Abstract:
During mitosis sister chromatids are accurately segregated into two daughter cells through a microtubule-based structure known as the mitotic spindle. Mitotic spindle assembly is a crucial machinery of cell proliferation that ensures the maintenance of genome integrity in eukaryotic cells. Errors in mitotic spindle formation have been associated with human diseases and tumorigenesis. Checkpoint kinase 1 (Chk1), a Serine/Threonine kinase, is a major regulator of DNA damage response. In our recent study, using confocal or time-lapse microscopy to analyse different cell lines in mitosis, we demonstrated that, in normally segregating cells without drug treatment, Chk1 is essential for optimal density and efficient polymerization of spindle microtubules in human cancer cells. Chk1 localizes to centrosomes in prometaphase and phosphorylates β-tubulin at Threonine 285. To investigate Chk1 activation at mitotic centrosomes, we focused on the role of ATR, ATRIP and TopBP1 in spindle formation. These proteins are known to activate Chk1 in response to DNA damage. First, we showed that ATR, ATRIP and TopBP1 localize to centrosomes in prometaphase, indicating a direct role of these proteins in mitosis but their role in mitosis is not yet fully understood. Second, depletion of ATR, ATRIP and TopBP1 reduced the relative intensity of active Chk1 (pS345) and phosphorylation of β-tubulin at Threonine 285 at centrosomes and impaired the formation of spindle microtubules. Impaired β-tubulin-T285 phosphorylation results in improper mitotic spindles, erroneous chromosome alignment and segregation, and unequal daughter cell-size. This novel ATR-Chk1 signaling pathway connects the molecular pathways between the cell cycle regulation and DNA damage response.
