Immunostaining and time-lapse analysis of vinblastine-induced paracrystal formation in human A549 cells.

Abstract

Vinblastine is a vinca alkaloid that binds to tubulin and inhibits microtubule formation in cells. Vinblastine treatment results in the formation of paracrystalline aggregates in the cells, which are formed from tightly packed tubulin molecules. Mitotic spindle assemblies in treated cells are disrupted and cell cycle progression is arrested at the mitosis phase. Vinblastine is therefore widely used for cancer treatment. However, the mechanism underlying paracrystal formation has not been fully elucidated. The present study attempted to observe paracrystal formation in human A549 cells. Initally, paracrystal formation was detected using the anti-tubulin antibody. Secondly, the exogenousuly expressed RFP-conjugated tubulin also formed paracrystals. Additionally, immunostaining with the anti-RBM8A antibody overlapped with paracrystal images obtained from RFP conjugated tubulin. This suggested that the localization of the RBM8A proteins was adjacent to the tubulin molecules prior to vinblastine treatment. Furthermore, a time-lapse analysis was developed for paracrystal formation in viable human A549 cells. This was achieved using exogenous expression of fluorescent proteins conjugated with tubulin and time-lapse microscopy. It may be concluded that the indicated method was successful for the real-time analysis of paracrystal formation in human cells.