周 如贇

The miniMOS technique has been widely used in the C. elegans community to generate single copy insertions. A worm is considered as a potential insertion candidate if it is resistant to G418 antibiotics and does not express a co-injected fluorescence marker. If the expression of the extrachromosomal array is very low, it is possible for a worm to be mistakenly identified as a miniMOS candidate, as this low expression level can still confer resistance to G418 without producing a detectable fluorescence signal from the co-injection marker. This may increase the workload for identifying the insertion locus in the subsequent steps. In the present study, we modified the plasmid platform for miniMOS insertion by incorporating a myo-2 promoter-driven TagRFP or a ubiquitous H2B::GFP expression cassette into the targeting vector and introducing two loxP sites flanking the selection cassettes. Based on this new miniMOS tool kit, the removable fluorescence reporters can be used to visualize the single copy insertions, greatly reducing insertion locus identification efforts. In our experience, this new platform greatly facilitates the isolation of the miniMOS mutants.

Oxysterol-binding protein (OSBP)-related protein (ORP) 6, a member of subfamily III in the ORP family, localizes to membrane contact sites between the endoplasmic reticulum (ER) and other organelles and functions in non-vesicular exchange of lipids including phosphatidylinositol-4-phosphate (PI4P) in neurons. In this study, we searched for the lipid counter-transported in exchange for PI4P by using molecular cell biology techniques. Deconvolution microscopy revealed that knockdown of ORP6 partially shifted localization of a phosphatidylserine (PS) marker but not filipin in primary cultured cerebellar neurons. Overexpression of ORP6 constructs lacking the OSBP-related ligand binding domain (ORD) resulted in the same shift of the PS marker. A PI4KⅢα inhibitor specifically inhibiting the synthesis and plasma membrane (PM) localization of PI4P, suppressed the localization of ORP6 and the PS marker at the PM. Overexpression of mutant PS synthase 1 (PSS1) inhibited transport of the PS marker to the PM and relocated the PI4P marker to the PM in Neuro-2A cells. Introduction of ORP6 but not the dominant negative ORP6 constructs, shifted the localization of PS back to the PM. These data collectively suggest the involvement of ORP6 in the counter-transport of PI4P and PS.