Dynein Light Chain LC8 Regulates Syntaphilin-Mediated Mitochondrial Docking in Axons

Abstract

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.

The expression of syntaphilin is down-regulated in the optic nerve after axonal injury

“…To our knowledge, the expression of syntaphilin in other neuronal systems has not been investigated. In this study, immunohistochemistry analyses revealed that syntaphilin is located in neural cell bodies in the brain and retina, which coincided with previous reports (Kang et al, 2008;Chen et al, 2009) that have shown syntaphilin expression in the cytoplasm of cultured neurons. Also, this study revealed that axonal injury by optic nerve transection induced mitochondrial accumulation in RGC soma, which co-localized with syntaphilin.…”

“…Syntaphilin mRNA, which is 5.5 kb in length, was prominently expressed in the brain, whereas syntaphilin mRNA expression was not detected in other tissues in the rat. Previous studies using neural cell cultures revealed that syntaphilin is expressed in axonal mitochondria of neurons (Kang et al, 2008;Chen et al, 2009;Zhu and Sheng, 2011). It is believed that syntaphilin is required for maintaining a large portion of axonal mitochondria in a stationary state through its interaction with the microtubule cytoskeleton in hippocampal neurons (Kang et al, 2008) and dorsal root ganglion neurons (Zhu and Sheng, 2011).…”

“…Later, in vitro studies demonstrated that syntaphilin was expressed in axonal mitochondria of neurons (Kang et al, 2008;Chen et al, 2009;Zhu and Sheng, 2011). Only one report has investigated the expression of syntaphilin in vivo.…”

Stoichiometry of scaffold complexes in living neurons - DLC2 as a dimerization engine for GKAP

“…Disruption of GKAP-DLC2 interactions in neuronal spines abolished the GKAP-GKAP association. Furthermore, after GKAP-DLC2 disruption, the DLC2-DLC2 interaction was re-localized to the membrane, probably through interaction with submembranous components of the actin-based membrane skeleton, emphasizing a reciprocal stabilization of the DLC2 homodimer by the interacting partners, as previously suggested for the DLC1-syntaphilin interaction (Chen et al, 2009). In their physiological environment, proteins rarely act in isolation but rather bind to other molecules to elicit specific subcellular responses.…”

Mitochondrial Trafficking in Neurons

“…Syntaphilin-GFP was observed preferentially on stationary mitochondria, but not if the microtubulebinding region was deleted. The dynein light chain LC8 also appears to be involved with syntaphilin-based docking (Chen et al 2009). Mice lacking syntaphilin were found to have substantially more mitochondria in the mobile fraction (Kang et al 2008).…”