The Physical Interaction of Myoblasts with the Microenvironment during Remodeling of the Cytoarchitecture

Abstract

Integrins, focal adhesions, the cytoskeleton and the extracellular matrix, form a structural continuum between the external and internal environment of the cell and mediate the pathways associated with cellular mechanosensitivity and mechanotransduction. This continuum is important for the onset of muscle tissue generation, as muscle precursor cells (myoblasts) require a mechanical stimulus to initiate myogenesis. The ability to sense a mechanical cue requires an intact cytoskeleton and strong physical contact and adhesion to the microenvironment. Importantly, myoblasts also undergo reorientation, alignment and large scale remodeling of the cytoskeleton when they experience mechanical stretch and compression in muscle tissue. It remains unclear if such dramatic changes in cell architecture also inhibit physical contact and adhesion with the tissue microenvironment that are clearly important to myoblast physiology. In this study, we employed interference reflection microscopy to examine changes in the close physical contact of myoblasts with a substrate during induced remodeling of the cytoarchitecture (de-stabilization of the actin and microtubule cytoskeleton and inhibition of acto-myosin contractility). Our results demonstrate that while each remodeling pathway caused distinct effects on myoblast morphology and sub-cellular structure, we only observed a ∼13% decrease in close physical contact with the substrate, regardless of the pathway inhibited. However, this decrease did not correlate well with changes in cell adhesion strength. On the other hand, there was a close correlation between cell adhesion and β1-integrin expression and the presence of cell-secreted fibronectin, but not with the presence of intact focal adhesions. In this study, we have shown that myoblasts are able to maintain a large degree of physical contact and adhesion to the microenvironment, even during shot periods (<60 min) of large scale remodeling and physiological stress, which is essential to their in-vivo functionality.

ROCK‐2 Is Associated With Focal Adhesion Maturation During Myoblast Migration

“…This acto-myosin force generated in the lamellipodia in response to RhoA/ROCK signalling induces the maturation of focal adhesions [Chrzanowska-Wodnicka and Burridge, 1996]. Y-27632 has previously been reported to decrease cell adhesion in myoblasts by 9% [Modulevsky et al, 2012]. This was confirmed by the results of the current study.…”

“…These adhesions also serve as key signalling points for motility regulation [Alvarez et al, 2008]. Actin contractility during motility requires the activation of myosin II downstream of ROCK [Choi et al, 2008;Modulevsky et al, 2012]. This acto-myosin force generated in the lamellipodia in response to RhoA/ROCK signalling induces the maturation of focal adhesions [Chrzanowska-Wodnicka and Burridge, 1996].…”

Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture

“…Cells were reliably imaged to a depth of approximately 120 µm below the upper surface of the scaffold. In all three cell types we also observed the presence of distinct F-actin stress fibres, which are a morphological characteristic of substrate-adhered cells [47], [77], [78]. Both fibroblasts and myoblasts cultured in our scaffolds possessed pronounced actin stress fibres, similar to those found in the same cells cultured on 2D substrates [46], [47].…”

Enzyme‐free cell detachment mediated by resonance vibration with temperature modulation

“…The mouse myoblast cell line, C2C12 (RCB0987, RIKEN BioResource Center, Ibaraki, Japan), was used to evaluate the proposed method as a representative adherent cell line, because C2C12 is one of the cells with strong adherence (Modulevsky et al, 2012;Yoshikawa et al, 2013). The myoblasts were cultured in growth medium (D-MEM/F12 supplemented with 10% fetal bovine serum [FBS]) in a 5% CO 2 humidified atmosphere incubator at 37 C. Cell passage was performed by trypsinization in 0.050% trypsin-EDTA (25300, Life Technologies, Carlsbad, CA) by pipetting.…”