DYNAMIC INTERPLAY BETWEEN CLEAVAGE FURROW PROTEINS IN CELLULAR MECHANORESPONSIVENESS

Cell shape changes associated with processes like cytokinesis and motility proceed on several second time-scales. However, they are derived from much faster molecular events occurring much faster, including protein-protein interactions, filament assembly, and force generation. How these fast molecular dynamics define cellular outcomes remain unknown. While accumulation of cytoskeletal elements during shape change is often 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 (FRAP) and fluorescence correlation spectroscopy (FCS), 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. Thus, the equatorial proteins are stabilized under mechanical stress, which likely enables them to generate contractility at the furrow. 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. Thus, cooperativity between This validates the high crosstalk occurring between various mechanosensitive elements through macromolecular assemblies may provide a new mechanism for regulating cellular contractility. Mechanical tuning of contractile protein dynamics provides robustness to the cytoskeletal framework responsible for regulating cell shape and contributes to the fidelity of cytokinesis

Understanding the Cooperative Interaction between Myosin II and Actin Cross-Linkers Mediated by Actin Filaments during Mechanosensation

AbstractMyosin II is a central mechanoenzyme in a wide range of cellular morphogenic processes. Its cellular localization is dependent not only on signal transduction pathways, but also on mechanical stress. We suggest that this stress-dependent distribution is the result of both the force-dependent binding to actin filaments and cooperative interactions between bound myosin heads. By assuming that the binding of myosin heads induces and/or stabilizes local conformational changes in the actin filaments that enhances myosin II binding locally, we successfully simulate the cooperative binding of myosin to actin observed experimentally. In addition, we can interpret the cooperative interactions between myosin and actin cross-linking proteins observed in cellular mechanosensation, provided that a similar mechanism operates among different proteins. Finally, we present a model that couples cooperative interactions to the assembly dynamics of myosin bipolar thick filaments and that accounts for the transient behaviors of the myosin II accumulation during mechanosensation. This mechanism is likely to be general for a range of myosin II-dependent cellular mechanosensory processes

Mechanoaccumulative elements of the mammalian actin cytoskeleton

To change shape, divide, form junctions, and migrate, cells reorganize their cytoskeletons in response to changing mechanical environments [1-4]. Actin cytoskeletal elements, including myosin II motors and actin crosslinkers, structurally remodel and activate signaling pathways in response to imposed stresses [5-9]. Recent studies demonstrate the importance of force-dependent structural rearrangement of α-catenin in adherens junctions [10] and vinculin's molecular clutch mechanism in focal adhesions [11]. However, the complete landscape of cytoskeletal mechanoresponsive proteins and the mechanisms by which these elements sense and respond to force remain to be elucidated. To find mechanosensitive elements in mammalian cells, we examined protein relocalization in response to controlled external stresses applied to individual cells. Here, we show that non-muscle myosin II, α-actinin, and filamin accumulate to mechanically stressed regions in cells from diverse lineages. Using reaction-diffusion models for force-sensitive binding, we successfully predicted which mammalian α-actinin and filamin paralogs would be mechanoaccumulative. Furthermore, a Goldilocks zone must exist for each protein where the actin-binding affinity must be optimal for accumulation. In addition, we leveraged genetic mutants to gain a molecular understanding of the mechanisms of α-actinin and filamin catch-bonding behavior. Two distinct modes of mechanoaccumulation can be observed: a fast, diffusion-based accumulation and a slower, myosin II-dependent cortical flow phase that acts on proteins with specific binding lifetimes. Finally, we uncovered cell-type and cell-cycle-stage-specific control of the mechanosensation of myosin IIB, but not myosin IIA or IIC. Overall, these mechanoaccumulative mechanisms drive the cell's response to physical perturbation during proper tissue development and disease

A Novel Role for Aquaporin-5 in Enhancing Microtubule Organization and Stability

Aquaporin-5 (AQP5) is a water-specific channel located on the apical surface of airway epithelial cells. In addition to regulating transcellular water permeability, AQP5 can regulate paracellular permeability, though the mechanisms by which this occurs have not been determined. Microtubules also regulate paracellular permeability. Here, we report that AQP5 promotes microtubule assembly and helps maintain the assembled microtubule steady state levels with slower turnover dynamics in cells. Specifically, reduced levels of AQP5 correlated with lower levels of assembled microtubules and decreased paracellular permeability. In contrast, overexpression of AQP5 increased assembly of microtubules, with evidence of increased MT stability, and promoted the formation of long straight microtubules in the apical domain of the epithelial cells. These findings indicate that AQP5-mediated regulation of microtubule dynamics modulates airway epithelial barrier properties and epithelial function

DYNAMIC INTERPLAY BETWEEN CLEAVAGE FURROW PROTEINS IN CELLULAR MECHANORESPONSIVENESS

Cell shape changes associated with processes like cytokinesis and motility proceed on several second time-scales. However, they are derived from much faster molecular events occurring much faster, including protein-protein interactions, filament assembly, and force generation. How these fast molecular dynamics define cellular outcomes remain unknown. While accumulation of cytoskeletal elements during shape change is often 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 (FRAP) and fluorescence correlation spectroscopy (FCS), 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. Thus, the equatorial proteins are stabilized under mechanical stress, which likely enables them to generate contractility at the furrow. 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. Thus, cooperativity between This validates the high crosstalk occurring between various mechanosensitive elements through macromolecular assemblies may provide a new mechanism for regulating cellular contractility. Mechanical tuning of contractile protein dynamics provides robustness to the cytoskeletal framework responsible for regulating cell shape and contributes to the fidelity of cytokinesis