Regulatory Mechanisms Governing the Establishment of Cell Polarity and Mitotic Spindle Orientation in the Drosophila Neuroblast
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The Drosophila neuroblast undergoes repeated asymmetric cell divisions that produce one daughter cell that assumes a neuronal fate and another that remains a neuroblast. During mitosis, the neuroblast polarizes the conserved Par polarity complex to the apical cortex, which is responsible for segregating fate determinants to the basal cell cortex. Polarity is accompanied by orientation of the mitotic spindle through the proteins Pins, Mud, and Dlg to ensure that the cleavage furrow properly segregates the fate determinants. The adaptor protein Inscuteable coordinates these two pathways. In my work, I have addressed how asymmetrically dividing cells are dynamically polarized during the cell cycle and how the resulting polarity is coupled to spindle position. To address how neuroblast polarity is dynamically controlled, I identified the protein Inscuteable as a continuously polarized cue for Par complex localization during mitosis. Inscuteable and Bazooka, a member of the Par complex, interact directly and form a complex that is regulated by the mitotic kinase Aurora A. Regulating this interaction allows for cell-cycle dependent establishment of polarity and for the subsequent loss of polarity after the cell divides. To investigate how Par complex directed polarity is connected to spindle position, I investigated the effect of Inscuteable binding on the spindle orientation ability of the protein Pins. When bound to Inscuteable, Pins' spindle orientation activity becomes repressed. Inscuteable competes with Mud for Pins binding and represses the Gai-Pins-Mud signaling pathway. Function of the parallel Pins-Dlg pathway remains unaffected. This repression behavior may allow differential timing of spindle attachment (through Dlg) and spindle shortening (through Mud) pathways that ensures correct alignment of the mitotic spindle. I was able to model the spindle orientation behavior of Pins using a synthetic protein containing activation sites that have different affinities for the activator. Changing the number and affinities of these activation sites leads to different response profiles that mimic the ultrasensitive behavior of Pins using a non-cooperative mechanism. Together, these regulatory mechanisms cooperate to allow for spatial and temporal control of polarity and for physical connection of polarity to the mitotic spindle. This dissertation includes previously published and unpublished co-authored material.