Localization of phosphorylated connexin 43 using serial section immunogold electron microscopy

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Gap junction turnover involves the formation of connexosomes, double membrane-enclosed vesicles derived from gap junction plaques.
• Phosphorylation is a key regulatory mechanism in gap junction dynamics, but precise protein associations require further investigation.
• Distinguishing between gap junctions and connexosomes, and identifying their associated proteins, is essential for understanding gap junction turnover.

Purpose of the Study:

• To investigate the formation and protein composition of connexosomes using advanced electron microscopy techniques.
• To determine the phosphorylation status of connexin 43 (GJA1) in gap junctions and connexosomes.
• To elucidate the role of specific connexin 43 phosphorylation sites in gap junction internalization and connexosome processing.

Main Methods:

• Utilized serial section electron microscopy with automated tape collecting ultramicrotome (ATUM) for high-resolution imaging of mouse ovarian follicles.
• Employed immunolabeling techniques on serial sections to identify and localize specific phosphorylated residues of connexin 43.
• Analyzed the three-dimensional organization of gap junctions and connexosomes within their native cellular environment.

Main Results:

• Connexosomes were observed to form from various cellular structures, including adjacent cell bodies, thin cell processes, and the same cell.
• Phosphorylation of connexin 43 at serine residue S368 was detected in both gap junctions and connexosomes.
• Phosphorylation of connexin 43 at serine residue S262 was specifically found on a subset of connexosomes, suggesting a role in their formation or processing.

Conclusions:

• Phosphorylation at S262 of connexin 43 is implicated in connexosome formation or subsequent processing.
• These findings provide precise evidence that phosphorylation plays a critical role in the internalization of gap junctions.
• Serial section electron microscopy of immunogold-labeled tissues is a powerful method for studying the 3D organization of cellular structures.