In neurons, proper distribution of mitochondria in axons and at synapses is critical for neurotransmission, synaptic plasticity, and axonal outgrowth. However, mechanisms underlying mitochondrial trafficking throughout the long neuronal processes have remained elusive. Here, we report that syntabulin plays a critical role in mitochondrial trafficking in neurons. Syntabulin is a peripheral membrane-associated protein that targets to mitochondria through its carboxyl-terminal tail. Using real-time imaging in living cultured neurons, we demonstrate that a significant fraction of syntabulin colocalizes and co-migrates with mitochondria along neuronal processes. Knockdown of syntabulin expression with targeted small interfering RNA or interference with the syntabulin–kinesin-1 heavy chain interaction reduces mitochondrial density within axonal processes by impairing anterograde movement of mitochondria. These findings collectively suggest that syntabulin acts as a linker molecule that is capable of attaching mitochondrial organelles to the microtubule-based motor kinesin-1, and in turn, contributes to anterograde trafficking of mitochondria to neuronal processes.

Different types of cargo vesicles containing presynaptic proteins are transported from the nerve cell body to the nerve terminal, and participate in the formation of active zones. However, the identity of the membranous cargoes and the nature of the motor-cargo interactions remain unsolved. Here, we report the identification of a syntaxin-1-binding protein named syntabulin. Syntabulin attaches syntaxin-containing vesicles to microtubules and migrates with syntaxin within the processes of hippocampal neurons. Knock-down of syntabulin expression with targeted small interfering RNAs (siRNAs) or interference with the syntabulin-syntaxin interaction inhibit attachment of syntaxin-cargo vesicles to microtubules and reduce syntaxin-1 distribution in neuronal processes. Furthermore, conventional kinesin I heavy chain binds to syntabulin and associates with syntabulin-linked syntaxin vesicles in vivo. These findings suggest that syntabulin functions as a linker molecule that attaches syntaxin-cargo vesicles to kinesin I, enabling the transport of syntaxin-1 to neuronal processes.

Mitochondria in the cell bodies of neurons are transported down neuronal processes in response to changes in local energy and metabolic states. Because of their extreme polarity, neurons require specialized mechanisms to regulate mitochondrial transport and retention in axons. Our previous studies using syntaphilin (snph) knock-out mice provided evidence that SNPH targets to axonal mitochondria and controls their mobility through its static interaction with microtubules (MTs). However, the mechanisms regulating SNPH-mediated mitochondrial docking remain elusive. Here, we report an unexpected role for dynein light chain LC8. Using proteomic biochemical and cell biological assays combined with time-lapse imaging in live snph wild-type and mutant neurons, we reveal that LC8 regulates axonal mitochondrial mobility by binding to SNPH, thus enhancing the SNPH-MT docking interaction. Using mutagenesis assays, we mapped a seven-residue LC8-binding motif. Through this specific interaction, SNPH recruits LC8 to axonal mitochondria; such colocalization is abolished when neurons express SNPH mutants lacking the LC8-binding motif. Transient LC8 expression reduces mitochondrial mobility in snph (ϩ/ϩ) but not (Ϫ/Ϫ) neurons, suggesting that the observed effect of LC8 depends on the SNPH-mediated docking mechanism. In contrast, deleting the LC8-binding motif impairs the ability of SNPH to immobilize axonal mitochondria. Furthermore, circular dichroism spectrum analysis shows that LC8 stabilizes an ␣-helical coiled-coil within the MT-binding domain of SNPH against thermal unfolding. Thus, our study provides new mechanistic insights into controlling mitochondrial mobility through a dynamic interaction between the mitochondrial docking receptor and axonal cytoskeleton.