Cell Cytoskeletal Behaviors on Curved Microtopography

Abstract

Physiological characteristics of cells are co-regulated by intracellular structures and extra-cellular matrix (ECM). Previous research demonstrated that both biophysical and biochemical properties of the ECM can determine cell behaviors including actomyosin-mediated cell contractility. Current studies on the biophysical factors have been largely related to the planar substrate rigidity and nano-roughness, yet how the three-dimensional ECM-coated substrate geometry co-affects cell behaviors with other ECM properties remains elusive.

In this research, we apply microfabrication and micro-contact printing technologies to develop arrays of ECM-coated microwells and microgrooves with different substrate radii of curvature as a high-throughput, generic assay for characterizing different cell properties as functions of the substrate curvature. We show that the substrate curvature is an essential biophysical factor in determining cell spreading and morphology. For more detailed studies, we focus on two primary human vascular cells, i.e. vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs), growing on different substrate radii of curvature. Interestingly, within radii of curvature ranging from 50 μm to 125 μm, we observe that some VSMCs can partially detach from the curved substrates, with the attachment of their ends along the major cell body axes and detachment of their central cell bodies – we name this partial cell detachment as the ‘freestanding’ behavior. On the other hand, all VECs conform to the substrate curvature.

We employ the micro-post array and immunofluorescence staining to quantify the actomyosin-mediated contractility and subcellular characteristics of intracellular cytoskeletal components including actomyosin, actin, and vinculin (a common protein of focal adhesions). Our results demonstrate that the cell freestanding behavior is closely related to actomyosin of the cells. Starting from the view of force interaction between cells and the ECM, a physical model built from the first principle reveals theoretically that actomyosin activity and organization are both critical for the occurrence of the freestanding behavior. More specifically, by mediating the activity and subcellular arrangement of actomyosin with two biochemical reagents separately (Y27632 for actomyosin suppression, Calyculin A mainly for actomyosin subcellular arrangement of VSMCs), we discover a necessary condition for the freestanding behavior among VSMCs, which requires sufficiently strong intracellular actomyosin expression and sufficient well-aligned arrangement of actomyosin.

Furthermore, we perform experiments to induce freestanding VECs for the first time using drug treatments in order to verify the roles and necessary conditions of actomyosin activity and cell morphology in the freestanding behavior, considering that the untreated-VECs are all freestanding-negative. In essence, we can induce the freestanding behavior of VECs by enhancing the actomyosin expression of VECs using Calyculin A and stabilizing actin polymerization of VECs using Jasplakinolide. Notably, application of either drug (i.e. solely Calyculin A or Jasplakinolide) fails to induce freestanding of VECs. Collectively, these results confirm the cytoskeleton-mediated freestanding behavior.

Considering the relevance to the vascular abnormality, we further employ the atherosclerosis-inducing oxidized low density lipoprotein (oxLDL) on VECs to examine whether the unconformity of cell attachment can be caused by oxLDL and is associated with alterations of cytoskeletal properties of VECs. Surprisingly, the oxLDL-treated freestanding VECs have their cell morphology and actomyosin expression agreeing with the necessary conditions for drug-treated freestanding VECs and VSMCs.

Taken together, since cell physiological and morphological dynamics in three-dimensional cell environments are closely related to tissue development and regeneration, for instance, angiogenesis, and diseases such as atherosclerosis, this research work can provide important insights for our further understandings on human tissue-related diseases as well as the effective medical strategies.

Date of Award	23 Jan 2017
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Hiu Wai Raymond LAM (Supervisor)