Centrosome duplication in mammalian cells arrested prior to entry into S phase and the role of tektin 2 in centrosome duplication, mid body formation and cytokinesis

Once every cell cycle, centrosomes duplicates precisely once. This facilitates the formation of a bipolar spindle, permitting equal segregation of chromosomes between both daughter cells. Yet little is known about the when the centrosome duplicates during the cell cycle, and few components have been identified that are critical in the generation of a new centrosome. Each centrosome is comprised of a pair of barrel shaped structures arranged in an orthogonal arrangement. EM studies have revealed the initial duplication event to be disorientation when the centriole pair lose their orthogonal arrangement with daughter centrioles forming from the proximal end of the pre-existing parent centriole. Yet when this event occurs is unclear. EM studies have suggested that procentrioles were first observed after transition into S phase. However, studies in sea urchin and yeast found that the centrosome could duplicate in cells that did not transit into S phase, providing support for the G1 phase possessing the necessary factors required for duplication. To investigate if duplication can occur during G1 arrest, CHO-K1 cells were stably transfected with GFP-centrin, a well known centriolar marker. When cells were arrested in G1 with mimosine, both individual and multiple rounds of duplication was observed in 97% of cells. This demonstrated that centrosomes could duplicate in G1 arrested cells. In addition to CHO cells, centrosome duplication was observed in the non-transformed CHEF cell-line, arrested in late G1 with mimosine. Yet, during a normal cell cycle, duplication was observed after cells have entered S phase. This demonstrates that factors necessary for duplication are present during G1, however new procentrioles are not observed during a normal cell cycle until after cells have transited into S phase. Although G1 appears to possess the necessary factors required for centrosomes to duplicate, few factors have been identified. In sea urchin, a group of axonemal proteins termed tektins were identified. Early studies in mammalian cells found that tektins could localize to centrosomes, however little work has been undertaken to investigate the role of tektins at the centrosome. Using GFP-tektin 2 in conjunction with a rabbit polyclonal antibody to tektin 2, tektin 2 localized to centrosomes, in a manner similar to γ-tubulin. In addition to the centrosome, tektin 2 also localizes to the mid body. When tektin 2 was knocked down by siRNA, levels of reduplication were decreased during S phase, suggesting a role for tektin 2 in duplication. Surprisingly, the loss of tektin 2 in cycling cells led to cells 're-fusing' after exiting mitosis. In the absence of tektin 2, the mid body condensed, however as a result of tektin 2 being absent, the mid body unravelled permitting cells to 'refuse'. In addition to tektin 2, GFP-tektin 1 was found to localize to the mid body and individual centrioles. These studies provide a starting point for examining the role of tektins at the centrosome and mid body.