and C, suggesting that a loss in the nucleus’ ability to dynamically remodel in response to stress could contribute to genome instability and aging. Mechanosensing by lamin-A is thus critical not only in embryonic development, but also in disease and aging of mature tissues.

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