The Dynamic Interplay between Cleavage Furrow Proteins in Cellular Mechanoresponsiveness

Abstract

Cell shape changes associated with processes like cytokinesis and motility proceed on several second time-scales. However, they are derived from molecular events occurring much faster, including protein-protein interactions, filament assembly, and force generation. How these fast dynamics define cellular outcomes remain unknown. While accumulation of cytoskeletal elements is often portrayed as being driven by signaling pathways, mechanical stresses also direct proteins. A myosin II-based mechanosensory system controls cellular contractility and shape during cytokinesis and under applied stress. In Dictyostelium, this system tunes myosin II accumulation under mechanical stress by feedback through the actin network, particularly through the crosslinker cortexillin I. Cortexillin-binding IQGAP proteins are major regulators of this system. We examined the dynamic interplay between these key cytoskeletal proteins using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy, defining the short time-scale dynamics of these players during cytokinesis and under mechanical stress. Actin and its polar cortex-enriched crosslinkers showed sub-second recovery, while equatorially enriched proteins including cortexillin I, IQGAP2, and myosin II recovered in 1-5 seconds. Mobility of these equatorial proteins was greatly reduced at the furrow, compared to their interphase dynamics. This mobility shift did not arise from a single biochemical event, but rather from global inhibition of protein dynamics by mechanical stress-associated changes in cytoskeletal structure. We further expanded our genetic and biochemical understanding of this mechanosensory system using a proteomics approach to identify relevant protein-protein interactions. We identified that, in addition to binding to each other, both cortexillin I and IQGAP2 also interact with myosin II under conditions that prevent myosin II-F-actin binding. This validates the high crosstalk occurring between various mechanosensitive elements. Mechanical tuning of contractile protein dynamics provides robustness to the cytoskeletal framework responsible for regulating cell shape and contributes to the fidelity of cytokinesis.