Roles of actin cytoskeletal dynamics on cyclic compression-induced autophagy in 3D collagen encapsulated human mesenchymal stem cells

Abstract

Hypothesis Cells can perceive and propagate external forces to their interior via membrane mechanosensors and actin reorganization, then promoting the biogenesis of physiological structures consisted of vesicular cargoes. Of these, a specialized vesicle called autophagosome, generated by a lyososomal degrading process known as autophagy involving in energy and nutrient recycling upon the mechanical loading. Dysfunction of autophagy impairs the cell adaptation and survival, eventually leading to cell death and diseases. Recent studies report the mechanical forces can induce autophagy and enhance their survival in 2D cell models but there is little evident reported on 3D models. In this regards, this study hypotheses that the dynamic compression loading can induce the autophagy via a change in actin dynamics in human mesenchymal stem cells entrapped in type I collagen (3D MSCs). Methods To examine this hypothesis, we exerted a constant 10% strain of dynamic compression loading on 3D hMSCs with 5e5 cells/ml cell density at different duration (2hrs, 5hrs and 9hrs) and harvested them immediately after loading for subsequent detection assays including quantitation of the candidate markers for autophagy and actin remodeling and their distribution within cells by qPCR analysis and immunofluorescent(IF) staining respectively. We also included 2D monolayer and 3D hMSCs without compression for normalization and comparisons. Results and conclusions The qPCR analysis reveals differential expressions of autophagic (BL1 and LC3) and actin related markers (ARP2) at different duration. For autophaic markers, there are no apparent change until 5+0 and then reach a maximum at 9+0 for BL1, and however, it prominently moves upward from 2+0 and 5+0 and get to the highest expression at 9+0 for LC3. For ARP2, it drops to a minimum at 5+0 and then recovers to the normal at 9+0. In protein analysis, it consistently exhibits increased LC3II/LC3I ratio but reduced ARP2 expression at 9+0 demonstrating a divergent expression between LC3II/I and ARP2. Taken together, it suggests actin related proteins as key regulators, presumably enhancing the compression-induced autophagy despite further studies on the downstream effectors along the processes of autophagy and actin cytoskeletal dynamics will be required to make a better understanding of their actual interactions on the cell metabolism.