Movement of the endoplasmic reticulum is driven by multiple classes of vesicles marked by Rab-GTPases.

Peripheral endoplasmic reticulum (ER) tubules move along microtubules to interact with various organelles through membrane contact sites (MCS). Traditionally, ER moves by either sliding along stable microtubules via molecular motors or attaching to the plus ends of dynamic microtubules through tip attachment complexes (TAC). A recently discovered third process, hitchhiking, involves motile vesicles pulling ER tubules along microtubules. Previous research showed that ER hitchhikes on Rab5- and Rab7-marked endosomes, but it is uncertain if other Rab-vesicles can do the same. In U2OS cells, we screened Rabs for their ability to cotransport with ER tubules and found that ER hitchhikes on post-Golgi vesicles marked by Rab6 (isoforms a and b). Rab6-ER hitchhiking occurs independently of ER-endolysosome contacts and TAC-mediated ER movement. Disrupting either Rab6 or the motility of Rab6-vesicles reduces overall ER movement. Conversely, relocating these vesicles to the cell periphery causes peripheral ER accumulation, indicating that Rab6-vesicle motility is crucial for a subset of ER movements. Proximal post-Golgi vesicles marked by TGN46 are involved in Rab6-ER hitchhiking, while other post-Golgi vesicles (Rabs 8/10/11/13/14) are not essential for ER movement. Our further analysis finds that ER to Golgi vesicles marked by Rab1 are also capable of driving a subset of ER movements. Taken together, our findings suggest that ER hitchhiking on Rab-vesicles is a significant mode of ER movement. SIGNIFICANCE STATEMENT: Peripheral endoplasmic reticulum tubules move on microtubules by either attaching to motors (cargo adaptor-mediated), dynamic microtubule-plus ends (tip attachment complexes) or motile vesicles (hitchhiking) but the prevalence of each mode is not clearPost-Golgi vesicles marked by Rab6/TGN46 and ER to Golgi vesicles marked by Rab1 drive ER movementsER hitchhiking on multiple classes of vesicles (endolysosomal, post-Golgi and ER to Golgi) marked by Rabs plays a prominent role in ER movement.

Modeling nociception in zebrafish: a way forward for unbiased analgesic discovery.

Acute and chronic pain conditions are often debilitating, inflicting severe physiological, emotional and economic costs and affect a large percentage of the global population. However, the development of therapeutic analgesic agents based primarily on targeted drug development has been largely ineffective. An alternative approach to analgesic development would be to develop low cost, high throughput, untargeted animal based behavioral screens that model complex nociceptive behaviors in which to screen for analgesic compounds. Here we describe the development of a behavioral based assay in zebrafish larvae that is effective in identifying small molecule compounds with analgesic properties. In a place aversion assay, which likely utilizes supraspinal neuronal circuitry, individually arrayed zebrafish larvae show temperature-dependent aversion to increasing and decreasing temperatures deviating from rearing temperature. Modeling thermal hyperalgesia, the addition of the noxious inflammatory compound and TRPA1 agonist allyl isothiocyanate sensitized heat aversion and reversed cool aversion leading larvae to avoid rearing temperature in favor of otherwise acutely aversive cooler temperatures. We show that small molecules with known analgesic properties are able to inhibit acute and/or sensitized temperature aversion.