Name: the microscopy-based assay to study and analyze the recycling endosomes using snare trafficking
Uploaded: 2022-02-12T15:00:00
Description: Watch this Scientific Journal Video about The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking at JoVE.com

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:...

<ol>
	<li>Dell'Angelica, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+building+BLOC(k)s+of+lysosomes+and+related+organelles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanosomes--dark+organelles+enlighten+endosomal+membrane+transport.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+understanding+the+molecular+basis+of+melanogenesis+in+melanocytes.">Recent advances in understanding the molecular basis of melanogenesis in melanocytes.</a> F1000Research. 9, (2020).</li><li>Theos, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+adaptor+protein+(AP)-3+and+AP-1+in+tyrosinase+sorting+from+endosomes+to+melanosomes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLOC-1+interacts+with+BLOC-2+and+the+AP-3+complex+to+facilitate+protein+trafficking+on+endosomes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLOC-1+is+required+for+cargo-specific+sorting+from+vacuolar+early+endosomes+toward+lysosome-related+organelles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AP-1+and+KIF13A+coordinate+endosomal+sorting+and+positioning+during+melanosome+biogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLOC-2,+AP-3,+and+AP-1+proteins+function+in+concert+with+Rab38+and+Rab32+proteins+to+mediate+protein+trafficking+to+lysosome-related+organelles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab4A+organizes+endosomal+domains+for+sorting+cargo+to+lysosome-related+organelles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLOC-2+targets+recycling+endosomal+tubules+to+melanosomes+for+cargo+delivery.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STX13+regulates+cargo+delivery+from+recycling+endosomes+during+melanosome+biogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab22A+recruits+BLOC-1+and+BLOC-2+to+promote+the+biogenesis+of+recycling+endosomes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+BLOC-1-AP-3+super-complex+sorts+a+cis-SNARE+complex+into+endosome-derived+tubular+transport+carriers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hermansky-Pudlak+syndrome:+a+disease+of+protein+trafficking+and+organelle+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+road+to+lysosome-related+organelles:+Insights+from+Hermansky-Pudlak+syndrome+and+other+rare+diseases.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syntaxin+13+mediates+cycling+of+plasma+membrane+proteins+via+tubulovesicular+recycling+endosomes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab9A+is+required+for+delivery+of+cargo+from+recycling+endosomes+to+melanosomes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recycling+endosome+tubule+morphogenesis+from+sorting+endosomes+requires+the+kinesin+motor+KIF13A.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARF+functions+as+a+melanoma+tumor+suppressor+by+inducing+p53-independent+senescence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Powering+membrane+traffic+in+endocytosis+and+recycling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+and+mechanisms+of+endocytic+recycling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+at+the+recycling+endosome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+recycling+endosomes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recycling+endosomes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosome-related+organelles+as+functional+adaptations+of+the+endolysosomal+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+active:+protein+sorting+in+endocytic+recycling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+tethering+complex+for+endosomal+recycling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanosome+biogenesis+in+the+pigmentation+of+mammalian+skin.">Melanosome biogenesis in the pigmentation of mammalian skin.</a> Integrated Computational Biology. 61 (4), 1517-1545 (2021).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

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 STX13&#916;129 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-STX13&#916;129 localized as ring-like structures in addition to the cell surface (Figure 1A). Further, intracellular ring-like GFP-STX13&#916;129 showed colocalization with the mel...

Watch this Scientific Journal Video about The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking at JoVE.com

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

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 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.

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...