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

Summary

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

Abstract

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Introduction

Biosynthesis of melanin pigments occurs in melanosomes, a melanocyte-specific lysosome-related organelle (LRO) that co-exists with conventional lysosomes. The endocytic system plays a key role in the biogenesis of melanosomes, required for skin color and photoprotection against ionizing radiation^1,2,3. During this process, the melanin synthesizing enzymes are sorted on early/sorting endosomes and then transported to premature melanosomes through tubular or vesicular endosomes called recycling endosomes (REs)^{4,5,6,7,8,9,10}. The targeting and fusion of these organelles regulate the maturation of fully functional pigmented melanosomes^{7,11,12,13,14}. Defects in the formation of these organelles or cargo sorting to these organelles cause oculocutaneous albinism and other clinical phenotypes, observed in Hermansky-Pudlak syndrome^15,16.

Here we describe a simple microscopy-based technique to study and analyze the REs. In this method, we have taken advantage of a transmembrane protein, Qa-SNARE Syntaxin (STX)13 that resides on recycling endosomes¹⁷ and cycles between sorting endosomes and melanosomes in melanocytes^12,18. Further, deletion of N-terminal unstructured regulatory domain (namely SynN or STX13^Δ129) allows the SNARE to get stuck in melanosomes, which measures the forward trafficking pathway towards the melanosome¹². We have used a known recycling endosomal marker Rab GTPase (Rab)11 in our studies^14,19. Fluorescence imaging of the proteins GFP-STX13^WT, GFP-STX13^Δ129, mCherry-Rab11, and TYRP1 in wild type melanocytes followed by quantification of their relative localization will provide the nature and dynamics of REs in addition to their targeting to melanosomes. Thus, this is a simple technique that can be used to visualize and measure the dynamics of REs in melanocytes.

Protocol

The protocol involves the seeding of melanocytes followed by transfection of the plasmids. Further steps include fixation, immunostaining, imaging, and analysis of the cells to measure the length and number of REs. The detailed description of the protocol is given below.

1. Seeding of mouse melanocytes on pre-treated coverslips

1. Coat the glass coverslips in a Petri dish (i.e., 4 - 5 in a 35 mm dish) with basement membrane matrix medium (1:20 in complete RPMI medium: RPMI + 10% heat inactivated FBS + 1x Glutamine + 1x Antibiotic mix) and dry it in the tissue culture hood for 15 min. Wash the coverslips once with 1x PBS before use.
2. Maintain the wild type mouse melanocytes (melan-Ink4a-Arf-1 from C57BL/6J, a/a, Ink4a-Arf^-/- mice, described in²⁰ and available at The Welcome Trust Functional Genomics Cell Bank) in a Petri dish (i.e., 35 or 60 mm dish) supplemented with complete RPMI medium.
   1. Wash the cells twice with 1x PBS and add 0.5 or 1 mL of Trypsin-EDTA (0.25%) solution for trypsinizing cells. Incubate the cells at 37 °C for 2 - 5 min for the detachment from the Petri dish.
   2. Add 1 - 2 mL of complete RPMI medium, suspend and then transfer the cells to a centrifuge tube.
3. Centrifuge the cell suspension at 4 °C, 376 x g for 5 min and then resuspend the pellet in 1x PBS.
4. Repeat the centrifugation step and resuspend the cells in 1 mL of complete RPMI medium.
5. Seed the cells on the basement membrane medium-coated coverslips at 50-60% confluency (approximately 6 x 10⁵ cells on a 35 mm cell culture dish containing 4 - 5 coverslips). Always add 200 nM of PMA (add 5 µL of working stock 40 µM phorbol 12-myristate 13-acetate) to the plated cell suspension in complete RPMI medium.
6. After seeding, incubate the plate at 37 °C for 12- 24 h.

2. Transfection of cells with the STX13 plasmids

1. Use the following reagents: pEGFP-C1-STX13^WT and pEGFP-C1-STX13^Δ129 (described in¹²). mCherry-Rab11, was a kind gift from Graça Raposo, Institut Curie, Paris (described in reference¹⁹). Anti-TYRP1 antibody from ATCC (TA99).
2. After 12 - 24 h of seeding, transfect the cells with plasmids using a lipid-based transfection reagent. For a 35 mm dish, take 5 µL of the transfection reagent in 250 µL of OPTI-MEM medium in a microcentrifuge tube and take approximately 200 ng of each plasmid in 250 µL of OPTI-MEM.
3. Incubate the tubes containing DNA and the transfection reagent for 5 min. Mix without repeated pipetting (total volume will be about 500 µL). Incubate for 30 min at room temperature. Perform hand tapping of the tube every 10 min for 30 min at RT.
4. During the incubation, wash the cells twice with 1x PBS, once with OPTI-MEM and then add 1 mL of OPTI-MEM to the cells.
5. Post 30 min of incubation, add the transfection reagent-DNA mix to the cells in a dropwise manner by covering the dish.
6. Incubate the cells at 37 °C for 6 h. Aspirate the OPTI-MEM medium with transfection reagent and add complete RPMI medium supplemented with 200 nM of PMA.
7. Incubate the cells at 37 °C for 48 h.

3. Fixation of the cells

NOTE: The following procedure is performed outside the tissue culture hood.

1. After 48 h of transfection, wash the cells twice with 1x PBS and then fix the cells with 3% formaldehyde (freshly prepared in 1x PBS) for 30 min.
2. 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 slides (see below) or stored at 4 °C.

4. Immunostaining of the cells

1. Prepare a humid chamber: place paraffin film cut piece on moist filter paper in a Petri dish, covered with aluminum foil.
2. Prepare 25 µL of primary antibody solution (0.2% saponin in 1x PBS, 0.1% BSA in 1x PBS and 0.02% Sodium azide in 1x PBS). Add antibody at a dilution of 1:200. Add this solution as a drop on a paraffin film in the humid chamber.
3. Carefully lift the coverslip with forceps, invert it on this drop of primary antibody staining solution and then cover the lid of the humid chamber. Incubate at room temperature for 30 min.
4. Similarly, prepare the secondary antibody solution at a dilution of 1:500 and place it on paraffin film next to the coverslip in the humid chamber. For staining the nucleus, add DAPI (1:20,000 to 1:30,000) to the solution.
5. Using forceps, carefully pick up the coverslip from the primary antibody solution and dip it thrice in 1x PBS (in a glass beaker).
6. Tap the coverslip on tissue paper to remove the excess PBS on the coverslip. Place it on secondary antibody staining solution in the humid chamber and do not expose it to light due to the presence of fluorescently tagged antibodies in the solution.
7. Incubate the coverslip again for 30 min at room temperature. Please always keep a note the side of the coverslip that has the cells throughout these steps.
8. After the incubation, carefully pick up the coverslip from the secondary antibody solution and then dip it thrice in 1x PBS. Further, tap the coverslip on tissue paper to remove the excess PBS on the coverslip.
9. Place 12 µL of Fluoromount-G mounting reagent onto a glass slide and carefully place the stained coverslip (facing towards the glass) on the mounting reagent. Invert the glass slide on tissue paper and then gently press.

5. Fluorescence microscopy of the cells

1. Image the stained cells under Bright-field (BF) and fluorescence (IF) filters using an inverted fluorescence microscope equipped with a CCD camera using 60x (oil) apochromatic objective or any other microscope with a similar configuration.

6. Quantification of overlap between the RE localized proteins and melanosomes:

NOTE: The following steps are followed for the quantification of Mander's overlap coefficient between the proteins using Fiji software (freely downloadable from the link: https://imagej.net/software/fiji/). Use the TIFF image with multiple channels.

1. Open the raw image. Go to the Image option, select Color | Split channels, and use the two channels for analysis.
2. Open the JACoP plugin in the Plugin option.
3. Set the threshold for both the channels such that all the bright spots are selected and the background is eliminated.
4. Go to Analysis option, select M1 and M2 coefficients for getting Mander's overlap coefficient.
5. Press the Analyze option in the JACoP Plugin and see the result that displays Mander's overlap coefficient.

7. Quantification of recycling endosomes' tubular number and length:

NOTE: The following steps are followed for the quantification of the number and length of the tubules using Fiji software.

1. Open the raw image Go to Image option, select Color | Split channels and use the desired channel for analysis.
2. Go to Image again, select Type | Convert to 8-bit image.
3. Then go to Plugins, select Analyze | Tubeness. Set sigma value at 0.1075 . Press Ok.
4. Go to Image again, select Type | Convert to 8 bit image.
5. Go to Image | Adjust | Threshold (Use the same threshold values for all the images. Images should have approximately equal intensity).
6. Go to Process | Binary | Convert to mask.
7. Go to Process, select Binary and then select Skeletonize.
8. Go to Analyze, select Skeleton and choose Analyze Skeleton. In result and output | select (a) Calculate the largest shortest path | (b) show detailed information | (c) display labeled skeleton. Press Ok.
   NOTE: The result will open in the tabulated format. The column of Average branch length shows the length of all the different tubules in the cell that is selected (set scale for getting values in micrometres).
9. For obtaining the number of tubules in the cell, go to Analyze, and select Analyze particle option. Press Ok.
   NOTE: In the obtained results, the count column shows the number of tubules in that particular cell.
10. Save the data and analyze.

Results

Quantification of STX13^Δ129 mutant localization to the melanosomes
Immunofluorescence microscopy of STX13 in mouse wild type melanocytes showed GFP-STX13^WT localized as vesicular and tubular structures and GFP-STX13^Δ129 localized as ring-like structures in addition to the cell surface (Figure 1A). Further, intracellular ring-like GFP-STX13^Δ129 showed colocalization with the mel...

Discussion

Recycling endosomes are a cohort of endocytic organelles, and they mediate the recycling of cargo to the cell surface in all cell types^{21,22,23,24,25}. In specialized cell types such as melanocytes, these organelles partly divert their trafficking route towards the melanosomes for their biogenesis^3,16

Materials

Name	Company	Catalog Number	Comments
anti-TYRP1 antibody (TA99)	ATCC	HB-8704
Fluoromount-G	Southern Biotech	0100-01
Lipofectamine 2000	ThermoFisher Scientific	11668-500
Matrigel matrix	BD Biosciences	356231
OPTI-MEM	ThermoFisher Scientific	022600-050
Phorbol 12-myristate 13-acetate	Sigma-Aldrich	P8139
RPMI Medium 1640	ThermoFisher Scientific	31800-022