Here, we demonstrate that the NGF/TrkA endosome is intimately associated with the actin cytoskeleton and that endosome constituents control actin severing, which is an obligate step for maturation of transport-competent signaling endosomes. Moreover, NGF/TrkA endosomes, but not NT3/TrkA endosomes, are differentially associated with key modulators of the actin cytoskeleton, explaining why NGF/TrkA endosomes are transported retrogradely to the cell body to support survival whereas NT3/TrkA endosomes are not. Our findings support a model in which the actin cytoskeleton imposes a physical barrier that restricts maturation of Rab5+ TrkA early endosomes into retrograde transport competent signaling endosomes, and that the actin-severing activity associated with NGF/TrkA early endosomes is essential for overcoming this barrier thereby enabling endosome maturation, association with the microtubule transport machinery, retrograde transport, and retrograde survival and synaptogenic signaling. Finally, we found that NGF and NT3 differ in their capacity to support formation of mature, transport-competent TrkA endosomes associated with actin modulators because of the differentially labile nature of the neurotrophin-TrkA interactions within the context of early endosomes. These findings define a novel function of actin modulation in the control of TrkA endosome maturation and signaling, and they explain how NGF produced by final target fields is the sole neurotrophin responsible for retrograde survival of developing sympathetic neurons.
Endosomal signals control actin dynamics
How do NGF/TrkA endosomes promote actin disassembly during their maturation into transport-competent signaling endosomes? We found that active forms of Cofilin and Rac1 are associated with and required for retrograde transport of NGF/TrkA endosomes. Moreover, both Rac1 and cofilin are necessary for neuronal survival when NGF is acting exclusively on distal axons, but not for survival signaling in response to NGF applied directly to cell bodies. Thus, Rac1 and cofilin are essential for propagation of retrograde signaling and not TrkA survival signaling. Furthermore, Rac1 inhibition and TrkA endosome/cofilin colocalization experiments place Rac1 upstream of cofilin. Therefore, an early endosome-associated TrkA–Rac1–cofilin–actin severing signaling module is required for NGF/TrkA endosome maturation and retrograde transport.
The precise temporal and spatial patterns of activation of Rac1 and cofilin are likely crucial to their functions during endosome maturation and signaling. We found that Rac1-GTP and cofilin are both required at a post-endocytic step for the formation of transport-competent TrkA endosomes. Recent findings in HeLa cells suggest that activation of Rac1 follows, and likely requires, clathrin-mediated endocytosis of receptor tyrosine kinases (Palamidessi et al., 2008
). Consistent with this, we found that Rac1 co-purifies with TrkA endosomes and that PAK binding sites are associated with Flag-TrkA+
endosomes, whereas little or no PAK binding is associated with the plasma membrane. Moreover, inhibition of TrkA endocytosis in sympathetic neurons using either a dominant negative form of dynamin or the dynamin inhibitor, dynasore, prevented Rac1 activation (Figure S6B-C
). Conversely, inhibition of Rac1 did not prevent internalization of TrkA. Thus, TrkA internalization is required for NGF-dependent production of Rac1-GTP, which is tethered to the NGF/TrkA endosome. On the other hand, internalization of TrkA is not sufficient for Rac1 activation since NT3 promotes formation of TrkA endosomes that are devoid of Rac1-GTP. Our results showing a requirement of Rac1 for TrkA signaling endosome maturation and retrograde transport are in agreement with previous studies in which perinuclear accumulation of TrkA following NGF treatment in PC12 cells (Valdez et al., 2007
) and EGF-dependent retrograde accumulation of an EGFR-TrkB chimeric receptor expressed in sympathetic neurons (Philippidou et al., 2011
) were blocked by RacN17. Thus, activation of Rac1 is essential for trafficking of Trk signaling endosomes.
Several questions remain as to how Rac1-GTP promotes recruitment of cofilin to the NGF/TrkA early endosome and how cofilin phosphorylation and activity are controlled. This is potentially broadly relevant since in other cell types Rac1 is also required for dephosphorylation of cofilin Ser3
following receptor activation (Pandey et al., 2009
; Sun et al., 2007
). In at least one known case, Rac1 promotes activation of the cofilin phosphatase, Slingshot (SSH), leading to dephosphorylation of cofilin Ser3
and catalytic activation (Kligys et al., 2007
). These findings also implicate a relief to inhibition of cofilin activity triggered by SSH and mediated by the binding of 14-3-3 to cofilin. Interestingly, our proteomic analysis identified both SSH and several isoforms of 14-3-3 proteins as TrkA signaling endosome-associated proteins (Supp. Table 1
). We propose that Rac1 controls the actin-severing activity of the NGF/TrkA endosome and actin depolymerization by endosomal recruitment and activation of cofilin, possibly through a mechanism involving SSH and 14-3-3 proteins.
Differential effects of NGF and NT3 on TrkA-mediated transport and survival
Although both NGF and NT3 promote TrkA autophosphorylation, activation of TrkA signaling events, TrkA-dependent axonal extension and, when applied directly to cell bodies, neuronal survival, it is remarkable that NT3 is completely incapable of supporting retrograde transport of TrkA and retrograde survival of sympathetic neurons. We found that NT3 does lead to internalization of Flag-TrkA within distal axons, to a similar extent as NGF. Since prior to visualization of Flag-TrkA endosomes the neurons were treated with a salt-acid wash to remove cell surface Flag antibody, the anti-Flag-labeled punctae detected in these experiments represents TrkA endosomes that have undergone a sufficient degree of endocytosis as to render them invulnerable to the surface antibody stripping conditions. Interestingly, the NT3/TrkA complexes that are internalized associate with the early endosome protein Rab5 but they are neither associated with Rac1-GTP or cofilin nor are they retrogradely transported to cell bodies. It is possible that NT3/TrkA is incapable of activating pathways required for either late stages of endocytosis, such as scission or translocation, or maturation of Rab5+ early endosomes into endosomes that associate with the microtubule transport machinery. In any case, NT3/TrkA complexes internalize but fail to form transport-competent endosomes associated with actin cytoskeleton modulators that enable endosome maturation and long-range retrograde survival.
The pH sensitive nature of NT3-mediated TrkA signaling
How do NGF and NT3 acting on the common receptor TrkA lead to differential activation of the Rac1-GTP–cofilin–actin signaling module? Our findings suggest that a key difference between NGF/TrkA and NT3/TrkA endosomes is the diminished ability of NT3 to bind to and support TrkA activity immediately following early endosome formation and acidification. We found that NT3 is incapable of activating TrkA and displays markedly reduced binding to TrkA at pH values below 7.0. Conversely, NT3 is capable of supporting the formation of TrkA endosomes associated with modulators of the actin cytoskeleton and retrograde transport of TrkA endosomes under conditions in which endosome acidification is prevented. We thus propose that differential maintenance of TrkA signaling within the context of the early endosome accounts for the distinct roles of NT3 and NGF during sympathetic neuron development (Figure S7
). The labile nature of NT3/TrkA complexes following endosome formation and acidification ensures a transient, local mode of action of NT3. This accounts for NT3's ability to support axonal extension but not retrograde survival (Belliveau et al., 1997
; Kuruvilla et al., 2004
; ). In contrast, acid-stable NGF/TrkA complexes promote activation of an endosomal TrkA–Rac1–cofilin–actin signaling module, which enables maturation of transport-competent signaling endosomes that propagate retrogradely to the cell body where they support survival and the formation of synapses with preganglionic partners. Interestingly, the ability of NT3 to promote retrograde transport of TrkA and retrograde survival in neurons expressing a constitutively active Rac1 suggests that there may be sufficient ligand-receptor engagement under acidic endosomal conditions to maintain the low levels of downstream signaling in the cell body that are necessary for survival. An alternate possibility is that RacV12 may enable survival signaling from NT3-formed endosomes. Ultimately, the differential sensitivity to endosomal pH accounts for the unique ability of NGF, and not NT3, to establish proper matching between the size of the neuronal population and the size and demands of the target field.
In summary, our findings indicate that NGF/TrkA endosomes employ a signaling pathway composed of Rac1 and cofilin that directs the breakdown of F-actin, a necessary step for maturation of retrograde transport-competent TrkA signaling endosomes. Moreover, the formation of TrkA endosomes associated with Rac1 and cofilin represents a divergence point for NGF and NT3 signaling in sympathetic neurons. Indeed, NT3/TrkA endosomes are devoid of Rac1-GTP and catalytically active cofilin and are incapable of maturing into retrograde transport endosomes. This is reconciled by NT3's inability to remain engaged with TrkA to support the recruitment of Rac1-GTP and cofilin to NT3/TrkA endosomes under the acidic conditions of the early endosome. We propose that differential activation of endosomal signaling pathways that culminate in actin severing and relief of an “actin block” on maturation of transport-competent TrkA signaling endosomes accounts for the differences between the local-axon growth promoting effects of the intermediate target-derived factor, NT3, and the long-distance, retrograde-mediated effects of final target-derived NGF.