Myosin light chain phosphorylation and growth cone motility

Abstract

According to the treadmill hypothesis, the rate of growth cone advance depends upon the difference between the rates of protrusion (powered by actin polymerization at the leading edge) and retrograde F-actin flow, powered by activated myosin. Myosin II, a strong candidate for powering the retrograde flow, is activated by myosin light chain (MLC) phosphorylation. Earlier results showing that pharmacological inhibition of myosin light chain kinase (MLCK) causes growth cone collapse with loss of F-actin-based structures are seemingly inconsistent with the treadmill hypothesis, which predicts faster growth cone advance. These experiments re-examine this issue using an inhibitory pseudosubstrate peptide taken from the MLCK sequence and coupled to the fatty acid stearate to allow it to cross the membrane. At 5-25 microM, the peptide completely collapsed growth cones from goldfish retina with a progressive loss of lamellipodia and then filopodia, as seen with pharmacological inhibitors, but fully reversible. Lower concentrations (2.5 microM) both simplified the growth cone (fewer filopodia) and caused faster advance, doubling growth rates for many axons (51-102 microm/h; p <.025). Rhodamine-phalloidin staining showed reduced F-actin content in the faster growing growth cones, and marked reductions in collapsed ones. At higher concentrations, there was a transient advance of individual filopodia before collapse (also seen with the general myosin inhibitor, butanedione monoxime, which did not accelerate growth). The rho/rho kinase pathway modulates MLC dephosphorylation by myosin-bound protein phosphatase 1 (MPP1), and manipulations of MPP1 also altered motility. Lysophosphatidic acid (10 microM), which causes inhibition of MPP1 to accumulate activated myosin II, caused a contracted collapse (vs. that due to loss of F-actin) but was ineffective after treatment with low doses of peptide, demonstrating that the peptide acts via MLC phosphorylation. Inhibiting rho kinase with Y27632 (100 microM) to disinhibit the phosphatase increased the growth rate like the MLCK peptide, as expected. These results suggest that: varying the level of MLCK activity inversely affects the rate of growth cone advance, consistent with the treadmill hypothesis and myosin II powering of retrograde F-actin flow; MLCK activity in growth cones, as in fibroblasts, contributes strongly to controlling the amount of F-actin; and the phosphatase is already highly active in these cultures, because rho kinase inhibition produces much smaller effects on growth than does MLCK inhibition.

The development of a rat in vitro model of spinal cord injury demonstrating the additive effects of rho and ROCK inhibitors on neurite outgrowth and myelination

“…The Rho and the ROCK effector proteins exert their growth inhibitory effects largely through regulation of myosin light chain phosphorylation (phosphomyosin), and activation upon injury results in the formation of bundled actin-phosphomyosin fibers (Coleman et al, 2001;Croft et al, 2005) and growth cone collapse (Schmidt et al, 2002). Therefore, inhibition of the Rho/ ROCK pathway should result in a reduction of phosphomyosin.…”

Activity‐driven sharpening of the retinotectal projection: The search for retrograde synaptic signaling pathways

“…Expression of constitutively active or dominant-negative rho family GTPases also profoundly affects the growth of both axons and dendrites of retinal ganglion cells (Ruechhoeft et al, 1999;Li et al, 2000). cdc42 and rac control F-actin polymerization to promote formation of filopodia and lamellipodia, respectively; whereas rho activity controls myosin II activation (powering retrograde flow) and must remain low for neurite outgrowth (Schmidt et al, 2002), as many collapsing agents such as ephrinA work via rho activation (Wahl et al, 2000). As one might expect, BDNF treatment results in activation of both cdc42 and rac, but leaves rho unaffected, thereby promoting extension in the direction of the gradient (Yuan et al, 2003).…”

Myosin IIC: A Third Molecular Motor Driving Neuronal Dynamics

“…The nonsarcomeric conventional motors, myosins IIA and IIB (Katsuragawa et al, 1989;Kawamoto and Adelstein, 1991;Simons et al, 1991), have variously been shown to be involved in cytokinesis (De Lozanne and Spudich, 1987;DeBiasio et al, 1996;Zang et al, 1997;Takeda et al, 2003;Jana et al, 2006), maintenance of cell morphology (Bialik et al, 2004;Ryu et al, 2006;Even-Ram et al, 2007) and cortical tension (Chrzanowska-Wodnicka and Burridge, 1996;van Leeuwen et al, 1999), adhesion (Wylie and Chantler, 2001;Conti et al, 2004;Cai et al, 2006;Giannone et al, 2007;Ma et al, 2007), locomotion (DeBiasio et al, 1996;Svitkina et al, 1997;Even-Ram et al, 2007), exocytosis-dependent membrane repair (Togo and Steinhardt, 2004) as well as cell guidance and migration in neuronal (Schmidt et al, 2002;Ma et al, 2004Ma et al, , 2006Turney and Bridgman, 2005), glioma (Gillespie et al, 1999), neutrophil (Eddy et al, 2000), fibroblast (Lo et al, 2004;Vicente-Manzanares et al, 2007), and endothelial (Kolega, 2003) cells. A third isoform, myosin IIC, has recently been established after a trawl of genomic databases , adding to the functional complexity.…”