Localization and distribution of connexin 43 in mitochondria of cervical cancer HeLa cells

Abstract

Objective To study the distribution of connexin 43 (Cx43) in cervical cancer HeLa cells, and to verify the localization of Cx43 in mitochondria. Methods HeLa cells were segregated into cytoplasm, cell nucleus, mitochondria and supernatant after segregation by using the method of homogenate and centrifuge. Immunoelectron microscope was used to observe the morphology of mitochondria and the localization as well as the distribution of Cx43 in HeLa cells. Voltage-dependent anion channel 1 (VDAC1) was used to confirm the localization of mitochondria. Immunofluorescence was used to costain HeLa cells with Cx43 and mitochondrial marker VDAC1 to verify mitochondria localization of Cx43 in cervical cancer HeLa cells. Then Western blot was used to quantify the expression of Cx43 in fractions (cytoplasmic fraction, nuclear, mitochondria and post mitochondrial supernatant). Mitochondrial markers including VDAC1 and cytochrome c oxidase Ⅳ (COX Ⅳ) were used to confirm mitochondria. Plasma membrane marker (LHR) was used to confirm plasma membrane. Results Immunoelectron microscope confirmed that the normal mitochondria or cystic swollen one could be seen in the complete HeLa cells and the detached HeLa cells mitochondria, with the presence of Cx43 and VDAC1 in detached mitochondria. Immunofluorescence showed Cx43 colocalized with VDAC1. There was a significant difference in the Cx43 expressions of the subcellular structure in the HeLa cells [cytoplasm (1.23±0.11), cell nucleus (0.39±0.09), mitochondria (3.67±0.59), supernatant after segregation (0.16±0.06); F = 84.17, P < 0.05]. It also showed that the relative amount of Cx43 in mitochondria was enriched. Conclusions Cx43 is enriched in mitochondria in cervical cancer HeLa cells. Therefore, Cx43 in mitochondria might be a potential target in diagnosis, therapy and prognosis of cervical cancer. Key words: HeLa cells; Connexin 43; Mitochondria; Localization; Distribution