Single-Axonal Organelle Analysis Method Reveals New Protein–Motor Associations

• Published in: ACS chemical neuroscience 2013-02, Vol.4 (2), p.277-284
• Main Authors: Sgro, Allyson E, Bajjalieh, Sandra M, Chiu, Daniel T
• Format: Article
• Language: English
• Subjects: Animals; Axonal Transport - physiology; Cells, Cultured; Fluorescent Antibody Technique; Hippocampus - cytology; Kinesin - metabolism; Kinesin - physiology; Membrane Glycoproteins - metabolism; Mice; Microfluidic Analytical Techniques; Microscopy, Confocal; Nerve Tissue Proteins - metabolism; Neurons - cytology; Neurons - metabolism; Neurons - physiology; Organelles - metabolism; Synaptic Vesicles - metabolism; Synaptophysin - metabolism; Transport Vesicles - metabolism; Transport Vesicles - physiology; Vesicle-Associated Membrane Protein 2 - metabolism; Vesicular Transport Proteins - metabolism
• ISSN: 1948-7193; 1948-7193
• DOI: 10.1021/cn300136y

Axonal transport of synaptic vesicle proteins is required to maintain neurons’ ability to communicate via synaptic transmission. Neurotransmitter-containing synaptic vesicles are assembled at synaptic terminals via highly regulated endocytosis of membrane proteins. These synaptic vesicle membrane proteins are synthesized in the cell body and transported to synapses in carrier vesicles that make their way down axons via microtubule-based transport utilizing kinesin molecular motors. Identifying the cargos that each kinesin motor protein carries from the cell bodies to the synapse is key to understanding both diseases caused by motor protein dysfunction and how synaptic vesicles are assembled. However, obtaining a bulk sample of axonal transport complexes from central nervous system (CNS) neurons to use for identification of their contents has posed a challenge to researchers. To obtain axonal carrier vesicles from primary cultured neurons, we fabricated a microfluidic chip designed to physically isolate axons from dendrites and cell bodies and developed a method to remove bulk axonal samples and label their contents. Synaptic vesicle protein carrier vesicles in these samples were labeled with antibodies to the synaptic vesicle proteins p38, SV2A, and VAMP2, and the anterograde axonal transport motor KIF1A, after which antibody overlap was evaluated using single-organelle TIRF microscopy. This work confirms a previously discovered association between KIF1A and p38 and shows that KIF1A also transports SV2A- and VAMP2-containing carrier vesicles.