Name: Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy (Video) | JoVE Journal
Uploaded: 2021-04-25T15:00:00.000+00:00
Duration: 14 min 4 s
Description: Learn fluorescence correlation spectroscopy for detecting protein aggregation in neurodegenerative disease research using ALS-associated TDP25 and mutant SOD1. This method enables sensitive analysis of diffusion properties in cell lysates and live cells.

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5 minutes

A.K. was supported by a Japan Society for Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (C) (#18K06201), by a grant-in-aid from the Nakatani Foundation for Countermeasures against novel coronavirus infections, by a grant from Hokkaido University Office for Developing Future Research Leaders (L-Station), and a grant-in-aid from Hoansha Foundation. M. K. was partially supported by a JSPS Grant-in-Aid for Scientific Research on Innovative Areas &#34;Chemistry for Multimolecular Crowding Biosystems&#34; (#20H04686), and a JSPS Grant-in-Aid for Scientific Research on Innovative Areas &#34;Information physics of living matters&#34; (#20H05522).

1. Materials and reagents
<ol>
	<li>Use pyrogenic free solutions and medium for cell culture (Table 1).</li>
	<li>Prepare solutions for the biochemical experiment using ultrapure water and use as DNase/RNase free.</li>
	<li>Select an appropriate FBS for the cell culture with a lot check process. Since the selected FBS lot changes regularly, the catalog and lot number for FBS cannot be represented here.</li>
	<li>Plasmid DNA
	<ol>
		<li>Prepare pmeGFP-N15 for eGFP monomer expression; pmeGFP-C1-TDP256 for GFP-TDP25 expression; pmeGFP-N1-SOD1-G85R7 for SOD1-G85R-GFP expression; and pCAGGS8 as a carrier. 
		NOTE: meGFP is a monomeric variant carrying A206K mutation of enhanced GFP (eGFP). TDP25 is an ALS-associated C-terminal fragment of TDP-43. SOD1-G85R is an ALS-associated mutant of SOD1.</li>
	</ol>
	</li>
	<li>Cell strain
	<ol>
		<li>Use murine neuroblastoma Neuro2a cells. 
		&#8203;NOTE: Neuro2a cells have high transfection efficiency and highly express exogenous proteins. For TDP25 expression, cell strains having high expression efficiencies such as Neuro2a or HEK293 cells are required. In HeLa cells, TDP25 was not able to be efficiently expressed.</li>
	</ol>
	</li>
</ol>
2. Cell culture and transfection
<ol>
	<li>Prepare a 100 mm plastic dish growing Neuro2a cells semi-confluently in normal growth medium. 
	NOTE: It is necessary to incubate at 37 &#176;C for approximately 48-72 hours after the previous seeding until semi-confluency is reached.</li>
	<li>Remove the medium.</li>
	<li>Add 0.5 mL of trypsin-EDTA solution into the cell-culture dish and incubate them at 37 &#176;C for 1 min.</li>
	<li>Add 9.5 mL of normal growth medium into the dish and suspend the detached cells.</li>
	<li>Using Trypan blue to stain the dead cells, count the number of cells using a cell counter or manually. Then dilute the cells in culture medium (1.0 &#215; 105/mL).</li>
	<li>Add 2 mL of cell suspension into a 35 mm plastic dish for cell lysis or a glass base dish for live-cell measurement.</li>
	<li>Incubate the dish at 37 &#176;C for 1 day.</li>
	<li>Start the following preparations 15 minutes before the day of the transfection.</li>
	<li>Prepare two 1.5 mL tubes and add 100 &#956;L of Opti-MEM I to each tube.</li>
	<li>In the first tube, mix 1.0 &#956;g of plasmid DNA (Solution A). In the second tube, mix 2.5 &#956;L of Lipofectamine 2000 (Solution B). 
	NOTE: To maintain transfection efficiency, keep the total DNA amount the same. To reduce the expression level for FCS measurement in live cells, the fraction amount of plasmid DNA for protein expression should decrease (e.g., 0.2 &#956;g of pmeGFP-N1-SOD1-G85R and 0.8 &#956;g of pCAGGS mixture). Moreover, keep the same ratio between the volume of Lipofectamine 2000 and the amount of plasmid DNA.</li>
	<li>Gently mix Solution A and B by adding one into either tube, and then incubate it for 1 min at room temperature (Solution C).</li>
	<li>Add the Solution C to the culture medium; and incubate the cells for 24 h at 37 &#176;C.</li>
</ol>
3. Cell lysis &#38; medium exchange
<ol>
	<li>Check the GFP expression using a routine microscope.</li>
	<li>Remove the medium in the dish.</li>
	<li>Add 2 mL of PBS at 25 &#176;C to wash out the medium. Remove the PBS.</li>
	<li>Place the dish on an aluminum plate on top of the crushed ice. 
	NOTE: We use a 1 mm thick aluminum plate cut in a Sunday tool store or commercially available one as laboratory equipment.</li>
	<li>Add 200 &#956;L of lysis buffer at 4 &#176;C. Shake the dish mildly so that the buffer is evenly distributed at the bottom of the dish.</li>
	<li>Scrape the dish using a cell scraper and recover the lysate with undissolved cell debris in a new 1.5 mL tube.</li>
	<li>Centrifuge the solution at 20,400 x g for 5 minutes at 4 &#176;C.</li>
	<li>Recover the supernatant in a new 1.5 mL tube and keep it at 4 &#176;C or on ice. 
	NOTE: Do not freeze the lysate.</li>
	<li>For live cell measurements, replace the medium before the measurement. Check the cell attachment using a phase-contrast microscope. Confirm expression using a fluorescence microscope.</li>
</ol>
4. Fluorescence correlation spectroscopy (FCS) calibration
<ol>
	<li>Start the system and run the operation software. Turn on the Argon+ laser (458/477/488/514 nm). Stabilize the system at least for 30 min.</li>
	<li>Set up the optical path: Main beam splitter: HFT488; Dichroic mirror: NFT600; Fluorescence barrier filter: a bandpass filter 505-540 (BP505-540); Detector: avalanche photodiode (APD)).</li>
	<li>Set the pinhole size by entering the value directly (66 &#956;m; 1 airy unit).</li>
	<li>Add the Rhodamine 6G (Rh6G) solution into a well of the cover glass chamber on the stage.</li>
	<li>Add the immersion ultrapure water on the objective. Do not use the immersion oil.</li>
	<li>Set the the chamber on the microscope stage. Move the stage to the appropriate position.</li>
	<li>Focus on the upper glass surface by measuring the scattered light from the glass surface. After lowering the objective lens to the bottom, turn the focus knob clockwise to adjust. 
	NOTE: Check the turning direction of the focus knob because it is opposite between those made in Japan and Germany.</li>
	<li>Raise the focal point 200 &#956;m above the upper glass surface to observe the inside of the solution.</li>
	<li>Click the Count rate and monitor the photon counts rate.</li>
	<li>Gradually increase the acousto-optic tunable filters (AOTF) value (e.g., excitation laser transmission) so that the count rate value is more than 10 kHz.</li>
	<li>Click the Pinhole Adjustment and open the pinhole adjustment wizard. Find the pinhole position with the highest (a peak) number of photons in both the x- and y-axis.</li>
	<li>Turn the correction ring of the objective lens so that the counts per molecule (CPM) value is the highest. 
	NOTE: Since the thickness of the cover glass of the chamber specified here is 0.12-0.17 mm, the correction ring is at around its minimum or just a few turns from it, where the CPM is at its maximum.</li>
	<li>Decrease the AOTF value gradually so that the CPM value becomes 2-3 kHz.</li>
	<li>Acquire the autocorrelation function (ACF) of Rh6G for 90 s.</li>
	<li>Once measurement is complete, click Fit to perform curve fitting analysis.</li>
	<li>Select a model for one-component three-dimensional (3D) diffusion with a triplet state. 
	NOTE: The Rh6G is monodisperse and one-component diffuse with a triplet state.</li>
	<li>Set the fitting start time by moving the red line. Click Fit All and check the fit deviation. After performing the curve fitting, make sure the diffusion time (DT) and the structure parameter (SP) are roughly in the range of 20-30 &#956;s and 4-8, respectively.</li>
	<li>Remember the structural parameter value. Use the SP value for curve fitting analysis of all ACFs measured on the same day, under the same optical conditions, and using the same type of glass base dish or chambered cover glass set on the microscope stage.</li>
</ol>
5. FCS measurement in cell lysate
<ol>
	<li>Prepare the cell lysates (see above).</li>
	<li>Place the lysate on the cover glass chamber. Place a lid to avoid drying and the stage lid for light shading.</li>
	<li>Set the acquisition condition, laser power, measurement time, and repetitions.</li>
	<li>Click Count rate and adjust the AOTF value of the laser so that the CPM value becomes more than 1 kHz.</li>
	<li>Perform a trial measurement for 1 min. Check whether the calculated ACF shows a positive amplitude and smooth decay.</li>
	<li>Perform the main measurement for 5 min. 
	NOTE: The total measurement time is gradually increased until the shape of the ACF does not change even if the measurement time is increased.</li>
	<li>Click Fit to perform curve fitting analysis.</li>
	<li>Select a model for two-component 3D diffusion with a triplet state. Remember to enter the SP value and change its setting to &#34;Fixed&#34; not &#34;Free&#34; before clicking Fit all.</li>
	<li>Export the fitted table as a tab-delimited text file.</li>
	<li>If necessary, export the ACF and Count rate record as a tab-delimited text file.</li>
</ol>
6. FCS measurement in live cells
<ol>
	<li>Replace the medium with a fresh one before the measurement.</li>
	<li>Use a heat stage incubator. Set the cell-culture dish on the microscope stage.</li>
	<li>Confirm the focus and position using the confocal micrope. Select the measuring cell. Zoom in and adjust the cell position. Acquire fluorescence images of a GFP-expressing cell using the slow scanning speed mode.</li>
	<li>Select an FCS-measuring position using Position in the &#34;FCS&#34; section.</li>
	<li>Select at least one FCS measurement point using a crosshair (Figure 1).</li>
	<li>Measure the ACF for 1 min. 
	NOTE: Because fluorescent proteins tend to be photobleached in live cells due to slow movement compared to the solution, the measurement time should be minimum. It is generally difficult to obtain ACF in the cell as smoothly as solution measurements because prolonged measurement will lead to photobleaching of fluorescent proteins.</li>
	<li>Click Fit to perform curve fitting analysis using a model for two-component 3D diffusion with a triplet state. 
	NOTE: For live cell measurements, a model for two-component 3D diffusion with a triplet state would be better because it is empirically difficult to decrease the chi-square value with a one-component 3D diffusion model because of the existence of various mobile components in the live cell. But even using a model for three-component 3D diffusion, the diffusion components are usually not separated compared to using the model for two-components.</li>
</ol>

<ol><li>Ross, C. A., Poirier, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+aggregation+and+neurodegenerative+disease%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Protein aggregation and neurodegenerative disease.</a> Nature Medicine. 10, Suppl 10-17 (2004).</li>
<li>Hipp, M. S., Kasturi, P., Hartl, F. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+proteostasis+network+and+its+decline+in+ageing%5BTitle%5D)+AND+%22Nature+Review+Molecular+Cell+Biology%22%5BJournal%5D)">The proteostasis network and its decline in ageing.</a> Nature Review Molecular Cell Biology. 20 (7), 421-435 (2019).</li>
<li>Kitamura, A., Nagata, K., Kinjo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conformational+analysis+of+misfolded+protein+aggregation+by+FRET+and+live-cell+imaging+techniques%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Science%22%5BJournal%5D)">Conformational analysis of misfolded protein aggregation by FRET and live-cell imaging techniques.</a> International Journal of Molecular Science. 16 (3), 6076-6092 (2015).</li>
<li>Rigler, R., Mets, U., Widengren, J., Kask, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+Correlation+Spectroscopy+with+High+Count+Rate+and+Low-Background+-+Analysis+of+Translational+Diffusion%5BTitle%5D)+AND+%22European+Biophysics+Journal+with+Biophysics+Letters%22%5BJournal%5D)">Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion.</a> European Biophysics Journal with Biophysics Letters. 22 (3), 169-175 (1993).</li>
<li>Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Partitioning+of+lipid-modified+monomeric+GFPs+into+membrane+microdomains+of+live+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.</a> Science. 296 (5569), 913-916 (2002).</li>
<li>Kitamura, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+RNA+with+a+C-terminal+fragment+of+the+amyotrophic+lateral+sclerosis-associated+TDP43+reduces+cytotoxicity%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Interaction of RNA with a C-terminal fragment of the amyotrophic lateral sclerosis-associated TDP43 reduces cytotoxicity.</a> Scientific Reports. 6, 19230(2016).</li>
<li>Kitamura, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dysregulation+of+the+proteasome+increases+the+toxicity+of+ALS-linked+mutant+SOD1%5BTitle%5D)+AND+%22Genes+to+Cells%22%5BJournal%5D)">Dysregulation of the proteasome increases the toxicity of ALS-linked mutant SOD1.</a> Genes to Cells. 19 (3), 209-224 (2014).</li>
<li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108 (2), 193-199 (1991).</li>
<li>Kitamura, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytosolic+chaperonin+prevents+polyglutamine+toxicity+with+altering+the+aggregation+state%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state.</a> Nature Cell Biology. 8 (10), 1163-1170 (2006).</li>
<li>Kitamura, A., Shibasaki, A., Takeda, K., Suno, R., Kinjo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+the+substrate+recognition+state+of+TDP-43+to+single-stranded+DNA+using+fluorescence+correlation+spectroscopy%5BTitle%5D)+AND+%22Biochemistry+and+Biophysics+Reports%22%5BJournal%5D)">Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy.</a> Biochemistry and Biophysics Reports. 14, 58-63 (2018).</li>
<li>Kinjo, M., Sakata, H., Mikuni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+steps+for+fluorescence+correlation+spectroscopy+of+living+cells%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">First steps for fluorescence correlation spectroscopy of living cells.</a> Cold Spring Harbor Protocols. 2011 (10), 1185-1189 (2011).</li>
<li>Kinjo, M., Sakata, H., Mikuni, S. Fluorescence correlation spectroscopy example: shift of autocorrelation curve. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKinjo%2C+M+%22Cold+Spring+Harbor+Protocols%22">Cold Spring Harbor Protocols</a>. 2011 (10), 1267-1269 (2011).</li>
<li>Kinjo, M., Sakata, H., Mikuni, S. Basic fluorescence correlation spectroscopy setup and measurement. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKinjo%2C+M+%22Cold+Spring+Harbor+Protocols%22">Cold Spring Harbor Protocols</a>. 2011 (10), 1262-1266 (2011).</li>
<li>Jazani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+alternative+framework+for+fluorescence+correlation+spectroscopy%5BTitle%5D)+AND+%22Nature+Communication%22%5BJournal%5D)">An alternative framework for fluorescence correlation spectroscopy.</a> Nature Communication. 10 (1), 3662(2019).</li>
<li>Pack, C., Saito, K., Tamura, M., Kinjo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microenvironment+and+effect+of+energy+depletion+in+the+nucleus+analyzed+by+mobility+of+multiple+oligomeric+EGFPs%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs.</a> Biophysical Journal. 91 (10), 3921-3936 (2006).</li>
<li>Ries, J., Chiantia, S., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accurate+determination+of+membrane+dynamics+with+line-scan+FCS%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Accurate determination of membrane dynamics with line-scan FCS.</a> Biophysical Journal. 96 (5), 1999-2008 (2009).</li>
<li>Fujioka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phase+separation+organizes+the+site+of+autophagosome+formation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Phase separation organizes the site of autophagosome formation.</a> Nature. 578 (7794), 301-305 (2020).</li>
<li>Sadaie, W., Harada, Y., Matsuda, M., Aoki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+in+vivo+fluorescence+cross-correlation+analyses+highlight+the+importance+of+competitive+effects+in+the+regulation+of+protein-protein+interactions%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">Quantitative in vivo fluorescence cross-correlation analyses highlight the importance of competitive effects in the regulation of protein-protein interactions.</a> Molecular and Cellular Biology. 34 (17), 3272-3290 (2014).</li>
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These authors have no conflicts of interest.

Regarding system calibration before measurements, the same glasswares as the one used to measure the sample should be used (e.g., the 8-wells cover glass chamber for cell lysate and the 35-mm glass base dish for live cells). Because of the adsorption of Rh6G on the glass, its effective concentration may sometimes decrease. If so, a highly concentrated Rh6G solution such as 1 &#956;M should be used just for the pinhole adjustment. Extremely high photon count rates must be avoided to protect the detector (e.g., more than 1...

Protein aggregations involving neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, and so on1 are known to be toxic and would disturb protein homeostasis (proteostasis) in the cells and organs, that could then lead to aging2. The clearance of protein aggregation is expected as a therapeutic strategy; however, chemicals that prevent protein aggregation formation and degrade protein aggregates (e.g., small molecules or drugs) have not been established yet. Moreover, how protein aggregation exerts toxicity remains elusive. Therefore, to promote research projects related to protein aggregation, it is important to introduce high throughput procedures to simply detect protein aggregation. Protein aggregation detection using antibodies recognizing the conformation of protein aggregation and aggregation-specific fluorescent dye has been widely used3. However, it is difficult to detect the aggregation, especially in live cells using such classical procedures. 
F&#246;rster resonance energy transfer (FRET) is a procedure to detect protein aggregation and structural change. However, FRET is unable to analyze protein dynamics (e.g., diffusion and oligomerization of protein in live cells)3. Therefore, we introduce here a simple protocol to detect protein aggregation in solution (e.g., cell lysate) and live cells using fluorescence correlation spectroscopy (FCS), which measures the diffusion property and brightness of fluorescent molecules with single molecule sensitivity4. FCS is a photon-counting method by using a laser scan confocal microscope (LSM). Using a highly sensitive photon-detector and calculation of autocorrelation function (ACF) of photon arrival time, the pass through time and brightness of the fluorescent molecules in the detection volume is measured. The diffusion slows with an increase of molecular weight; thus, intermolecular interaction can be estimated using FCS. Even more powerfully, an increase in the brightness of the fluorescent molecule indicates homo-oligomerization of the molecules. Therefore, FCS is a powerful tool to detect such protein aggregation.

<table><tbody><tr><td>0.25% (w/v) Trypsin-1 mmol/L EDTA&amp;middot;4Na Solution with Phenol Red (Trypsin-EDTA)</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>201-16945</td><td></td></tr><tr><td>100-mm plastic dishes</td><td>CORNING</td><td>430167</td><td></td></tr><tr><td>35-mm glass base dish</td><td>IWAKI</td><td>3910-035</td><td>For live cell measurement</td></tr><tr><td>35-mm plastic dishes</td><td>Thermo Fisher Scientific</td><td>150460</td><td></td></tr><tr><td>Aluminum plate</td><td>Bio-Bik</td><td>AB-TC1</td><td></td></tr><tr><td>C-Apochromat 40x/1.2NA Korr. UV-VIS-IR M27</td><td>Carl Zeiss</td><td></td><td>Objective</td></tr><tr><td>Cell scraper</td><td>Sumitomo Bakelite Co., Ltd.</td><td>MS-93100</td><td></td></tr><tr><td>Cellulose acetate filter membrane (0.22 mm)</td><td>Advantech Toyo</td><td>25CS020AS</td><td></td></tr><tr><td>Cover glass chamber 8-wells</td><td>IWAKI</td><td>5232-008</td><td>For solution measurement</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Sigma-Aldrich</td><td>D5796</td><td>basal medium</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>biosera</td><td></td><td>Lot check required</td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>11668019</td><td></td></tr><tr><td>LSM510 META + ConfoCor3</td><td>Carl Zeiss</td><td></td><td>FCS system</td></tr><tr><td>Murine neuroblastoma Neuro2a cells</td><td>ATCC</td><td>CCL-131</td><td>Cell line</td></tr><tr><td>Opti-MEM I</td><td>Thermo Fisher Scientific</td><td>31985070</td><td></td></tr><tr><td>pCAGGS</td><td>RIKEN</td><td>RDB08938</td><td>Plasmid DNA for the transfection carrier</td></tr><tr><td>Penicillin-Streptomycin Solution (&amp;times;100 )</td><td>Fujifilm Wako Pure Chemical Corp.</td><td>168-23191</td><td></td></tr><tr><td>pmeGFP-C1-TDP25</td><td></td><td></td><td>Plasmid DNA for TDP25 tagged with monomeric eGFP</td></tr><tr><td>pmeGFP-N1</td><td></td><td></td><td>Plasmid DNA for eGFP monomer expression</td></tr><tr><td>pmeGFP-N1-SOD1-G85R</td><td></td><td></td><td>Plasmid DNA for ALS-linked G85R mutant of SOD1 tagged with monomeric eGFP</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P8304</td><td></td></tr></tbody></table>

detection of protein aggregation using fluorescence correlation spectroscopy

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We performed FCS measurement of GFP-TDP25 in cell lysate and SOD1-G85R-GFP in live cells. In both cases, a positive amplitude and smooth ACFs were able to be acquired. We have shown that a portion of GFP-TDP25 expressed in Neuro2a cells was recovered in the soluble fraction under the indicated condition6. In the soluble fraction of the cell lysate, extremely bright fluorescence molecules were detected in the photon count rate record using FCS (Figure 2A, top, arrow). ...

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

detection-protein-aggregation-using-fluorescence-correlation

Research

JoVE Journal

Biology

In this video, we demonstrate a cell-based protein aggregation assay using proteasome inhibitors, which block proteasome activity, preventing misfolded, mutant proteins, fused to a fluorescent label, from undergoing ubiquitin-dependent proteasomal degradation, leading to their accumulation within the cell cytoplasm. The protein aggregates are then visualized and quantified by fluorescence microscopy.

Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor

This video demonstrates the technique of fluorescence fluctuation spectroscopy (FFS) to study protein homo-oligomerization. Fluorescently-labeled proteins in a sample are dimerized using an agent when studying protein oligomerization using FFS. Using a confocal microscope, as the proteins move in and out of the small observation volume, the analysis of the fluctuations in the brightness of the fluorescent molecules is performed to determine the oligomeric state of the proteins.

Fluorescence Fluctuation Spectroscopy to Study Protein Homo-Oligomerization

In this video, we describe the fluorescence anisotropy technique to study the interactions between the fluorophore-tagged Shwachman-diamond syndrome (SBDS) protein and the elongation factor-like 1 GTPase (EFL1). On incubating SBDS proteins with gradually increasing concentrations of EFL1, a steady increase in anisotropy is observed, indicating a successful interaction between the two proteins.

Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions

Cellular viability depends on timely and efficient management of protein misfolding.  Here we describe a method for visualizing the different potential fates of a misfolded protein:  refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

4D Imaging of Protein Aggregation in Live Cells

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in α-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with Förster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

Learn fluorescence correlation spectroscopy for detecting protein aggregation in neurodegenerative disease research using ALS-associated TDP25 and mutant SOD1. This method enables sensitive analysis of diffusion properties in cell lysates and live cells.

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

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