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.

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

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 the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

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 particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

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 visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

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 labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

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 (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

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

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

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 function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

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

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

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 for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

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.

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

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

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 RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

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

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.