Abstract
T antigen, mucin-type core 1 O-glycan, is highly expressed in the embryonic central nervous system (CNS) and co-localizes with a Drosophila CNS marker, BP102 antigen. BP102 antigen and Derailed, an axon guidance receptor, are localized specifically in the proximal axon segment of isolated primary cultured neurons, and their mobility is restricted at the intra-axonal boundary by a diffusion barrier. However, the preferred trafficking mechanism remains unknown. In this study, the major O-glycan T antigen was found to localize within the proximal compartments of primary cultured Drosophila neurons, whereas the N-glycan HRP antigen was not. Ultrastructural analysis by atmospheric scanning electron microscopy revealed that microtubule bundles cross one another at the intra-axonal boundary, and that T antigens form circular pattern before the boundary. We then identified Short stop (Shot), a crosslinker protein between F-actin and microtubules, as a mediator for the proximal localization of T antigens; null mutation of shot cancelled preferential localization of T antigens. Moreover, F-actin binding domain of Shot was required for their proximal localization. Together, our results allow us to propose a novel trafficking pathway where Shot crosslinks F-actin and microtubules around the intra-axonal boundary, directing T antigen-carrying vesicles toward the proximal plasma membrane.
Highlights
T antigen, mucin-type core 1 O-glycan, is highly expressed in the embryonic central nervous system (CNS) and co-localizes with a Drosophila CNS marker, BP102 antigen
We found that T antigen, mucin-type core 1 O-glycan, is localized in the proximal axon segment of primary cultured neurons, whereas the Drosophila neuronal marker horse-radish peroxidase (HRP) antigen[20,21], a type of N-glycan, is localized in the whole axon fibre
We previously reported that T antigen (Fig. 1a), a major O-glycan in Drosophila, is expressed in the embryonic CNS and co-localized with BP102 antigen, a marker of the Drosophila CNS6 (Supplementary Fig. S1)
Summary
T antigen, mucin-type core 1 O-glycan, is highly expressed in the embryonic central nervous system (CNS) and co-localizes with a Drosophila CNS marker, BP102 antigen. BP102 antigen and Derailed, an axon guidance receptor, are localized in the proximal axon segment of isolated primary cultured neurons, and their mobility is restricted at the intra-axonal boundary by a diffusion barrier. Our results allow us to propose a novel trafficking pathway where Shot crosslinks F-actin and microtubules around the intraaxonal boundary, directing T antigen-carrying vesicles toward the proximal plasma membrane. We analysed the precise localization of Drosophila CNS marker BP102 antigen, F-actin, and microtubules near the boundary of the intra-axonal segment by atmospheric scanning electron microscopy (ASEM)[3]. In Drosophila, Shot is required for the extension of axons to target muscles[17], midline axon repulsion[18], and microtubule organization during neuronal growth[19] It is still unclear whether Shot is involved in axonal trafficking
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