Journal: PLOS Biology
Article Title: MSP-tracker: A versatile vesicle tracking software tool used to reveal the spatial control of polarized secretion in Drosophila epithelial cells
doi: 10.1371/journal.pbio.3003099
Figure Lengend Snippet: A) Diagram of the RUSH system. The Halo-tagged cargo is retained in the ER by the binding of the Streptavidin binding peptide (SBP) to Streptavidin (StrepA), which is fused to the ER retention signal, KDEL. The cargo is synchronously released upon the addition of biotin, which outcompetes SBP for binding to Streptavidin. B) Diagram of the tagged cargo constructs used in this study. A signal sequence is followed by the SBP, then the tag, and the cargo. Cadherin 99C (Cad99c) was used as an apical cargo, and Nidogen (Ndg) as a basal cargo. C) Steady state expression of UAS-SBP-Halo-Cadherin99c (magenta) under the control of traffic jam-Gal4, showing its localization to the apical microvilli. Phalloidin staining of F-actin (green) labels the apical microvilli in the follicle cells and the oocyte. Scale bar 10 µm. D) Steady state expression of UAS-SBP-SNAP-Ndg (magenta) under the control of traffic jam-Gal4, showing its localization to the basement membrane. Actin is shown in green. Scale bar 10 µm. E) En face view of SBP-SNAP-Ndg in the basement membrane. Scale bar 10 µm. F) Time course of SBP-Halo-Cadherin99C trafficking in fixed samples. At 0 min, Cad99c localizes to the ER throughout the cytoplasm. Fifteen min after release from the ER with biotin, Cad99c (green) has accumulated in the Golgi, marked by Golgin245 (magenta). By 30 min, almost all Cad99c has reached the apical membrane. Scale bar 10 μm. G) 25 min after release from the ER, Cad99c (green) localizes to subapical puncta that are labeled by Rab11 (magenta). Scale bar 10 µm. In all figures with a cross-section of the follicle cells, apical is toward the top of the image and basal toward the bottom.
Article Snippet: UAST-SBP-Halo-Cad99c was created by cloning the signal peptide of the Cad99c followed by a GSGSGSG linker, the sequence for streptavidin binding peptide (SBP), Pro-Ala-Gly, then the HaloTag sequence, another GSGSGSG linker, and the rest of Cad99c into pUAST(AttB) using NEB HiFi assembly.
Techniques: Binding Assay, Construct, Sequencing, Expressing, Control, Staining, Membrane, Labeling