Visualizing Direct Interactions in the Mechanobiome

Cytokinesis, the end point of cell division, is controlled by a complex system that coordinates the mitotic spindle signals with mechanoresponsive proteins to drive contractility and furrow ingression. These feedback loops endow the cell with the ability to respond to mechanical stress, allowing it to divide and regulate cell shape in the presence of various environmental stresses (Kee et al. 2012, Srivastava and Robinson 2015). In the social amoeba Dictyostelium, the scaffolding protein IQGAP2 is found at a node between spindle signals and the contractile machinery, integrating the feedback mechanisms. We applied a mass spectrometric approach to identify the composite network of proteins that define the mechanosensory modules involved in cytokinesis. By using IQGAP2 and the actin crosslinker cortexillin I (an IQGAP2-binding partner) as bait, we identified myosin II as a biochemical interactor of both proteins, independent of actin association. Interestingly, we identified a few unusual hits, such as RNP1 and methylmalonyl semialdehyde dehydrogenase (mmsdh), that have been previously identified in genetic suppressor screens from our lab, providing even greater evidence for their function in the network (Zhou et al. 2010, Ren et al. 2014). Here, we use fluorescence cross-correlation spectroscopy (FCCS) to measure these biochemical interactions in vivo. Cortexillin I and myosin II indeed show cross-correlation, suggesting their association in the same complex at a key node of the feedback controller. We are currently using FCCS to confirm and visualize protein associations with and without mechanical stress in vivo, yielding a broader picture of the interactions that are critical within the mechanobiome.