After injury to the mammalian CNS, CSPGs are one of the major components that create a barrier to axonal growth, preventing effective regeneration and recovery of function. Growing axons respond to CSPGs by altering their rate and direction of extension, both of which require coordinated cytoskeleton reorganization in neurons. We present several lines of evidence showing that myosin II plays pivotal roles in initiation, outgrowth and guidance of neurites in response to inhibitory CSPGs. First, CSPGs induced phosphorylation of RMLC, which activates myosin II and is one of the main factors involved in the regulation of cytoskeleton dynamics. Second, pharmacological inhibition of myosin II activity by blebbistatin caused neurites to ignore the negative guidance cues presented by CSPGs, resulting in increased crossing at the interface between CSPGs and PLL. Third, blebbistatin promoted neurite outgrowth irrespective of the presence of inhibitory CSPGs as substrates. Fourth, gene silencing of individual isoforms of myosin II revealed that both myosin IIA and IIB are involved in axonal guidance and neurite outgrowth. Finally, although blebbistatin did not alter neuronal cell attachment to PLL and CSPGs, the blockage of myosin II activity accelerated the initiation of neurites.
There are three isoforms of myosin II: IIA, IIB and IIC and myosin IIB is predominant in neurons. Which isoforms are involved in control of neurite growth and response to guidance cues are still a matter of intense investigation. Using gene silencing approaches, we found that the combination of individual myosin isoforms and substrates used for neuron culture is important: knockdown of myosin IIA promoted neurite outgrowth while knockdown of myosin IIB inhibited outgrowth on permissive cues such as PLL and laminin, consistent with previous publications (Bridgman et al. 2001
; Wylie & Chantler 2003
; Wylie et al. 1998
; Turney & Bridgman 2005
; Hur et al. 2011
). In contrast, knockdown of either myosin IIA or IIB reduced the response to CSPGs, which is in agreement with a very recent report by Hur et al. (2011)
on the role of myosin II in DRG neurons plated onto laminin. Other data suggest that the role of myosin II in the response to inhibitory molecules may not be consistent: Kubo et al. (2008)
reported that knockdown of myosin IIA, but not IIB, reduced the inhibition of neurite outgrowth by RGMa, while Brown et al. (2009)
reported neurons with a knockout of myosin IIB showed a significantly reduced retraction rate in response to semaphorin 3A. Overall, these findings indicate that myosin IIA is generally involved in slowing growth both on permissive or non-permissive substrates, while myosin IIB has opposite actions depending upon the substrate.
Time-lapse microscopy revealed a surprising synergism in neurite initiation between the inhibition of myosin II activity by blebbistatin and inhibition of cell spreading by CSPGs. Similarly, after blebbistatin treatment, axons of DRG neurons on CSPGs grow longer than axons growing on laminin (Hur et al. 2011
). Myosin II activity accelerates retrograde actin flow and actin depolymerization (Medeiros et al. 2006
), and is also necessary for the stabilization of focal contacts (Chrzanowska-Wodnicka & Burridge 1996
). Both processes inhibit neurite growth: stabilization of focal adhesions reduces mobility, and retrograde actin flow inhibits the extension of microtubules into the periphery of the growth cone and is involved in growth cone retraction (Kalil & Dent 2005
). Blebbistatin antagonizes both actions of myosin II, reducing adhesion and retrograde actin flow (Koch et al. 2011
). CSPGs also inhibit cellular adhesion and cells on CSPGs do not form the actin-rich lamella that is the basis for retrograde actin flow. It appears that under the circumstances of poor adhesion, microtubule protrusions are not prevented from extending, and so neurites are generated much earlier than on more adhesive substrates. This action, coupled with a reduction in adhesion, then may explain the ability of blebbistatin and myosin II inhibitors to promote neurite outgrowth on CSPGs. Further studies are required to clarify the correlation between the extent of cell spreading and the speed of neurite initiation.
Unlike many other inhibitory molecules which induce growth cone collapse, such as myelin-associated inhibitors and semaphorins (Bandtlow et al. 1993
; Jin & Strittmatter 1997
), growth cones that encounter CSPG boundaries turn to avoid the inhibitory cue but do not collapse (Snow & Letourneau 1992
; Wang et al. 2008
). Growth cone turning requires the coordination of the dynamics of both actin localized at the periphery and microtubule at the central region of the growth cone. This implies the presence of a local signaling cascade triggered by CSPGs in the growth cone to guide the direction of growing neurite. Our previous data demonstrate such a local mechanism for tubulin polymerization at a boundary (Kelly et al. 2010
). Attractive guidance cues do initiate a local promotion of actin polymerization on the side of the attractive stimulus (Marsick et al. 2010
); a similar increase in F-actin can be achieved by altering retrograde actin flow (Schaefer et al. 2008
). It might be expected that repulsive cues would have an opposite effect, either locally decreasing F-actin polymerization or increasing retrograde actin flow. Since myosin II increases retrograde actin flow in growth cones (Lin et al. 1996
), a local activation of myosin II by CSPGs would cause growth cone turning away from the stimulus. In contrast, blebbistatin reduces F-actin levels (Hur et al. 2011
), which is consistent with a requirement of polymerized actin for turning.
The mechanisms by which CSPGs or other repulsive molecules slow neurite growth are not well understood. Recently, the receptor tyrosine phosphatases σ (RPTPσ) and LAR have been identified as putative receptors for CSPGs (Fry et al. 2010
; Shen et al. 2009
; Fisher et al. 2011
). However, the intracellular signal transduction pathway downstream of RPTPσ is not known. We found that exposure to soluble CSPGs produced a sustained increase in myosin light chain phosphorylation. Many guidance cues affect the contraction of myosin II by modulating the balance between Rho and Rac and/or Cdc42 activities (Amano et al. 1998
; Driessens et al. 2001
). However, it was recently shown that lysophosphatidic acid (LPA), which enhances Rho-dependent myosin contractility, induces growth cone retraction independent of peripheral retrograde actin flow but rather driven by the contraction of more central actin structures (Zhang et al. 2003
). We found that treatment with the Rho kinase inhibitor Y-27632 or the myosin light chain kinase inhibitor ML-7 did not alter myosin light chain phosphorylation in response to CSPGs (P. Y, unpublished). A lack of involvement of Rho is also supported by the minimal change in axonal growth on CSPGs when neurons are treated with Y-27632 (Hur et al. 2011
). These results suggest that CSPGs may affect myosin light chain kinase or phosphatase through pathways that do not involve Rho or MLCK.
Our results, together with others, indicate that myosin II activity is involved in growth cone turning and neurite outgrowth in response to both positive and negative guidance cues. Although blockage of myosin II activity overcomes inhibition caused by CSPGs and other inhibitors which prevent axonal regeneration after CNS injury, it might not be a good target for promoting axonal regeneration, since myosin II also mediates the positive response towards permissive cues like laminin. Further investigations into more selective inhibitors of myosin II isoforms or other members of the signaling cascade that involves myosin II are therefore warranted.