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The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking
In This Article
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 radiation1,2,3. 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,<....
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
- 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:.......
Representative Results
Quantification of STX13Δ129 mutant localization to the melanosomes
Immunofluorescence microscopy of STX13 in mouse wild type melanocytes showed GFP-STX13WT localized as vesicular and tubular structures and GFP-STX13Δ129 localized as ring-like structures in addition to the cell surface (Figure 1A). Further, intracellular ring-like GFP-STX13Δ129 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 types21,22,23,24,25. In specialized cell types such as melanocytes, these organelles partly divert their trafficking route towards the melanosomes for their biogenesis3,16
Acknowledgements
This work was supported by the Department of Biotechnology (BT/PR32489/BRB/10/1786/2019 to SRGS); Science and Engineering Research Board (CRG/2019/000281 to SRGS); DBT-NBACD (BT/HRD-NBA-NWB/38/2019-20 to SRGS) and IISc-DBT partnership program (to SRGS). Infrastructure in the department was supported by DST-FIST, DBT, and UGC. AMB was supported by DBT-JRF (DBT/2015/IISc/NJ-02).
....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 |
References
- Dell'Angelica, E. C. The building BLOC(k)s of lysosomes and related organelles. Current Opinion in Cell Biology. 16 (4), 458-464 (2004).
- Raposo, G., Marks, M. S. Melanosomes--dark organelles enlighten endosomal membrane transpo....
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