Name: subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment
Uploaded: 2017-09-25T15:00:00
Description: Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

This work was supported by an Ellison/AFAR Postdoctoral Fellowship in Aging Research Program to SJK, and a Glenn Foundation Award and NIH grants to PC (1P01AG034906, 1R01GM 090311, 1R01ES 020812). All authors appear in the film.

1. Peptide Treatment to Cells
<ol>
	<li>Plate two million SH-SY5Y or HEK293 cells (2 x 106) into a 10 cm dish and grow them for 2 days</li>
	<li>(Optional) The next day, wash the cells with serum-free Dulbecco&#39;s Modified Eagle Medium (DMEM) media once and incubate with serum free DMEM overnight if the treatment needs to be done in serum free media.</li>
	<li>On day 3, dissolve S14G-humanin peptides in 0.2 &#181;m filtered, distilled water, and reconstitute them as 1mM stock solution. 
	NOTE: The pep...

<ol>
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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=Humanin:+a+novel+central+regulator+of+peripheral+insulin+action.">Humanin: a novel central regulator of peripheral insulin action.</a> PloS one. 4 (7), 6334 (2009).</li><li>Lee, C., Yen, K., Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanin:+a+harbinger+of+mitochondrial-derived+peptides.">Humanin: a harbinger of mitochondrial-derived peptides.</a> Trends Endocrinol. Metab. 24 (5), 222-228 (2013).</li><li>Kim, S. J., Guerrero, 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=The+mitochondrial-derived+peptide+humanin+activates+the+ERK1/2,+AKT,+and+STAT3+signaling+pathways+and+has+age-dependent+signaling+differences+in+the+hippocampus.">The mitochondrial-derived peptide humanin activates the ERK1/2, AKT, and STAT3 signaling pathways and has age-dependent signaling differences in the hippocampus.</a> Oncotarget. 7 (30), 46899-46912 (2016).</li><li>Ying, G., Iribarren, P., 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=Humanin,+a+newly+identified+neuroprotective+factor,+uses+the+G+protein-coupled+formylpeptide+receptor-like-1+as+a+functional+receptor.">Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor.</a> J. 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Here, we demonstrated that humanin peptide-mediated ERK1/2 activation occurs in two different cell types, and the subcellular localization of activated ERK1/2 can be different depending on the conditions (e.g., dose of peptide, time point, and cell type). It has been shown that humanin signals through two different receptors22,23, which may explain the differences in signaling between the two cell lines as well as the requirement for different doses of h...

Emerging studies show that mitochondrial-derived peptides (MDPs) play important roles in cytoprotection and metabolism1,2,3. Understanding the signal transduction pathway in the presence of MDPs gives us insight into the mechanism by which MDPs modulate various functions. The first identified MDP, humanin, has been shown to increase extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation through its receptor binding4,5. However, the downstream effect of ERK1/2 activation is still underexplored.
The ERK1/2 cascade serves as an essential mediator in a variety of cellular processes including proliferation, cell migration, cellular metabolism, survival, and apoptosis6,7,8. To orchestrate all these distinct cellular processes, the activity and subcellular localization of ERK1/2 are tightly regulated by phosphatases and scaffold proteins9,10. In addition to post-translational modification, the dynamic shuttling of ERK1/2 also regulates its signaling function, activity, and specificity11,12. ERK1/2 is primarily localized in the cytosol13. A set of anchoring and scaffold proteins help retain ERK1/2 in cytoskeletal elements, on the surface of organelles, or diffused in the cytoplasm13. Upon stimulation, ERK1/2 is phosphorylated and becomes dissociated from its anchoring proteins, allowing the translocation of ERK1/2 to other subcellular compartments, including the nucleus, mitochondria, Golgi, and lysosomes14,15,16.
Although humanin is known to activate the ERK1/2 signaling pathway, the activation of ERK1/2 is only observed in the total cell lysates. As described previously, since ERK1/2 subcellular localization plays a crucial role in its downstream effect, the analysis of both the subcellular localization and the total levels of phosphorylated ERK1/2 is necessary to provide a comprehensive understanding of humanin-induced ERK1/2 activation and the activation of downstream targets.
To understand the target organelles of activated ERK1/2, subcellular fractionation followed by western blotting for phosphorylated ERK1/2 was performed. This method can be easily implemented as it utilizes standard laboratory equipment and reagents. The isolated subcellular compartments are of high purity, allowing the results to be straightforwardly interpreted. Immunostaining of ERK1/2 may produce similar results. However, certain subcellular compartments are relatively hard to visualize and require special fixation and permeabilization methods. ERK1/2 levels vary in subcellular compartments, and this variation could cause false positive and false negative signals when looking at whole cell lysates. Therefore, an immunoblot using isolated subcellular compartments gives us a better understanding of ERK1/2 localization.
The versatility of the method enables modifications of the protocol to investigate effects of other stimulants including other MDPs or translocation of other signaling molecules such as STAT3. Recently discovered small humanin-like peptides (SHLPs) are encoded from 16S rRNA region where humanin is encoded, and they have similar but distinct properties compared to HN17. For example, SHLP2 and SHLP3 activate ERK1/2 after 8 h although humanin activates ERK1/2 within 5 min. Examining subcellular localization of ERK1/2 in response to different peptides will give us a better understanding of the biology of these peptides. Emerging evidence showed that the subcellular localization of signaling molecules plays a crucial role in their downstream effects. For instance, STAT3 traditionally is known to be primarily localized in the cytosol in resting cells, and then it translocates into the nucleus to activate gene expression in response to cytokines18. STAT3 also translocates to mitochondria and regulates the TCA cycle and ATP production19. Regarding autophagy regulation, different subcellular localization of STAT3 modulates autophagy in various ways20. For example, nuclear STAT3 transcriptionally regulates autophagy-related genes and acts as an autophagy modulator. Cytoplasmic STAT3 constitutively inhibits autophagy by interacting with autophagy signaling molecules. Mitochondrial STAT3 inhibits and prevents mitophagy by suppressing oxidative stress induced autophagy. Therefore, this subcellular compartment isolation method is crucial for understanding the role of other signaling molecules as well as ERK1/2.

<table>
	<tbody>
		<tr>
			<td>p44/42 MAPK (ERK1/2)</td>
			<td>Cell signaling</td>
			<td>9102</td>
			<td>Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)</td>
			<td>Cell signaling</td>
			<td>4370</td>
			<td>Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>Lamin B1</td>
			<td>Cell signaling</td>
			<td>12586</td>
			<td>Nuclear Marker, Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>Cell signaling</td>
			<td>5174</td>
			<td>Cytoplasmic marker, Dilution 1:2,000</td>
		</tr>
		<tr>
			<td>Tom20</td>
			<td>Santa cruz</td>
			<td>SC-17764</td>
			<td>Mitochondria marker, Dilution 1:2,000</td>
		</tr>
		<tr>
			<td>anti-Rabbit-HRP conjugated</td>
			<td>Cell signaling</td>
			<td>7074</td>
			<td>Dilution 1:30,000</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Culture Dish</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>12556002</td>
			<td></td>
		</tr>
		<tr>
			<td>HNG peptide</td>
			<td>Genescript</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25mm sylinge filter</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>09-719A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>3609</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HCL</td>
			<td>Sigma</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>FALCON</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt&#8482; Protease and Phosphatase Inhibitor Cocktail (100X)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>78440</td>
			<td></td>
		</tr>
		<tr>
			<td>Thomas Pestle Tissue Grinder Assemblies with Smooth Pestles</td>
			<td>Thomas Scientific</td>
			<td>3432S90</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>8-16% Mini-PROTEAN TGX Precast Protein Gels</td>
			<td>BIO RAD</td>
			<td>4561104</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Module</td>
			<td>BIO RAD</td>
			<td>1658030</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer System</td>
			<td>BIO RAD</td>
			<td>1704150</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td>
			<td>BIO RAD</td>
			<td>1704272</td>
			<td></td>
		</tr>
		<tr>
			<td>Clarity Western ECL Blotting Substrates</td>
			<td>BIO RAD</td>
			<td>1705060</td>
			<td></td>
		</tr>
		<tr>
			<td>Restore Western blot stripping buffer</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>21059</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator, Medel: FB120</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>695320-07-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the mitochondrial genome. MDPs have a wide variety of biological effects such as protecting neurons from apoptosis, improving metabolic markers, and protecting cells from chemotherapy. Humanin was the first MDP to be discovered and is the most studied peptide among the MDP family. The membrane receptors and downstream signaling pathways of humanin have been carefully characterized. Additional MDPs such as MOTS-c and SHLP1-6 have been more recently discovered and the signaling mechanisms have yet to be elucidated. Here we describe a cell culture based method to determine the function of these peptides. In particular, cell fractionation techniques in combination with western blotting allow for the quantitative determination of activation and translocation of important signaling molecule. While there are other methods of cell fractionation, the one described here is an easy and straightforward method. These methods can be used to further elucidate the mechanism of action of these peptides and other therapeutic agents.

Using the procedure presented here, we treated HEK293 and SH-SY5Y cells with 1 &#956;M and 100 &#956;M S14G-humanin, a potent humanin analog21, respectively, in complete media for the indicated time periods (Figure 1A and Figure 1B). We then examined the total and phosphorylated form of ERK1/2 at Thr202/Tyr204 from total protein extracts. S14G-humanin...

Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

This protocol describes how to stimulate cells with mitochondrial-derived peptides and assess the signaling cascade and localization of phospho-proteins.

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

The overall goal of this experiment is to identify the downstream signaling cascade involved in cells stimulated with mitochondrial-derived peptides. This method can answer key questions in the mitochondrial-derived peptide field such as what signaling pathways are involved, and where this occurs. The main advantage of this technique is that it is straight forward and relatively easy to perform.We first had the idea for this method when we examined humanin-mediated ERK signaling which gave us limited information on the downstream facts. The Subscale Kersher method gave us a better idea of the role of humanin-mediated signaling. On the third day after plating and washing two times ten to the sixth, SH-SY5Y or HEK293 cells according to the text protocol.Dissolve S14G humanin peptides in zero point two micron filtered distilled water. And re-constitute them as a one millimolar stock solution. Aliquot the pre-warmed serum-containing or serum-free medium in a 15 milliliter conical tube and add room temperature humanin peptide stock solution to make a working concentration.Immediately replace the medium in the cells with six milliliters of the peptide solution. And incubate the cells for the appropriate amount of time. Use 10 milliliters of ice-cold PBS to wash the cells twice.Then scrape the cells off the plate in five milliliters of ice-cold PBS. And transfer the cell suspension into a 15 milliliter conical tube. Centrifuge the cells at 500 times G and four degrees Celsius for five minutes.Re-suspend the pellet in 200 microliters of ice-cold fractionation buffer and incubate on ice for 15 minutes. Centrifuge the tube at 250 times G and four degrees Celsius for five minutes. Then collect the supernatant as the cytoplasmic fraction and keep the pellet for the nuclear fraction.Centrifuge the supernatant to remove cellular debris and other contaminants at 18000 times G and four degrees celsius for ten minutes. And then transfer the supernatant into a new micro-centrifuge tube. This is the cytisol fraction.Re-suspend the pellet in 200 microliters of ice-cold wash buffer and centrifuge the solution at 250 times G and four degrees Celsius for five minutes. Remove the supernatant and use 100 microliters of ice-cold nuclear extraction buffer to re-suspend the pellet. Then sonicate the sample on ice 10 times.Centrifuge the sample again. Then transfer the supernatant to a new microcentrifuge tube. To isolate the crude mitochondrial fraction, use 10 milliliters of ice-cold PBS to wash the cells in each 10 centimeter dish.Then add five milliliters of ice-cold PBS and use a cell-scraper to detach the cells. Transfer the cell-suspensions into a single 15 milliliter conical tube. Then centrifuge the cells at 600 times G and four degrees Celsius for 10 minutes.Aspirate the PBS and use one milliliter of ice-cold mitochondrial isolation buffer to re-suspend the pellet. Then with a polytetrafluoroethylene-coated pestle on ice, homogenize the cells with 25 strokes of a two milliliter homogenizer. Transfer the homogenate to a micro-centrifuge tube and centrifuge the sample at 600 times G and four degrees Celsius for 10 minutes to remove the nuclei and unbroken cells.Then collect the supernatant, transfer it to a new micro-centrifuge tube and centrifuge the solution at 7000 times G and four degrees Celsius for 10 minutes. After removing the supernatant, re-suspend the pellet with 200 microliters of ice-cold mitochondria isolation buffer and transfer the solution to a new micro-centrifuge tube. Centrifuge the tube at 7000 times G and four degrees Celsius for 10 minutes.After repeating the wash, use 15 microliters of RIPA buffer to re-suspend the pellet and incubate the suspension on ice for 10 minutes. Then centrifuge the sample at 16000 times G and four degrees Celsius for 15 minutes. Transfer the supernatant, which is the mitochondrial fraction, into a new tube.Quantify and carry out western blotting according to the text protocol. Using the procedure demonstrated in this video, HEK293 and SH-SY5Y cells retreated with one and 100 micromolar S14 G humanin, a potent humanin analog. And the total and phosphorylated form of ERK12 at theanine 202, tyrosine 204 from total protein extracts were analyzed.As seen here, S14G humanin treatment showed an increase in phosphorylation of ERK12 and its regulatory theanine-202, tyrosine-204 site. To understand the target of S14G humanin mediated ERK12 activation, the sub-cellular localization of total and phosphorylated ERK12 following S14G humanin treatment, was analyzed. As shown in these western blots, in HEK293 cells, the phosphorylated ERK increased in both the cytoplasm and nucleus but not in the mitochondria.In SH-SY5Y cells however, phosphorylated ERK decreased in the cytoplasm nucleus and mitochondria. Suggesting that S14G humanin-mediated ERK phosphorylation may play a different role in different cell types and different conditions. Once mastered, the cell nuclear fractionation can be done in one hour and the mitochondrial fractionation can be done in one hour and 30 minutes, if it is performed properly.While attempting this procedure, it is important to remember to keep all samples on ice. Following this procedure, other methods including co-immunal precipitation and mass spectrometry can be performed in order to understand the downstream targets of the signaling molecules.

subcellular-fractionation-for-erk-activation-upon-mitochondrial

Research

JoVE Journal

Biology

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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, ...

RhoC GTPase Activation Assay

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

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 ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...