We have demonstrated a required function of the conserved CAP-Gly domain of dynactin in facilitating the efficient initiation of transport from the distal axon. We show that the CAP-Gly domain of p150Glued is necessary to enrich dynactin in distal neurites and that this enrichment promotes the flux of cargo out of the neurite tip. Kinesin-1 delivers dynactin to the distal neurite, while EBs retain dynactin distally and may also promote the initiation of transport by recruiting dynactin onto the MT plus-end. Once transport is initiated, the CAP-Gly domain is not necessary for transport of cargo along the axon.
Here we show that while full-length p150Glued
is enriched on vesicles, the CAP-Gly domain does not contribute to the motility of these vesicles along the axon. Rather, we propose a model in which the CAP-Gly domain serves a specialized function at the neurite tip (). The domain is necessary to enrich dynactin the distal neurite and promote the efficient initiation of retrograde transport. Previous studies have suggested that both p150Glued
and the related CAP-Gly protein, CLIP-170, may be important in the capture of dynamic MTs for the initiation of minus-end directed transport (Lomakin et al., 2009
; Vaughan et al., 2002
We find that the distal accumulation of dynactin is dependent on kinesin-1-mediated transport. Dynactin may be delivered to the distal neurite via fast axonal transport, on anterograde-moving vesicles, or via slow axonal transport, which delivers cytoplasmic cargo and is also kinesin-1 dependent (Scott et al., 2011
). Neither mechanism is likely to involve a direct interaction with dynactin. While dynein does interact with kinesin-1 (Ligon et al., 2004
), dynein does not accumulate distally.
The pool of distally enriched dynactin is highly stable, suggesting a mechanism of active retention at the neurite tip. We show that the end-binding proteins, EB1 and EB3, are necessary to maintain this distal pool. Although the length of a single EB comet is 0.5–2 μm, enrichment of +TIP proteins in a spatially restricted domain may provide a platform for spatial organization in the cell (Akhmanova and Steinmetz, 2008
). Thus the increased EB3 comet density we observe in the distal neurite leads to the preferential enrichment and retention of dynactin in the distal neurite. In an interesting parallel, dynactin is observed to accumulate in the distal hyphal tip of filamentous fungi. Further, this localization is dependent on the MT plus-end binding protein, Peb1, which binds to the CAP-Gly domain (Lenz et al., 2006
; Schuster et al., 2011
), paralleling our observations in neurons. However, in fungi, dynein also accumulates in the hyphal tips. We did not observe the distal enrichment of dynein in neurons, suggesting there are likely key differences in the regulation of the motor in these two systems.
We propose a model in which the distal enrichment of dynactin enhances the coupling of dynein to the cargo and the MT to increase the efficient initiation of transport (). The CAP-Gly domain is necessary to enrich and retain dynactin distally where dynactin can directly interact with cargos such as late endosomes and lysosomes as well as dynein and MTs (Johansson et al., 2007
; Karki and Holzbaur, 1995
; Waterman-Storer et al., 1995
). Thus, dynactin may be the key mediator in the formation of a motile motor-cargo complex. The distal enrichment of dynactin may promote the initial interaction of dynactin with the MT and cargo followed by the recruitment of the dynein motor. Dynein is the limiting step in the initiation of retrograde transport of the motor-cargo complex in our model; consistent with this interpretation we do not observe dynein enrichment in the distal neurite.
Axons are longer than any single MT, so cargos must switch MT tracks to efficiently transit along the axon. It is possible that dynactin also promotes this switching for vesicles in transit, by promoting the efficient formation of a cargo-motor-MT complex following interruption of motility along the axon caused by a gap in the MT track. However, our observations that a ΔCAP-Gly construct could fully rescue transport along the mid-axon suggests that this activity is not strongly required to maintain normal transport.
The importance of the CAP-Gly domain in dynactin to neuronal function is highlighted by the multiple disease-causing point mutations identified in this motif to date. Here, we show that the mechanisms driving the pathogenesis of HMN7B and Perry syndrome are distinct. The HMN7B mutation affects a residue important for maintaining the structure of the CAP-Gly domain so the mutation promotes misfolding and aggregation (Levy et al., 2006
). This aggregation decreases the stability of the dynactin complex, preventing effective association between dynein and dynactin and ultimately disrupts axonal transport (). The Perry syndrome mutations, in contrast, are surface-exposed and more specifically disrupt protein-protein interactions. The Perry syndrome mutations phenocopy
in all our assays, which suggests that the primary pathogenic mechanism in Perry syndrome is a loss of CAP-Gly function. Consistent with this, we observe a decrease in the efficiency of cargo flux from the distal neurite in Perry syndrome ().
Our data on the HMN7B and Perry syndrome mutations are consistent with the pathology observed in patients and in available mouse models (Chevalier-Larsen et al., 2008
; Lai et al., 2007
; Laird et al., 2008
). HMN7B patients have significant deposits of dynactin in motor neurons (Puls et al., 2005
), while minimal aggregates of dynactin are observed Perry syndrome patients (Farrer et al., 2009
). These data support a model in which the HMN7B mutation decreases p150Glued
stability due to the critical location of glycine-59 for maintaining domain structure. In contrast, the Perry syndrome mutants cause a loss of function with no change in protein stability. Initial studies examining the effects of the Perry syndrome mutations on MT binding have yielded conflicting results (Ahmed et al., 2010
; Farrer et al., 2009
). However our data clearly show that the Perry syndrome mutations cause a loss of CAP-Gly function, resulting in a decrease in transport initiation from the distal neurite.
How do these distinct mechanisms result in the disease phenotypes associated with HMN7B and Perry syndrome? Defects in axonal transport have been observed in models of motor neuron disease and other neurodegenerative diseases (Perlson et al., 2009
; Perlson et al., 2010
). We speculate that multiple factors play a role in the selectivity of cell death. The HMN7B mutant protein is preferentially degraded in vivo
(Lai et al., 2007
), suggesting that most cells recognize the decreased stability of the protein and can effectively target the polypeptide for degradation. However, motor neurons may not effectively induce a stress response to protein misfolding (Batulan et al., 2003
), leaving them vulnerable to the dominant-negative effect of the G59S mutation. In contrast, the distinct morphology of dopaminergic neurons may make these cells uniquely vulnerable to defects in the initiation of retrograde transport. The immense axonal arborizations of dopaminergic neurons (Matsuda et al., 2009
) suggest that the loss-of-function effects of the Perry mutations may critically affect this cell type.
Thus, our data inform normal dynein-dynactin function as well as the selective vulnerabilities of discrete populations of neurons to specific perturbations in the cellular function of these proteins. The two distinct mechanisms we propose for the pathogenesis of HMN7B and Perry syndrome highlight the specialized function of a single domain of dynactin and provide a model for the function of the CAP-Gly domain of p150Glued in neurons.