Name: single-cell quantification of protein degradation rates by time-lapse fluorescence microscopy in adherent cell culture
Uploaded: 2018-02-04T15:00:00
Description: Watch this Scientific Journal Video about Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture at JoVE.com

Time-lapse microscopy experiments were performed at the Biomolecular Screening Facility (BSF), EPFL. We thank Marc Delachaux (Service Audiovisuel, EPFL) for the videography and editing of the movie.

Note: In this study, the E14 ES cell line was used. However, this protocol is directly applicable to any other mouse ES cell line expressing a protein of interest fused to a SNAP-tag, either by tagging the endogenous protein or by using overexpression. For the examples shown in the results section, doxycycline-inducible SNAP-tag fusion cell lines were used (SNAP-tag fused to the following proteins: Nanog, Oct4, Srsf11, or to the fluorescent proteins mOrange2 and sfGFP, and put under the control of a doxycycline-inducible...

<ol>
	<li>Zhou, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+Protein+Half-Lives.">Determining Protein Half-Lives.</a> Signal Transduct Protoc. , 67-77 (2004).</li><li>Schwanh&#228;usser, B., 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=Global+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473 (7347), 337-342 (2011).</li><li>Plachta, N., Bollenbach, T., Pease, S., Fraser, S. E., Pantazis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oct4+kinetics+predict+cell+lineage+patterning+in+the+early+mammalian+embryo.">Oct4 kinetics predict cell lineage patterning in the early mammalian embryo.</a> Nature Cell Biol. 13 (2), 117-123 (2011).</li><li>Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H., Johnsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+method+for+the+covalent+labeling+of+fusion+proteins+with+small+molecules+in+vivo.">A general method for the covalent labeling of fusion proteins with small molecules in vivo.</a> Nature Biotechnol. 21 (1), 86-89 (2002).</li><li>Keppler, A., Pick, H., Arrivoli, C., Vogel, H., Johnsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+of+fusion+proteins+with+synthetic+fluorophores+in+live+cells.">Labeling of fusion proteins with synthetic fluorophores in live cells.</a> Proc Nat Acad Sci U S A. 101 (27), 9955-9959 (2004).</li><li>Gronemeyer, T., Chidley, C., Juillerat, A., Heinis, C., Johnsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+O6-alkylguanine-DNA+alkyltransferase+for+applications+in+protein+labeling.">Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling.</a> Protein Eng Design Select. 19 (7), 309-316 (2006).</li><li>Jansen, L. E. T., Black, B. E., Foltz, D. R., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+centromeric+chromatin+requires+exit+from+mitosis.">Propagation of centromeric chromatin requires exit from mitosis.</a> J Cell Biol. 176 (6), 795-805 (2007).</li><li>Bojkowska, 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=Measuring+In+Vivo+Protein+Half-Life.">Measuring In Vivo Protein Half-Life.</a> Chem Biol. 18 (6), 805-815 (2011).</li><li>Mandic, A., Strebinger, D., Regali, C., Phillips, N. E., Suter, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+quantitative+measurements+of+gene+expression+in+single+living+cells.">A novel method for quantitative measurements of gene expression in single living cells.</a> Methods. 120, 65-75 (2017).</li><li>Komatsu, T., 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=Real-Time+Measurements+of+Protein+Dynamics+Using+Fluorescence+Activation-Coupled+Protein+Labeling+Method.">Real-Time Measurements of Protein Dynamics Using Fluorescence Activation-Coupled Protein Labeling Method.</a> J Am Chem Soc. 133 (17), 6745 (2011).</li><li>Deluz, 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=A+role+for+mitotic+bookmarking+of+SOX2+in+pluripotency+and+differentiation.">A role for mitotic bookmarking of SOX2 in pluripotency and differentiation.</a> Genes Dev. 30 (22), 2538-2550 (2016).</li><li>Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-end+rule+pathway+and+regulation+by+proteolysis.">The N-end rule pathway and regulation by proteolysis.</a> Protein Sci. 20 (8), 1298-1345 (2011).</li><li>Tamm, C., Pijuan Galit&#243;, S., Anner&#233;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparative+Study+of+Protocols+for+Mouse+Embryonic+Stem+Cell+Culturing.">A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing.</a> PLoS ONE. 8 (12), e81156 (2013).</li><li>Nagaoka, 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=E-Cadherin-Coated+Plates+Maintain+Pluripotent+ES+Cells+without+Colony+Formation.">E-Cadherin-Coated Plates Maintain Pluripotent ES Cells without Colony Formation.</a> PLoS ONE. 1 (1), e15 (2006).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Meth. 9 (7), 676-682 (2012).</li><li>Hilsenbeck, O., 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=Software+tools+for+single-cell+tracking+and+quantification+of+cellular+and+molecular+properties.">Software tools for single-cell tracking and quantification of cellular and molecular properties.</a> Nat Biotech. 34 (7), 703-706 (2016).</li><li>Blanchoud, S., Nicolas, D., Zoller, B., Tidin, O., Naef, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAST:+An+automated+segmentation+and+tracking+tool+for+the+analysis+of+transcriptional+kinetics+from+single-cell+time-lapse+recordings.">CAST: An automated segmentation and tracking tool for the analysis of transcriptional kinetics from single-cell time-lapse recordings.</a> Methods. 85, 3-11 (2015).</li><li>Peng, T., 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+BaSiC+tool+for+background+and+shading+correction+of+optical+microscopy+images.">A BaSiC tool for background and shading correction of optical microscopy images.</a> Nature Comm. 8, (2017).</li><li>Smith, 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=CIDRE:+an+illumination-correction+method+for+optical+microscopy.">CIDRE: an illumination-correction method for optical microscopy.</a> Nature Methods. 12 (5), 404-406 (2015).</li><li>Chae, H. D., Lee, M. R., Broxmeyer, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Aminoimidazole-4-carboxyamide+Ribonucleoside+Induces+G1/S+Arrest+and+Nanog+Downregulation+via+p53+and+Enhances+Erythroid+Differentiation.">5-Aminoimidazole-4-carboxyamide Ribonucleoside Induces G1/S Arrest and Nanog Downregulation via p53 and Enhances Erythroid Differentiation.</a> Stem Cells. 30 (2), 140-149 (2012).</li><li>Lin, Y., 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=Reciprocal+Regulation+of+Akt+and+Oct4+Promotes+the+Self-Renewal+and+Survival+of+Embryonal+Carcinoma+Cells.">Reciprocal Regulation of Akt and Oct4 Promotes the Self-Renewal and Survival of Embryonal Carcinoma Cells.</a> Mol Cell. 48 (4), 627-640 (2012).</li><li>Wei, F., Scholer, H. R., Atchison, 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=Sumoylation+of+Oct4+Enhances+Its+Stability,+DNA+Binding,+and+Transactivation.">Sumoylation of Oct4 Enhances Its Stability, DNA Binding, and Transactivation.</a> J Biol Chem. 282 (29), 21551-21560 (2007).</li><li>Gautier, 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=An+Engineered+Protein+Tag+for+Multiprotein+Labeling+in+Living+Cells.">An Engineered Protein Tag for Multiprotein Labeling in Living Cells.</a> Chem Biol. 15 (2), 128-136 (2008).</li><li>Los, G. V., 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=HaloTag:+A+Novel+Protein+Labeling+Technology+for+Cell+Imaging+and+Protein+Analysis.">HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis.</a> ACS Chem Biol. 3 (6), 373-382 (2008).</li></ol>

The most crucial step when using a SNAP-tag to monitor protein decay is to ensure that no residual unbound dye is left in the medium or in the cells after washing, as otherwise it might bind to newly produced SNAP-tag molecules later in the course of the experiment and thereby compromise the decay curve. This is on one hand achieved by carefully performing all the described washing steps. On the other hand, the dye concentration should be kept as low as possible, while still being in a range that allows for obtaining a g...

It is well known that cellular proteins undergo extensive turnover, with synthesis and degradation rates being specific for each protein and subject to physiological regulation. Traditionally, protein degradation rates have been measured using bulk methods, such as radioactive pulse chase analysis, or involving protein synthesis inhibitors such as cycloheximide1. More recently, stable isotope labeling with amino acids in cell culture (SILAC) in combination with mass spectrometry has been established to quantify protein turnover on a global scale2. However, these methods are limited by population averaging, and information about cell-to-cell variability is therefore lost. Furthermore, transient changes in protein degradation that are unsynchronized across the cell population cannot be identified.
Alternatively, protein half-lives can also be determined by fluorescence-based approaches, which often have the advantage of providing single-cell resolution. For example, a photoactivatable green fluorescent protein (paGFP) has been used to determine Oct4 half-life in the early mammalian embryo3. Another method to monitor protein decay in living cells is the use of a SNAP-tag in combination with fluorescence time-lapse imaging. The SNAP-tag is a mutant version of the DNA repair enzyme O6-alkylguanine DNA-alkyltransferase (AGT) that specifically reacts with benzylguanine (BG) derivatives, which can be coupled to molecular probes4,5,6. Therefore, any SNAP-tag fusion protein can be irreversibly labeled with a fluorescent, cell permeable dye. Pulse labeling of a protein of interest fused to the SNAP-tag, followed by washout of the residual dye, allows for monitoring the degradation of the labeled protein population and thus for determining protein half-life. SNAP-tags have been successfully used for pulse-chase labeling of proteins and for determining protein half-lives in adherent cell culture and in vivo5,7,8,9. A large variety of SNAP-tag substrates covering commonly used fluorescent spectra are commercially available, enabling the selection of the optimal dye for each specific application. Thus, SNAP-tags can also be used for multicolor imaging in combination with other fluorescent fusion proteins or dyes. Cell-impermeable dyes are suitable for labeling of membrane-tethered proteins, whereas cell-permeable dyes are applicable for monitoring both intracellular and membrane-bound proteins. Furthermore, some of these probes exhibit almost no basal fluorescence and only start emitting a strong fluorescent signal upon binding to a SNAP-tag10.
This protocol describes how to measure the degradation rates of different proteins of interest in single cells using a SNAP-tag. Here we apply this method to mouse embryonic stem (ES) cells cultured on E-cadherin, but it should be possible to use it with any adherent cultured cell type. We show that pulse labeling of SNAP-tag fusion proteins followed by fluorescence time-lapse imaging allows for determining the single cell half-lives of various proteins of interest and provides an estimate for the cell-to-cell variability of half-lives in a population of cultured cells.

<table border="0" cellpadding="0" cellspacing="0" width="803">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Equipment</td>
			<td style="width:189px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">ES cell line expressing a SNAP-tag fusion protein of interest</td>
			<td style="width:189px;">-</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 100 mm TC-Treated Cell Culture Dish</td>
			<td style="width:189px;">Corning</td>
			<td style="width:119px;">353003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">96 Well, Black/Clear, Tissue Culture Treated Plate</td>
			<td style="width:189px;">Corning</td>
			<td style="width:119px;">353219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Neubauer-improved counting chamber, 0.1 mm</td>
			<td style="width:189px;">Marienfeld-superior</td>
			<td style="width:119px;">640030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">CO2 Incubator</td>
			<td style="width:189px;">Panasonic</td>
			<td style="width:119px;">MCO-170AICUV-PE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Centrifuge 5804 R</td>
			<td style="width:189px;">Eppendorf</td>
			<td>5804000528</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">InCell Analyzer 2200 Cell Imaging System</td>
			<td style="width:189px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">29027886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name</td>
			<td style="width:189px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Reagents</td>
			<td style="width:189px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Glasgow Minimum Essential Medium</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td>G5154</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Fetal Bovine Serum, embyonic stem cell-qualified</td>
			<td style="width:189px;">ThermoFisher</td>
			<td style="width:119px;">16141-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Sodium pyruvate solution</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">113-24-6</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Minimum Essential Medium&#160; Non-Essential Amino Acids</td>
			<td style="width:189px;">ThermoFisher</td>
			<td style="width:119px;">11140-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin</td>
			<td style="width:189px;">BioConcept</td>
			<td style="width:119px;">4-01F00H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">L-Glutamine 200mM</td>
			<td style="width:189px;">ThermoFisher</td>
			<td style="width:119px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2-Mercaptoethanol</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">63689-25ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Leukemia Inhibitory factor</td>
			<td style="width:189px;">-</td>
			<td style="width:119px;">-</td>
			<td>Produced in the lab by transient transfection of HEK-293T cells, followed by collection and filtering of the supernatant.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">CHIR99021 (GSK-3 Inhibitor XVI)</td>
			<td style="width:189px;">Merck Millipore</td>
			<td style="width:119px;">361559</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">PD 0325901</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">391210-10-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Gelatin from bovine skin</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">9000-70-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dulbecco&#39;s PBS 10x concentrated</td>
			<td style="width:189px;">BioConcept</td>
			<td style="width:119px;">3-05K00-I</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dulbecco&#39;s PBS Without Ca++/Mg++</td>
			<td style="width:189px;">BioConcept</td>
			<td style="width:119px;">3-05F29-I</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Trypsin-EDTA-Solution 0.25%</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Recombinant Mouse E-Cadherin Fc Chimera protein</td>
			<td style="width:189px;">R&#38;D systems</td>
			<td style="width:119px;">748-EC-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Doxycycline hyclate</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">SNAP-Cell 647-SiR</td>
			<td style="width:189px;">New England BioLabs</td>
			<td style="width:119px;">S9102S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">FluoroBrite DMEM</td>
			<td style="width:189px;">ThermoFisher</td>
			<td style="width:119px;">A18967-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name</td>
			<td style="width:189px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Software</td>
			<td style="width:189px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">FIJI</td>
			<td style="width:189px;">-</td>
			<td style="width:119px;">-</td>
			<td>Open-source image analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">MATLAB R2014a</td>
			<td style="width:189px;">Mathworks</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Microsoft Excel</td>
			<td style="width:189px;">Microsoft</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell quantification of protein degradation rates by time-lapse fluorescence microscopy in adherent cell culture

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

The described protocol provides an estimate of the cell-to-cell variability in half-life for any given protein fused to a SNAP-tag. The use of recombinant E-cadherin-Fc for coating of the imaging plate allows for single cell resolution in ES cells, which otherwise grow in colonies. Single cells can be tracked separately throughout the course of the movie ( Figure 1A).
In order to determine the protein half-life for each single cell, it is crucial to measure the in...

Watch this Scientific Journal Video about Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture at JoVE.com

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

This protocol describes a method to determine protein half-lives in single living adherent cells, using pulse labeling and fluorescence time-lapse imaging of SNAP-tag fusion proteins.

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

Research

JoVE Journal

Developmental Biology

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...