Figure 1.

Nsg1 and Nsg2 are type II membrane proteins and endocytose from the cell surface into early endosomes. (A–F) Nsg1 and Nsg2 co-localize substantially. (A) DIV9 neurons were fixed and stained against MAP2 (blue) and endogenous Nsg1 (green) and Nsg2 (red). Bar = 10 µm. The boxed region is shown larger in (B). Co-localization is extensive in the zoomed-in panel (B). (C) shows a small stretch of dendrite (along arrow in B) with individual channels shown separately. A line scan is shown in (D). Even though co-extensive peaks are prevalent, some non-colocalizing puncta are evident (green arrows). These tend to be smaller and fainter compartments. (E) The extent of co-localization was quantified for endogenous Nsg1 and Nsg2 from confocal Z-stacks. Each data point corresponds to multiple dendrites in a single microscopy field containing multiple neurons. (F) Airyscan super-resolution imaging was used to obtain improved spatial resolution. A dendritic segment (MAP2-positive; blue) is shown with a zoomed panel below. Endosomes are crisply resolved using Airyscan. Co-incidence of Nsg1 and Nsg2 is high in endosomes which contain both proteins, but some puncta contain only one or the other (red only). (G) Dual live imaging of DIV9 neuron after transfection with Nsg1-mcherry and Nsg2-GFP. A still frame of one dendritic segment is shown with the kymograph displaying time on the y-axis below. Even though the relative ratios of Nsg1 and Nsg2 differ in distinct endosomes, they are greatly co-localizing to the same endosomes. (H) Nsg2 also exists as a type II membrane protein. Endocytosis of endogenous Nsg2 (red) and Nsg1 (green) can be detected using C-terminal directed antibodies for live uptake. The boxed region is shown enlarged on the right and a line scan along it is shown in (I). The line scan illustrates near-complete coincidence of peaks (I). Bar = 10 µm.