A key step in mitosis is the separation of sister chromatids at the metaphase-anaphase transition, this step is controlled by the anaphase-promoting complex (APC) and it's regulatory proteins. On the other hand, several earlier studies showed that Ca²⁺ signaling is involved in regulating the metaphase-anaphase transition in meiosis. The present study is aimed at investigating the signaling transduction pathway in regulating the metaphase-anaphase transition in mitosis in mammalian cells. We tried to answer three questions: ( 1 ) Is Cdk1 inactivation required for the metaphase-anaphase transition in mammalian cells? (2) Is calcium signaling required for the activation of APC in mitosis in mammalian somatic cells? (3) How does calcium signaling play its role in mitosis? Using a GFP gene f...[ Read more ]

A key step in mitosis is the separation of sister chromatids at the metaphase-anaphase transition, this step is controlled by the anaphase-promoting complex (APC) and it's regulatory proteins. On the other hand, several earlier studies showed that Ca²⁺ signaling is involved in regulating the metaphase-anaphase transition in meiosis. The present study is aimed at investigating the signaling transduction pathway in regulating the metaphase-anaphase transition in mitosis in mammalian cells. We tried to answer three questions: ( 1 ) Is Cdk1 inactivation required for the metaphase-anaphase transition in mammalian cells? (2) Is calcium signaling required for the activation of APC in mitosis in mammalian somatic cells? (3) How does calcium signaling play its role in mitosis? Using a GFP gene fusion technique and living cell imaging methods, we have conducted a series of experiments to answer those key questions. First, we measured the temporal and spatial dependent proteolysis of both securin and cyclin B and found that both of them are degraded before the onset of anaphase. By quantify the protein level of nondegradable cyclin B (Δ85) compared with endogenous cyclin B, we found that a low level of nondegradable cyclin B can cause cells arrested before M/A transition. These results indicate that Cdk1 inactivation is required for sister chromatids separation. Second, by examining the differential effects of Ca²⁺ signaling blockers (BAPTA/AM, BAPTA, KN-93, heparin) on the metaphase-anaphase transition in HeLa cells, we found that the activation of APC depends on Ca²⁺ signaling via Ca²⁺/CaM-dependent protein kinase II (CaMKII). The inhibition of APC by Ca²⁺ signal blockers may have two parallel pathways, one is through the spindle checkpoint via the destruction of mitotic spindle, while the another is suppression of CaMKII activity. Finally, we examined other important signaling molecules involved in the metaphase-anaphase transition, Mad2, Mad1 and Polo-like kinase 1. By measuring the dynamics of Mad2/Mad1 and cyclin B proteolysis in intact living cells, we demonstrated the degradation of cyclin B is related to the translocation of Mad2/Mad1. The detailed molecular mechanisms of the signaling transduction pathway in the metaphase-anaphase transition in mitosis mammalian cells were discussed.