Ordered Recruitment of Dynactin to the Microtubule Plus-End is Required for Efficient Initiation of Retrograde Axonal Transport

Abstract

Long-range retrograde axonal transport in neurons is driven exclusively by the microtubule motor cytoplasmic dynein. The efficient initiation of dynein-mediated transport from the distal axon is critical for normal neuronal function, and neurodegenerative diseaseassociated mutations have been shown to specifically disrupt this process. Here, we examine the role of dynamic microtubules and microtubule plus-end binding proteins (ϩTIPs) in the initiation of dynein-mediated retrograde axonal transport using live-cell imaging of cargo motility in primary mouse dorsal root ganglion neurons. We show that end-binding (EB)-positive dynamic microtubules are enriched in the distal axon. The ϩTIPs EB1, EB3, and cytoplasmic linker protein-170 (CLIP-170) interact with these dynamic microtubules, recruiting the dynein activator dynactin in an ordered pathway, leading to the initiation of retrograde transport by the motor dynein. Once transport has initiated, however, neither the EBs nor CLIP-170 are required to maintain transport flux along the mid-axon. In contrast, the ϩTIP Lis1 activates transport through a distinct mechanism and is required to maintain processive organelle transport along both the distal and mid-axon. Further, we show that the EB/CLIP-170/dynactin-dependent mechanism is required for the efficient initiation of transport from the distal axon for multiple distinct cargos, including mitochondria, Rab5-positive early endosomes, late endosomes/lysosomes, and TrkA-, TrkB-, and APP-positive organelles. Our observations indicate that there is an essential role for ϩTIPs in the regulation of retrograde transport initiation in the neuron.

Dynamic allostery governs cyclophilin A–HIV capsid interplay

“…In fact, dynamic allostery as a regulatory mechanism is commonly used in many biological processes (33), including, but not limited to, receptor activation in signaling (34), enzymatic catalysis (35,36) including viral maturation involving proteolysis (37), intracellular transport (38)(39)(40), and others.…”

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

“…Lis1 is well known to be involved in the initiation of dynein‐dependent transport in cells (Lenz et al , 2006; Egan et al , 2012; Moughamian et al , 2013). Reducing Lis1 levels in fungi and mammalian cells inhibits dynein‐dependent transport of several organelles (Pandey & Smith, 2011; Egan et al , 2012; Dix et al , 2013; Moughamian et al , 2013; Klinman & Holzbaur, 2015) and BicD2‐N carrying vesicles (Splinter et al , 2012) in agreement with the simultaneous requirement of Lis1 and BicD2 for transport initiation as observed in our minimal system.…”

“…Different pathways are known to be responsible for microtubule plus‐end accumulation of dynein. Kinesin‐dependent transport has been observed in fungi and neurons of metazoans, whereas EB1 family protein (EB)‐dependent end tracking has been described for non‐neuronal cultured mammalian cells (Carvalho et al , 2004; Moughamian et al , 2013; Roberts et al , 2014). …”

“…Dynactin‐dependent plus‐end localisation of dynein has been demonstrated to be involved in the control of transport initiation in distal neurites (Moughamian et al , 2013; Nirschl et al , 2016). In non‐polarised cells, the role of dynactin in transport initiation is, however, not so well understood (Kim et al , 2007; Dixit et al , 2008).…”

“…Lis1 has been reported to regulate both initiation of minus‐end‐directed motility (Egan et al , 2012; Splinter et al , 2012; Moughamian et al , 2013) and plus‐end tracking of dynein (Coquelle et al , 2002; Splinter et al , 2012) in cells. However, Lis1 was not required for dynein end tracking in a minimal in vitro reconstitution (Duellberg et al , 2014), raising the question as to why Lis1 is needed for dynein end tracking in cells.…”