The ADF/Cofilin family of proteins plays a critical role in actin filament turnover essential to all forms of eukaryotic cell motility and normal brain development. Despite a vast literature on signaling pathways controlling cofilin activity, assessing cofilin function in living cells has been hampered by lack of real time assays of cofilin activity.
In Aim I will take advantage of the intrinsic ATPase activity of actin filaments and high affinity of active cofilin for ADP-actin subunits to implement an assay for cofilin activity and dynamics in living neurons using quantitative fluorescent speckle microscopy (qFSM). Aplysia cofilins will be derivatized at specific sites with AlexaFluor tags and their biochemical activity and functionality verified in vitro. AlexaFluor-apCofilins and AlexaFluor-G-actin will then be injected into Aplysia neurons and low levels for qFSM and speckle dynamics recorded under conditions of varying apCofilin activity. apCofilin vs actin speckle kinematics, speckle lifetimes, and turnover dynamics will be analyzed. Effects on cofilin activity will be correlated with actin filament structure assessed by light and electron microscopy. Myosin II dependent mechanical forces have been reported to affect cofilin severing activity;thus, we will investigate whether Myosin II activity directly affect cofilin activity and actin dynamics during neurite outgrowth. Ths robust cofilin activity assay is portable to other cells types and will provide a valuable new tool for addressing regulation of cell motility processes including axon growth and regeneration. Neurite outgrowth is characterized by coordinated advance of the central (C) and peripheral (P) cytoplasmic growth cone domains. We recently reported that serotonin (5-HT) accelerates rates of neurite outgrowth by ~300% via a mechanism involving phospholipase C (PLC) dependent Ca release and calcineurin (CN) dependent activation of cofilin in the growth cone P domain. 5-HT stimulated outgrowth was accompanied by CN dependent increases in retrograde actin filament flow in the P domain. When background non-muscle Myosin II activity was inhibited, 5-HT continued to trigger cofilin activation and increases in retrograde actin flow but C domain advance no longer occurred. Thus, myosin II activity is necessary for functionally coupling increases in actin treadmilling in the P domain with advance of the C domain.
In Aim II -III we address why this is so. We have previously implicated Rho kinase (ROCK) in regulation of Myosin II dependent C domain contractility and will investigate a role for ROCK in coordination of C and P domain function. Experiments are proposed to generalize the cytoskeletal mechanisms being studied to the many other growth factor receptors that utilize PLC signaling. These studies are predicted to have significant clinical implications for understanding neurodegenerative disease and nerve regeneration related to brain and/or spinal cord injury.

Public Health Relevance

The constant assembly and disassembly of actin filament networks creates an actin filament treadmilling system at the front of motile cells that must be coordinated with contractile forces in the cell rear to promote normal cell advance, cancer cell metastasis and neuronal growth. New evidence suggests there is functional compartmentalization of the F-actin severing protein Cofilin and non-muscle Myosin II to the front and rear of neuronal growth cones, respectively. This project investigates how this functional compartmentalization facilitates acceleration of neuronal growth in response to chemotropic factors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS028695-23A1
Application #
8631268
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Riddle, Robert D
Project Start
1990-08-01
Project End
2018-08-31
Budget Start
2013-09-15
Budget End
2014-08-31
Support Year
23
Fiscal Year
2013
Total Cost
$363,948
Indirect Cost
$145,198
Name
Yale University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Hyland, Callen; Dufresne, Eric R; Dufrense, Eric R et al. (2014) Regeneration of Aplysia bag cell neurons is synergistically enhanced by substrate-bound hemolymph proteins and laminin. Sci Rep 4:4617
Hyland, Callen; Mertz, Aaron F; Forscher, Paul et al. (2014) Dynamic peripheral traction forces balance stable neurite tension in regenerating Aplysia bag cell neurons. Sci Rep 4:4961
Mejean, Cecile O; Schaefer, Andrew W; Buck, Kenneth B et al. (2013) Elastic coupling of nascent apCAM adhesions to flowing actin networks. PLoS One 8:e73389
Yang, Qing; Zhang, Xiao-Feng; Van Goor, David et al. (2013) Protein kinase C activation decreases peripheral actin network density and increases central nonmuscle myosin II contractility in neuronal growth cones. Mol Biol Cell 24:3097-114
Van Goor, David; Hyland, Callen; Schaefer, Andrew W et al. (2012) The role of actin turnover in retrograde actin network flow in neuronal growth cones. PLoS One 7:e30959
Craig, Erin M; Van Goor, David; Forscher, Paul et al. (2012) Membrane tension, myosin force, and actin turnover maintain actin treadmill in the nerve growth cone. Biophys J 102:1503-13
Mejean, Cecile O; Schaefer, Andrew W; Millman, Eleanor A et al. (2009) Multiplexed force measurements on live cells with holographic optical tweezers. Opt Express 17:6209-17
Zhang, Xiao-Feng; Forscher, Paul (2009) Rac1 modulates stimulus-evoked Ca(2+) release in neuronal growth cones via parallel effects on microtubule/endoplasmic reticulum dynamics and reactive oxygen species production. Mol Biol Cell 20:3700-12
Schaefer, Andrew W; Schoonderwoert, Vincent Th G; Ji, Lin et al. (2008) Coordination of actin filament and microtubule dynamics during neurite outgrowth. Dev Cell 15:146-62
Burnette, Dylan T; Ji, Lin; Schaefer, Andrew W et al. (2008) Myosin II activity facilitates microtubule bundling in the neuronal growth cone neck. Dev Cell 15:163-9

Showing the most recent 10 out of 30 publications