We thank Dr. Seth Robia at Loyola University Chicago for his valuable comments on this manuscript. This work was supported by NIH grant 1R01NS073967-01A1 to Joanna C. Bakowska.

<ol>
	<li>Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobility+measurement+by+analysis+of+fluorescence+photobleaching+recovery+kinetics.">Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.</a> Biophysical Journal. 16 (9), 1055-1069 (1976).</li><li>Peters, R., Peters, J., Tews, K. H., Bahr, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluorimetric+study+of+translational+diffusion+in+erythrocyte+membranes.">A microfluorimetric study of translational diffusion in erythrocyte membranes.</a> Biochimica et Biophysica Acta. 367 (3), 282-294 (1974).</li><li>Patterson, G., Day, R. N., Piston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+spectra.">Fluorescent protein spectra.</a> Journal of Cell Science. 114, 837-838 (2001).</li><li>Rabut, G., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching+techniques+to+study+mobility+and+molecular+dynamics+of+proteins+in+live+cells:+FRAP,+iFRAP,+and+FLIP.">Photobleaching techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, iFRAP, and FLIP.</a> Live Cell Imaging: A Laboratory Manual (1st ed). , 101-126 (2004).</li><li>Fritzsche, M., Charras, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+protein+reaction+dynamics+in+living+cells+by+fluorescence+recovery+after+photobleaching.">Dissecting protein reaction dynamics in living cells by fluorescence recovery after photobleaching.</a> Nature Protocols. 10 (5), 660-680 (2015).</li><li>Goodwin, J. S., Kentworthy, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching+approaches+to+investigate+diffusional+mobility+and+trafficking+of+Ras+in+living+cells.">Photobleaching approaches to investigate diffusional mobility and trafficking of Ras in living cells.</a> Methods. 37 (2), 154-164 (2005).</li><li>Hildick, K. L., Gonz&#224;lez-Gonz&#224;lez, I. M., Jaskolski, F., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+diffusion+and+exocytosis+of+membrane+proteins+in+cultured+neurons+assessed+using+fluorescence+recovery+and+fluorescence-loss+photobleaching.">Lateral diffusion and exocytosis of membrane proteins in cultured neurons assessed using fluorescence recovery and fluorescence-loss photobleaching.</a> Journal of Visualized Experiments. (60), e3747 (2012).</li><li>Nissim-Rafinia, M., Meshorer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching+assays+(FRAP+&amp;+FLIP)+to+measure+chromatin+protein+dynamics+in+living+embryonic+stem+cells.">Photobleaching assays (FRAP &amp; FLIP) to measure chromatin protein dynamics in living embryonic stem cells.</a> Journal of Visualized Experiments. (52), e2696 (2011).</li><li>Zheng, C., Petralia, R. S., Wang, Y., Kachar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+recovery+after+photobleaching+(FRAP)+of+fluorescence+tagged+proteins+in+dendritic+spines+of+cultured+hippocampal+neurons.">Fluorescence recovery after photobleaching (FRAP) of fluorescence tagged proteins in dendritic spines of cultured hippocampal neurons.</a> Journal of Visualized Experiments. (50), e2568 (2011).</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Transactions on Image Processing. 7 (1), 27-41 (1998).</li><li>Ishikawa-Ankerhold, H. C., Ankerhold, R., Drummen, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+fluorescence+microscopy+techniques--FRAP,+FLIP,+FLAP,+FRET+and+FLIM.">Advanced fluorescence microscopy techniques--FRAP, FLIP, FLAP, FRET and FLIM.</a> Molecules. 17 (4), 4047-4132 (2012).</li><li>Cabe, M., Rademacher, D. J., Karlsson, A. B., Cherukuri, S., Bakowska, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PB1+and+UBA+domains+of+p62+are+essential+for+aggresome-like+induced+structure+formation.">PB1 and UBA domains of p62 are essential for aggresome-like induced structure formation.</a> Biochemical and Biophysical Research Communications. 503 (4), 2306-2311 (2018).</li><li>Brown, E. B., Wu, E. S., Zipfel, W., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+molecular+diffusion+in+solution+by+multiphoton+fluorescence+photobleaching+recovery.">Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery.</a> Biophysical Journal. 77 (5), 2837-2849 (1999).</li><li>Centonze, V., Pawley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tutorial+on+practical+confocal+microscopy+and+the+use+of+the+confocal+test+specimen.">Tutorial on practical confocal microscopy and the use of the confocal test specimen.</a> Handbook of biological confocal microscopy. , 549-570 (1995).</li><li>Chudakov, D. M., Matz, M. V., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+and+their+applications+in+imaging+living+cells+and+tissues.">Fluorescent proteins and their applications in imaging living cells and tissues.</a> Physiological Reviews. 90 (3), 1103-1163 (2010).</li></ol>

The authors have no conflict of interest to disclose.

We provide a comprehensive, practical, and simple step-by-step protocol for FRAP experiments with live cells. Herein, the protocol was used to measure the mobility of YFP-p62 in ALIS in RAW264.7 macrophages, but it can be applied to many of the laser scanning confocal microscope systems and genetically encoded fluorescent proteins that are now available. For any microscopy system, pilot experiments are critical for determining the optimal FRAP parameters, including acquisition, bleach, and control ROI sizes, laser intensity for photobleaching, and prebleach and postbleach image acquisition. It is reasonable to expect the optimal FRAP parameters to differ for each genetically encoded fluorescent protein and cell line.
Important factors to consider when conducting a FRAP experiment include (a) achieving suitable bleach depth, (b) the use of a brief bleaching step, (c) allowing sufficient time postbleach to observe the full recovery function, (d) photobleaching efficiency, (e) cytotoxicity with repeated FRAP, and (f) the inclusion of a control for fluorescence loss due to repeated imaging. We recommend that bleach depth, which can be calculated according to the equation provided in section 3.3.1, be &#8805;9013. When bleach depth is &#60;90, the degree of postbleach fluorescence recovery will be underestimated, and the values of If, Mf, and t1/2 will be incorrect. Although the duration and intensity of the bleach-inducing laser pulses may vary between FRAP experiments, it is important that the photobleaching step be brief and substantially faster than the fluorescence recovery function. If it is not, then a significant amount of fluorescence recovery could occur during the bleaching step. With a long bleach time, fluorescence recovery during the bleaching step would not be measured, and it would lead to incorrect measurements of If, Mf, and t1/2. In addition, to obtain correct values for If, Mf, and t1/2, the acquisition ROI should be observed postbleach until the fluorescence level in the bleach ROI has reached a plateau. For example, in our FRAP experiments, there was no difference between the If, Mf,&#160;and t1/2 values when we observed YFP-p62 fluorescence in the bleach ROI for 32.2 min postbleach versus when we observed YFP-p62 in the bleach ROI for 15.1 min postbleach; thus, we concluded that the recovery function reached a plateau at 15.1 min postbleach12. With regard to photobleaching efficiency, photobleaching increases with the square of the optical zoom factor14. Thus, the use of high optical zoom lenses is favorable for rapid photobleaching but can result in undesirable photobleaching during acquisition; the latter can be accounted for by imaging a control ROI. Repeated photobleaching is to be avoided as it can lead to the generation of cytotoxic reactive oxygen species (ROS). However, the degree of ROS generation due to exposure to a high-intensity laser is lower for genetically encoded fluorescent proteins than for chemical fluorophores (e.g., fluorescent antibodies)15, and the ROS generated are more likely to react within the genetically encoded fluorescent protein than with other molecules in the cell4. In addition to the increased probability of generating cytotoxic ROS, repeated photobleaching is to be avoided as it is difficult to control. Finally, although low laser transmission is used to acquire all non-bleach images, some photobleaching will invariably occur, which must be controlled. Possible controls for this include monitoring fluorescence in a control ROI within the acquisition ROI, obtaining control images in a neighboring unbleached cell, and performing control experiments with the identical settings for those used in the photobleaching experiments but without the photobleaching event.

Fluorescence recovery after photobleaching (FRAP) is a microscopy technique that can be used to quantify protein dynamics in live cells1,2. The popularity of FRAP has increased because of the widespread commercial availability of laser scanning confocal microscopes with high resolution, speed, and sensitivity, and a &#34;rainbow&#34; of genetically encoded fluorescent proteins, such as green fluorescent protein (GFP) and yellow fluorescent protein (YFP)3. Genetically encoded fluorescent proteins are fused to a protein of interest to allow for the subcellular localization of the protein of interest. In a typical FRAP experiment, the steady-state fluorescence in an acquisition region of interest (ROI) within a cell is observed via repeated imaging of that ROI with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired in a predefined subset of the acquisition ROI, hereafter referred to as the bleach ROI, by brief exposure to high-intensity laser light. As new unbleached proteins replenish bleached proteins over time, the speed and intensity of fluorescence recovery in the bleach ROI provides information about protein mobility (Figure 1)4.
Our interest in determining the mobility of the ubiquitin binding protein p62 (also known as sequestosome-1) in aggresome-like induced structures (ALIS) in murine RAW264.7 macrophages after stimulation with lipopolysaccharide (LPS) led us to review the FRAP literature. Unfortunately, many of the existing FRAP protocols are incomplete or inordinately complex5,6,7,8,9. Some do not provide detailed information about the laser settings, beam path configuration, and image acquisition parameters5,6,7,8,9. Others omitted key details regarding data analysis, such as how to address the issue of bleach ROI drift6,9 or how to calculate important recovery parameters, including the mobile fraction (Mf), immobile fraction (If), and half-time of recovery (t&#189;)5,7. Conversely, others placed too much emphasis on complex mathematical formulas used to calculate Mf, If, and t1/25,6,8,9. Thus, our purpose is to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Hyclone</td><td>SH30022.01</td><td>High Glucose (4.5 g/L)</td></tr><tr><td>Fetal bovine serum</td><td>Gemini Bio-Products</td><td>100-106</td><td></td></tr><tr><td>Glass bottom dishes</td><td>MatTek</td><td>P35G-1.5-14-C</td><td>35 mm</td></tr><tr><td>ImageJ software</td><td>National Institutes of Health</td><td>N.A.</td><td></td></tr><tr><td>Lipopolysaccharide (LPS)</td><td>Sigma</td><td>L4391</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma</td><td>M1404</td><td></td></tr><tr><td>Penicillin-streptomycin solution</td><td>Corning</td><td>30-002-Cl</td><td>100&amp;times;</td></tr><tr><td>Plan-Apochromat 63&amp;times;/1.40 Oil objective</td><td>Carl Zeiss MicroImaging, Inc</td><td>4407629904000000</td><td></td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polysciences</td><td>23966-1</td><td>linear (MW 25,000)</td></tr><tr><td>RAW 264.7 murine macrophages</td><td>ATCC</td><td>TIB-71</td><td></td></tr><tr><td>Zeiss confocal microscope</td><td>Carl Zeiss MicroImaging, Inc</td><td>LSM 510</td><td></td></tr></tbody></table>

fluorescence recovery after photobleaching of yellow fluorescent protein tagged p62 in aggresome-like induced structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

Shown here are the results of a typical experiment in which we used FRAP to examine the degree of mobility of p62 in ALIS in RAW264.7 cells treated with LPS for 3-5 h12. Figure 3A shows the raw data obtained from a bleach, control, and background ROI after the stack of images had been aligned to correct for small amounts (&#60; ~3 &#181;m) of image drift of the ALIS. YFP-p62 fluorescence in this ALIS at prebleach and postbleach is shown in Figure 3E and Supplementary Video 1. Figure 3B shows these data after background-correction. Figure 3C shows these data after correcting for fluorescence in the control ROI. Figure 3D shows these data normalized to the mean fluorescence of the 5 prebleach values in the bleach ROI. The degree of bleaching was sufficient in this experiment (bleach depth = 91.94). YFP-p62 fluorescence within this ALIS recovered slowly, as t1/2 = 128.27 s (2.14 min). YFP-p62 was not a very mobile protein in this experiment, as Mf = 21.97 and If = 78.03.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59288/59288fig1.jpg" /> 
Figure 1: A diagram of a RAW264.7 cell and steps for image analysis after the FRAP images have been aligned to correct for image drift of the ALIS. (A) Diagram of a RAW264.7 cell depicting several ALIS and the bleach, control, and background ROIs within the acquisition ROI. (B) An idealized depiction of raw FRAP data obtained in the bleach, control, and background ROIs in panel A. (C) An idealized graph of raw FRAP data that have been background-corrected. (D) An idealized graph of background-corrected FRAP data that have been normalized to fluorescence in the control ROI. (E) An idealized graph of normalized and background-corrected FRAP data that have been normalized to the prebleach fluorescence in the bleach ROI. Abbreviations: Con = control; BG = background. This figure was modified from Rabut and Ellenberg4. <a href="https://www.jove.com/files/ftp_upload/59288/59288fig1large.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/59288/59288fig2.jpg" /> 
Figure 2:&#160;Images of the user interface in the AIM software (Table of Materials) for laser selection, beam path configuration, and image acquisition parameters for the FRAP experiments. (A) Laser control screen showing the Argon/2 laser lines, output, and tube current used. (B) Configuration control screen showing the beam splitter and emission filter settings used. (C)&#160;Scan control screen showing the setting used for image acquisition.&#160;(D) Scan control screen showing the pinhole, detector gain, amplifier offset, amplifier gain, and transmission settings for the Argon/2 514 nm laser line. (E) Time series control screen showing the time series settings used. (F) Bleach control screen showing the prebleach and postbleach parameters used. Abbreviations: HFT = Haupt Farb Teiler (primary dichroic beam splitter); NT1 = Neben Farb Teiler 1 (first secondary dichroic beam splitter); NT2 = Neben Farb Teiler 2 (second secondary dichroic beam splitter); EF = emission filter. <a href="https://www.jove.com/files/ftp_upload/59288/59288fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59288/59288fig3.jpg" /> 
Figure 3: A representative FRAP experiment to study YFP-p62 dynamics in ALIS in RAW264.7 cells. (A) Raw fluorescence intensity data for a bleach, control, and background ROI. (B) The data given in panel A after the bleach and control ROI had been corrected for fluorescence in the background ROI. (C) The data given in panels A and B after normalizing the fluorescence intensity in the bleach ROI to account for fluorescence in the background-corrected control ROI. (D) The data given in panels A-C after normalizing the fluorescence intensity in the normalized and background-corrected bleach ROI to the average of the first 5 prebleach values in the bleach ROI. Mf = 21.97, If = 78.03, t1/2 = 128.27 s (2.14 min), and bleach depth = 91.94. (E) An image of an ALIS at prebleach (1.5 min) and postbleach (0, 6, and 16 min). Scale bar = 5 &#181;m and is valid for all panels. Abbreviations: Con = control; BG = background; Corr. = corrected; Norm. = normalized; Avg. = average. This figure was modified from Cabe et al.12 <a href="https://www.jove.com/files/ftp_upload/59288/59288fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Video 1" src="/files/ftp_upload/59288/59288supvideo1.jpg" /> 
Supplementary Video 1: A typical ALIS in a RAW264.7 macrophage before and after photobleaching. YFP-p62 fluorescence is slow to recover after the photobleach; hence, p62 is not a very mobile protein. For this experiment, Mf = 25.62, If = 74.38, t1/2 = 442.79 s (7.38 min), and bleach depth = 90.19. Scale bar = 5 &#181;m. This video was modified from Cabe et al.12 <a href="https://www.jove.com/files/ftp_upload/59288/Fig_4-Vid_FINAL.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures at JoVE.com

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...

fluorescence-recovery-after-photobleaching-yellow-fluorescent-protein

Research

JoVE Journal

Biology

14.8K Views.  Loyola University Chicago. Bestaand fluorescentieherstel na fotobleaching, FRAP-protocollen, zijn onvolledig of te complex. Het protocol gepresenteerd in deze video is belangrijk in die zin dat het is uitgebreid, praktisch en eenvoudig. Het belangrijkste voordeel van deze FRAP-techniek is dat het mogelijk maakt voor de kwantificering van eiwitmobiliteit binnen en tussen subcellulaire compartimenten in levende cellen.FRAP wordt bestudeerd voor zowel therapeutische als diagnostische doeleinden. Een dergelijk gebru...

Video: Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Neuroscience

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

Biochemistry

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...

Bioengineering

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

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 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 ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Samenvatting

Meer video's verkennen