Actin Waves Do Not Boost Neurite Outgrowth in the Early Stages of Neuron Maturation.

Simone Mortal,Vincent Torre,Andrea Perissinotto,Elisa D'Este,Luisa M R Napolitano,Federico Iseppon,Dan Cojoc

doi:10.3389/fncel.2017.00402

Abstract

During neurite development, Actin Waves (AWs) emerge at the neurite base and move up to its tip, causing a transient retraction of the Growth Cone (GC). Many studies have shown that AWs are linked to outbursts of neurite growth and, therefore, contribute to the fast elongation of the nascent axon. Using long term live cell-imaging, we show that AWs do not boost neurite outgrowth and that neurites without AWs can elongate for several hundred microns. Inhibition of Myosin II abolishes the transient GC retraction and strongly modifies the AWs morphology. Super-resolution nanoscopy shows that Myosin IIB shapes the growth cone-like AWs structure and is differently distributed in AWs and GCs. Interestingly, depletion of membrane cholesterol and inhibition of Rho GTPases decrease AWs frequency and velocity. Our results indicate that Myosin IIB, membrane tension, and small Rho GTPases are important players in the regulation of the AW dynamics. Finally, we suggest a role for AWs in maintaining the GCs active during environmental exploration.

Highlights

The actin cytoskeleton is a highly dynamical system that facilitates the transduction of mechanical signals and generates the intracellular forces required for many cellular functions such as cell motility, polarization, active cell shape control, neurite outgrowth, and exocytosis (Madden and Snyder, 1998; Morales et al, 2000; Pollard and Borisy, 2003)
We performed long-term live cell imaging of Actin Waves (AWs) on rat hippocampal cells plated on Matrigel/Laminin at 1–2 days in vitro (1–2 DIV) using the actin label mCherryLifeAct (Riedl et al, 2008; Winans et al, 2016)
After having assessed the presence of Myosin IIB in both AWs and Growth Cone (GC), we investigated the mechanisms regulating the pulling effect of the approaching AW on the GC, since this appears to be a basic feature of their dynamics

Summary

Introduction

The actin cytoskeleton is a highly dynamical system that facilitates the transduction of mechanical signals and generates the intracellular forces required for many cellular functions such as cell motility, polarization, active cell shape control, neurite outgrowth, and exocytosis (Madden and Snyder, 1998; Morales et al, 2000; Pollard and Borisy, 2003). AWs have been observed mainly in growing neurons and are present in cultured primary hippocampal neurons as well as in organotypic slices (Ruthel and Banker, 1998; Flynn et al, 2009; Katsuno et al, 2015) They have been proposed to be associated with outbursts of neurite growth and to constitute, in concert with microtubules, a transport mechanism that brings actin and actin associated proteins toward the GC (Flynn et al, 2009; Katsuno et al, 2015; Winans et al, 2016)

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Conclusion