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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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

  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:.......

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

NameCompanyCatalog NumberComments
anti-TYRP1 antibody (TA99)ATCCHB-8704
Fluoromount-GSouthern Biotech0100-01
Lipofectamine 2000ThermoFisher Scientific11668-500
Matrigel matrixBD Biosciences356231
OPTI-MEMThermoFisher Scientific022600-050
Phorbol-12-myristate-13-acetateSigma-AldrichP8139
RPMI Medium 1640ThermoFisher Scientific31800-022

References

  1. Dell'Angelica, E. C. The building BLOC(k)s of lysosomes and related organelles. Current Opinion in Cell Biology. 16 (4), 458-464 (2004).
  2. Raposo, G., Marks, M. S. Melanosomes--dark organelles enlighten endosomal membrane transpo....

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