It is well established that mechanical forces around cells and cellular functions are closely related to each other under both physiological and pathological conditions (1, 2). These cellular forces are either generated from cells and then transmitted through actin stress fibers referred to as endogenous forces or transmitted from outside cells referred to as external forces (3). Advancements in the field of mechanobiology have provided much evidence that cells are able to sense and adapt to these mechanical forces around their microenvironment (4). It has been shown that mechanotransduction through cell-extracellular matrix (ECM) adhesions (5), cell-cell junctions (6), plasma membrane (7), glycocalyx (8), and nucleus (9) can modulate various cell behaviors such as cell spreading (10), proliferation (11), differentiation (12-14), migration (15, 16), morphogenesis (7), cancer progression (17), and ECM remodeling (18, 19). Therefore, developing novel tools to discover how cells could dynamically sense and respond to these mechanical forces would be of great importance to understand the physiology and pathology in life science and bioengineering fields.
Over the years, studies on mechanical forces have extensively exploited functions of integrin-mediated FAs that can act as mechanotransducers between actomyosin stress fibers and ECMs-/polymer-based cell culture substrates with varying stiffness (13, 20). Moreover, it has been well established that the interplay among focal adhesions, cell surface integrins, and the stiffness of ECMs could play a significant role in regulating cell adhesion and spreading (21-23). For examples, it has been reported that the density of ECM ligands could control the spreading behavior of cells through focal adhesion (FA) assembly and that subsequent degree of cell spreading could regulate cellular functions through changes in cell shape, cytoskeletal tension, and Ras homolog family member A (RhoA) mediation (10, 24). In addition, seminal studies pioneered by Discher and Engler have reported that the stiffness of PAA-based hydrogels as cell culture substrates with tunable mechanical properties could determine the fate of human mesenchymal stem cells (hMSCs) by remodeling focal adhesion and cytoskeleton (12, 25). These hMSCs adhered onto either “soft” or “stiff” matrix could sense biophysical and mechanical cues of the matrix having a native tissue-like stiffness, resulting in undergoing lineage-specific differentiation of hMSCs into various cell types depending on tissue-like elasticity. They also further reported that mechanotransduction for regulating stem cell fates could be primarily determined by matrix stiffness, not by ECM tethering or porosity of substrates (12, 26). Indeed, a comprehensive understanding of mechanobiology requires novel tools to measure the forces between cells and ECMs, which are termed as traction forces and the methods to quantify these forces using microscopy-based techniques are known as traction force microscopy (TFM) (27). Therefore, in this review, we will highlight recent advancements in TFM-based methods for understanding multiple aspects of cellular forces exerted by cells at cell-ECM interfaces as well as at junctional intracellular domains within cellular microenvironment. Specifically, we will also discuss how the TFM-based methods can further elucidate the roles of mechanical forces at interfaces of cell-cell/cell-ECM in controlling various cellular functions. The different approaches and methods introduced in this review are summarized in Table 1 and 2.
The first approach to determine cellular traction force using TFM was reported by Harris
Very recently, Razafiarison
As an alternative to TFM using PAA hydrogel-based flat and continuous substrates, Chen and his colleagues have developed microfabricated post-array-detectors (mPADs) to manipulate spatial characteristics of substrates with tunable mechanical compliance (Fig. 1C and 1D). Subcellular traction force was calculated based on one-dimensional (1D) Hooke's law by measured deflection and spring constant of deformable posts (36). Very importantly, this study firstly suggested the possibility of tunable mechanical properties of micropost-based substrates by varying heights of deformable posts without changing their surface chemistry. In their follow-up studies using mPADs, Fu
In another study, Kiran
Similar approaches have been applied to understand how traction forces could mediate cell shape changes such as cell spreading and flatting of human mesenchymal stem cells (hMSCs) and their differentiation into osteogenic lineage through RhoA/ROCK activation and cytoskeletal tension (38). In that study, Wang
In combination, these diverse reports indicate that there is a strong correlation between matrix stiffness and adhered cell-induced traction forces. The degree of traction forces could become one of the determinants for switching stem cell fates through cell spreading.
Cellular forces are known to predominantly occur in tangential (in-plane) directions (X, Y) with an assumption that there are no normal (out-of-plane) forces to the substrates beneath cells (Fig. 1E) (4). Therefore, TFM has been extensively used to calculate two-dimensional (2D) traction forces generated by adhered cells onto 2D substrates. More recently, however, several studies have reported 3D TFM methods to quantify both tangential and normal forces against 3D ECM by utilizing z-stacked 3D images obtained from confocal microscopy (33, 39, 40). For example, Hur
Although mapping multi-dimensional traction forces with spatiotemporal manners is highly demanded, much less is known about how to quantify 3D traction forces exerted by cells within 3D microenvironments. To solve these issues, novel approaches have been reported to quantify the spatiotemporal nature of 3D traction forces exerted by cells within 3D hydrogels, exhibiting linear elastic properties (41, 42). Legant
As an alternative approach to measure 3D traction forces in native nonlinear and viscoelastic connective tissue-like microenvironments, Steinwachs
Soon after substantial progresses have been made in analyzing cellular traction forces via TFM, it has been suggested that the same principle as TFM could be extended to interpret average cell-cell junctional or intracellular forces by applying the same force balance principle. These methods are known as intracellular force microscopy (IFM) and monolayer stress microscopy (MSM) (45-47). Emerging evidences have suggested that adherent cells could exert normal forces to beneath substrates and that these forces are no longer ignorable. Thus, there have been numerous attempts to decipher spatiotemporal regulations of 3D forces around cells (33, 48). Furthermore, recent advances in IFM have unraveled important attributes of force transmission through cell-ECM and cell-cell adhesions or intercellular junctions-mediated force transmission to the ECM (49, 50). In addition, these IFM methods offer new opportunities to assess intracellular and intercellular forces in a group of cells such as cell-cell doublets (46, 51) and monolayers of cells.
For example, to better understand endogenous intracellular forces, cell-cell tugging junctional forces between pairs of ECs, Liu
In a similar approach using IFM-based methods, Ng
It has been shown that IFM-based methods have great advantages with little assumptions required for mechanical properties of cellular materials such as nuclei, plasma membranes, actin cytoskeletons, and cell-cell junctions to calculate intercellular or intracellular forces at adherens junctions. However, intercellular or intracellular tensions measured by IFM-based methods are averaged in-plane (in 2D) and mapped linearly (in 1D). Therefore, stresses could not be mapped on a 2D plane (53, 54). To resolve these challenges, Tambe
Very recently, Serrano
The past two decades have seen the development of a variety of methods to measure cell-generated forces on FAs, AJs, and intracellular organelles via actin stress fibers. These methods have elucidated many aspects of mechanisms through which cells migrate, proliferate, differentiate, remodel, and mechanosense their microenvironment. In this review, we provided an overview of recent advancements of TFM quantifying cell-ECM forces (or traction forces) exerted on integrin-based focal adhesions and IFM and MSN quantifying cell-cell and intracellular forces applied through E-cadherin-based adherens junctions. As mechanobiology becomes more important in life science and engineering, TFM and IFM will play a fundamental role in elucidating cell functions related to mechanical force responses in biological research field. There is no doubt that these TFM-/IFM-based novel methods for understanding roles of biomechanical forces at interfaces of cell-cell/cell-ECM will open doors to breakthrough technologies for revolutionizing regenerative medicine, disease modelling, and drug discovery.
This research was supported by a grant (2019H1D3A2A02102074) of Brain Pool Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT. It was also partially supported by Soonchunhyang University Research Fund.
The authors have no conflicting interests.
Summary of each TFM-based cellular force measurement analysis
|TFM Methods||Target Forces||Dimension & Image acquisition||Substrate Materials||Advantages||Disadvantages||Refs|
|Deformable material-based 2D TFM||
||PAA, PDMS, PEG||
||(27, 31, 32, 34)|
|Micropost-based 2D TFM||
|Deformable material-based 3D (2.5D) TFM||
|Deformable material-based 3D TFM||
||PEG, type I collagen||
Summary of each IFM-or MNM-based cellular force measurement analysis
|IFM & MSN Methods||Target Forces||Dimension & Image acquisition||Basic method for IFM & MSN||Advantages#||Disadvantages#||Refs|
|Deformable material-based 2D IFM||
||Deformable material-based 2D TFM||
|Micropost-based 2D IFM||
||Micropost-based 2D TFM||
|Deformable material-based 3D IFM||
||Deformable material-based 3D (2.5D) TFM||
|Deformable material-based 2D MSM||
||TFM 2D Micropost-based||
|Deformable material-based 3D MSM||
||Deformable material-based 3D (2.5D) TFM||
#IFM and MSN analyses are proceeded using the basic information acquired from TFM, therefore IFM and MSN inherit advantages and disadvantages of TFM-based force measurement analysis.