Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay

Abstract

Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was tagged with either CFP or YFP. Given that GalT is recycled to the cisterna where it is located at steady state, GalT-containing vesicles target GalT-containing cisternal membranes. Golgi membranes were therefore isolated from GalT-CFP expressing cells, while vesicles were prepared from GalT-YFP expressing ones. Incubating CFP-labelled Golgi with YFP-labelled vesicles in the presence of cytosol and an energy regeneration mixture at 37 °C produced a significant increase in CFP-YFP co-localization upon fluorescent imaging of the mixture compared to incubation on ice. The assay was validated to require energy, proteins and physiologically important trafficking components such as Rab GTPases and the conserved oligomeric Golgi tethering complex. This assay is useful for the investigation of both physiological and pathological changes that affect the Golgi trafficking machinery, in particular, vesicle tethering.

Keywords: Conserved oligomeric Golgi complex; Fluorescent imaging; Galactosyltransferase; Golgi apparatus; Vesicle tethering; Vesicle trafficking.

Figure 1.. Execution of the assay.

GalT-YFP vesicles and GalT-CFP Golgi are mixed together in a total reaction volume of 50 μl containing the desired reaction conditions. The mixture is equally divided into two tubes. One is incubated at 0 °C for 40 min as an internal control, and the other is incubated at 37 °C for the same period. After incubation, 5 micron silica beads are mixed with each sample, then 3 μl of the mixture is immediately delivered in two roughly equally-sized spots onto a microscope slide. The sample is covered with a 22 x 22 mm coverslip, sealed around the edges with clear nail varnish with the silica beads acting as spacers. The slide is kept in the dark at 4 °C until imaging by epifluorescence microscopy at room temperature.

Figure 2.. Shielding the mounted sample during imaging.

Although low-light room conditions are used during imaging, extraneous light on the sample is also minimized by shielding. Matt black card is utilized as follows: 1. a wide collar is fitted around the objective lens to block light from below; 2. a sheet with a rectangular hole is placed over the objective to cover gaps; and 3. a shallow box lid is placed on top to block light from above. The slide is placed in the universal mounting frame on top of the rectangular sheet in image 2.

Figure 3.. Image processing workflow

(Cottam, 2012). A. Raw images of Golgi and vesicles are treated with background subtraction. This also corrects slight uneven illumination of the sample generating the images in (B). B. An intensity threshold is applied to images to select particles above the average background noise. C. The selection is converted to a binary image and re-colored to magenta and green for Golgi and vesicles respectively. D. The binary images are overlaid to reveal colocalized areas as white pixels. E. A hysteresis operation is applied to the overlaid image which completely fills any particle containing white pixels to become a single colocalization event. The number of colocalization events is expressed as a percentage of the total number of particles (colocalization events/(Golgi + vesicles)) to give the measure of assay activity. In our previous work, we have used the calculation (Golgi + vesicles + colocalization events) for total particle numbers, which is not correct but should be considered when comparing data to our published results. Given the generally low number of colocalization events (Figure 4), the distortion by the incorrect particle total does not change the biological conclusions when comparing results for different assay conditions. Scale bar = 10 μm.

Figure 4.. Typical data sheet of the image processing results.

Particle numbers as well as the numbers of co-localizing particles are shown for each image.