Name: The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking (Video) | JoVE Journal
Uploaded: 2022-02-12T15:00:00.000+00:00
Duration: 8 min 51 s
Description: Learn microscopy-based analysis of recycling endosome dynamics using GFP-STX13 in mouse melanocytes. This method reveals SNARE trafficking mechanisms underlying melanosome biogenesis and pigmentation.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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).

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
<ol>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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 &#176;C for 2 - 5 min for the detachment from the Petri dish.</li>
		<li>Add 1 - 2 mL of complete RPMI medium, suspend and then transfer the cells to a centrifuge tube.</li>
	</ol>
	</li>
	<li>Centrifuge the cell suspension at 4 &#176;C, 376 x g for 5 min and then resuspend the pellet in 1x PBS.</li>
	<li>Repeat the centrifugation step and resuspend the cells in 1 mL of complete RPMI medium.</li>
	<li>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 &#181;L of working stock 40 &#181;M phorbol 12-myristate 13-acetate) to the plated cell suspension in complete RPMI medium.</li>
	<li>After seeding, incubate the plate at 37 &#176;C for 12- 24 h.</li>
</ol>
2. Transfection of cells with the STX13 plasmids
<ol>
	<li>Use the following reagents: pEGFP-C1-STX13WT and pEGFP-C1-STX13&#916;129 (described in12). mCherry-Rab11, was a kind gift from Gra&#231;a Raposo, Institut Curie, Paris (described in reference19). Anti-TYRP1 antibody from ATCC (TA99).</li>
	<li>After 12 - 24 h of seeding, transfect the cells with plasmids using a lipid-based transfection reagent. For a 35 mm dish, take 5 &#181;L of the transfection reagent in 250 &#181;L of OPTI-MEM medium in a microcentrifuge tube and take approximately 200 ng of each plasmid in 250 &#181;L of OPTI-MEM.</li>
	<li>Incubate the tubes containing DNA and the transfection reagent for 5 min. Mix without repeated pipetting (total volume will be about 500 &#181;L). Incubate for 30 min at room temperature. Perform hand tapping of the tube every 10 min for 30 min at RT.</li>
	<li>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.</li>
	<li>Post 30 min of incubation, add the transfection reagent-DNA mix to the cells in a dropwise manner by covering the dish.</li>
	<li>Incubate the cells at 37 &#176;C for 6 h. Aspirate the OPTI-MEM medium with transfection reagent and add complete RPMI medium supplemented with 200 nM of PMA.</li>
	<li>Incubate the cells at 37 &#176;C for 48 h.</li>
</ol>
3. Fixation of the cells
NOTE: The following procedure is performed outside the tissue culture hood.
<ol>
	<li>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.</li>
	<li>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 &#176;C.</li>
</ol>
4. Immunostaining of the cells
<ol>
	<li>Prepare a humid chamber: place paraffin film cut piece on moist filter paper in a Petri dish, covered with aluminum foil.</li>
	<li>Prepare 25 &#181;L 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Using forceps, carefully pick up the coverslip from the primary antibody solution and dip it thrice in 1x PBS (in a glass beaker).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place 12 &#181;L 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.</li>
</ol>
5. Fluorescence microscopy of the cells
<ol>
	<li>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.</li>
</ol>
6. Quantification of overlap between the RE localized proteins and melanosomes:
NOTE: The following steps are followed for the quantification of Mander&#39;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.
<ol>
	<li>Open the raw image. Go to the Image option, select Color | Split channels, and use the two channels for analysis.</li>
	<li>Open the JACoP plugin in the Plugin option.</li>
	<li>Set the threshold for both the channels such that all the bright spots are selected and the background is eliminated.</li>
	<li>Go to Analysis option, select M1 and M2 coefficients for getting Mander&#39;s overlap coefficient.</li>
	<li>Press the Analyze option in the JACoP Plugin and see the result that displays Mander&#39;s overlap coefficient.</li>
</ol>
7. Quantification of recycling endosomes&#39; tubular number and length:
NOTE: The following steps are followed for the quantification of the number and length of the tubules using Fiji software.
<ol>
	<li>Open the raw image Go to Image option, select Color | Split channels and use the desired channel for analysis.</li>
	<li>Go to Image again, select Type | Convert to 8-bit image.</li>
	<li>Then go to Plugins, select Analyze | Tubeness. Set sigma value at 0.1075 . Press Ok.</li>
	<li>Go to Image again, select Type | Convert to 8 bit image.</li>
	<li>Go to Image | Adjust | Threshold (Use the same threshold values for all the images. Images should have approximately equal intensity).</li>
	<li>Go to Process | Binary | Convert to mask.</li>
	<li>Go to Process, select Binary and then select Skeletonize.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Save the data and analyze.</li>
</ol>

<ol><li>Dell'Angelica, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+building+BLOC%28k%29s+of+lysosomes+and+related+organelles%5BTitle%5D)+AND+%22Current+Opinion+in+Cell+Biology%22%5BJournal%5D)">The building BLOC(k)s of lysosomes and related organelles.</a> Current Opinion in Cell Biology. 16 (4), 458-464 (2004).</li>
<li>Raposo, G., Marks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Melanosomes--dark+organelles+enlighten+endosomal+membrane+transport%5BTitle%5D)+AND+%22Nature+Reviews+in+Molecular+Cell+Biology%22%5BJournal%5D)">Melanosomes--dark organelles enlighten endosomal membrane transport.</a> Nature Reviews in Molecular Cell Biology. 8 (10), 786-797 (2007).</li>
<li>Ohbayashi, N., Fukuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+understanding+the+molecular+basis+of+melanogenesis+in+melanocytes%5BTitle%5D)+AND+%22F1000Research%22%5BJournal%5D)">Recent advances in understanding the molecular basis of melanogenesis in melanocytes.</a> F1000Research. 9, F1000 Faculty Rev-608 (2020).</li>
<li>Theos, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functions+of+adaptor+protein+%28AP%29-3+and+AP-1+in+tyrosinase+sorting+from+endosomes+to+melanosomes%5BTitle%5D)+AND+%22Molecular+Biology+of+the+Cell%22%5BJournal%5D)">Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes.</a> Molecular Biology of the Cell. 16 (11), 5356-5372 (2005).</li>
<li>Di Pietro, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BLOC-1+interacts+with+BLOC-2+and+the+AP-3+complex+to+facilitate+protein+trafficking+on+endosomes%5BTitle%5D)+AND+%22Molecular+Biology+of+the+Cell%22%5BJournal%5D)">BLOC-1 interacts with BLOC-2 and the AP-3 complex to facilitate protein trafficking on endosomes.</a> Molecular Biology of the Cell. 17 (9), 4027-4038 (2006).</li>
<li>Setty, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BLOC-1+is+required+for+cargo-specific+sorting+from+vacuolar+early+endosomes+toward+lysosome-related+organelles%5BTitle%5D)+AND+%22Molecular+Biology+of+the+Cell%22%5BJournal%5D)">BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles.</a> Molecular Biology of the Cell. 18 (3), 768-780 (2007).</li>
<li>Delevoye, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AP-1+and+KIF13A+coordinate+endosomal+sorting+and+positioning+during+melanosome+biogenesis%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.</a> Journal of Cell Biology. 187 (2), 247-264 (2009).</li>
<li>Bultema, J. J., Ambrosio, A. L., Burek, C. L., Di Pietro, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BLOC-2%2C+AP-3%2C+and+AP-1+proteins+function+in+concert+with+Rab38+and+Rab32+proteins+to+mediate+protein+trafficking+to+lysosome-related+organelles%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles.</a> Journal of Biological Chemistry. 287 (23), 19550-19563 (2012).</li>
<li>Sitaram, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+recognition+of+a+dileucine-based+sorting+signal+by+AP-1+and+AP-3+reveals+a+requirement+for+both+BLOC-1+and+AP-3+in+delivery+of+OCA2+to+melanosomes%5BTitle%5D)+AND+%22Molecular+Biology+of+the+Cell%22%5BJournal%5D)">Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes.</a> Molecular Biology of the Cell. 23 (16), 3178-3192 (2012).</li>
<li>Nag, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rab4A+organizes+endosomal+domains+for+sorting+cargo+to+lysosome-related+organelles%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">Rab4A organizes endosomal domains for sorting cargo to lysosome-related organelles.</a> Journal of Cell Science. 131 (18), (2018).</li>
<li>Dennis, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BLOC-2+targets+recycling+endosomal+tubules+to+melanosomes+for+cargo+delivery%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery.</a> Journal of Cell Biology. 209 (4), 563-577 (2015).</li>
<li>Jani, R. A., Purushothaman, L. K., Rani, S., Bergam, P., Setty, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STX13+regulates+cargo+delivery+from+recycling+endosomes+during+melanosome+biogenesis%5BTitle%5D)+AND+%22Journal+Cell+Science%22%5BJournal%5D)">STX13 regulates cargo delivery from recycling endosomes during melanosome biogenesis.</a> Journal Cell Science. 128 (17), 3263-3276 (2015).</li>
<li>Shakya, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rab22A+recruits+BLOC-1+and+BLOC-2+to+promote+the+biogenesis+of+recycling+endosomes%5BTitle%5D)+AND+%22EMBO+Reports%22%5BJournal%5D)">Rab22A recruits BLOC-1 and BLOC-2 to promote the biogenesis of recycling endosomes.</a> EMBO Reports. 19 (12), 45918(2018).</li>
<li>Bowman, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+BLOC-1-AP-3+super-complex+sorts+a+cis-SNARE+complex+into+endosome-derived+tubular+transport+carriers%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">A BLOC-1-AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers.</a> Journal of Cell Biology. 220 (7), 202005173(2021).</li>
<li>Wei, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hermansky-Pudlak+syndrome%3A+a+disease+of+protein+trafficking+and+organelle+function%5BTitle%5D)+AND+%22Pigment+Cell+Research%22%5BJournal%5D)">Hermansky-Pudlak syndrome: a disease of protein trafficking and organelle function.</a> Pigment Cell Research. 19 (1), 19-42 (2006).</li>
<li>Bowman, S. L., Bi-Karchin, J., Le, L., Marks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+road+to+lysosome-related+organelles%3A+Insights+from+Hermansky-Pudlak+syndrome+and+other+rare+diseases%5BTitle%5D)+AND+%22Traffic%22%5BJournal%5D)">The road to lysosome-related organelles: Insights from Hermansky-Pudlak syndrome and other rare diseases.</a> Traffic. 20 (6), 404-435 (2019).</li>
<li>Prekeris, R., Klumperman, J., Chen, Y. A., Scheller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Syntaxin+13+mediates+cycling+of+plasma+membrane+proteins+via+tubulovesicular+recycling+endosomes%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes.</a> Journal of Cell Biology. 143 (4), 957-971 (1998).</li>
<li>Mahanty, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rab9A+is+required+for+delivery+of+cargo+from+recycling+endosomes+to+melanosomes%5BTitle%5D)+AND+%22Pigment+Cell+Melanoma+Research%22%5BJournal%5D)">Rab9A is required for delivery of cargo from recycling endosomes to melanosomes.</a> Pigment Cell Melanoma Research. 29 (1), 43-59 (2016).</li>
<li>Delevoye, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recycling+endosome+tubule+morphogenesis+from+sorting+endosomes+requires+the+kinesin+motor+KIF13A%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Recycling endosome tubule morphogenesis from sorting endosomes requires the kinesin motor KIF13A.</a> Cell Reports. 6 (3), 445-454 (2014).</li>
<li>Ha, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ARF+functions+as+a+melanoma+tumor+suppressor+by+inducing+p53-independent+senescence%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Science+U.+S.+A%22%5BJournal%5D)">ARF functions as a melanoma tumor suppressor by inducing p53-independent senescence.</a> Proceedings of the National Academy of Science U. S. A. 104 (26), 10968-10973 (2007).</li>
<li>Soldati, T., Schliwa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Powering+membrane+traffic+in+endocytosis+and+recycling%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Powering membrane traffic in endocytosis and recycling.</a> Nature Reviews Molecular Cell Biology. 7 (12), 897-908 (2006).</li>
<li>Grant, B. D., Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathways+and+mechanisms+of+endocytic+recycling%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Pathways and mechanisms of endocytic recycling.</a> Nature Reviews Molecular Cell Biology. 10 (9), 597-608 (2009).</li>
<li>Hsu, V. W., Prekeris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transport+at+the+recycling+endosome%5BTitle%5D)+AND+%22Current+Opinion+in+Cell+Biology%22%5BJournal%5D)">Transport at the recycling endosome.</a> Current Opinion in Cell Biology. 22 (4), 528-534 (2010).</li>
<li>Taguchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emerging+roles+of+recycling+endosomes%5BTitle%5D)+AND+%22Journal+of+Biochemistry%22%5BJournal%5D)">Emerging roles of recycling endosomes.</a> Journal of Biochemistry. 153 (6), 505-510 (2013).</li>
<li>Goldenring, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recycling+endosomes%5BTitle%5D)+AND+%22Current+Opinion+in+Cell+Biology%22%5BJournal%5D)">Recycling endosomes.</a> Current Opinion in Cell Biology. 35, 117-122 (2015).</li>
<li>Delevoye, C., Marks, M. S., Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysosome-related+organelles+as+functional+adaptations+of+the+endolysosomal+system%5BTitle%5D)+AND+%22Current+Opinion+in+Cell+Biology%22%5BJournal%5D)">Lysosome-related organelles as functional adaptations of the endolysosomal system.</a> Current Opinion in Cell Biology. 59, 147-158 (2019).</li>
<li>Hsu, V. W., Bai, M., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Getting+active%3A+protein+sorting+in+endocytic+recycling%5BTitle%5D)+AND+%22Nature+Reviews+in+Molecular+Cell+Biology%22%5BJournal%5D)">Getting active: protein sorting in endocytic recycling.</a> Nature Reviews in Molecular Cell Biology. 13 (5), 323-328 (2012).</li>
<li>Desfougeres, Y., D'Agostino, M., Mayer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modular+tethering+complex+for+endosomal+recycling%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">A modular tethering complex for endosomal recycling.</a> Nature Cell Biology. 17 (5), 540-541 (2015).</li>
<li>Le, L., Sires-Campos, J., Raposo, G., Delevoye, C., Marks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Melanosome+biogenesis+in+the+pigmentation+of+mammalian+skin%5BTitle%5D)+AND+%22Integrated+Computational+Biology%22%5BJournal%5D)">Melanosome biogenesis in the pigmentation of mammalian skin.</a> Integrated Computational Biology. 61 (4), 1517-1545 (2021).</li>
</ol>

The authors declare that they have no conflict of interest.

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<s...

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)4,5,6,7,8,9,10. The targeting and fusion of these organelles regulate the maturation of fully functional pigmented melanosomes7,11,12,13,14. Defects in the formation of these organelles or cargo sorting to these organelles cause oculocutaneous albinism and other clinical phenotypes, observed in Hermansky-Pudlak syndrome15,16.
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 melanocytes12,18. Further, deletion of N-terminal unstructured regulatory domain (namely SynN or STX13&#916;129) 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 studies14,19. Fluorescence imaging of the proteins GFP-STX13WT, GFP-STX13&#916;129, 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.

<table><tbody><tr><td>anti-TYRP1 antibody (TA99)</td><td>ATCC</td><td>HB-8704</td><td></td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>ThermoFisher Scientific</td><td>11668-500</td><td></td></tr><tr><td>Matrigel matrix</td><td>BD Biosciences</td><td>356231</td><td></td></tr><tr><td>OPTI-MEM</td><td>ThermoFisher Scientific</td><td>022600-050</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate</td><td>Sigma-Aldrich</td><td>P8139</td><td></td></tr><tr><td>RPMI Medium 1640</td><td>ThermoFisher Scientific</td><td>31800-022</td><td></td></tr></tbody></table>

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

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.

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 melanosome protein T...

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.

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

the-microscopy-based-assay-to-study-analyze-recycling-endosomes-using

Research

JoVE Journal

Biology

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase.

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on Förster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

A High-content Assay for Monitoring AMPA Receptor Trafficking

Endocytic and retrograde transport of proteins from the plasma membrane to the trans-Golgi network is essential to maintaining membrane homeostasis and regulating signaling. Here, we describe a method to image and quantify endocytic transport of transmembrane cargo proteins by live-cell microscopy using derivatized anti-GFP nanobodies in HeLa cells.

Live-cell Imaging of Endocytic Transport using Functionalized Nanobodies in Cultured Cells

Learn microscopy-based analysis of recycling endosome dynamics using GFP-STX13 in mouse melanocytes. This method reveals SNARE trafficking mechanisms underlying melanosome biogenesis and pigmentation.

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

Explore More Videos