The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

The microscopy-based assay to study and analyze the recycling endosomes using SNARE trafficking. These are skin melanocytes. The black-colored organelles that you see inside the cells are called melanosomes.

Melanosomes are known as a class of organelles called lysozome related organelles or LRO. Recycling endosomes are tubular vesicular organelles from early or sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysozome related organelle produced by melanocytes.

Recycling endosomes deliver the melanocyte-specific cargo to premature melanosomes during their formation. the generation of recycling endosomes observed in several syndrome is cells in hyperpigmentation of skin, hair, and eye. Therefore, studying the dynamics of recycling endosomes is useful to understand the function of these organelles in normal and disease conditions.

This study aims to measure the recycling endosomes'dynamics using arresting SNARE syntaxin-13. The first step is the seeding of mouse melanocytes on pre-treated coverslips. Coat the glass coverslips in Petri dish in basement membrane matrix medium.

And dry it in the tissue culture hood for 15 minutes. Wash the coverslips once with 1X PBS before use. Seed the cells on the basement membrane medium coated coverslips at 50 to 60%competency.

Always add 200 nanomolars of PMA to the plated cell suspension in complete RPMI media. After seeding the cells, incubate the dish containing cells in CO2 incubator for 12 to 24 hours. The second step is the transfection of cells with syntaxin-13 plasmids.

Incubate the tips containing DNA and transfection reagent, mix for five minutes and mix without repeated pipetting. Tap the tube with hand every ten minutes for 30 minutes at room temperature. During the incubation, wash the cells twice with 1X PBS, once with OPTI-MEM, and then add 1 mil of OPTI-MEM to the cells.

Post 30 minutes of incubation, add the transfection reagent DNA mix to the cells in a drop-wise manner by covering the dish. Incubate the cells at 37 degrees Celsius for six hours. Aspirate the OPTI-MEM medium with transfection reagent and add complete RPMI medium supplemented with 200 nanomolars of of PMA.

Incubate the cells at 37 degrees Celsius for 48 hours. The third step is fixation of the cells. Please note, the following procedure is performed outside the tissue culture hood.

After 48 hours of transfection, wash the cells twice with 1X PBS and then fix the cells with 3%formaldehyde for 30 minutes. After fixation, wash the cells twice with 1X PBS and store the coverslips in 1X PBS until further use. Alternatively, cells can be mounted on glass plates or stored at four degrees Celsius.

The fourth step is immunostaining of the cells. Prepare a humid chamber. Place parafilm cut piece on a mouse filter paper in a Petri dish.

Cover with aluminum foil. Prepare 25 microliters of primary antibody solution. Add antibody at a dilution of one to 200.

Add this solution as a drop on the parafilm in the humid chamber. Carefully lift the coverslip with forceps. Invert it on the drop of primary antibody staining solution, and then cover the lid of the humid chamber.

Incubate at room temperature for 30 minutes. Similarly, prepare the secondary antibody solution at a dilution of one to 500 and place it on the parafilm next to the coverslip in the humid chamber. For staining the nucleus, add DAPI at a dilution of 120, 000 to 130, 000 to the solution.

Using forceps, carefully pick up the coverslip from the primary antibody solution and dip it twice in 1X PBS. Tap the coverslip on tissue paper to remove the excess PBS on the coverslip. Place it on the secondary antibody staining solution in the humid chamber and do not expose it to the light, due to the presence of fluorescently dyed antibodies in the solution.

After the incubation, carefully pick up the coverslip from the secondary antibody solution and then dip it twice in 1X PBS. Further, tap the coverslip on tissue paper to remove the excess PBS on the coverslip. Place 12 microliters of imaging reagent onto a glass slide and carefully place the stained coverslip on the mounting reagent.

Invert the glass slide on the tissue paper and then gently press. Next, image the coverslip using a fluorescence microscope and analyze the images using ImageJ. The sixth step is the quantification of overlap between the recycling endosome localized proteins and melanosomes.

Quantification of syntaxin-13 delta-129 colocalization with the melanosomes. Immunofluorescence microscopy of syntaxin-13 in mouse wild type melanocytes showed GFP-syntaxin-13 wild-type localized as vesicular and tubular structures. And GFP-syntaxin-13 delta-129 localizes ring-like structures in addition to the cell surface.

Further, intracellular ring-like GFP-syntaxin-13 delta-129 showed colocalization with the melanosome protein TYRP1 in bright field-imaged melanosomes. As shown before, a cohort of overexpressed GFP-syntaxin-13 wild type is observed in melanosomes. To measure the relative localization of GFP-syntaxin-13 wild type and GFP-syntaxin-13 delta-129 to melanosomes, Fiji software was used and analysis was done with JACOP plugin.

The measured Mander's overlap coefficient, that is MOC, between GFP-syntaxin-13 delta-129 with TYRP1 is approximately 1.5 folds higher compared to GFP-syntaxin-13 wild type with TYRP1. Interestingly, TYRP1 showed 2.9 folds higher MOC values with GFP-syntaxin-13 delta-129 compared to GFP-syntaxin-13 wild type. These data indicate that the localization of GFP-syntaxin-13 delta-129 melanosomes is relatively higher compared to GFP-syntaxin-13 wild type at a steady state.

Quantification of syntaxin-13 wild type localization of the recycling endosomes. Immunofluorescence microscopy of GFP-syntaxin-13 wild type showed colocalization with the known recycling endosomal protein Rab11. The MOC between GFP-syntaxin-13 wild type with mCherry-Rab11 is approximately 1.4 folds higher compared to mCherry-Rab11 with GFP-syntaxin-13 wild type.

To measure the number and length of GFP-syntaxin-13 wild type positive endosomal tubules, Fiji software was used as described in the protocol section. mCherry-Rab11 is used a positive control in the experiments. Melanocytes transfected with GFP-syntaxin-13 wild type showed a higher number of tubules per cell compared to cells expressing mCherry-Rab11.

However, the tubules are reduced upon coexpression of both GFP-syntaxin-13 wild type and mCherry-Rab11 in the cells. The seventh step is the quantification of recycling endosomes'tubular number and length. The tubules are reduced upon coexpression of both GFP-syntaxin-13 wild type and mCherry-Rab11 in the cells.

Interestingly, the average tubule length from both GFP-syntaxin-13 wild type and mCherry-Rab11 is comparable to each other in cells expressing individually or together. Together, these data suggest that GFP-syntaxin-13 wild type localizes to recycling endosomes as similar to Rab11. This study showed that the N-terminal delivery of syntaxin-13 mutant, that is GFP-syntaxin-13 delta-129, localizes to melanosomes GFP-syntaxin-13 delta-129 can possibly be used as a reporter to vesicular trafficking from recycling endosomes to the cell surface and LRO.

In contrast, GFP-syntaxin-13 wild type localizes to recycling endosomes as similar to Rab11. This study illustrated that GFP-syntaxin-13 wild type could also be used for marking recycling endosomes in melanocytes. GFP-syntaxin-13 wild type may be a better recycling endosomal marker than Rab11, since Rab11 alters the endosomal dynamics.

All together, GFP-syntaxin-13 wild type acts a potential recycling endosomal marker to study the dynamics at steady state conditions.