1. Role of nucleoporin Nup153 in spindle checkpoint regulation
The nucleoporin Nup153 is a component of the nuclear basket of the NPC and known to play important roles in nuclear protein import as well as most RNA export pathways. It interacts with virtually every nuclear transport receptor studied. Nup153 is essential for cell viability and it is required for nuclear envelope breakdown at the onset of mitosis. Moreover, Nup153 is a dynamic nucleoporin that associates with the NPC in a transcription-dependent manner and that binds chromatin. In the past, we have studied more nucleocytoplasmic transport-related aspects of Nup153 function and have now shifted our interest to presumably non-transport functions of this nucleoporin. We found that altered expression of Nup153 in human cell lines causes abnormal mitoses with phenotypes that resemble defects in the spindle assembly checkpoint (SAC) and cytokinesis. The SAC assures that cells only divide after completion of chromosome segregation to prevent their mis-segregation and aneuploidy. Two SAC proteins, Mad1 and Mad2 had previously been shown to localise to nuclear pores in interphase cells, and our lab could show that Mad1 binds directly to Nup153 and that both proteins co-localise within the NPC. On the cellular level, enhanced levels of Nup153 in HeLa cells lead to inactivation of the SAC and consequently abnormal mitoses. SAC activity can be restored by simultaneous expression of Nup153 and Mad1. On the molecular level, enhanced levels of Nup153 are accompanied by hypophosphorylation of Mad1, which likely induces SAC impairment, as Mad1 hyperphosphorylation is a prerequisite for SAC activity. Moreover, in cells over-expressing Nup153 the localization of Mad1 in interphase is disrupted. Depletion of Nup153 from HeLa cells by RNA interference (RNAi) also causes abnormal mitoses, especially an increase in the number of cells in cytokinesis. Cytokinesis is the process in which the cytoplasm of two newly formed daughter cells is separated at the late stage of mitosis. It is highly linked to the SAC, and in Nup153 depleted cells the SAC remains functional with no effect on Mad1's phosphorylation status. The localization of Mad1, however, is altered during interphase and mitosis in cells lacking Nup153. In interphase cells, less Mad1 is found at NPCs in cells lacking Nup153. During mitosis, Mad1 is usually found at kinetochores in prometaphase and dissociates from kinetochores in metaphase. In Nup153-depleted cells, Mad1 remains associated with kinetochores during metaphase, which may lead to prolonged activity of the SAC and delayed exit from mitosis, which is consistent with the observed increase in cells that have undergone failed or aborted cytokinesis. We are now aiming to arrive at a better mechanistic understanding how Nup153 alters mitotic checkpoint and cytokinesis.