Mechanical tugging force regulates the size of cell-cell junctions

Abstract

Actomyosin contractility affects cellular organization within tissues in part through the generation of mechanical forces at sites of cell-matrix and cell-cell contact. While increased mechanical loading at cell-matrix adhesions results in focal adhesion growth, whether forces drive changes in the size of cell-cell adhesions remains an open question. To investigate the responsiveness of adherens junctions (AJ) to force, we adapted a system of microfabricated force sensors to quantitatively report cell-cell tugging force and AJ size. We observed that AJ size was modulated by endothelial cell-cell tugging forces: AJs and tugging force grew or decayed with myosin activation or inhibition, respectively. Myosin-dependent regulation of AJs operated in concert with a Rac1, and this coordinated regulation was illustrated by showing that the effects of vascular permeability agents (S1P, thrombin) on junctional stability were reversed by changing the extent to which these agents coupled to the Rac and myosin-dependent pathways. Furthermore, direct application of mechanical tugging force, rather than myosin activity per se, was sufficient to trigger AJ growth. These findings demonstrate that the dynamic coordination of mechanical forces and cell-cell adhesive interactions likely is critical to the maintenance of multicellular integrity and highlight the need for new approaches to study tugging forces.

Fig. 1.

An approach to measure tugging forces (A) Endothelial cells exert contractile forces that strongly deflect underlying microneedles (phase contrast image; scale bar, 50 μm). (B and C) The vector sum of individual traction forces, T_n, (Red Arrows) exerted by a cell is zero (Eq. 1) (28, 29). (D and E) For a pair of contacting cells, the net force encompasses both traction forces (Red Arrows) and the intercellular tugging force, F_c, (Blue Arrows). (F) The intercellular tugging force is equal in magnitude and opposite in direction to the measured net traction force reported on the microneedle array (Eq. 2). Vector F_c plotted over cell A is defined as the net tugging force that cell A is exerting on cell B at the cell–cell contact. Cell B is expected to pull on cell A with an equal and opposite force. (G and H) Cells were constrained to a bowtie pattern using microcontact printing of fibronectin (Cyan) (G) and formation of adherens junctions in green (anti-β-catenin, H). Microneedles and cell nuclei were counterstained with DiI (Blue) and DAPI (Red), respectively. Scale bar, 10 μm. (I and J) Representative force vector plots of single (I) and paired (J) cells. Force vectors are plotted as arrows showing the direction of force and magnitude (arrow length; reference arrow for 10 nN shown above scale bar). Individual traction forces are shown in red and tugging force is shown in white. Small, force imbalances are observed due to uncertainties in microneedle deflection measurements. The error in the vector sum of individual traction forces equals the error of the individual forces multiplied by the square root of the number of tractions. The microneedles typically have an error of ±3 nN, and cells span on average 15 microneedles, leading to an expected noise level of 12 nN in the determination of F_c. (K) Average F_c experienced in bowtie pairs of HPAECs. * p < 0.05 indicates comparison against single cells. (L) Average traction force per microneedle was similar in singlet or contacting cells. (M–O) Tugging forces require coupling between VE-cadherin and the actin cytoskeleton. Cells expressing adeno-VE-Δ, a truncated VE-cadherin mutant lacking the β-catenin binding domain (residues 703–784), form adherens junctions (anti-β-catenin, Green, N) but show greatly diminished tugging force compared to adeno-GFP infected cells (M and O). * p < 0.05 indicates comparison against GFP. (P) Average traction force per microneedle was unaffected by VE-Δ expression. (Q and R) AJ (anti-β-catenin) in HPAECs are heterogeneous in size. Tugging force is shown in yellow. (S) AJ size is linearly correlated with F_c, with a correlation coefficient R² ≈ 0.9. Each data point represents one pair of cells. (T) AJ size is not correlated with the average traction force per microneedle. Error bars on all graphs denote standard error of the mean.

Fig. 2.

Tugging force regulates the size of adherens junctions. (A and B) Tension antagonists and agonists modulate average traction force per microneedle and F_c. The following antagonists were used: blebbistatin (30 μM, Blebbi), Y27632 (25 μM, Y27), siRNA against myosin IIA (siMyoIIA), myosin IIB (siMyoIIB). Tension agonists included nocodazole (1 μM, Noc) calyculin-A (1nM, Calyc), lentivirally encoding phosphomimetic light chain (ppMLC). Control conditions included vehicle control (Ctrl), control siRNA (siCyclophilin), no treatment (N/T), or lentiviral wild-type MLC (wtMLC). * p < 0.05 indicates comparison against vehicle control, # p < 0.05 indicates comparison against control siRNA, and Δ p < 0.05 indicates comparison against wtMLC. (C) AJ size is regulated by changes in actomyosin-mediated tension. Bowtie pairs exposed to the conditions in (A and B) and stained for β-catenin. (D–G) Relationship between AJ size and F_c. Inhibition of F_c by blebbistatin, Y27632 (D) or treatment with siRNA against myosin II A or B (E) leads to reduced junction size, whereas elevated F_c following stimulation with nocodazole, calyculin-A (F), or infected with lentivirus encoding phosphomimetic mutants (G) increased junction size. AJ size and F_c measurements (at least 10 bowtie pairs per condition) were clustered and plotted as an elliptical fit (D–G). Statistical significance between conditions is reported in Table S1. (H) A scatter plot showing the correlation between AJ size and F_c across all conditions. (I–L) Tension manipulations affect AJs in monolayers. Immunofluorescence images showing changes in AJs (anti- β -catenin) in HPAECs monolayers exposed to tension-manipulating conditions (I). Quantification of monolayer junctional response (J–L). * p < 0.05 indicates comparison against control monolayers: Ctrl (J), siCyclophilin (K), or wtMLC (L). All scale bars are 10 μm. Error bars denote standard error of the mean.

Fig. 3.

Modulation of force-AJ relationship by soluble factors. (A and B) Vasoactive compounds S1P and thrombin coordinately upregulate and downregulate adherens junctions. (A) Monolayers exposed to vehicle only (Ctrl), thrombin (0.1 μM, Thrb), or S1P (1 μM, S1P) show S1P-induced AJ growth and thrombin-induced AJ disassembly (anti-β-catenin staining). (B) Quantification of changes in AJs in monolayer cultures shown in A. * p < 0.05 indicates comparison against control. (C) Thrombin, but not S1P, induces cellular tension. Bar graph showing average traction force per microneedle reported in bowtie pairs stimulated with vasoactive compounds. (D and E) Effects of thrombin and S1P on junctional size-tugging force relationship. S1P and thrombin alter AJ formation (anti-β-catenin) in bowtie pairs (D). Relationship between AJ and F_c data plotted as an elliptical fit (E). The trend band and control condition from Fig. 2 are replotted here for reference. (F) RhoA and Rac1 activity assay for Thrombin and S1P using Rho G-LISA and Rac G-LISA kits, respectively. *, # p < 0.05 indicates comparison against baseline RhoA (*) or Rac (#) activity. (G) Manipulations of Rac activity do not alter cellular tension, as assayed by average traction force per microneedle. In contrast, inhibition of Rho kinase (Y27632, 25 μM) blocks thrombin-induced tension. * p < 0.05 indicates comparison against control. (H and I) Altering the coupling of S1P and thrombin to Rac and tension-dependent pathways reverse the effects of these vasoactive compounds on junctions. S1P-induced AJ growth is inhibited by treatment with NSC23766 (10 μM), whereas thrombin-induced AJ disassembly is blocked by expression of constitutively active Rac (RacV12). AJ junctions visualized by β-catenin localization (H). Decoupling thrombin from cellular tension or forcibly linking it to constitutively active Rac restores the linear relationship between AJ and tugging force. Similarly, inhibition of Rac1 with NSC23766 restores the normal AJ-tugging force balance in S1P-stimulated cells. (J) Myosin-manipulation regulates cellular tension independently of Rac activity levels. Blebbistatin (30 μM) downregulates average traction force per microneedle even in the presence of RacV12, whereas phosphomimetic myosin upregulates average traction force, even in the context of reduced Rac activity (NSC23766 treatment). * p < 0.05 indicates comparison against RacV12; and # p < 0.05 indicates comparison against NSC23766. (K– M) Interdependence of Rac-induced and tension-induced AJ assembly. Inhibition of myosin activity (blebbistatin, 30 μM) reduced the growth of AJs stimulated by constitutively active Rac (RacV12). Loss of Rac activity reduced the growth of AJs stimulated by phosphomimetic myosin. AJs visualized by β-catenin localization. (L and M) Effects of double Rac/myosin manipulations in J on AJ-T_c relationship. (N and O) Rac and tension-dependent pathways control the size of AJs in monolayer culture. Representative immunofluorescent micrographs of monolayers of cells exposed to the treatments in (G–M). AJs are visualized by β-catenin staining (N). Quantification of changes in junction size in monolayers (O). * p < 0.05 indicates comparison against Ctrl; # p < 0.05 indicates comparison against RacV12; and Δ p < 0.05 indicates comparison against NSC23766. (P) Schematic of how myosin-mediated tugging force and Rac activity coordinate to determine the ultimate size and stability of AJs. We suggest three general regimes: a low stress regime (Shaded Green) wherein high Rac activity support AJs assembly independently of tugging force, a moderate stress regime (White Region) wherein Rac activity and myosin-mediated tugging force coordinate to mediate mechanosensitive growth of AJs, and a high stress regime (Shaded Red) wherein high tugging forces/myosin activity initiate breaking/disassembly of junctions due to insufficient Rac-mediated junction assembly. All scale bars are 10 μm. Error bars on all graphs denote standard error of the mean. Statistical significance between conditions E, I, L, and M is reported in Table S1.

Fig. 4.

Microinjection of constitutively active RhoA and direct mechanical tugging leads to junction assembly. (A and B) Time-series plots showing enhancement of traction force in response to microinjection of constitutively active Rho protein (RhoA-Q63L) (B) but not wild-type Rho protein (wt) (A) for each of the two cells in bowtie pairs. Dotted arrow indicates the time point of microinjection, and only Cell I (Purple Line) was microinjected. Whiskers show the range of traction force observed in two independent bowtie movies. (C and D) Microinjection of RhoA-Q63L (D) but not wild-type Rho protein (C), acutely boosts tugging forces for each of the two cells in bowtie pairs. Dotted arrow indicates the time point of microinjection. Whiskers show the range of two microinjected bowtie pairs across all time points. (E) Representative image frames showing assembly of GFP-VE-cadherin following microinjection of RhoA-Q63L, 10 minutes before and 16 minutes after microinjection, respectively. Dotted white lines indicate the location of the pipette and the red arrowheads point to locations of increased AJ size. Scale bar is 10 μm. (F) RhoA-Q63L induced AJ assembly in a tension-dependent manner. The RhoA-microinjected pairs (Orange Squares) showed an increase in junction size while pretreatment with Y27632 (25 μM) blocked this effect (Blue Squares). Dotted arrow indicates the time point of microinjection. Whiskers show the range of two replicated experiments across all time points. (G) Exogenous tugging force stimulated VE-cadherin assembly. Fluorescence micrograph of GFP-VE-cadherin distribution prior to micropipet-mediated mechanically tugging of the cell–cell junction. Dotted white lines indicate the location of the pipette and the white arrow points to the direction of pipette movement. Region of interest (Green Box) shows distribution of junctional VE-cadherin before (0 min) and after tugging (4 min), pseudocolored green and red, respectively in the overlay image. Yellow boxes denote the regions of the AJ showing the strongest cadherin recruitment. Scale bar is 10 μm. (H) Quantification of AJ growth in response to mechanical tugging. AJ size of bowtie pairs over 10 min intervals plotting for baseline (Green Line), with pipette on top of the cell but no tugging (Blue Line), or before and after tugging one cell of the bowtie (Red). Black arrow indicates the time point of tugging with the micropipet. Whiskers show the standard error of the mean across six independent experiments.