Connexon-mediated cell adhesion drives microtissue self-assembly

Abstract

Microtissue self-assembly is thought to be driven primarily by cadherins, while connexons have been examined mainly in intercellular coupling. We investigated whether connexon 43 (Cx43)-mediated cell adhesion modulates self-assembly of human KGN granulosa cells, normal human fibroblasts (NHFs), and MCF-7 breast cancer cells seeded into nonadhesive agarose gels. We found that treatment with anti-Cx43 E2 (112 μg/ml), which suppresses Cx43 docking, significantly inhibited the kinetics of KGN and NHF self-assembly compared to the preimmune sera control (41.1 ± 4.5 and 24.5 ± 10.4% at 8 h, respectively). Likewise, gap junction inhibitor carbenoxolone also inhibited self-assembly of KGN, NHF, and MCF-7 cells in a dose-dependent manner that was specific to cell type. In contrast, Gap26 connexin mimetic peptide, which inhibits channel permeability but not docking, accelerated self-assembly of KGN and NHF microtissues. Experiments using selective enzymatic digestion of cell adhesion molecules and neutralizing N-cadherin antibodies further showed that self-assembly was comparably disrupted by inhibiting connexin- and cadherin-mediated adhesion. These findings demonstrate that connexon-mediated cell adhesion and intercellular communication differentially influence microtissue self-assembly, and that their contributions are comparable to those of cadherins.

Figure 1.

Production of self-assembled microtissues. A) Monodispersed KGN cells were seeded into micromolded gels with circular or trough-shaped recesses and allowed to self-assemble. B, C) Twenty-four-hour time-lapse phase-contrast images of KGN spheroid (B) and rod (C) microtissue formation. Scale bars = 100 μm (B); 400 μm (C).

Figure 2.

Microtissues express Cx43 at sites of cell-cell contact and perinuclear regions. Cx43 immunostaining and confocal microscopy of KGN and NHF spheroids show positive Cx43 expression at sites of cell-cell contact (plaques) and perinuclear regions. Cx43 plaque expression appears higher in the microtissue interior compared to the surface. H/E and DAPI staining are provided as reference for microtissue morphology and nuclei, respectively. Scale bars = 50 μm.

Figure 3.

Microtissues demonstrate gap junction coupling by 3 h postseeding. Five percent NHF cells double-labeled with DiI and calcein AM were mixed with 95% unlabeled NHF cells and seeded into spheroid gels. Epifluorescent images at 0 h postseeding demonstrate colocalization of DiI and calcein AM, showing that coupling has not occurred yet. At 3 h, significant radial diffusion of calcein AM, but not DiI, indicates that coupling via gap junctions has occurred. Control microtissues composed of double-labeled/unlabeled NHF cells seeded into 200 μM carbenoxolone showed no evidence of coupling at 3 h. Scale bar = 100 μm

Figure 4.

Gap junction inhibitors carbenoxolone (CBX) and 1-heptanol inhibit microtissue self-assembly and self-sorting. A) Carbenoxolone inhibited KGN rod self-assembly in a dose-dependent manner, with increased inhibition on pretreatment. B) 1-Heptanol also inhibited self-assembly in a dose-dependent manner. C) Live/dead analysis confirms that microtissue viability was not compromised, n = 35. D) Percentage inhibition of rod contraction by carbenoxolone varied with postseeding time and cell type; n = 18 (KGN), 11 (MCF-7), and 19 (NHF). KGN cells labeled with CellTracker Green (CMFDA) were mixed with HeLa cells labeled with CellTracker Blue (CMAC) at a 1:1 ratio and seeded into spheroid gels. E) Cells were clearly segregated by 16 h postseeding, with the connexin-expressing cells (KGN) sorting to the center and the connexin-negative cells (HeLa) sorting to the periphery. Treatment with carbenoxolone (50 μM) disrupted sorting, with an increased degree of mixing. EF, epifluorescence; CF, confocal. Scale bar = 200 μm.

Figure 5.

Selective protease digestion of CAMs shows comparable inhibition of rod self-assembly by removal of CADs and CIDs. A–C) KGN (A) and MCF-7 (C) rod contraction were inhibited by enzymatic removal of both CADs and CIDs, while NHF rod contraction (B) was inhibited by removal of CADs only. Condition A: positive control, all CAMs intact. Condition B: CIDs only. Condition C: CADs only. Condition D: negative control, no CAMs intact. D) Western blot analysis confirmed high Cx43, N-cadherin, and total cadherin expression in KGN and NHF cells, the two rapidly aggregating cell types. Slower aggregating MCF-7 cells showed minimal Cx43 and total cadherin levels, with undetectable N-cadherin expression. Blots were immunolabeled for actin to ensure equal loading.

Figure 6.

Connexon-mediated adhesion and intercellular communication differentially modulate self-assembly of high-Cx43-expressing cell types. A, B) KGN (A) and NHF (B) rod self-assembly is significantly inhibited by anti-Cx43 (56 μg/ml) compared to preimmune serum control; n = 16, P < 0.05. C, D) Increased anti-Cx43 E2 concentration (112 μg/ml) further inhibited self-assembly of KGN cells (C) but not NHF cells (D). In contrast, Gap26 connexin mimetic peptide (300 μM) accelerated self-assembly of KGN (C) and NHF cells (D); n = 10. E, F). Inhibition by anti-Cx43 E2 is comparable to that seen with neutralizing anti-N-cadherin (40 μg/ml); n = 10.