To study the role of fodrin, a non erythroid spectrin, in cell cycle and mitosis

Abstract

Fodrin or non erythroid spectrin is composed of an α (240kDa) and a β (235kDa) subunit that form a tetrameric complex. It was isolated from goat brain as a part of cytoplasmic gamma tubulin ring complex, which is the nucleating agent of microtubules in mammalian cells. Gamma tubulin is present in cytoplasm as well as centrosomes but it is from the centrosome that gamma tubulin ring complex nucleates microtubules. The nucleation ability increases manifold for mitotic cells compared to interphase cells. In order to study the role of fodrin on HEK293 cells that has very low amount of fodrin, we overexpressed an α-fodrin EGFP plasmid in HEK293 cells. α-fodrin overexpression led to an increase in the level of acetylated tubulin whereas the total tubulin or gamma-tubulin amount remained the same. It has been earlier shown that the down regulation of α-fodrin has effects on the cell cycle, specifically a G1-S arrest. In light of this, cell cycle analysis upon overexpression of α-fodrin in HEK 293 cells revealed that there was a shift towards S phase with simultaneous decrease in the G1 phase. Besides, overexpression of α-fodrin led to a significant increase in the number of cells with mitotic abnormalities in the form of unattached chromosomes. Analysis of the spindle assembly checkpoint proteins revealed that the level of the protein MAD2 had significantly reduced which possibly explained the chromosome detachment. Further, a limited study showed a significant increase of gamma tubulin at the centrosomes when α-fodrin was overexpressed.