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 peptides should be dissolved in the appropriate solvent, which can be determined by the sequence characteristics of the peptide (e.g., overall charge) and may be provided by the manufacturer if the peptides are commercially available. For example, S14G-humanin, SHLP2, and SHLP6, and MOTS-c dissolve in water. Aliquot the peptides in an appropriate volume (e.g., 50 &#956;L) to avoid freeze and thaw cycles. Once thawed, do not use them again.</li>
	<li>Aliquot the pre-warmed serum-containing or serum-free media in a 50-mL conical tube and add room temperature 1 mM stock solution to make it into a working concentration (e.g., 1 &#181;M, 10 &#181;M).</li>
	<li>Replace the media with 6 mL peptide solution from step 1.4. and incubate the cells for the appropriate amount of time. 
	NOTE: Choose the incubation time depending on your condition which activates ERK.</li>
</ol>
2. Subcellular Fractionation for Cytosol and Nucleus
<ol>
	<li>Wash the cells twice with 10 mL of ice-cold PBS, scrape the cells off the plate in 5 mL of ice-cold PBS, and transfer the cell suspension into a 15 mL conical tube.</li>
	<li>Centrifuge the cells at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the PBS and re-suspend the pellet in 200 &#181;L of ice-cold, fractionation buffer (10 mM HEPES pH = 7.6, 3 mM MgCl2, 10 mM KCl, 5% (v/v) glycerol, 1% non-ionic surfactant (C13H22O(C2H4O)n), protease/phosphatase inhibitors). 
	NOTE: To keep the proteins in their phosphorylation status, the phosphatase inhibitor as well as protease inhibitor should be freshly added and samples should be kept on ice at all times. Repeating freeze and thaw cycles of samples should be avoided. Aliquot samples into small amount (e.g. 50 &#956;g) before freezing them at -80 &#176;C.</li>
	<li>Incubate the re-suspended pellet on ice for 15 min and then centrifuge at 250 x g for 5 min at 4 &#176;C.</li>
	<li>Collect the supernatant as the cytoplasmic fraction and keep the pellet for the nuclear fraction.</li>
	<li>Centrifuge the supernatant to remove cellular debris and other contaminants at 18,000 x g for 10 min at 4 &#176;C and then transfer the supernatant into a new microcentrifuge tube. This is the cytosol fraction.</li>
	<li>Resuspend the pellet in 200 &#956;L ice-cold wash buffer (10 mM HEPES pH = 7.6, 1.5 mM MgCl2, 420 mM NaCl, 25% (v/v) glycerol, 0.2 mM EDTA, protease/phosphatase inhibitors) and centrifuge at 250 x g for 5 min at 4 &#176;C</li>
	<li>Remove the supernatant and resuspend the pellet in 100 &#956;L ice-cold, nuclear extraction buffer (20 mM HEPES pH = 7.6, 1.5 mM MgCl2, 420 mM NaCl, 25% (v/v) glycerol, 0.2 mM EDTA, protease/phosphatase inhibitors) and sonicate 10 times (5 s on, 10 s off, 30% amp.) on ice.</li>
	<li>Centrifuge at 18,000 x g for 10 min at 4 &#176;C.</li>
	<li>Transfer the supernatant to a new microcentrifuge tube. This is the nuclear fraction.</li>
	<li>Quantify the protein amount using a BCA assay</li>
</ol>
3. Crude Mitochondrial Fraction
<ol>
	<li>Wash the cells in each 10 cm dish with 10 mL of ice-cold PBS, add 5 mL ice-cold PBS, and detach the cells using a cell scraper. 
	NOTE: Use three 10 cm dishes of cells to get a good yield of mitochondria for Western Blotting.</li>
	<li>Transfer the cell suspension to a 15 mL conical tube, and combine all from three 10 cm dishes into one 15 ml conical tube.</li>
	<li>Centrifuge the cells at 600 x g for 10 min at 4 &#176;C.</li>
	<li>Aspirate the PBS and re-suspend the pellet in 1 mL of ice-cold mitochondria isolation buffer (10 mM Tris-MOPS, 1 mM EGTA/Tris, 200 mM Sucrose, adjust to pH = 7.4).</li>
	<li>Homogenize the cells with 25 strokes of a 2 mL homogenizer with polytetrafluoroethylene coated pestle on ice. 
	NOTE: This step is critical to maintain mitochondrial integrity and maximize the yield of the mitochondrial fraction. The number of strokes should be optimized for each cell type. Precool the homogenizer before starting the procedure.</li>
	<li>Transfer the homogenate to a microcentrifuge tube and centrifuge it at 600 x g for 10 min at 4 &#176;C to remove nuclei and unbroken cells.</li>
	<li>Collect the supernatant, transfer it to a new microcentrifuge tube, and centrifuge it at 7,000 x g for 10 min at 4 &#176;C. 
	NOTE: The pellet is loose, collect the supernatant with care and try not to disturb the pellet.</li>
	<li>Remove the supernatant, re-suspend the pellet with 200 &#956;L of ice-cold mitochondria isolation buffer, and transfer the solution to a new microcentrifuge tube. Centrifuge the tube at 7,000 x g for 10 min at 4 &#176;C.</li>
	<li>Repeat step 3.8 to wash the pellet. It is not necessary to transfer the supernatant to a new microcentrifuge tube in this step.</li>
	<li>Remove the supernatant from the washed pellet and re-suspend the pellet containing mitochondria with 50 &#956;L of RIPA buffer.</li>
	<li>Incubate the suspension on ice for 10 min.</li>
	<li>Centrifuge the suspension at 16,000 x g for 15 min at 4 &#176;C.</li>
	<li>Transfer the supernatant to a new microcentrifuge tube. This is the mitochondrial fraction.</li>
	<li>Quantify the protein amount using a BCA assay.</li>
</ol>
4. Western Blotting for Phospho-Specific Proteins
<ol>
	<li>Perform an SDS-polyacrylamide gel electrophoresis (8-16% premade gel) and transfer the protein to a PVDF membrane4.</li>
	<li>Incubate the membrane with 5% BSA in TBST (0.1% Polysorbate 20) for 30 min at room temperature to block background non-specific binding sites. 
	NOTE: For phosphorylated protein detection, block the membrane with BSA not Milk. Milk contains casein, an abundant phosphoprotein, which results in high nonspecific signal.</li>
	<li>Incubate the membrane with the primary antibody (anti-phospho-ERK1/2) at 4 &#176;C overnight.</li>
	<li>The next day, wash the membrane with TBST (0.1% polysorbate 20) three times for 5 min at room temperature.</li>
	<li>Incubate the membrane with secondary antibody (anti-rabbit HRP) for 1 h at room temperature.</li>
	<li>Wash the membrane with TBST (0.1% polysorbate 20) three times for 5 min at room temperature.</li>
	<li>Incubate the membrane with ECL solution and image the membrane in the image analyzer.</li>
	<li>Strip the membrane with stripping buffer for 15 min at room temperature and wash the membrane with TBST three times for 5 min.</li>
	<li>Repeat steps 4.2 to 4.7 using the anti-total ERK1/2 antibody.</li>
	<li>To check the purity of each fraction, run SDS-PAGE and perform immunoblots using antibodies recognizing markers for each compartment (e.g., Lamin B1 for nucleus, GAPDH for cytosol, and TOM20 for mitochondria).</li>
</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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Rep. 6 (1), 39517 (2016).</li><li>You, L., Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+STAT3+in+autophagy.">The role of STAT3 in autophagy.</a> Autophagy. 11 (5), 729-739 (2015).</li><li>Hashimoto, Y., Niikura, 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=Detailed+characterization+of+neuroprotection+by+a+rescue+factor+humanin+against+various+Alzheimer's+disease-relevant+insults.">Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults.</a> The J Neurosci. 21 (23), 9235-9245 (2001).</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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Pinchas Cohen is a shareholder and consultant for CohBar, Inc. Kelvin Yen has served as a consultant for CohBar, Inc.

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 humanin. The physiological implications of this have not been fully studied but it may suggest one possible mechanism for tissue dependent responses to HN. 
Phosphorylation of ERK1/2 plays a crucial role in ERK1/2 signaling and the phosphorylation status of ERK1/2 shows dynamic changes. It is important to optimize the conditions to find when ERK1/2 is phosphorylated under a specific condition/stimulus. To achieve this, try different stimulation conditions (e.g., dose and medium) and conduct time courses to find when the highest level of phosphorylation is achieved. A majority of studies have only focused on ERK1/2 phosphorylation in total cell lysates. However, the results may provide limited functional information as ERK1/2 can interact with different targets and modulate different downstream cascades in specific subcellular compartments. Therefore, examining the subcellular localization of phosphorylated ERK1/2 gives a better understanding of the role of ERK1/2 activation. 
Subcellular fractionation after ERK1/2 activation can be used as a source of further study (e.g., co-immunoprecipitation) to identify novel downstream targets because the subcellular fractionation can enrich for downstream targets of ERK1/2. This method can be applied to other signaling molecules that are translocated into different subcellular compartments upon stimuli after optimization of the conditions. 
Although various methods to isolate mitochondria have been proposed in the past decades, there is partial contamination from other subcellular compartments. The crude mitochondrial isolation method proposed here also contains limited amount of cytoplasm contaminants. As the amount of contamination is small, we consider that it is still a valid method for detecting the localization of ERK1/2 into mitochondria upon activation. As peptides are not necessarily stable in solution, it is sometimes difficult to maintain consistency between experiments. Be sure to make small aliquots of peptide solution to avoid freeze-thaw effects and to improve the consistency of peptide activity.

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&amp;trade; 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&#039;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 treatment showed increase in phosphorylation of ERK1/2 at its regulatory Thr202/Tyr204 Site. The subcellular localization of ERK1/2 plays an important role in its signaling function, particularly in getting its specificity. To understand the target of S14G-humanin-mediated ERK1/2 activation, we analyzed the subcellular localization of total and phosphorylated ERK1/2 following S14G-humanin treatment. In HEK293 cells, the phosphorylated ERK increased in the both cytoplasm and nucleus, but not in the mitochondria (Figure 2A). Interestingly, phosphorylated ERK decreased in the cytoplasm, nucleus, and mitochondria in SH-SY5Y cells (Figure 2B). These results suggest that S14G-humanin-mediated ERK phosphorylation may play a different role in different cell types and different conditions. Therefore, further studies are required to analyze the localization of S14G-humanin-mediated phosphorylation of ERK1/2 in other subcellular compartments, as phosphorylated ERK1/2 translocates into the nucleus, mitochondria, endosomes/lysosomes, and various membranes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56496/56496fig1.jpg" /> 
Figure 1: S14G-humanin increases the phosphorylation of ERK1/2 in HEK293 and SH-SY5Y cells. Quantification and representative western blots of ERK activation in (A) HEK293 and (B) SH-SY5Y cells. Total cell lysates following (A) 1 &#181;M or (B) 100 &#181;M S14G-humanin treatment in DMEM supplemented with 10% FBS for the indicated time periods were immunoblotted using anti-phospho- and total-ERK 1/2 (Thr202/Tyr204). This figure has been modified from Kim et al.4 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56496/56496fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56496/56496fig2.jpg" /> 
Figure 2: Phosphorylated ERK1/2 is localized to cytosol, nucleus, and other subcellular compartments upon S14G-humanin treatment in HEK293 and SH-SY5Y cells. Representative western blots showing ERK1/2 subcellular localization (n = 3). (A) HEK293 cells were treated with 1 &#181;M S14G-humanin. Cytosolic (15 &#181;g), nuclear (15 &#181;g and 50 &#181;g), and mitochondrial (15 &#956;g) lysates were immunoblotted using phospho- and total-ERK antibody. (B) SH- SY5Y cells were treated with 100 &#181;M S14G-humanin. Mitochondrial fractions were obtained 30 min after S14G-humanin treatment. Cytosolic, nuclear, and mitochondrial (15 &#181;g) lysates were immunoblotted using phospho- and total-ERK antibody. Anti-GAPDH, Anti-Lamin B, anti-Tom20 antibody were used as the cytosolic, nuclear, mitochondrial fraction markers, respectively. This figure has been modified from Kim et al.4. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56496/56496fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/56496/56496table1v2.jpg" /> 
Table 1: Buffers for cytosol, nuclear, and Mitochondrial fraction.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

7.8K Views.  University of Southern California. Dit protocol wordt beschreven hoe stimuleren van cellen met mitochondriale afkomstige peptiden en beoordelen van de signalering cascade en lokalisatie voor fosfoproteÃ¯ne.

Video: Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Biochemistry

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Developmental Biology

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Neuroscience

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

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