Dataset Information

Substrate Stiffness Mediates Formation of Novel Cytoskeletal Structures in Fibroblasts during Cell-Microspheres Interaction.

ABSTRACT: It is well known that living cells interact mechanically with their microenvironment. Many basic cell functions, like migration, proliferation, gene expression, and differentiation, are influenced by external forces exerted on the cell. That is why it is extremely important to study how mechanical properties of the culture substrate influence the cellular molecular regulatory pathways. Optical microscopy is one of the most common experimental method used to visualize and study cellular processes. Confocal microscopy allows to observe changes in the 3D organization of the cytoskeleton in response to a precise mechanical stimulus applied with, for example, a bead trapped with optical tweezers. Optical tweezers-based method (OT) is a microrheological technique which employs a focused laser beam and polystyrene or latex beads to study mechanical properties of biological systems. Latex beads, functionalized with a specific protein, can interact with proteins located on the surface of the cellular membrane. Such interaction can significantly affect the cell's behavior. In this work, we demonstrate that beads alone, placed on the cell surface, significantly change the architecture of actin, microtubule, and intermediate filaments. We also show that the observed molecular response to such stimulus depends on the duration of the cell-bead interaction. Application of cytoskeletal drugs: cytochalasin D, jasplakinolide, and docetaxel, abrogates remodeling effects of the cytoskeleton. More important, when cells are plated on elastic substrates, which mimic the mechanical properties of physiological cellular environment, we observe formation of novel, "cup-like" structures formed by the microtubule cytoskeleton upon interaction with latex beads. These results provide new insights into the function of the microtubule cytoskeleton. Based on these results, we conclude that rigidity of the substrate significantly affects the cellular processes related to every component of the cytoskeleton, especially their architecture.

SUBMITTER: Adamczyk O

PROVIDER: S-EPMC7835802 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Substrate Stiffness Mediates Formation of Novel Cytoskeletal Structures in Fibroblasts during Cell-Microspheres Interaction.

Adamczyk Olga O Baster Zbigniew Z Szczypior Maksymilian M Rajfur Zenon Z

International journal of molecular sciences 20210119 2

It is well known that living cells interact mechanically with their microenvironment. Many basic cell functions, like migration, proliferation, gene expression, and differentiation, are influenced by external forces exerted on the cell. That is why it is extremely important to study how mechanical properties of the culture substrate influence the cellular molecular regulatory pathways. Optical microscopy is one of the most common experimental method used to visualize and study cellular processes ...[more]

PMID: 33478069

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Project description:Restoration of epidermal organization and function in response to a variety of pathophysiological insults is critically dependent on coordinated keratinocyte migration, proliferation, and stratification during the process of wound healing. These processes are mediated by the reconfiguration of both cell-cell (desmosomes, adherens junctions) and cell-matrix (focal adhesions, hemidesmosomes) junctions and the cytoskeletal filament networks that they serve to interconnect. In this study, we investigated the role of substrate elasticity (stiffness) on keratinocyte colony formation in vitro during the process of nascent epithelial sheet formation as triggered by the calcium switch model of keratinocyte culture. Keratinocytes cultured on pepsin digested type I collagen coated soft (nominal E = 1.2 kPa) polyacrylamide gels embedded with fluorescent microspheres exhibited (i) smaller spread contact areas, (ii) increased migration velocities, and (iii) increased rates of colony formation with more cells per colony than did keratinocytes cultured on stiff (nominal E = 24 kPa) polyacrylamide gels. As assessed by tracking of embedded microsphere displacements, keratinocytes cultured on soft substrates generated large local substrate deformations that appeared to recruit adjacent keratinocytes into joining an evolving colony. Together with the observed differences in keratinocyte kinematics and substrate deformations, we developed two ad hoc analyses, termed distance rank (DR) and radius of cooperativity (RC), that help to objectively ascribe what we perceive as increasingly cooperative behavior of keratinocytes cultured on soft versus stiff gels during the process of colony formation. We hypothesize that the differences in keratinocyte colony formation observed in our experiments could be due to cell-cell mechanical signaling generated via local substrate deformations that appear to be correlated with the increased expression of β4 integrin within keratinocytes positioned along the periphery of an evolving cell colony.

Project description:Pulmonary arterial adventitial fibroblasts (PAAFs) are important regulators of fibrotic vascular remodeling during the progression of pulmonary arterial hypertension (PAH), a disease that currently has no effective anti-fibrotic treatments. We conducted in-vitro experiments in PAAFs cultured on hydrogels attached to custom-made equibiaxial stretchers at 10% stretch and substrate stiffnesses representing the mechanical conditions of mild and severe stages of PAH. The expression of collagens α(1)I and α(1)III and elastin messenger RNAs (Col1a1, Col3a1, Eln) were upregulated by increased stretch and substrate stiffness, while lysyl oxidase-like 1 and α-smooth muscle actin messenger RNAs (Loxl1, Acta2) were only significantly upregulated when the cells were grown on matrices with an elevated stiffness representative of mild PAH but not on a stiffness representative of severe PAH. Fibronectin messenger RNA (Fn1) levels were significantly induced by increased substrate stiffness and transiently upregulated by stretch at 4 h, but was not significantly altered by stretch at 24 h. We modified our published computational network model of the signaling pathways that regulate profibrotic gene expression in PAAFs to allow for differential regulation of mechanically-sensitive nodes by stretch and stiffness. When the model was modified so that stiffness activated integrin β3, the Macrophage Stimulating 1 or 2 (MST1\2) kinases, angiotensin II (Ang II), transforming growth factor-β (TGF-β), and syndecan-4, and stretch-regulated integrin β3, MST1\2, Ang II, and the transient receptor potential (TRP) channel, the model correctly predicted the upregulation of all six genes by increased stiffness and the observed responses to stretch in five out of six genes, although it could not replicate the non-monotonic effects of stiffness on Loxl1 and Acta2 expression. Blocking Ang II Receptor Type 1 (AT1R) with losartan in-vitro uncovered an interaction between the effects of stretch and stiffness and angiotensin-independent activation of Fn1 expression by stretch in PAAFs grown on 3-kPa matrices. This novel combination of in-vitro and in-silico models of PAAF profibrotic cell signaling in response to altered mechanical conditions may help identify regulators of vascular adventitial remodeling due to changes in stretch and matrix stiffness that occur during the progression of PAH in-vivo.

Dataset Information

Substrate Stiffness Mediates Formation of Novel Cytoskeletal Structures in Fibroblasts during Cell-Microspheres Interaction.

Publications

Substrate Stiffness Mediates Formation of Novel Cytoskeletal Structures in Fibroblasts during Cell-Microspheres Interaction.

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