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Abstract

Precise spindle orientation during mitosis is essential for determining both cell fate and tissue organization. Proper alignment of chromosomes is a result of many processes that have to be orchestrated in a precise manner. Although some of the molecular mechanisms that underlie spindle orientation have been described recently, many aspects of this fundamental process remain unknown. Our protein of interest, MISP (mitotic interactor and substrate of Plk1, C19orf21), which was first characterized by our group as a substrate of Polo-like kinase 1 (Plk1), also seems to play a role in spindle orientation and positioning and in metaphase-to-anaphase transition (Zhu et al. 2013). In a mass spectrometrical screen aiming at identifying MISP-interacting proteins I identified IQGAP1, a multidomain scaffolding protein that is believed to link the microtubule network with the actin cytoskeleton, as a potential binding partner. By using co-immunoprecipitation experiments the interaction between MISP and IQGAP1 was confirmed both after their overexpression and endogenously. Functionally, I discovered that depletion of MISP leads to increased accumulation of IQGAP1 at the cell cortex in mitosis. The cortical accumulation of IQGAP1 seems to be dependent on Cdc42, since overexpression of Cdc42 can revert the cortical accumulation of IQGAP1. Cdc42 is a small signaling molecule belonging to the Rho family of GTPases and it is a well-characterized binding partner of IQGAP1. The altered localization of IQGAP1 also coincides with a decrease in its Cdc42 binding capacity and thereby reduced active Cdc42 levels upon MISP knock-down. Furthermore, I found that MISP shows a preferential binding to active Cdc42 similar to IQGAP1. Not surprisingly, I could show that this interaction is not direct and is indeed mediated by IQGAP1. Interestingly, overexpression of IQGAP1 can rescue the mitotic defects caused by MISP downregulation including spindle misorientation, loss of astral microtubules, prolonged mitosis and cortical accumulation of the dynactin subunit p150glued. In addition, it also restores active Cdc42 levels. Importantly, MISP-depletion leads to a reduction in active Cdc42 levels in wild-type but not in IQGAP1 knock-out cells pointing to the effector role of IQGAP1 in regulating active Cdc42 levels upon MISP depletion. Altogether, stabilization of active Cdc42 by IQGAP1 can restore mitotic defects upon MISP silencing. In conclusion, I found that IQGAP1 acts downstream of MISP in regulating active Cdc42 levels, astral microtubule dynamics and the localization of p150glued. Collectively, these results identify a novel pathway, namely that MISP regulates IQGAP1 and Cdc42 to ensure proper mitotic progression and correct spindle orientation.