Connexin 43-mediated modulation of polarized cell movement and the directional migration of cardiac neural crest cells

Abstract

Connexin 43 knockout (Cx43α1KO) mice have conotruncal heart defects that are associated with a reduction in the abundance of cardiac neural crest cells (CNCs) targeted to the heart. In this study, we show CNCs can respond to changing fibronectin matrix density by adjusting their migratory behavior,with directionality increasing and speed decreasing with increasing fibronectin density. However, compared with wild-type CNCs, Cx43α1KO CNCs show reduced directionality and speed, while CNCs overexpressing Cx43α1 from the CMV43 transgenic mice show increased directionality and speed. Altered integrin signaling was indicated by changes in the distribution of vinculin containing focal contacts, and altered temporal response of Cx43α1KO and CMV43 CNCs to β1 integrin function blocking antibody treatment. High resolution motion analysis showed Cx43α1KO CNCs have increased cell protrusive activity accompanied by the loss of polarized cell movement. They exhibited an unusual polygonal arrangement of actin stress fibers that indicated a profound change in cytoskeletal organization. Semaphorin 3A, a chemorepellent known to inhibit integrin activation, was found to inhibit CNC motility, but in the Cx43α1KO and CMV43 CNCs, cell processes failed to retract with semaphorin 3A treatment. Immunohistochemical and biochemical analyses suggested close interactions between Cx43α1,vinculin and other actin-binding proteins. However, dye coupling analysis showed no correlation between gap junction communication level and fibronectin plating density. Overall, these findings indicate Cx43α1 may have a novel function in mediating crosstalk with cell signaling pathways that regulate polarized cell movement essential for the directional migration of CNCs.