Mechanical catalysis of calcineurin dependent cofilin activity during chemotropic axon growth: a new role for PKC in coordinating actin dynamics and myosin II contractility

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The ADF/cofilin family of proteins play a critical role in actin filament turnover essential to all forms of eukaryotic cell motility. Despite a vast literature on signaling pathways controlling cofilin activity, assessing the function of this important protein in living cells has been hampered by lack of real time assays of cofilin function. Using a novel real time assay for assessing cofilin activity and actin dynamics simultaneously by quantitative fluorescent speckle microscopy (qFSM), we have discovered that mechanical stress imposed on treadmilling actin networks increases cofilin activity with dramatic effects on actin turnover rates that depend on the level of stress. Low stress levels are associated with increases in actin turnover and treadmilling rates that are associated with chemotropic growth; in contrast, stress levels above a critical threshold lead to catastrophic decreases in actin network density resulting in neurite retraction. We have been studying mechanical effects on cofilin activity in the context of serotonin (5-HT) evoked neurite growth responses mediated by classical G(q) subtype GPCRs, which activate phospholipase C to generate IP3 and DAG signals. 5-HT evokes IP3 dependent Ca release from intracellular stores and cofilin activation by a Ca→calcineurin signaling cascade. We now have evidence that DAG production results in PKC dependent increases in non-muscle myosin II activity. This in turn generates local network stress and mechano-catalytic activation of cofilin resulting in local alteration of F-actin structure and network turnover rates. Effects on actin structure also depend on the level of PKC activation. PKC has other known roles including regulation of microtubule (MT) dynamics in growth cones and since MTs are the transport substrate for ER/Ca stores, MT dynamics regulate the functional topography of IP3 dependent Ca release involved in 5-HT dependent growth. PKC can also potentiate integrin based cell adhesion and thereby affect traction forces involved in neuronal growth. We propose to investigate PKC as a signaling node that coordinates: 1) myosin II contractility, 2) actin turnover via cofilin mechano-catalysis, 3) Ca release topography via regulation of microtubule/ER dynamics, and 4) ultimately, traction force production during axon growth. These studies will provide a mechanistic framework for understanding how cofilin enables functional crosstalk between actin dynamics and myosin II contractility during chemotropic growth responses. The results will have interesting implications regarding the key role PKC plays in neuronal growth and neurodegenerative disease.