Dorsal Root Ganglion Neurons React to Semaphorin 3A Application through a Biphasic Response that Requires Multiple Myosin II Isoforms

Abstract

Growth cone responses to guidance cues provide the basis for neuronal pathfinding. Although many cues have been identified, less is known about how signals are translated into the cytoskeletal rearrangements that steer directional changes during pathfinding. Here we show that the response of dorsal root ganglion (DRG) neurons to Semaphorin 3A gradients can be divided into two steps: growth cone collapse and retraction. Collapse is inhibited by overexpression of myosin IIA or growth on high substrate-bound laminin-1. Inhibition of collapse also prevents retractions; however collapse can occur without retraction. Inhibition of myosin II activity with blebbistatin or by using neurons from myosin IIB knockouts inhibits retraction. Collapse is associated with movement of myosin IIA from the growth cone to the neurite. Myosin IIB redistributes from a broad distribution to the rear of the growth cone and neck of the connecting neurite. High substrate-bound laminin-1 prevents or reverses these changes. This suggests a model for the Sema 3A response that involves loss of growth cone myosin IIA to facilitate actin meshwork instability and collapse, followed by myosin IIB concentration at the rear of the cone and neck region where it associates with actin bundles to drive retraction. INTRODUCTIONCorrect responses to extracellular signaling molecules during development are essential for the formation of the mammalian central and peripheral nervous systems (Sobeih and Corfas, 2002;Hagg, 2005). Growth cones navigate through the tissue environment in vivo by integrating growth-promoting signals with guidance cue signals at choice points and then responding through two opposing processes: extension and retraction (Buettner, 1994;Dontchev and Letourneau, 2003). This steers the growing axon to the correct target location. Cues are classified as either attractive or repulsive depending on their ability to invoke responses. In vitro attractants generally stimulate growth and turning toward the source of the cue, whereas repulsive cues induce turning away from the cue (Lohof et al., 1992;Fan and Raper, 1995). Repulsive cues can also cause growth cone collapse and temporary cessation of outgrowth (Luo et al., 1993;Wahl et al., 2000). It is unclear to what degree full or partial collapse or even retractions are associated with growth cone steering in vivo (Knobel et al., 1999;Mason and Erskine, 2000;Kalil et al., 2000). The complex in vivo environment is likely to result in a variety of behaviors that reflect the integration of signals from multiple cues within a short time period. Some of these interactions occur at the receptor level (Stein and Tessier-Lavigne, 2001;Halloran and Wolman, 2006), but others may converge on common growth cone effector mechanisms. To understand growth cone behavior responsible for pathfinding in vivo, it is important to fully dissect the responses to individual cues in vitro.Sema 3A is secreted by cells in the developing spinal cord and repels dorsal root ganglion (DRG) neurons that are sensitive to nerv...

Engineering neuronal growth cones to promote axon regeneration over inhibitory molecules

“…On laminin, blebbistatin has been shown to reduce, rather than to promote axon growth of peripheral neurons (28,39). The seemingly apparent discrepancy between previous studies (28, Representative images of axons at the permissive-inhibitory border.…”

“…39) and ours might be due to differences in the substratum. In our study, neurons were cultured on polylysine (100 μg/mL) plus 5-10 μg/mL of laminin, whereas other studies applied a higher concentration of laminin (25 μg/mL) without polylysine (39) or with a less adhesive substrate, polyornithine (28). The final outcome of NMII inhibition on axon growth over permissive substrates might depend on the adhesiveness of the substrates, but further studies are required to address this issue.…”

“…On aggrecan, knocking down either NMIIA or NMIIB promoted axon growth, and knocking down both isoforms accounted for nearly all of the axon growth-promoting effect of blebbistatin ( Fig. 2 D and E), ruling out the possible role of NMIIC, which is also expressed in DRG growth cones (28). These results suggest that, on permissive substrates, blockade of NMIIA activity is responsible for the axon growth-promoting effect of blebbistatin, whereas on inhibitory substrates axon growth promotion is attributed to inhibition of both NMIIA and NMIIB.…”

Cytoskeletal dynamics in growth-cone steering

“…Experiments with knockout mice might help to address this problem; however, retrograde flow rate in growth cones from myosin-IIBknockout mice is higher than in wild-type mice, possibly because of compensation by myosin IIA (Brown and Bridgman, 2003). This functional redundancy between isoforms complicates the interpretation of these results (Bridgman et al, 2001;Brown et al, 2009). Growth cones from myosin-IIB-knockout mice turn less efficiently at sharp substrate borders in culture than growth cones that contain all isoforms of myosin II, whereas treatment of wildtype growth cones with blebbistatin severely inhibits turning, suggesting that more than one isoform is involved (Turney and Bridgman, 2005).…”

Building Blocks of Functioning Brain: Cytoskeletal Dynamics in Neuronal Development

“…In DRG neurons, however, overexpression of myosin IIA but not myosin IIB could prevent growth-cone collapse and growthcone retraction in response to Sema3A. Inhibiting all myosin II isoforms with blebbistatin completely abolished retraction in response to Sema3A; and deletion of myosin IIB alone did not block retraction, solidifying a role for other isoforms (Brown et al, 2009). Myosin IIB is shown to further enhance the process of retraction by localizing to the neck and rear of these growth cones.…”