Interaction with a Kinesin‐2 Tail Propels Choline Acetyltransferase Flow Towards Synapse

Abstract

Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin based movement of large protein-aggregates aids this process. Choline acetyl-transferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates towards synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. Here, we show that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution.

Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila

“…The latter is implicated in the assembly of T-bars marked by Bruchpilot at the presynaptic compartment (Tsurudome et al, 2010). The observed increase in the ChAT enrichment at the neuropile, which is transported by Kinesin-2 (Sadananda et al, 2012), is also consistent with this hypothesis. Further, the contents of the nascent Rab4-associated vesicles could regulate the membrane recycling at the nerve termini.…”

“…4H–J; S3, Movie S4A). Previous reports showed that the KLP64DΔT forms a functional heterotrimeric motor complex (Sadananda et al, 2012). The presence of KLP64DΔT-GFP marginally improved the anterograde traffic as compared to the homozygous Klp64D −/− background.…”

“…Recent studies have, however, stated that the motor subunits could independently interact with soluble proteins through the tail domain (Girotra et al, 2016; Sadananda et al, 2012). Here, we showed that the Kinesin-2α tail could also bind to a membrane-associated protein, Rab4.…”

The C‐terminal tails of heterotrimeric kinesin‐2 motor subunits directly bind to α‐tubulin1: Possible implications for cilia‐specific tubulin entry

“…FRAP kymographs indicate that both kinesin‐2 and tubulin are transported in the ciliary outer segment in a non‐particulate form. A similar recovery pattern was observed for the choline acetyltransferase in the axon . The latter directly binds to the C‐terminal tails of the kinesin‐2α subunit.…”

Seeing the Unseen

“…Terada et al injected a bolus of fluorescently-labeled creatine kinase (a soluble protein conveyed in SCb) into the squid giant axon, and saw a slow anterogradely-biased movement of the labeled protein population at rates reminiscent of SCb (Terada et al, 2000, 2010). Using a drosophila model, Sadananda et al have also recently shown a kinesin-dependent slow, anterogradely-biased flow of the soluble protein choline acetyltransferase in axons (Sadananda et al 2012). Thus it appears that the biased flow of cytosolic cargoes in slow axonal transport is a conserved phenomenon, and collective efforts in different model-systems should provide clarity of underlying mechanisms in the future.…”