Name: nanogold labeling of the yeast endosomal system for ultrastructural analyses
Uploaded: 2014-07-14T15:00:00
Description: Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

The authors thank Ren&#233; Scriwanek for the assistance with the preparation of the Figure. F.R. is supported by ECHO (700.59.003), ALW Open Program (821.02.017 and 822.02.014), DFG-NWO cooperation (DN82-303) and ZonMW VICI (016.130.606) grants.

1. Spheroplast Preparation
<ol>
	<li>Incubate the yeast overnight at 30 &#176;C in 10 ml of the appropriate medium determined by the design of the experiment.</li>
	<li>The day after, measure the optical density of the culture at 600 nm (OD600) using a photometer, Dilute cells in the same culture medium to an OD600 of 0.2-0.4 and grow them to an exponential growth phase unless the design of the experiment requires a different condition. Cells are in the exponential phase when the culture has an OD...

<ol>
	<li>Gruenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endocytic+pathway:+a+mosaic+of+domains.">The endocytic pathway: a mosaic of domains.</a> Nat Rev Mol Cell Biol. 2, 721-730 (2001).</li><li>Sachse, M., Ramm, G., Strous, G., Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomes:+multipurpose+designs+for+integrating+housekeeping+and+specialized+tasks.">Endosomes: multipurpose designs for integrating housekeeping and specialized tasks.</a> Histochem Cell Biol. 117, 91-104 (2002).</li><li>Luzio, J. P., Pryor, P. R., Bright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomes:+fusion+and+function.">Lysosomes: fusion and function.</a> Nature Rev Mol Cell Biol. 8, 622-632 (2007).</li><li>Meel, E., Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+imagination:+understanding+the+endo-lysosomal+system.">Imaging and imagination: understanding the endo-lysosomal system.</a> Histochem Cell Biol. 129, 253-266 (2008).</li><li>Bonangelino, C. J., Chavez, E. M., Bonifacino, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+screen+for+vacuolar+protein+sorting+genes+in+Saccharomyces+cerevisiae.">Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae.</a> Mol Biol Cell. 13, 2486-2501 (2002).</li><li>Griffith, J., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+nanogold-labeled+endocytic+compartments+of+yeast+Saccharomyces+cerevisiae+using+a+cryosectioning+procedure.">Ultrastructural analysis of nanogold-labeled endocytic compartments of yeast Saccharomyces cerevisiae using a cryosectioning procedure.</a> J Histochem Cytochem. 57, 801-809 (2009).</li><li>Babst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESCRT-III:+an+endosome-associated+heterooligomeric+protein+complex+required+for+MVB+sorting.">ESCRT-III: an endosome-associated heterooligomeric protein complex required for MVB sorting.</a> Dev Cell. 3, 271-282 (2002).</li><li>Griffith, J., Mari, M., De Maziere, A., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryosectioning+procedure+for+the+ultrastructural+analysis+and+the+immunogold+labelling+of+yeast+Saccharomyces+cerevisiae.">A cryosectioning procedure for the ultrastructural analysis and the immunogold labelling of yeast Saccharomyces cerevisiae.</a> Traffic. 9, 1060-1072 (2008).</li><li>Lewis, M. J., Nichols, B. J., Prescianotto-Baschong, C., Riezman, H., Pelham, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+retrieval+of+the+exocytic+SNARE+Snc1p+from+early+yeast+endosomes.">Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes.</a> Mol Biol Cell. 11, 23-38 (2000).</li><li>Prescianotto-Baschong, C., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordering+of+compartments+in+the+yeast+endocytic+pathway.">Ordering of compartments in the yeast endocytic pathway.</a> Traffic. 3, 37-49 (2002).</li><li>Odorizzi, G., Babst, M., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fab1p+PtdIns(3)P+5-kinase+function+essential+for+protein+sorting+in+the+multivesicular+body.">Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body.</a> Cell. 95 (3), 847-858 (1998).</li><li>Donselaar, E., Posthuma, G., Zeuschner, D., Humbel, B. M., Slot, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogold+labeling+of+cryosections+from+high-pressure+frozen+cells.">Immunogold labeling of cryosections from high-pressure frozen cells.</a> Traffic. 8, 471-485 (2007).</li><li>Lah, J. J., Hayes, D. M., Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neutral+pH+silver+development+method+for+the+visualization+of+1-nanometer+gold+particles+in+pre-embedding+electron+microscopic+immunocytochemistry.">A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry.</a> J Histochem Cytochem. 38, 503-508 (1990).</li><li>Shah, N., Zhang, S., Harada, S., Smith, R. M., Jarett, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+visualization+of+insulin+translocation+into+the+cytoplasm+and+nuclei+of+intact+H35+hepatoma+cells+using+covalently+linked+Nanogold-insulin.">Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin.</a> Endocrinol. 136, 2825-2835 (1995).</li><li>Weipoltshammer, K., Schofer, C., Almeder, M., Wachtler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+enhancement+at+the+electron+microscopic+level+using+Nanogold+and+gold-based+autometallography.">Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography.</a> Histochem Cell Biol. 114, 489-495 (2000).</li><li>Tokuyasu, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+ultracryotomy+of+cell+suspensions+and+tissues.">A technique for ultracryotomy of cell suspensions and tissues.</a> J Cell Biol. 57, 551-565 (1973).</li><li>Cabrera, 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=Phosphorylation+of+a+membrane+curvature-sensing+motif+switches+function+of+the+HOPS+subunit+Vps41+in+membrane+tethering.">Phosphorylation of a membrane curvature-sensing motif switches function of the HOPS subunit Vps41 in membrane tethering.</a> J Cell Biol. 191, 845-859 (2010).</li><li>Harner, 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=The+mitochondrial+contact+site+complex,+a+determinant+of+mitochondrial+architecture.">The mitochondrial contact site complex, a determinant of mitochondrial architecture.</a> EMBO J. 30, 4356-4370 (2011).</li><li>Nair, U., 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=SNARE+proteins+are+required+for+macroautophagy.">SNARE proteins are required for macroautophagy.</a> Cell. 146, 290-302 (2011).</li><li>Jacquier, N., 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=Lipid+droplets+are+functionally+connected+to+the+endoplasmic+reticulum+in+Saccharomyces+cerevisiae.">Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae.</a> J Cell Sci. 124, 2424-2437 (2011).</li><li>Karanasios, E., 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=Regulation+of+lipid+droplet+and+membrane+biogenesis+by+the+acidic+tail+of+the+phosphatidate+phosphatase+Pah1p.">Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p.</a> Mol Biol Cell. 24, 2124-2133 (2013).</li><li>Mari, 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=An+Atg9-containing+compartment+that+functions+in+the+early+steps+of+autophagosome+biogenesis.">An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis.</a> J Cell Biol. 190, 1005-1022 (2010).</li><li>Nair, U., 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+role+for+Atg8-PE+deconjugation+in+autophagosome+biogenesis.">A role for Atg8-PE deconjugation in autophagosome biogenesis.</a> Autophagy. 8, 780-793 (2012).</li><li>Vaart, A., Griffith, J., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exit+from+the+Golgi+is+required+for+the+expansion+of+the+autophagosomal+phagophore+in+yeast+Saccharomyces+cerevisiae.">Exit from the Golgi is required for the expansion of the autophagosomal phagophore in yeast Saccharomyces cerevisiae.</a> Mol Biol Cell. 21, 2270-2284 (2010).</li><li>Erpapazoglou, Z., 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+dual+role+for+K63-linked+ubiquitin+chains+in+multivesicular+body+biogenesis+and+cargo+sorting.">A dual role for K63-linked ubiquitin chains in multivesicular body biogenesis and cargo sorting.</a> Mol Biol Cell. 23, 2170-2183 (2012).</li><li>Tuinen, E., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+glyceraldehyde-3-phosphate+dehydrogenase,+hexokinase,+and+carboxypeptidase+Y+in+yeast+cells+at+the+ultrastructural+level.">Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase, hexokinase, and carboxypeptidase Y in yeast cells at the ultrastructural level.</a> J Histochem Cytochem. 35, 327-333 (1987).</li><li>Slot, J. W., Geuze, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+and+immunolabeling.">Cryosectioning and immunolabeling.</a> Nat Protoc. 2, 2480-2491 (2007).</li><li>Prescianotto-Baschong, C., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+the+yeast+endocytic+pathway.">Morphology of the yeast endocytic pathway.</a> Mol Biol Cell. 9, 173-189 (1998).</li><li>Vida, T. A., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+vital+stain+for+visualizing+vacuolar+membrane+dynamics+and+endocytosis+in+yeast.">A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast.</a> J Cell Biol. 128, 779-792 (1995).</li><li>Hainfeld, J. F., Powell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+frontiers+in+gold+labeling.">New frontiers in gold labeling.</a> J Histochem Cytochem. 48, 471-480 (2000).</li></ol>

Immuno-electron-microscopy is a technique that allows combining localization of proteins with the ultrastructural resolution of the carriers and organelles where these proteins reside. This is particularly crucial when studying the yeast endosomal system because its compartments appear as punctuate structures in fluorescence microscopy. It is therefore difficult to distinguish them. For this reason the use of a probe detectable by EM and entering the endocytic route in a time dependent manner is crucial to specifically m...

The endosomal system is a major membrane sorting apparatus that plays multiple crucial cellular roles including trafficking of lysosomal enzyme sorting receptors and the recycling of plasma membrane (PM) receptors1,2. Endosomes are divided in three different compartments, i.e. the early endosomes (EE), the late endosomes (LE) and the recycling endosomes. This classification is based on the time it takes for endocytosed material to reach them, on specific marker proteins and on their morphology. Membranes, i.e. protein and lipid bilayers, internalized from the PM can either be delivered to lysosomes via endosomes for degradation or be recycled back. Membranes are also transported to endosomes from the Golgi and similarly, either continue to lysosomes or be retrieved back to the Golgi. Furthermore, proteins can be sorted into luminal vesicles budding inward from the endosomal limiting membrane, a process that leads to the formation of a subcategory of LE, the multivesicular bodies.
The yeast endosomal system is relatively less complex than the one of high eukaryotic cells. Yeast endosomes are divided into EE and LE. In contrast to mammalian cells they do not contain recycling endosomes but also tissue-specific lysosome-related organelles. Consequently they have a less complex network of endosomal trafficking routes3,4. Therefore yeast has represented and still represents an advantageous experimental system to study some of the principles underlying membrane traffic in the endosomal system. This advantage is emphasized by the fact that numerous genes involved in the endosomal pathways have been initially isolated with genetic screens in yeast5. While the yeast endosomal system in wild type and mutant cells has been extensively studied using biochemical and fluorescence microscopy approaches, its investigation at the ultrastructural level has only been minimal. Morphological analyses are particularly relevant in yeast because most of the endosomal organelles are detected as punctuate structures by fluorescence microscopy, which make difficult their unequivocal identification6. Unfortunately only a limited number of antisera recognizing yeast endosomal protein markers is working in immuno-electron-microscopy (IEM) preparations7-10. For some proteins, this problem has been circumvented by the endogenous tagging of the gene of interest and the use of an antibody recognizing the tag to detect it7,11,12. Often, however, proteins are undetectable by IEM because of their low expression levels. Their overexpression is not a solution because this approach can induce mis-localizations and/or alterations in the organelle morphology/functions. Thus the labelling of the endocytic compartments with a probe detectable by electron microscopy EM is an effective option. This is an optimal solution especially if the probe is entering the endocytic route in a time-dependent manner, which allows to know when it will mark a specific organelle6.
The uptake of positively charged nanogold by yeast spheroplasts (i.e. yeast where the cell wall has been enzymatically removed) has successfully been used to identify the yeast endosomal compartments10. These particles strongly bind to the negatively charged lipids composing the biological membranes. Thus the positively charged nanogold associates with the PM, penetrates the cell by endocytosis and passes through the EE and LE before reaching the vacuole. These small gold particles, however, do not have an appropriate size to be seen by EM. To render them visible, their size can be enlarged by chemical reactions that lead to the deposition of silver or gold around the gold probe13-15. We have developed and successfully applied an IEM approach based on the Tokuyasu method to perform subcellular localization studies8,16. This method allows performing immunogold labelling on yeast preparations with an excellent resolution of the morphology8,17-24. We have also established a procedure combining this IEM protocol with the nanogold labelling of the yeast endosomal system compartments6. Using this approach we have morphologically characterized different subclasses of endosomes and ultrastructurally examined mutants with an endosomal trafficking defect6,25. Moreover, we have demonstrated that this nanogold labelling can be combined with immunogold labelings providing the possibility to explore the distribution of a protein of interest on the different endosome subpopulations. Here we present how the labelling of the yeast endosomal system with positively charged nanogold is practically performed.

<table border="0" cellpadding="0" cellspacing="0" width="754">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">PIPES (piperazin-1,4-bis (2-ethansulfensaure)</td>
			<td style="width:188px;">Merck, Darmstadt, Germany</td>
			<td style="width:119px;">1,102,200,250</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">HEPES</td>
			<td style="width:188px;">Merck, Darmstadt, Germany</td>
			<td style="width:119px;">391,340</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">EGTA</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">E4378</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:280px;">MgCl2.6H2O</td>
			<td style="width:188px;">Merck, Darmstadt, Germany</td>
			<td style="width:119px;">1,058,330,250</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">DL-Dithiothreitol</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">D0632</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sorbitol</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">S1876</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Positively charged Nanogold</td>
			<td style="width:188px;">Nanoprobes, Yaphank, NY</td>
			<td style="width:119px;">2022</td>
			<td style="width:167px;">store at -20 &#778;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">HQ-silver&#8482; enhancement kit</td>
			<td style="width:188px;">Nanoprobes, Yaphank, NY</td>
			<td style="width:119px;">2012</td>
			<td style="width:167px;">store at -20 &#778;C</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:280px;">Paraformaldehyde (PFA)</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">441244</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:280px;">Glutaraldehyde 8% EM grade</td>
			<td style="width:188px;">Polyscience, Inc. , Warrington, PA</td>
			<td style="width:119px;">216</td>
			<td style="width:167px;">store at 4 &#778;C</td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:280px;">Lytic enzyme</td>
			<td style="width:188px;">MP Biomedicals, Santa Ana, CA</td>
			<td style="width:119px;">153526</td>
			<td style="width:167px;">store at 4 &#778;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:280px;">Parafilm M</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">P7793</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">50 meshes nickel grids&#160;</td>
			<td style="width:188px;">Agar Scientific, Stansted, United Kingdom</td>
			<td style="width:119px;">G209N</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;">Cryo-immuno diamond knife 35 &#186;</td>
			<td style="width:188px;">Diatome AG, Biel, Switzerland</td>
			<td style="width:119px;">N.A.</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">UCT ultramicrotome&#160;</td>
			<td style="width:188px;">Leica, Solms, Germany</td>
			<td style="width:119px;">N.A.</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;">Formvar 15/95 resine</td>
			<td style="width:188px;">Electron Microscopy Sciences, Hatfield, PA</td>
			<td style="width:119px;">15800</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">50 meshes nickel grids&#160;</td>
			<td style="width:188px;">Agar Scientific, Stansted, United Kingdom</td>
			<td style="width:119px;">AGG2050N</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Parafilm</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">P7793</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Grids storage box</td>
			<td style="width:188px;">Leica, Solms, Germany</td>
			<td style="width:119px;">162-50</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Falcon 100mmx15mm petri dishe&#160;</td>
			<td style="width:188px;">Corning, Corning, NY</td>
			<td style="width:119px;">351029</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pasteur capillary pipettes 150 mm</td>
			<td style="width:188px;">Van Bruggen Glas, Rotterdam, The Netherlands</td>
			<td style="width:119px;">10216234</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">1.5 ml microcentrifuge&#160;</td>
			<td style="width:188px;">Sarstedt, N&#252;mbrecht, Germany</td>
			<td style="width:119px;">72690001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">50 ml tube</td>
			<td style="width:188px;">Bio-one, Alphen aan den Rijn, The Netherlands</td>
			<td style="width:119px;">210296</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">Benchkote surface protector</td>
			<td style="width:188px;">Whatman, Maidstone, United Kingdom&#160;</td>
			<td style="width:119px;">1159201</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

nanogold labeling of the yeast endosomal system for ultrastructural analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

According to the presented protocol, the morphology of the yeast endosome system can be access by transmission EM. Figure 1 shows different types of endosomal compartments that have been reached and therefore labeled with nanogold. The silver-enhanced nanogold can be clearly seen as electron dense particles. The optimal settings established for the silver enhancement reaction allows having gold particles in a homogenous size range between 5 and 15 nm, which does not interfere with the ultrastructure of t...

Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Research

JoVE Journal

Biology

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Fluorescent Labeling of Drosophila Heart Structures

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...