The impact of smooth muscle cell ageing upon actin cytoskeleton organisation, adhesion and motility

Abstract

Vascular smooth muscle cell (VSMC) phenotypic switching, from a contractile to a migratory phenotype, is essential for vascular repair and is compromised in ageing. Phenotypic transition involves dramatic actin reorganisation which is regulated by the linker of the nucleoskeleton and cytoskeleton (LINC) complex that spans the nuclear envelope (NE). The LINC complex physically anchors cytoskeletal filaments to the nucleoplasm via nesprin-SUN-nuclear lamina connections which enable rapid biophysical signalling between the nuclear interior and exterior. The nuclear lamina consists of A and B-type lamins that maintain nuclear architecture, however, the lamin A precursor protein, prelamin A, is a biomarker of VSMC ageing and this project investigates the impact of prelamin A upon LINC complex function.
During VSMC ageing in-vitro, prelamin A accumulates at the presenescent growth phase which is associated with cellular elongation and focal adhesion reorganisation. Interference reflection imaging (IRM) and time-lapse microscopy revealed that presenescent VSMCs exhibit increased focal adhesion turnover with enhanced migratory speed and persistence. Importantly, prelamin A accumulation induced by siRNA-mediated knockdown of its processing enzyme, FACE-1, reiterates these morphological changes and enhances migratory persistence. Moreover, RhoA and Rac1 are well-established regulators of cell motility and their expression and activity diminishes in presenescent and FACE-1 depleted VSMCs. Fluorescence recovery after photobleaching (FRAP) also revealed that nuclear lamina disruption increases nesprin-2 dynamics at the nuclear exterior. Thus, we suggest that prelamin A impacts on actin-regulated processes including cell shape and motility via the LINC complex.
We utilised an siRNA-mediated approach to investigate the importance of other LINC complex components in regulating cell morphology and migration. Interestingly, SUN2 levels decrease during in-vitro VSMC ageing and SUN2 knockdown enhances the migratory speed of VSMCs and fibroblasts similarly to presenescent VSMCs. The role of nesprins in regulating cell phenotype varies between different cells, highlighting that LINC complex organisation and function is flexible and cell-type specific.
Together, our data reveal that the LINC complex is a versatile structure that is specialised to cell function and is an important regulator of cellular morphology, focal adhesion organisation, Rho GTPase activity and migration. VSMC ageing is associated with prelamin A accumulation and loss of SUN2 expression which consequently deregulates LINC complex organisation and functioning. Therefore, LINC complex disruption gives rise to an aged VSMC phenotype which we predict may underlie cardiovascular diseases such as atherosclerosis.
In addition, IRM captured the release of adhesion-like structures, termed cell traces, from the rear of migrating VSMCs that outline their migratory path. Cell traces form physical tracks that enhance the speed and migrational directionality of neighbouring VSMCs. Therefore, we predict that cell traces support VSMC migration to injury sites and are important for vessel repair.

Date of Award	2014
Original language	English
Awarding Institution	King's College London
Supervisor	Derek Warren (Supervisor) & Cathy Shanahan (Supervisor)