Microtubule nucleation at the Golgi-related membrane organelles of mouse neurons

Microtubules are often nucleated at non-centrosomal sites in some differentiated cell types. We previously reported that microtubules are nucleated at the cytoplasm in cultured mouse cortical neurons. It is unclear, however, what organelle is the site of such nucleation. In this study, we examined the possibility that recently discovered neuron-specific Golgi-like structures called Golgi satellites are the nucleation sites. Microtubule nucleation was tested by observing microtubule regrowth after nocodazole depolymerization. First, the spatial association between microtubule nucleation and membrane organelles was investigated. Organelle markers including GM130 (cis Golgi and Golgi outpost marker), Golgin97 (trans-Golgi network marker), transferrin receptor (recycling endosome marker), TOMM40 (mitochondria marker), and syntaxin 6 (early endosome and Golgi satellite marker) were examined. Microtubule regrowth was observed in cytoplasmic regions where TOMM40-positive and syntaxin 6-positive organelles were rich. Triple immunostaining showed that γ-tubulin at one end of regrown microtubules was attached to syntaxin 6-organelles but not to TOMM40-organelles, indicating that syntaxin 6-organelles are the microtubule nucleation sites. To address the possibility that the microtubule-nucleating syntaxin 6-organelles were Golgi satellites, we transfected neurons with plasmid vector caring FLAG-tagged Golt sequence, a marker for Golgi satellites, and subsequently performed microtubule regrowth experiments. We found regrown microtubules on FLAG-positive organelles in dendrites. This observation suggests that Golgi satellites are microtubule nucleation sites. Microtubules from the Golgi satellites might guide transport vesicles generated at rough endoplasmic reticulum in dendrites.