During cell division, the precise positioning of the cell-cleavage plane at the center of a ~ 10-15 um long cell is essential for maintaining genome integrity. Consistently, cytokinesis failures can lead to aneuploidy, a hallmark of tumorigenesis. This motivates a fundamental question: How does a dividing cell find its center? It is now well established that an overlapping array of antiparallel microtubules, known as the spindle midzone, plays a critical role. The midzone acts as a micron scale ‘mark’ for the cell center to direct the signaling reactions that further precisely define the site of cell-cleavage. An essential signaling protein that relocates from the chromosomes to the midzone microtubules in anaphase is the AuroraB kinase, which is part of a 4-protein complex called Chromosome Passenger Complex (CPC). AuroraB is maximally activated proximal to the midzone in anaphase. Its activity is important for the phosphorylation of key cytokinesis factors that direct the formation of cell cleavage furrow. However, despite an appreciation for its critical functions in anaphase, our knowledge of how intrinsic biophysical and microtubule binding properties of CPC contribute to localization and activation at antiparallel arrays is limited. Additionally, how the CPC is specifically concentrated on a specific subset of microtubules in the spindle remains confounding. The overarching goal of the work proposed here is to decipher how CPC is specifically localized to a narrow region of overlap formed by the plus ends of antiparallel microtubules, and how the kinase activity of AuroraB is maximally activated at such structures. To achieve this goal, we build on previous research where we reconstituted antiparallel microtubule bundles, the minimal structural units of the midzone, from the collective activity of conserved midzone motor and non-motor microtubule associated proteins. We will first determine the intrinsic dynamics and microtubule binding properties of recombinant full-length CPC using biophysical methods. We then take a small-scale systems biochemistry to elucidate how the collective activity of key midzone motors and MAPs regulate the microtubule localization of CPC. Finally, we will test key findings from our biochemical studies in cells using quantitative analysis of CPC localization and activity in anaphase cells. In addition to fundamental mechanistic insights on AuroraB, these studies have the potential to uncover new therapeutic strategies to modulate its activity. The proposed in vitro reconstitutions, combined with structural and cellular analysis, promises to provide significant new insights into the broader question of specific subsets are microtubules are located by dense networks by signaling proteins for spatially confined activity within the cellular cytoplasm.