Self-propelling vesicles define glycolysis as the minimal energy machinery for neuronal transport.

María-Victoria Hinckelmann,Amandine Virlogeux,Frédéric Saudou,Diana Zala,Daniel Choquet,Bernard Hoflack,Christel Poujol,Christian Niehage

doi:10.1038/ncomms13233

Abstract

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) facilitates fast axonal transport in neurons. However, given that GAPDH does not produce ATP, it is unclear whether glycolysis per se is sufficient to propel vesicles. Although many proteins regulating transport have been identified, the molecular composition of transported vesicles in neurons has yet to be fully elucidated. Here we selectively enrich motile vesicles and perform quantitative proteomic analysis. In addition to the expected molecular motors and vesicular proteins, we find an enrichment of all the glycolytic enzymes. Using biochemical approaches and super-resolution microscopy, we observe that most glycolytic enzymes are selectively associated with vesicles and facilitate transport of vesicles in neurons. Finally, we provide evidence that mouse brain vesicles produce ATP from ADP and glucose, and display movement in a reconstituted in vitro transport assay of native vesicles. We conclude that transport of vesicles along microtubules can be autonomous.

Highlights

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) facilitates fast axonal transport in neurons
We found that enriched vesicular fractions can produce ATP when incubated with the substrates of GAPDH, and that increasing the stoichiometric load of GAPDH on vesicles increased Fast axonal transport (FAT); we could not exclude the role of additional factors such as NADH, which is produced by GAPDH, or diffusion within the cytoplasm, in providing the substrates necessary for ATP production
Vesicles associated to p50-green fluorescent protein (GFP) can be considered as motile vesicles as they are likely to associate with the molecular motors

Summary

Introduction

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) facilitates fast axonal transport in neurons. Fast axonal transport (FAT) is a very efficient mode of delivery in neurons that is mediated by the ATPases kinesin and dynein[1]. It is characterized by high velocity and processivity over long distances. We recently reported that in addition to these mechanisms, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) localized on vesicles may promote efficient FAT by optimizing the energy supply to the molecular motors[6]. Given that the entire glycolytic enzyme complex is associated with the plasma membrane of red blood cells[7] and the locally produced ATP fuels Na þ /K þ and Ca2 þ pumps[8], this metabolic organization may be present on other membranes such as vesicles, even though these are motile. Using a combination of label-free quantitative proteomics on neuronal motile vesicles and functional transport assays in neurons and in vitro, we determine the minimal energyproducing machinery that is sufficient to propel vesicles in neurons

Methods

Results

Conclusion