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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Principal Investigator: PAUL  FORSCHER
Organization: YALE UNIVERSITY
Fiscal Year: 2024
Award: $389,283
Funding agency: National Institute of Neurological Disorders and Stroke

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.

Terms: <5-HT><5-Hydroxytryptamine><5HT><Actin Filaments><Actins><Address><Adhesions><Affect><Assay><Bathing><Baths><Behavior><Bioassay><Biological Assay><Calcineurin><Catalysis><Cell Adhesion><Cell Body><Cell Communication and Signaling><Cell Locomotion><Cell Migration><Cell Movement><Cell Signaling><Cells><Cellular Adhesion><Cellular Matrix><Cellular Migration><Cellular Motility><Cues><Cytoskeletal System><Cytoskeleton><Degenerative Neurologic Disorders><Development><Enteramine><Eukaryotic Cell><Exposure to><F-Actin><Filamentous Actin><G Protein-Complex Receptor><G Protein-Coupled Receptor Genes><G-Protein-Coupled Receptors><GPCR><Generalized Growth><Growth><Growth Cones><Hippophaine><Integrins><Integrins Extracellular Matrix><Intracellular Communication and Signaling><Knowledge><Lead><Lecithinase C><Literature><Malignant Cell><Mechanical Stress><Mechanics><Mediating><Metastasis><Metastasize><Metastatic Lesion><Metastatic Mass><Metastatic Neoplasm><Metastatic Tumor><Micro-tubule><Microfilaments><Microscopy><Microtubules><Motor><Myofilaments><Myosin II><Myosin Type II><Neoplasm Metastasis><Nervous System Degenerative Diseases><Neural Degenerative Diseases><Neural degenerative Disorders><Neurites><Neurodegenerative Diseases><Neurodegenerative Disorders><Neurologic Degenerative Conditions><Normal Cell><P Domain><P-Type Domain><PP2B><Pattern><Pb element><Phospholipase C><Play><Production><Protein Family><Protein Phosphatase-2B><Proteins><Regulation><Role><Secondary Neoplasm><Secondary Tumor><Serotonin><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Stimulus><Stress><Structure><System><TFF Domain><Testing><Time><Tissue Growth><Traction><Trefoil Motif><Visualization><axon growth><axonal growth><biological signal transduction><cancer cell><cancer metastasis><cell motility><cofilin><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><density><developmental><experiment><experimental research><experimental study><experiments><heavy metal Pb><heavy metal lead><intracellular skeleton><lipophosphodiesterase I><mechanic><mechanical><neurite growth><neurodegenerative illness><neuronal growth><non-muscle myosin><nonmuscle myosin><novel><ontogeny><phosphatidylcholine cholinephosphohydrolase><response><shear stress><social role><spatiotemporal><treadmill><tumor cell metastasis>