Cell–cell mechanical communications in distance have been much documented in recent decade, which has shown remote mechanosensitive responses among cells (Alisafaei et al., 2021; Long et al., 2022). In tracking the development of this discovery back, biologists had observed the outgrowth of separated neuronal tissues toward each other in early last century (Weiss, 1929; Katzberg, 1959). In 1981, Harris et al. reported that traction force from fibroblast tissue explants induced fibrillary morphogenesis of type I collagen (COL), in which separation reached a distance of 1.5–4 cm (Harris et al., 1981; Stopak and Harris, 1982). The work is followed with a mechanistic study on the collagen fiber self-organization driven by cell and matrix movements (Sawhney and Howard, 2002). Over 10 years ago, single-cell studies reported mechanical interactions within neighboring cells (Reinhart-King et al., 2008; Winer et al., 2009). Later, studies established long-range force-induced communications among mammary cell clusters, which causes COL fiber remodeling and helps assembly of tissue patterns (Guo et al., 2012; Brownfield et al., 2013; Shi et al., 2014).

In recent decade, cell long-range mechanical communications have been shown in different scenarios. Cardiomyocytes can reserve a beating frequency induced by a mechanical probe nearby or achieve synchronized beating (Nitsan et al., 2016; Sapir and Tzlil, 2017). Fibroblast cells show an ability to remotely mechanosense the lateral boundaries when seeding on floating collagen gel (Mohammadi et al., 2014). Collective migration of epithelial sheet is promoted by long-range intercellular force transmission on the gradient-stiffness substrate (Sunyer et al., 2016). Fibroblasts can collectively align through long-range interactions arising from modification of underlying matrix (Li et al., 2017). Long-range mechanotransduction via collagen fibers enables myofibroblast–fibroblast interactions during fibrosis expansion (Liu et al., 2020). Fibroblasts display remote attraction of macrophages on collagen hydrogel via distant force transmission (Pakshir et al., 2019). Neural crest cell group achieves collective migration in a supracellular fashion with persistent contractility at the rear of the cluster, and self-generates a stiffness gradient within the tissue in vivo (Shellard et al., 2018; Shellard and Mayor, 2021). These studies have demonstrated long-range mechanical communications as a common feature of different cells.

At the same time, the relevant mechanism of long-range force transmission and COL fibrillary modeling have been investigated based on experimental data and computational simulations (Ma et al., 2013; Wang et al., 2014; Baker et al., 2015). Clinical biopsy check and in vitro assays both show cells aligning collagen fibers to guide metastatic migrations of breast cancer cells (Han et al., 2016). The property of non-linear elastic extracellular matrix (ECM) from tissue can facilitate distant force transmission (Sopher et al., 2018), and cell-generated stresses are capable of buckling COL filaments in ECM at large spatial extent (Han et al., 2018; Mann et al., 2019). Our recent data have shown that motile cells can actively recruit soluble environmental COL to assemble filaments for tissue-level assembly (Wang et al., 2020).

These observations also lead to an interesting topic on how cells mechanosense each other in distance or at large spatial scales. Some progresses have emerged in recent years, for example, mechanosensitive integrin signals and ion channels in cells are necessary components in mechanical communications (Pakshir et al., 2019; Liu et al., 2020). Another mechanistic advancement is the demonstration that cells are capable of rapidly sensing traction force in the matrix or substrate deformation, which results in directional migration and tissue-level assembly (Zarkoob et al., 2018; Ouyang et al., 2020). It has also been proven that cells can directly sense the mechanical force from the substrate besides conventional stiffness (Panzetta et al., 2019). From computational simulation along with experimental verification, COL fiber bundles re-organized by cell contraction bear the major tensile force in the ECM, which can be transmitted to distant cells (Fan et al., 2021). During migration of epithelial cell sheet, there is also long-range force transmission through cell–cell junctions, and ERK signal wave is activated in cells to promote the migration direction (Sunyer et al., 2016; Hino et al., 2020). Cell mechanical communications also play a role in tissue pattern formation (Guo et al., 2012). Our work has shown that cells can form stable connections by mechanical interactions on the elastic ECM, but not on coated glass, and the assembled connections rely on cell contraction force by the ECM substrate (Ouyang et al., 2020). The cellular mechanism for distant mechanical communications and resulted cell connections still remains largely to be elucidated.

In this work, we applied the experimental model of mechanical communication from our recent study (Ouyang et al., 2020) and investigated the mechanosensitive pathways in regulating the mechanical attractions and cell connection stability. Cell mechanotransduction has been shown involving a bunch of molecules such as the typical membrane receptors integrins and cadherins and ion channels (Martino et al., 2018). Here, we screened the roles of integrin, cadherin, and calcium channels in cell distant mechanical communications and branching assembly. In regards, specifically, cells communicate with the microenviromental matrix largely through ligations between integrins and matrix proteins (Li et al., 2016; Sun et al., 2016); cadherins located at the cell–cell junctions are mechanosensitive molecules between cells (Leckband and de Rooij, 2014); membrane calcium channels have been demonstrated critical for cells in transducing external mechanics (Kim et al., 2015; Fang et al., 2022). Through experimental characterizations, our work demonstrated the importance of calcium channels on the ER membranes and integrin signal along with actomyosin contraction in cell distant mechanical attractions, and N-cadherin in stabilizing cell–cell connections.

Cell–cell mechanical communications in long or large spatial scale have been increasingly recognized in the past decade. It has been further revealed recently that cells are able to communicate via traction force mechanosensation through the matrix substrates. The mechano-responsive mechanism is still to be investigated. In this work, we tried to study the primary mechanosensing components for cell–cell distant mechanical interactions. Based on the experimental model from our recent work (Ouyang et al., 2020), we characterized cell directional migration and network assembly to measure cell–cell mechanical attractions, which was applied to identify cellular mechanosensitive components.

ASM cells assembled into network structures within 1 day on the hydrogel, while stayed isolated individually on the glass surface without force transmission (Figure 1A, Supplementary Figure S1; Supplementary Movie S1). Migration quantifications showed much quicker movements on the hydrogel than on the glass, indicating a significant promoting role in driving cell directional migration by cell–cell mechanical interactions (Figure 1B). The integrity of actin cytoskeleton and actomyosin activity are essential for the mechanical interactions (Figures 1D–H; Supplementary Movie S2), verifying the interaction force originated from cellular contraction. Hence, we have validated the experimental model based on migration and branching assembly for cell–cell mechanical communications.

Recent studies reported that Piezo ion channel (one type of mechanosensitive calcium channel) on the plasma membrane plays an important role in cell mechanosensation of force (Pakshir et al., 2019; Liu et al., 2020). Here, we further explored the relevance of calcium channels on plasma and ER membranes during the cell–cell distant mechanical interactions. The experimental data demonstrated that the IP₃R calcium channel and SERCA pump on the ER membrane are important for cell directional migration and essential for branching assembly (Figures 2A–E; Supplementary Movie S3). This observation is consistent with the previous reports that the calcium signal from ER storage is mechanosensitive to mechanical stimulation (Kim et al., 2015; Kim et al., 2020), which also supports the cell–cell distant attractions due to mechanical communications.

Integrins are mechanosensitive receptors on the plasma membrane in response to ECM mechanics (Alenghat and Ingber, 2002; Isomursu et al., 2019). Two recent studies have shown that integrin signaling mediates intracellular mechanical interactions (Pakshir et al., 2019; Liu et al., 2020). Here, we tried to validate the role of integrin signaling in this experimental model. Inhibition of integrin β1 with siRNA in ASM cells, which is a representative subunit forming dimer with variable α subunits, led to significantly reduced cell–cell remote attractions as shown by the inhibited directional migrations and branching formations (Figures 3A–E; Supplementary Movie S4). In considering that integrins are basic components in assembling focal adhesions (Kanchanawong et al., 2010), deficiency in integrin may hinder the mechano-response for directional migrations toward the neighboring cells. From these few studies, it is conclusive that integrins are mechanosensitive signals in the cell–cell distant mechanical communications.

There is an interesting observation in our work that ASM cells formed one-to-one connections to assemble a continuous network structure, but no stable connections occurred on the glass without traction force transmission (Supplementary Figure S1; Figures 1A,B) (Ouyang et al., 2020). N-cadherin is one mechanosensitive molecule at cell–cell junctions (Leckband and de Rooij, 2014) and also shows the role in promoting cell directional movements (Choi et al., 2021). In this work, selective knockdown of N-cadherin with siRNA did not have apparent impact on cell–cell remote attraction as measured by the move speeds, but inhibited one-to-one stable cell connections for branching assembly (Figures 4A–D, Supplementary Movie S5). This result implies that N-cadherin is critical for traction force-induced stable cell–cell connections.

From this work, we have not understood the correlations of these few factors during cell–cell mechanical communications. From previously known information and this study, calcium channels mediate actomyosin contraction by calcium signaling, which drives cell migration (Kuo and Ehrlich, 2015); integrin is important in transducing biomechanical signaling at the cell–matrix interface, and promoting cell protrusion at the migratory front (Huttenlocher and Horwitz, 2011); N-cadherin is not directly responsive in this distant mechanical communication, but maintains the branching through cell–cell junctions adjacent with intracellular cytoskeleton. There are cross talks between integrin signaling and cadherin, as both interacting closely with actin cytoskeleton (Weber et al., 2011).

In summary, our work identified the importance of the IP₃R calcium channel and SERCA pump on the ER membrane and also verified the integrin signal in the cell–cell distant mechano-attractions, and further demonstrated junctional N-cadherin in stabilizing traction force-induced cell–cell connections. The molecular mechanism or subsequential relations of these components during the mechanosenation need more studies.