We thank Zehavit Frenkel and Sandra Tolchinsky for technical assistance. Research related to this work is supported by grants from the Israel Science Foundation (1229/07) and German-Israeli Project Cooperation (DIP-DFG).

The following protocol is intended for analysis of sugar chains of total glycoproteins or of a specific glycoprotein of interest. The procedure is basically the same for both cases with a few alterations as stated throughout the protocol.
<ol>
 <li> For metabolic labeling of N-linked glycans one needs to use a subconfluent culture of mammalian cells grown overnight in a 90 mm dish for each sample (samples may represent different time points or treatments). This protocol is optimized for NIH 3T3 cells. </li>
 <li> Starve the cells for glucose by incubation in 5 ml of freshly prepared pre-warmed (37&deg;C) glucose-free medium, supplied with 10% dialyzed FCS and 4 mM sodium pyruvate for 5-15 minutes. </li>
 <li> Replace the starvation medium with 1 ml of pre-warmed (37&deg;C) glucose-free medium containing 400 &mu;Ci of [2-3H]-labeled mannose (lower concentration of 65 &mu;Ci/ml is used for labeling of total glycoproteins), and incubate the cells in the normal tissue culture conditions for 1 hour. </li>
 <li> Remove the labeling medium from each sample, and carefully add 2 ml of PBS at 4&deg;C to the samples corresponding to pulse and place them on ice (these samples are referred to as pulses), while the samples corresponding to the chase need to be rinsed 3 times with 2 ml of pre-warmed (37&deg;C) normal complete culture medium, and then placed in a CO2 incubator at 37&deg;C with 5 ml of pre-warmed regular medium for the desired chase periods. </li>
 <li> Rinse the pulse samples (previously placed on ice) 3 times with 2 ml of ice-cold PBS. The cells are then scraped off the dish in 2 ml PBS using a cell scraper, and placed into a 2 ml Eppendorf tube. </li>
 <li> Short-spin the cells at 16000Xg (6-10 sec), discard the supernatant and freeze the cell pellet at -80&deg;C. </li>
 <li> Perform steps 5 and 6 also for the chase samples at the end of the chase time. </li>
 <li> Remove the cells from the freezer and lyse them by addition of 300 &mu;l of buffer A, vortexing briefly and incubating for 20 min on ice, or in the case of total glycoproteins analysis, by incubation 20 min on ice with buffer B, followed by sonication (4 times, 10 sec, maximum amplitude), then boil the samples for 5min. </li>
 <li> Centrifuge the lysates at 16000Xg for 20 min at 4&deg;C. Transfer the supernatant to a new Eppendorf tube and discard the pellet. </li>
 <li> If analyzing total glycoproteins, proceed to molecular filtration and deglycosylation (step 17). For immunoprecipitation of the H2a glycoprotein demonstrated here, add 20l/sample of (Repligen) protein A-agarose beads 1:1 suspension and 3 &mu;l/sample of our rabbit polyclonal anti H2a antibody for the desired glycoprotein (in our example anti-H2a antibody), to the supernatant from step 9. </li>
 <li> Incubate the samples for 4-16 hours at 4&deg;C, constantly mixing by slow rotation. </li>
 <li> Spin down the beads at 16000Xg for 30 seconds, and carefully remove the supernatant using vacuum. </li>
 <li> Rinse the beads, by adding 500 &mu;l buffer D and vortexing. Spin as in step 12 and remove the supernatant. Repeat the wash 3 times. </li>
 <li> Add 10&mu;l denaturing buffer (supplied with the NEB endo-H kit) to the bead pellet and boil the samples for 5 min (when total proteins are analyzed, the deglycosylation is performed on the Microcon filter, step 17). </li>
 <li> Spin down the beads (16000Xg for 30 seconds), and transfer the supernatant into a new Eppendorf tube. Then 0.5&mu;l of endo H enzyme are added to each sample along with 10 &mu;l of the reaction buffer (also supplied with the NEB endo H kit), and the samples are incubated at 37&deg;C for 3 hours. </li>
 <li> To separate the released glycans from the proteins, the sample is diluted 5 times with deionized water and placed on top of a molecular filter (Microcon Ultracel YM30), with a 30kDa cut-off, then centrifuged at 14000Xg for 3 min. Apply 50&mu;l of deionized water and repeat centrifugation of the samples. This washout is repeated 2 more times, keeping the flow-through. </li>
 <li> If you are analyzing total glycoproteins, apply the supernatant of the lysates (from step 9) to the Microcon filter. Wash the extract with 100 &mu;l of buffer E, 3 times by repeated centrifugation at 14000Xg for 3 min. Add 3 &mu;l of 10X endo H reaction buffer and 1.5 &mu;l of the endo H enzyme to the Microcon retentate, and incubate for 3 hours at 37&deg;C. The released glycans are eluted with deionized water by 3 repeated centrifugations, as in step 16. </li>
 <li> If desired, retentates from steps 16 and 17, containing endo H-resistant glycoproteins are then incubated with 200mU of N-glycosidase F in 15 &mu;l of buffer C, at 37&deg;C for 16 h. Elution with deionized water is done as in step 16. </li>
 <li> Place the tube containing the endo H reaction flow-through (steps 16 and 17) in a SpeedVac concentrator, and dry the samples completely (this can be done up to 45&deg;C to accelerate this process). </li>
 <li> To prepare the samples for the HPLC, resuspend the dry pellets in 12 &mu;l of the HPLC solvent (acetonitrile:water, 60:40, 1% phosphoric acid). </li>
 <li> Adjust the HPLC device (connected to the Spherisorb column) to constant flow of the solvent (1 ml/min) and pressure (a particular value between 1000 and 2000 psi), and place a fraction collector to change a tube every 1 minute. </li>
 <li> Load the samples into the HPLC device and start the fraction collector simultaneously. </li>
 <li> Collect 48 fractions of 1 ml from each sample run and from a run of a standard oligosaccharide mixture. </li>
 <li> Transfer 500 &mu;l from each fraction to a scintillation vial, and mix the contents with 3 ml of water-miscible scintillation fluid. </li>
 <li> Load the vials and read in a scintillation counter. </li>
 <li> The cpm readout is plotted as a function of fraction number. </li>
 <li> The cpm values within a peak represent a specific glycan species as follows: fractions 18-21 correspond to M5, fractions 22-26 to M6, fractions 27-31 to M7, fractions 32-35 to M8, fractions 36-39 to M9, fractions 40-42 to G1M9 and fractions 43-45 to G2M9. </li>
 <li>The sum of cpm values for the fractions within each peak reflects the absolute amount of a specific glycan species. In order to compensate for the fact that species containing more mannose residues acquire more label, the "relative molar amount" of each glycan species is calculated as follows: the absolute amount of each glycan species is divided by the number of mannose residues that it contains. This value is then divided by the sum of the relative molar amounts of all glycan species obtained to obtain the "percent of total" for each specific glycan species.</li>
</ol> 
<table border="1">
	<tbody>
		<tr>
			<td style="text-align:center;">Chase (h)</td>
 <td style="text-align:center;">0</td>
 <td style="text-align:center;">4</td>
		</tr>
		<tr>
 <td>Released with endo H (cpm) a</td>
 <td>90000</td>
 <td>16800</td>
		</tr>
		<tr>
 <td>Released with N-glycosidase F (cpm) b</td>
 <td>20400</td>
 <td>3900</td>
		</tr>
		<tr>
 <td>Glycoproteins in retentate (cpm) c</td>
 <td>371700</td>
 <td>127695</td>
 </tr>
 <tr>
 <td>Dolichol-oligosaccharides in retentate (cpm) d</td>
 <td>4350</td>
 <td>540</td>
 </tr>
 <tr>
 <td>Dolichol-oligosaccharides in retentate (%) e</td>
 <td>1.4 &plusmn; 0.7</td>
 <td>0.2 &plusmn; 0.2</td>
 </tr>
	</tbody>
</table>
Table 1. Analysis of release of N-linked sugar chains with deglycosidases and of the presence of dolichol-oligosaccharides in glycoprotein samples.
We applied the pulse-chase procedure to a lysate from NIH 3T3 cells. After Microcon filtration, labeled N-linked oligosaccharides were released mainly with endo H after pulse labeling (high mannose type) and with further treatment with N-glycosidase F after a chase period, which would correspond to complex type (Golgi-processed glycans), endo H resistant oligosaccharides. This quantitative analysis determined that dolichol-oligosaccharides, accounted for an insignificant fraction of the label in the initial retentate.
Notes:
<ol class="lalpha">
<li>NIH 3T3 cells were pulse labeled with [2-3H] mannose and chased with complete medium. After cell lysis and filtration through Microcon YM30, high mannose N-linked oligosaccharides were released from retentates by incubation with endo H.</li>
<li>Endo H-resistant material in Microcon retentates from (a) was then incubated with N-glycosidase F and released oligosaccharides measured in a scintillation counter.</li>
<li>Microcon retentates obtained as in (a) before endo H treatment, were extracted 4 times with chloroform:methanol:water 10:10:3 to remove precursor glycolipids, and the unextracted material solubilized in 1N NaOH and counted in a scintillation counter.</li>
<li>Dolichol-oligosaccharides were purified from extracts from (c) as described in 1 and counted in a scintillation counter.</li>
<li>Average of 3 experiments for percent of dolichol-oligosaccharides (d) of total cpm in retentate (c+d).</li>
</ol>
Representative Results
<img src="/files/ftp_upload/1899/1899fig1.jpg" alt="Figure 1" /> Figure 1. Pulse-chase analysis of total NIH 3T3 glycoproteins in untreated cells and upon proteasomal inhibition. After 1h pulse-labeling with [2-3H] we obtained an expected profile with some glucosylated precursors remaining (Glc2Man9GlcNAc2 (G2M9) and G1M9), but most of the label present in M9 and M8 species, the latter being the result of trimming of all the glucose and one mannose residue (Figure 1 A). No free mannose or other small precursors were detected, attesting to the thoroughness of the purification. Following a rather long 8h chase there was more extensive trimming to M7-6 and a small amount of M5, but the major species continued to be M9 and M8 (Figure 1 B). If the chase was done in the presence of a proteasomal inhibitor (30 &mu;M MG-132), there was only a minor accumulation of these same trimmed species (Figure 1 C).
<img src="/files/ftp_upload/1899/1899fig2.jpg" alt="Figure 2" /> Figure 2. Quantitative analysis of sugar chains of an ERAD substrate compared to total glycoproteins. (A) In an experiment similar to the one in Figure 1, relative molar amounts of each oligosaccharide species were calculated based on mannose content. We converted the cpm values obtained for each glycan species, the percent of each species relative to the total sum of the relative molar amounts of all species present was then plotted as a function of chase time for an average of two experiments. (B) Similar to (A) except that glycans released from the ERAD substrate ASGPR H2a were analyzed after the pulse labeling and chase for up to 4h, in the presence or absence of the proteasomal inhibitor MG-132. (C) Values for 4h chase in the presence of MG-132 from (A) and (B) are compared for ASGPR H2a and the glycoprotein pool. (D) Scheme of N-linked sugar chain trimming processes in the ER. Sugar trimming processes that lead to M6-5 linked to proteins (R) that are targeted to ERAD compared to M9-8 on those that exit to the Golgi and beyond. The results indicate that the ERAD process is associated with the mannose trimming of N-glycans to yield species with 5-6 mannose residues remaining.

<ol>
	<li>Parodi, A. J., Behrens, N. H., Leloir, L. F., Carminatti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+polyprenol-bound+saccharides+as+intermediates+in+glycoprotein+synthesis+in+liver.">The role of polyprenol-bound saccharides as intermediates in glycoprotein synthesis in liver.</a> Proc Natl Acad Sci U S A. 69 (11), 3268-3272 (1972).</li><li>Avezov, E., Frenkel, Z., Ehrlich, M., Herscovics, A., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+(ER)+mannosidase+I+is+compartmentalized+and+required+for+N-glycan+trimming+to+Man5-6GlcNAc2+in+glycoprotein+ER-associated+degradation.">Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation.</a> Mol Biol Cell. 19 (1), 216-225 (2008).</li><li>Frenkel, Z., Gregory, W., Kornfeld, S., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-associated+degradation+of+mammalian+glycoproteins+involves+sugar+chain+trimming+to+Man6-5GlcNAc2.">Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2.</a> J Biol Chem. 278 (36), 34119-34124 (2003).</li><li>Hosokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+endoplasmic+reticulum+(ER)+degradation+of+misfolded+Null+Hong+Kong+alpha1-antitrypsin+by+human+ER+mannosidase+I.">Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.</a> J Biol Chem. 278 (28), 26287-26294 (2003).</li><li>Jakob, C. A., Burda, P., Roth, J., Aebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+misfolded+endoplasmic+reticulum+glycoproteins+in+Saccharomyces+cerevisiae+is+determined+by+a+specific+oligosaccharide+structure.">Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure.</a> J Cell Biol. 142 (5), 1223-1233 (1998).</li><li>Tolchinsky, S., Yuk, M. H., Ayalon, M., Lodish, H. F., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-bound+versus+secreted+forms+of+human+asialoglycoprotein+receptor+subunits.+Role+of+a+juxtamembrane+pentapeptide.">Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide.</a> J Biol Chem. 271 (24), 14496-14503 (1996).</li></ol>

The pulse-chase analysis of glycans in live cells with HPLC separation provides a method for studying the dynamics of oligosaccharide structural alterations throughout the life of a glycoprotein. There is growing evidence that such alterations are involved in producing the signals for ER folding, quality control and trafficking systems 2-5. The method can be applied not only for a specific glycoprotein of interest but also for analyzing the dynamics of the structure of total glycoproteins, as illustrated in th...

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<td>Merck Bioscience</td>
<td>1.0003</td>
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<td>Microcon Amicon Ultra 0.5 ml 30K or Centricon ultracel YM-30</td>
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<td>5301 000.016</td>
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<td>04-011-1A</td>
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<td>Buffer C: N-glycosidase F reaction buffer: 200mM Na3PO4, pH 8.0, 4mM EDTA, 1.5% N-octylglucoside</td>
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<td>&nbsp;</td>
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<td>NIH 3T3 stably expressing H2a 6</td>
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pulse-chase analysis of n-linked sugar chains from glycoproteins in mammalian cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

Watch this Scientific Journal Video about Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells at JoVE.com

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

pulse-chase-analysis-n-linked-sugar-chains-from-glycoproteins

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15.1K Views.  Tel Aviv University. We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Video: Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...