Drosophila Growth Cones Advance by Forward Translocation of the Neuronal Cytoskeletal Meshwork In Vivo

Abstract

In vitro studies conducted in Aplysia and chick sensory neurons indicate that in addition to microtubule assembly, long microtubules in the C-domain of the growth cone move forward as a coherent bundle during axonal elongation. Nonetheless, whether this mode of microtubule translocation contributes to growth cone motility in vivo is unknown. To address this question, we turned to the model system Drosophila. Using docked mitochondria as fiduciary markers for the translocation of long microtubules, we first examined motion along the axon to test if the pattern of axonal elongation is conserved between Drosophila and other species in vitro. When Drosophila neurons were cultured on Drosophila extracellular matrix proteins collected from the Drosophila Kc167 cell line, docked mitochondria moved in a pattern indicative of bulk microtubule translocation, similar to that observed in chick sensory neurons grown on laminin. To investigate whether the C-domain is stationary or advances in vivo, we tracked the movement of mitochondria during elongation of the aCC motor neuron in stage 16 Drosophila embryos. We found docked mitochondria moved forward along the axon shaft and in the growth cone C-domain. This work confirms that the physical mechanism of growth cone advance is similar between Drosophila and vertebrate neurons and suggests forward translocation of the microtubule meshwork in the axon underlies the advance of the growth cone C-domain in vivo. These results highlight the need for incorporating en masse microtubule translocation, in addition to assembly, into models of axonal elongation.

Role of kinesin-1–based microtubule sliding in Drosophila nervous system development

“…Additionally, we have observed MT sliding in axons as well as MTs pushing on the axon tip (9). Recent studies from other groups have also implicated MT translocation in axon extension and dendritic organization (10)(11)(12). Based on these studies, we hypothesize that kinesin-1 drives neurite extension by sliding MTs against the plasma membrane.…”

Cytoplasmic dynein pushes the cytoskeletal meshwork forward during axonal elongation

“…We have previously shown that neurons grow by axonal stretching and bulk forward movement of the cytoskeletal meshwork (Miller and Sheetz, 2006;O'Toole et al, 2008;Lamoureux et al, 2010a;Lamoureux et al, 2010b;Roossien et al, 2013). To test whether dynein contributes to this process, we tracked the movement of docked mitochondria (Morris and Hollenbeck, 1993;Saxton and Hollenbeck, 2012) after microinjection of IgG and aDIC antibodies into chick sensory neurons.…”

“…Because this movement occurs at a rate of ,1000 times slower than fast transport, it is straightforward to distinguish between this 'low velocity transport' and fast axonal transport in kymographs (Miller and Sheetz, 2006). Using this approach, we have shown that axonal stretching occurs during axonal elongation in vivo (Roossien et al, 2013), in cultured neurons from various species (Miller and Sheetz, 2006;O'Toole et al, 2008;Lamoureux et al, 2010a;Lamoureux et al, 2010b;Roossien et al, 2013) and in response to forces generated in or applied to the growth cone (O'Toole et al, 2008;Lamoureux et al, 2010a). Furthermore, in the field of nonneuronal cell biophysics, docked mitochondria are well-accepted fiducial markers for bulk subcellular deformation (Wang et al, 2001;Knight et al, 2006;Gilchrist et al, 2007;Wang et al, 2007;del Alamo et al, 2008;Bell et al, 2012;Silberberg and Pelling, 2013).…”

“…By convention, the axonal cytoskeleton was considered to be stationary during elongation and it was thought that the new axon was formed by the addition of new material at the growth cone, either through cytoskeleton polymerization or the deposition of material by motor-driven transport (Goldberg and Burmeister, 1986;Dent and Gertler, 2003;Lowery and Van Vactor, 2009). Recently, however, there have been several studies of bulk translocation in multiple model systems, such as Aplysia growth cones (Lee and Suter, 2008), cultured chick sensory neurons (Miller and Sheetz, 2006;Lamoureux et al, 2010a) and Drosophila motoneurons in vivo (Roossien et al, 2013). They indicate that the classic findings of bulk microtubule translocation in Xenopus neurons (Reinsch et al, 1991;Chang et al, 1998) were not a species-specific phenomenon, but rather a broadly conserved mechanism for elongation.…”

“…This is paired with stretching of the axon, which is followed by intercalated mass addition along the axon to prevent thinning (Lamoureux et al, 2010). In terms of the cytoskeleton, stretching presumably occurs because filaments are sliding apart either through pulling or pushing forces generated by molecular motors (Suter and Miller, 2011;Lu et al, 2013;Roossien et al, 2013). It is worth noting that because adhesions along the axon dissipate forces generated in the growth cone (O'Toole et al, 2008), these en masse movements of the cytoskeleton occur only in the distal axon, whereas the cytoskeletal framework is stationary near the cell body (Lim et al, 1990;Okabe and Hirokawa, 1990).…”