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Apr 2019

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Studying Protein Aggregation in the Context of Liquid-liquid Phase Separation Using Fluorescence and Atomic Force Microscopy, Fluorescence and Turbidity Assays, and FRAP
利用荧光和原子力显微镜、荧光和浊度分析以及FRAP研究液-液相分离中的蛋白质聚集   

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Abstract

Liquid-liquid phase separation (LLPS) underlies the physiological assembly of many membrane-less organelles throughout the cell. However, dysregulation of LLPS may mediate the formation of pathological aggregates associated with neurodegenerative diseases. Here, we present complementary experimental approaches to study protein aggregation within and outside the context of LLPS in order to ascertain the impact of LLPS on aggregation kinetics. Techniques described include imaging-based approaches [fluorescence microscopy, atomic force microscopy (AFM), fluorescence recovery after photobleaching (FRAP)] as well as plate reader assays [Thioflavin-T (ThT) fluorescence intensity and turbidity]. Data and conclusions utilizing these approaches were recently reported for the low complexity domain (LCD) of the transactive response DNA binding protein of 43 kDa (TDP-43).

Keywords: Liquid-liquid phase separation (液相分离), Amyloid (淀粉样), Protein aggregation (蛋白质凝聚), TDP-43 (TDP-43), Thioflavin-T (硫黄素-T), Turbidity (浊度), Fluorescence recovery after photobleaching (荧光漂白恢复), Atomic force microscopy (原子力显微镜), Fluorescence microscopy (荧光显微镜)

Background

Many proteins that aggregate in neurodegenerative disease also undergo LLPS to form dynamic, reversible liquid-like droplets (Molliex et al., 2015; Conicella et al., 2016; Hofweber et al., 2018). LLPS is commonly mediated by the presence of intrinsically disordered regions (IDRs) that enable transient, weak, multivalent interactions (Lin et al., 2017). These IDRs are often composed of only a few amino acid types and may be arranged in short, repetitive motifs. The low complexity domain (LCD) of TDP-43 forms pathologically-associated amyloid aggregates and was recently shown to phase separate (Conicella et al., 2016; Lim et al., 2016). However, any relationship between these two processes remained unclear. We recently demonstrated that amyloid formation by the LCD can occur within the context of LLPS (Babinchak et al., 2019). In this work, we implemented a combinatorial experimental approach to assess the kinetic role of LLPS in the formation of amyloids that can be applied to other aggregation-prone proteins that phase separate (Figure 1). This approach includes four major components: (1) imaging of amyloid and droplet-like species using fluorescence microscopy and atomic force microscopy (AFM); (2) assessment of LLPS propensity using turbidity measurements; (3) monitoring amyloid aggregation via Thioflavin-T (ThT) fluorescence intensity measurements; and (4) assessment of maturation of liquid-like droplets using fluorescence recovery after photobleaching (FRAP). Analysis of these results can provide quantitative insights for comparing aggregation under LLPS conditions and in the absence of LLPS.



Figure 1. Methods for studying the formation of TDP-43 LCD amyloid aggregates from liquid-like droplets. A. Atomic force microscopy (AFM) imaging of amyloid aggregates formed by the TDP-43 LCD in the absence of LLPS, which often appear as individual, non-clumped fibers. B. Fluorescence microscopy of liquid-like droplets formed by the TDP-43 LCD. C. With time, fibrils emanating from within mature droplets can be observed on AFM imaging and fibrils often appear clumped or intertwined. Complementary methods for studying aggregation within the context of liquid-liquid phase separation are presented. Turbidity measurements and fluorescence microscopy are optimal methods for studying liquid-like droplets, while ThT assays can be used to study amyloid formation. The transition from liquid-like droplets to amyloids can be captured using a combination of FRAP, AFM, and ThT assays. All images were originally published in J. Biol. Chem. (Babinchak et al., 2019).

Materials and Reagents

  1. Pipette tips
  2. FluoroDish Cell Culture Dish (35 mm dish, 23 mm well, glass thickness: 0.17 mm) (World Precision Instruments, catalog number: FD35-100)
  3. Amicon® Ultra–0.5 ml Centrifugal Filters (Ultracel–100,000 nominal molecular weight limit, NMWL) (Millipore, catalog number: UFC510096)
  4. ScotchTM Tape
  5. Adhesive tabs (Ted Pella, Inc., catalog number: 16079)
  6. AFM specimen discs, 15 mm diameter (Ted Pella, Inc., catalog number: 16218)
  7. Mica discs, 9.9 mm diameter (Ted Pella, Inc., catalog number: 50)
  8. Corning® Assay Plate, 96-well half area (non-treated, no lid; black with clear flat bottom; polystyrene) (Corning, catalog number: 3880)
  9. Sealing Tape, Advanced Polyolefin (Certified DNase-, RNase-, and Nucleic Acid-free) (Thermo ScientificTM, catalog number: 235307)
  10. Millex®–GV filter unit, low protein binding Durapore® (PVDF) membrane, 0.22 μm (Millipore, catalog number: SLGV033RS)
  11. Zeba spin desalting column (2 ml; 7 kDa molecular weight cut off) (Thermo ScientificTM, catalog number: 89890)
  12. Microscope Cover Glass 12CIR-1 (Thermo ScientificTM, catalog number: 1254580)
  13. Alexa Fluor 488TM C5 Maleimide (InvitrogenTM, catalog number: A10254)
  14. Potassium phosphate monobasic, KH2PO4 (Fisher Scientific, catalog number: 7778-77-0)
  15. Potassium phosphate dibasic, K2HPO4 (Fisher Scientific, catalog number: 7758-11-4)
  16. Sodium acetate trihydrate, CH3COONa·3H2O (Fisher Scientific, catalog number: 127-09-3)
  17. Acetic acid (glacial), CH3COOH (Fisher Scientific, catalog number: A38-212)
  18. Urea (Fisher Scientific, catalog number: BP 169-500)
  19. NaCl, 5 M solution (Sigma, catalog number: S5150-1L)
  20. Imidazole (Fisher Scientific, catalog number: O3196)
  21. Thioflavin-T (Sigma-Aldrich, catalog number: T-3516)
  22. Tris (2-carboxyethyl) phosphine hydrochloride, TCEP (GoldBio, catalog number: TCEP25)
  23. Trizma Base, Tris (Sigma-Aldrich, catalog number: T1503)
  24. Hydrochloric acid, HCl (Fisher Scientific, catalog number: A144)
  25. Type F immersion liquid (Leica Microsystems, catalog number: 11513859)
  26. Silicon tip on nitride lever probe (Bruker, SCANASYST-AIR)
  27. Milli-Q H2O (from Milli-Q Reference Water Purification System, MilliPore Sigma)
  28. 100 mM Phosphate and Acetate Stock Buffers (10x) (see Recipes)
  29. Thioflavin-T stock buffer (see Recipes)
  30. Labeling buffer (see Recipes)
  31. Equilibration buffer (see Recipes)

Equipment

  1. Plate Reader (Tecan Life Sciences, model: Infinite M1000)
  2. Pipette
  3. EppendorfTM Microcentrifuge (Fisher Scientific, model: 5424)
  4. Computer
  5. Vortex-Genie 2 Lab Mixer (Scientific Industries, Inc., model: G-560)
  6. pH/mV meter (Fisher Scientific, model: Accumet AB150)
  7. NanoDropTM Spectrophotometer (Thermo ScientificTM, model: 2000)
  8. Leica TCS SP8 confocal microscope (Leica Microsystems)
  9. All-in-one fluorescence microscope (Keyence, model: BZ-X710)
  10. 100x PlanApoλ objective (NA 1.45, oil immersion) (Nikon, catalog number: MRD31905)
  11. GFP filter cube (Keyence, catalog number: OP-87763)
  12. Multimode Atomic Force Microscope with Nanoscope V (Bruker)
  13. Incubator (Fisher Scientific, model: 516D)

Software

  1. i-controlTM 1.10 (for infinite reader) software (Tecan Life Sciences)
  2. Leica Application Suite X (LAS X, Leica Microsystems)
  3. Nanoscope 9.1 (Bruker)
  4. Nanoscope Analysis 1.5 (Bruker)
  5. BZ-X Viewer (Keyence)
  6. BZ-X Analyzer (Keyence)

Procedure

Note (Introduction to Procedure A): Wild type and single-cysteine mutant proteins were expressed and purified as described in detail previously (Babinchak et al., 2019). In brief, this involves a two-step purification using nickel nitrilotriacetic acid affinity chromatography under denaturing conditions followed by C4 reverse phase HPLC. Purified protein is flash-frozen and lyophilized. Procedure A is designed to pick up after lyophilization and to remove any potential aggregates that may have formed. Procedure A is crucial to ensuring that experiments are formed with highly soluble, non-aggregated protein. In addition to the TDP-43 LCD, this protocol may be used to study other proteins that may phase separate and aggregate; however, users may need to amend Procedures A and D based on the specific purification protocol for their protein of interest.

  1. Protein preparation
    1. Suspend lyophilized protein in ~200-250 μl of Milli-Q H2O. Briefly vortex.
    2. Pass soluble protein solution through 0.5 ml 100 kDa NMWL Amicon® centrifugal filter via centrifugation at > 21,000 x g for 7 min.
    3. Collect “flow-through solution” that passes through the filter.
    4. Measure “flow-through solution” absorbance at 280 nm (A280) to determine stock molar concentration using appropriate extinction coefficient [17,990 M-1·cm-1 for LCD protein used in Babinchak et al. (2019)] and the Beer-Lambert law.

    Note (Introduction to Procedure B): Because induction of phase separation results in the scattering of light, the process can be quantified using turbidity measurements. These types of measurements provide a method for comparing a variety of conditions that promote varying degrees of phase separation. For the TDP-43 LCD, LLPS is regulated by both NaCl concentration and pH. Therefore, recipes to prepare solutions with different buffer conditions are provided (see Recipes).

  2. Induction of phase separation and turbidity measurements
    1. Calculations and dilutions from stocks in order to study phase separation by altering pH and NaCl concentration: Using a known protein stock concentration (see Procedure A), calculate the volume of 100 mM buffer (Recipe 1), 5 M NaCl, Milli-Q H2O, and protein stock solution necessary to create a final solution containing 10 mM buffer, the desired salt concentration (0-300 mM NaCl), and the desired protein concentration. To allow for technical replicates, we recommend a total volume of 300 μl that can be evenly distributed into 3 wells of the 96-well plate.
    2. Experimental Solutions: Mix by pipetting reagents in the following order, mixing thoroughly at each step: (1) Milli-Q H2O, (2) buffer, (3) additional reagents (e.g., NaCl, polyethylene glycol, 1,6-hexanediol). Protein will be added last and just before taking measurements.
    3. Blank Solutions: As in Steps B1-B2, calculate and prepare “blank” solutions using the same conditions but in the absence of protein (which will be replaced by water). Three technical replicates may also be used.
    4. Add protein to experimental solutions. Mix thoroughly. Immediately add solutions to 96-well plate and measure absorbance at 600 nm (A600).
    5. For experiments at 37 °C, prepare solutions as described in Steps B1-B3. Pre-heat plate reader to 37 °C. Pre-heat all solutions, remaining stocks, and materials (i.e., 96-well plate, pipette tips) using a 37 °C incubator for 15 min to minimize cooling during sample preparation. Once warmed, add protein to reaction buffer and measure A600. To minimize additional cooling effects during sample preparation, incubate the plate (with samples) for 5-10 min in the pre-heated plate reader before measuring A600.

    Note (Introduction to Procedure C): Thioflavin-T has been used as a fluorescent probe to monitor amyloid formation for a number of proteins as well as to detect amyloids in histopathology samples from patient brains (Nielsen et al., 2001). Upon binding to amyloids, the intensity of fluorescence emission increases substantially, allowing for Thioflavin-T to be used to monitor the in vitro polymerization reaction. We found that the molecule itself does not appear to alter conditions under which phase separation is observed and its fluorescence intensity is not greatly altered by the presence of droplets, allowing us to assess amyloid formation within the context of phase separation. We typically monitor polymerization for 48-72 h, though the time to reaction completion is likely to be different for each protein.

  3. Thioflavin-T (ThT) measurements to monitor aggregation
    1. Pre-set plate reader temperature to 25 °C or 37 °C, depending on the temperature at which the user would like to study aggregation. This will be kept constant throughout the entirety of the experiment. Set up experimental parameters for Thioflavin-T aggregation kinetics in Tecan i-control software (Table 1).
    2. Prepare solutions as described in Steps B1-B3 with the addition of 15 μM ThT (from 1.5 mM ThT stock; Recipe 2) that is added just before protein. Mix thoroughly.
    3. Immediately add solutions to a 96-well plate and cover with advanced polyolefin sealing tape to prevent evaporation during time course of experiment.
    4. Initiate protocol to measure fluorescence intensity every 10 min at a constant temperature. Tecan i-control 1.10 (for infinite reader) software will populate in real-time a Microsoft Excel file containing raw fluorescence intensity data as measurements are taken.

      Table 1. Experimental parameters for Thioflavin-T aggregation kinetics


    Note (Introduction to Procedure D): The use of a fluorescently-labeled molecule is critical to performing fluorescence microscopy. The exact choice of dye (i.e., Alexa FluorTM 488, Alexa FluorTM 594, etc.) is up to the user.

  4. Fluorescent labeling of single-cysteine mutant protein
    1. Perform Alexa FluorTM 488 (AF488) labeling using Alexa Fluor 488TM C5 maleimide according to manufacturer’s protocol in the presence of Labeling Buffer (Recipe 3).
    2. Remove excess fluorescent label by passing protein through 7 kDa molecular weight cut-off (MWCO) Zeba spin desalting column (2 ml) equilibrated with Equilibration Buffer (Recipe 4). Follow manufacturer’s protocol guidelines for centrifugation and volumetric parameters.
    3. Determine the concentration of AF488-labeled protein by measuring A280 and correcting for contribution of AF488 to absorbance (instructions provided in the manufacturer’s online manuals).

    Note (Introduction to Procedure E): When performing microscopy imaging of droplets, we recommend the technique of “doping” unlabeled protein with a small amount of fluorescently-labeled protein, which can help to minimize costs by saving labeled protein. Which microscope is used depends on user preference and access provided by the laboratory; however, it is crucial to use a microscope with the capability of exciting and detecting emission of the user’s choice fluorophore (i.e., a GFP filter cube for AF488-labeled protein).

  5. Imaging technique: Fluorescence microscopy of droplets
    1. Prepare sample utilizing unlabeled protein from Procedure A and labeled protein from Procedure D with a ratio of 1:200-1:500 AF488-labeled to unlabeled protein. Steps in Procedure B should be used to design conditions. Pre-mix labeled and unlabeled protein before adding to remaining solution components. Mix thoroughly.
    2. Add ~15 μl of protein solution to the surface of FluoroDish and cover with coverslip.
    3. Image sample on BZ-X710 via bright field and with GFP filter cube. Use of 100x oil immersion objective can provide optimal resolution of droplets with diameters ranging from 1-5 μm on the BZ-X710, though other microscopes may provide adequate imaging at lower magnification. Imaging can be performed at the surface of the dish where droplets will sediment or in the solution above the dish surface. Imaging should not be performed near the edges of the coverslip, as evaporation of solution may cause artifacts. Droplets may be imaged immediately or over a long duration of time (for example, through use of time-lapse imaging). Typically, droplets will undergo growth, sedimentation, and fusion to eventually reach an equilibrium. Therefore, the timing of imaging should be consistent across conditions if a comparison is being performed.

    Note (Introduction to Procedure F): Atomic force microscopy can provide more detailed insight into morphological differences between phase-separated and aggregated species. This is most clearly evident when fibrillar species can be identified within or surrounding droplets–a level of detail that is difficult to observe using fluorescence microscopy but is more easily seen with AFM. Importantly, AFM can therefore distinguish between droplets with and without fibrils; however, an inherent shortcoming should be noted wherein the provided protocol images species that are dried on mica. Whether artifacts may be introduced during this drying process is unclear. Therefore, it is helpful to perform both complementary atomic force microscopy and fluorescent microscopy imaging. Additionally, liquid-like character of droplets cannot be assessed by AFM in this manner (see Procedure G for more information on how to do this).

  6. Imaging technique: Atomic force microscopy of aggregates and droplets
    1. Prepare fibrils or droplets as described in Procedures A-C.
    2. Attach a single mica disc to the surface of an AFM specimen disc using an adhesive tab. Use ScotchTM tape to strip mica surface (adhere tape to the surface of mica and press down firmly. Smoothly remove the tape from the surface of mica). Strip mica until the surface is smooth.
    3. Add ~10 μl of sample to mica surface. Cover sample to prevent dust particles from entering. Incubate sample at ambient temperature for 5 min.
    4. Rinse mica surface with 100 μl of Milli-Q H2O 4-5 times.
    5. Dry mica surface for ~1 min using a gentle stream of nitrogen gas.
    6. Image sample on multimode atomic force microscope using ScanAsyst in Air mode (with SCANASYST-AIR silicon tip on nitride lever).
      Recommended parameters for imaging: 0.977-1.95 Hz scan rate; 512 samples/line; feedback gain: 5.289; 0.1 V peak force set point; ScanAsyst autocontrol: ON.

    Note (Introduction to Procedure G): While fluorescence microscopy and AFM imaging can help to distinguish fibrillar from droplet-like species, maturation or aggregation within liquid droplets might occur before fibrils can be observed via imaging. Therefore, fluorescence recovery after photobleaching (FRAP) is ideal for assessing the dynamicity of phase-separated protein during this intermediary time period. This procedure provides a general approach to photobleaching liquid droplets to ascertain their dynamic nature. A number of works have utilized this approach but include slight variations in methodology and analysis. We recommend the following reference for users who might be interested in learning more (Taylor et al., 2019).

  7. Fluorescence recovery after photobleaching (FRAP) to monitor droplet maturation
    1. Turn on Leica HyVolution SP8 confocal microscope according to manufacturer’s guidelines. Allow 488 nm argon laser to warm up ~30 min.
    2. Prepare a sample in a solution condition under which phase separation is observed. Liquid droplets are necessary to perform a FRAP experiment. Add sample to FluoroDish as described in Steps E1-E2.
    3. Initial sample imaging and post-bleach frames should be collected using a laser intensity that does not bleach the sample. The absence of bleaching can be ascertained by recording 3-5 pre-bleach frames at the beginning of each FRAP experiment.
    4. Identify droplets that sediment on the bottom of the microscope dish as these will move minimally and allow for ease in data analysis.
    5. Using a consistent region of interest (ROI) diameter, bleach sample (2.4 mW or similar laser intensity) for 10 consecutive frames. Minimize time in between each bleach frame. Changes in ROI size may influence observed recovery and therefore should be kept consistent across samples.
    6. Monitor recovery over 300 s (1 frame per 5 s) or over sufficient time to observe recovery.

Data analysis

Note (Introduction to Data Analysis): Data analysis is designed to emphasize identification of amyloids primarily through use of atomic force microscopy and of liquid droplets using fluorescence microscopy. FRAP experiments help to ascertain whether droplets are more dynamic (i.e., liquid-like) or less dynamic (i.e., potential aggregates). Quantitative assessment of amyloid formation and the extent of phase separation is performed using data from Thioflavin-T and turbidity assays, respectively.

  1. Imaging Analysis
    Atomic Force Microscopy
    1. Open AFM image in Nanoscope Analysis 1.5. A ‘height’ and ‘peak force error’ image should be present.
    2. For the ‘height’ image, apply image flattening filter (Filter → Flatten) using the following recommended parameters: Flatten order: 1st; Flatten Z thresholding direction: no thresholding; Use histogram: OFF. Example imaging can be seen in Figure 1A.
    3. In some cases, the height of dried droplets may be so great that a flattened ‘height’ image may not be best for visualization. In this case, ‘peak force error’ images can also provide information on sample morphology (Figure 1C).
    4. Export image using Analysis → Journal Quality Export (recommended parameters: export type: TIFF; dots per inch: 300 or higher).

    Fluorescence Microscopy
    1. Open images in BZ-X analyzer software. All imaging analysis will be performed in this software.
    2. For bright field images, a haze reduction filter may be used to facilitate better visualization of droplets. Recommended haze reduction settings: Blur size, 8; brightness, 3.9; reduction rate, 0.75.
    3. For fluorescence images, a black background correction may be used to allow greater clarity of droplets (Figure 1B).

  2. Turbidity and Thioflavin-T data analysis using Microsoft Excel
    General Analysis (Turbidity measurements and ThT traces)
    1. In Microsoft Excel, average each set of technical replicates separately. For a specific condition, subtract the average value of blanks from the average value of the respective experimental traces to calculate a corrected value (A600 for turbidity measurements and fluorescence intensity values for ThT traces).
    2. Calculate standard deviation from raw values.
    3. To assess the effect of NaCl concentration on LLPS propensity, plot the average turbidity as a function of NaCl concentration (this can be applied to other experimental parameters, such as temperature).
    4. To evaluate aggregation from Thioflavin-T measurements, for each condition plot the average fluorescence intensity as a function of time. Discrete lag and elongation phases should be readily apparent if aggregation has occurred (Figure 2).

    Lag Time Analysis (Thioflavin-T Measurements)
    1. Lag times should be calculated using non-corrected and non-averaged fluorescence intensity traces (i.e., individual raw ThT traces). Plot each trace as a function of time.
    2. Using Microsoft Excel, insert a trend line through the lag phase data points and a second trend line through the elongation phase data points for a single trace. This can be performed in Excel by separating each individual trace into two datasets, one of which contains lag phase data points and the second of which contains elongation phase data points. We recommend minimizing the number of data points from the transition between lag and elongation phase, if possible (Figure 2). Do not include plateau phase data points.
    3. Use Excel to calculate the slope and y-intercept of the trend line drawn through each set of data points. Calculate the x-value (time point) at which both lines intersect. This is the lag time for a single trace (Nielsen et al., 2001).
    4. After calculating for multiple traces, average lag times within a single condition and calculate the standard deviation.


      Figure 2. Representative fluorescence intensity trace of amyloid formation. Amyloid formation can be described as having three distinct phases: (1) nucleation/lag phase, (2) elongation/exponential growth phase, and (3) plateau phase. Kinetic differences can be quantified by measuring the lag time. This is performed by generating a trend line through the lag phase data points (blue) and a second line through the elongation phase data points (red). The time point at which these two lines intersect is defined as the lag time (Nielsen et al., 2001).

  3. FRAP analysis using Leica LAS X and Microsoft Excel
    1. After FRAP experiment completion, LAS X built-in software procures a fluorescence intensity trace for the specific ROI used. Within the software, draw an additional ROI on an unbleached (control) droplet to monitor fluctuations in intensity as a result of z-axis drift. Intensity traces can be exported as ASCII or Excel files to be analyzed using Microsoft Excel.
    2. In Excel, plot each individual bleached and control trace as a function of time (Figure 3A). If significant drift is present, subtraction of the control droplet intensity trace from the bleached droplet intensity trace can be used to reduce the effects of drift (Figure 3B).
    3. Normalize each individual droplet intensity trace across pre-bleach and post-bleach frames.
    4. Calculate the average and standard deviation from recovery traces (Figure 3C).


      Figure 3. Analysis of FRAP Experiments. A. Individual traces commonly contain fluctuations in fluorescence intensity due to z-axis drift (black). This can be parsed out by drawing an ROI on a non-bleached droplet (grey). B. Subtraction of the unbleached ROI fluorescence trace from the bleached ROI fluorescence trace can correct for z-axis drift. C. Normalization and averaging of multiple FRAP experiments should be performed to assess the dynamicity of droplets under a specific condition.

Notes

  1. Imaging
    The number and observed size of droplets on the FluoroDish surface may be greatly influenced by incubation time. For more insight, please see McGuire et al., 1995. We therefore recommend using a constant time window for imaging across sample conditions.

  2. Turbidity and Thioflavin-T Fluorescence Assays
    1. For Thioflavin-T aggregation traces, there was a tendency for the number of wells measured to have an impact on the lag phase length, possibly due to varying degrees of agitation involved with taking a higher number of fluorescence intensity readings by the plate reader at each time point. To work around this, we recommend standardizing all plate reader experiments to the same number of wells measured (i.e., 96 wells, even if sample is not present in all wells) and the same time interval between reads (10 min).
    2. Introduction of bubbles or an air pocket into a well of a 96-well plate can cause artifact. We recommend tapping the side of the plate in order to remove bubbles. Measurements should not be considered valid if bubbles are present.

  3. Fluorescence recovery after photobleaching
    1. If many droplets are present within a single frame, multiple droplets can be bleached at one time to increase the number of technical repeats within an experiment.
    2. When evaluating the effect of time on fluorescence recovery, we recommend preparing a reaction solution of sufficient volume in order to add a sample multiple times to a new dish for each time point of interest. As a result, the effect of droplet maturation on FRAP can be observed for droplets aging in the test tube and this minimizes potential aging effects resulting from droplet stasis on the bottom of the dish or interaction with the glass surface.

Recipes

  1. 100 mM Phosphate and Acetate Stock Buffers (10x)
    1. Prepare 1 M stocks of KH2PO4, K2HPO4, CH3COONa, and CH3COOH in Milli-Q H2O. Prepare secondary stocks by diluting 10-fold in Milli-Q to reach a final concentration of 100 mM for each
    2. To prepare phosphate buffers (pH 6, 7, or 7.3), mix 1:1 100 mM KH2PO4 and 100 mM K2HPO4 in the presence of a pH meter while stirring. Adjust to the desired pH by adding additional 100 mM KH2PO4 (to make more acidic) or 100 mM K2HPO4 (to make more basic). Once the desired pH is reached, ensure that pH remains constant for more than 15 min
    3. To prepare acetate buffer (pH 4), mix 1:1 100 mM CH3COONa and 100 mM CH3COOH in the presence of a pH meter while stirring. Add additional 100 mM CH3COOH (to make more acidic) or 100 mM CH3COONa (to make more basic) until the desired pH is reached
    4. Filter freshly made buffers before use with a Millex®-GV filter unit (0.22 μm). Store at ambient temperature
  2. Thioflavin-T stock buffer
    1. Prepare a fresh 1.5 mM Thioflavin-T stock solution in Milli-Q H2O by adding an appropriate volume of Milli-Q H2O to a known mass of Thioflavin-T powder
    2. Filter before use with a Millex®–GV filter unit (0.22 μm). Store at 4 °C
  3. Labeling Buffer
    1. Prepare 1 M stock of Tris. Prepare 500 mM stock of TCEP
    2. To prepare 1 L of labeling buffer (20 mM Tris buffer at pH 8 containing 8 M urea, 200 mM NaCl, 250 mM imidazole, and 1 mM TCEP), combine 20 ml of 1M Tris, 480.48 g urea, 17.02 g imidazole, 2 ml of 500 mM TCEP, and 40 ml of 5 M NaCl. Fill with Milli-Q H2O to 1 L and adjust pH using HCl
  4. Equilibration Buffer
    1. Prepare 1 M stock of Tris
    2. To prepare 50 ml of equilibration buffer (20 mM Tris buffer at pH 8, containing 8 M urea and 150 mM NaCl), combine 1 ml of 1 M Tris, 24.02 g urea, and 1.5 ml of 5 M NaCl. Fill with Milli-Q H2O to 50 ml and adjust pH using HCl

Acknowledgments

This research was originally published in the Journal of Biological Chemistry: Babinchak, WM; Haider, R; Dumm, BK; Sarkar, P; Surewicz, K; Choi, JK; Surewicz, WK. J. Biol. Chem. 2019; 294 (16): 6306-6317. This work was supported by NIH grants F30 AG059350 (W.M.B.), T32 NS077888, T32 GM 007250, P01 AI106705 (W.K.S.), R01 NS103848 (W.K.S.), and RF1 AG061797 (W.K.S.). Confocal microscopy work was supported by NIH ORIP grant S10 OD024996. We thank Benjamin Dumm and Raza Haider for critical evaluation and proofreading of this work.

Competing interests

The authors declare no competing interests.

References

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  9. Taylor, N., Wei, M., Stone, H., Brangwynne, C. (2019). Quantifying dynamics in phase-separated condensates using fluorescence recovery after photobleaching. Biophysical Journal 117(7): 1285-1300.

简介

[摘要] 液相-液相分离(LLPS)是整个细胞中许多无膜细胞器生理组装的基础。但是,LLPS失调可能介导与神经退行性疾病相关的病理性聚集体的形成。在这里,我们提出了补充的实验方法来研究LLPS内外的蛋白质聚集,以确定LLPS对聚集动力学的影响。在所描述的技术CLUDE基于成像的方法[ 漂白后的荧光显微镜,原子力显微镜(AFM),荧光恢复(FRAP)] 一š以及读板器测定[ 硫磺素T(ThT的 [荧光强度和浊度] 。最近报道了使用这些方法的数据和结论用于43 kDa (TDP-43)交易反应DNA结合蛋白的低复杂性域(LCD )。
[背景] 许多在神经退行性疾病中聚集的蛋白质也会经历LLPS形成动态的,可逆的液体状液滴(Molliex 等,2015; Conicella 等,2016; Hofweber 等,2018)。LLPS通常由内在无序区(IDR)的存在介导,这些内在无序区能够实现短暂,微弱的多价相互作用(Lin 等人,2017)。这些IDR通常仅由几种氨基酸类型组成,并且可以排列成短的重复基序。TDP-43的低复杂度结构域(LCD)形成与病理相关的淀粉样蛋白聚集体,并且最近显示出相分离(Conicella 等人,2016; Lim 等人,2016)。但是,这两个过程之间的任何关系仍然不清楚。我们最近证明了LCD产生的淀粉样蛋白可以在LLPS的背景下发生(Babinchak 等,2019)。在这项工作中,我们实施了一种组合实验方法,以评估LLPS在淀粉样蛋白形成中的动力学作用,该作用可应用于相分离的其他易于凝集的蛋白质(图1)。该方法包括四个主要部分:(1)使用荧光显微镜和原子力显微镜(AFM)对淀粉样蛋白和液滴样物质进行成像;(2)使用浊度测量评估LLP S倾向;(3)通过硫黄素-T(ThT )荧光强度测量监测淀粉样蛋白的聚集;(4)利用光漂白后的荧光恢复(FRAP)评估液体状液滴的成熟度。这些结果的分析可以提供定量的见解,以比较LLPS条件下和不存在LLPS时的聚集。





图1.研究从液体状液滴形成TDP-43 LCD淀粉样蛋白聚集体的方法。A.在没有LLPS的情况下,由TDP-43 LCD形成的淀粉状蛋白聚集体的原子力显微镜(AFM)成像,该聚集体通常表现为单独的非聚集纤维。B.由TDP-43LCD形成的液体状液滴的荧光显微镜检查。C.随着时间的流逝,可以在AFM成像中观察到从成熟液滴内部散发的原纤维,并且原纤维经常看起来结块或缠绕在一起。提出了在液-液相分离的背景下研究聚集的补充方法。浊度测量和荧光显微镜检查是研究液体状液滴的最佳方法,而ThT 分析可用于研究淀粉样蛋白的形成。可以使用FRAP,AFM和ThT 分析相结合来捕获从液体样液滴到淀粉样蛋白的过渡。所有图像最初发表在J. Biol。化学。(Babinchak 等人,2019)。

关键字:液相分离, 淀粉样, 蛋白质凝聚, TDP-43, 硫黄素-T, 浊度, 荧光漂白恢复, 原子力显微镜, 荧光显微镜

材料和试剂


 


移液器技巧
FluoroDish 细胞培养皿(35毫米培养皿,23毫米孔,玻璃厚度:0.17毫米)(World Precision Instruments,目录号:FD35-100)
的Amicon ® 超– 0.5毫升离心过滤器(Ultracel – 100,000额定分子量极限,NMWL)(密理博,目录号:UFC510096)
苏格兰TM 胶带
粘性标签(Ted Pella,Inc.,目录号:16079)
直径15 mm的AFM样品盘(Ted Pella,Inc.,目录号:16218)
直径9.9毫米的云母盘(Ted Pella,Inc.,目录号:50)
康宁®检测板,半孔96孔(未处理,无盖子;黑色,底面透明;聚苯乙烯)(Corning,目录号:3880)
密封胶带,高级聚烯烃(不含认证的DNase,RNase 和核酸)(Thermo Scientific TM ,目录号:235307)
的Millex ® - GV过滤器单元,低蛋白质结合的Durapore ® (PVDF)膜,0.22μ米器(Millipore,目录号:SLGV033RS)
Zeba 旋转脱盐柱(2 ml;截留分子量为7 kDa )(Thermo Scientific TM ,目录号:89890)
显微镜盖玻片12CIR-1(Thermo Scientific TM ,目录号:1254580)
Alexa Fluor 488 TM C 5 马来酰亚胺(Invitrogen TM ,目录号:A10254)
磷酸氢二钾,KH 2 PO 4 (Fisher Scientific,目录号:7778-77-0)
磷酸氢二钾,K 2 HPO 4 (Fisher Scientific,目录号:7758-11-4)
三水合乙酸钠CH 3 COONa 3H 2 O(Fisher Scientific,目录号:127-09-3)
乙酸(冰),CH 3 COOH(Fisher Scientific,目录号:A38-212)
尿素(Fisher Scientific,目录号BP 169-500)
NaCl 5 M溶液(Sigma,目录号:S5150-1L)
咪唑(Fisher Scientific,目录号:O3196)
硫黄素-T(Sigma-Aldrich,目录号:T-3516)
三(2-羧乙基)膦盐酸盐TCEP(GoldBio ,目录号:TCEP25)
Trizma Base,Tris(Sigma-Aldrich,目录号:T1503)
盐酸盐酸(Fisher Scientific,目录号:A144)
F型浸没液体(Leica Microsystems,目录号:11513859)
氮化物杠杆探针上的硅尖端(布鲁克,SCANASYST-AIR)
Milli-Q H 2 O(来自Milli-Q参考水净化系统,MilliPore Sigma)
100 mM磷酸盐和乙酸盐库存缓冲液(10x)(请参阅食谱)
硫黄素-T库存缓冲液(请参见食谱)
标签缓冲液(请参见配方)
平衡缓冲液(请参见配方)
 


设备


 


酶标仪(Tecan Life Sciences,型号:Infinite M1000)
吸管
Eppendorf TM 微量离心机(Fisher Scientific,型号:5424)
电脑
Vortex-Genie 2实验室混合器(科学工业公司,型号:G-560)
pH / mV计(Fisher Scientific,型号:Accumet AB150)
NanoDrop TM 分光光度计(Thermo Scientific TM ,型号:2000)
徕卡TCS SP8共焦显微镜(Leica Microsystems)
多合一荧光显微镜(Keyence,型号:BZ-X710)
100X PlanApo λ 物镜(NA 1.45,浸油)(尼康,目录号:MRD31905)
GFP滤镜立方体(Keyence,目录号:OP-87763)
配备Nanoscope V的多模原子力显微镜(布鲁克)
孵化器(Fisher Scientific,型号:516D)
 


软件


 


i-control TM 1.10(用于无限阅读器)软件(Tecan Life Sciences)
Leica Application Suite X(LAS X,Leica Microsystems)
Nanoscope 9.1(布鲁克)
Nanoscope Analysis 1.5(布鲁克)
BZ-X Viewer(Keyence)
BZ-X分析仪(Keyence)
 


程序


 


注(1.产品离子过程A ):野生型和单半胱氨酸突变蛋白表达和纯化如前面详细描述的(Babinchak等人,2019。) 。简而言之,这涉及在变性条件下使用次氮基三乙酸镍亲和色谱进行两步纯化,然后进行C 4 反相HPLC。将纯化的蛋白质快速冷冻并冻干。程序A设计为在冻干后拾取,并去除可能形成的任何潜在聚集体。程序A对于确保使用高度可溶的非聚集蛋白形成实验至关重要。除TDP-43 LCD外,该协议还可用于研究其他可能发生相分离和聚集的蛋白质。但是,用户可能需要根据其感兴趣的蛋白质的特定纯化方案修改步骤A和D。


蛋白质p 赔偿
暂停在〜200-250冻干蛋白μ 升的Milli-Q H的2 O.简言之涡流。
通过0.5毫升通过可溶性蛋白溶液100 kDa的NMWL 的Amicon ® 经由离心离心过滤> 21000 ×g离心7分钟。
收集通过过滤器的“流通解决方案”。
测量在280nm处的“流过溶液”吸光度(A 280 )使用,以确定库存摩尔浓度适当的消光系数[1799 0 中号-1 · 厘米-1 为在使用LCD蛋白(Babinchak 等人,2019)] 和啤酒朗伯定律。
 


注(1.产品离子方法B ):由于相分离导致的光散射的诱导,该过程可以是曲antified使用浊度测量。这些类型的测量提供了一种用于比较各种条件的方法,这些条件促进了不同程度的相分离。对于TDP-43 LCD,LLPS受NaCl浓度和pH值的调节。因此,提供了用于准备具有不同缓冲液条件的溶液的配方(请参见配方)。


诱导相分离和浊度测量
为了通过改变pH和NaCl浓度来研究相分离,需要对储备液进行计算和稀释:使用已知的蛋白质储备液浓度(请参见步骤A),计算100 mM缓冲液(配方1),5 M NaCl,Milli-Q H 的体积2 O,以及创建最终溶液所需的蛋白质储备溶液,该最终溶液包含10 mM缓冲液,所需的盐浓度(0-300 mM NaCl)和所需的蛋白质浓度。为了允许技术复制,我们建议300的总体积μ 升,可均匀地分布到96孔平板的3个孔中。
实验解决方案:按以下顺序移液以混合试剂,并在每个步骤中彻底混合:(1)Milli-Q H 2 O,(2)缓冲液,(3)其他试剂(例如NaCl,聚乙二醇,1,6-己二醇)。蛋白质将在测量前最后添加。
空白溶液:与步骤B1-B 2一样,使用相同的条件但没有蛋白质(将被水代替),计算并准备“空白”溶液。也可以使用三个技术复制。
将蛋白质添加到实验溶液中。彻底混合。立即将溶液添加到96孔板中,并在600 nm(A 600 )处测量吸光度。
F 或在37进行实验℃下,如在步骤B1-B3中所述制备溶液。将读板器预热至37 °C 。使用37 °C的培养箱预热所有溶液,剩余储备液和材料(即96孔板,移液器吸头)15分钟,以最大程度减少样品制备过程中的冷却。加热后,将蛋白质添加到反应缓冲液中并测量A 600 。为了最大程度地减少样品制备过程中的额外冷却效果,在测量A 600 之前,将板(与样品)在预热的板读数器中孵育5-10分钟。
 


注(1.产品离子方法C ):硫磺素-T已被用作荧光探针来监控淀粉样蛋白形成为许多蛋白质以及检测来自患者的大脑组织病理学样品中淀粉状蛋白(Nielsen等,2001)。与淀粉样蛋白结合后,荧光发射强度显着增加,从而使硫黄素-T可用于监测体外聚合反应。我们发现分子本身似乎没有改变观察相分离的条件,并且其荧光强度不会因液滴的存在而大大改变,这使我们能够在相分离的背景下评估淀粉样蛋白的形成。我们通常监测聚合反应48-72小时,尽管每种蛋白质的反应完成时间可能不同。


硫黄素-T(ThT )测量以监测聚集
将板读取器的预设温度设置为25 °C 或37 °C,具体取决于用户希望研究聚集的温度。在整个实验过程中,这将保持恒定。在Tecan i- control软件中为硫黄素T聚集动力学设置实验参数(表1 )。
如在步骤B1-B3中所述制备溶液通过加入15 μ 中号的ThT (从1.5毫米的ThT 库存; RECI PE 2),这只是蛋白质之前添加。彻底混合。
立即加入溶液到一个96孔板中,并盖有先进的聚烯烃密封带防止时间期间蒸发实验当然。
发起协议来测量荧光强度每10米在一个恒定的温度。Tecan i -control 1.10(用于无限阅读器)软件将实时填充包含测量时原始荧光强度数据的Microsoft Excel文件。
 


表1.硫黄素-T聚集动力学的实验参数


测量参数


仪器设定


运动间隔


10分钟


间歇性搅拌


没有


激发波长(带宽)


440纳米(5纳米)


发射波长(带宽)


485纳米(5纳米)


获得


100


闪烁次数


50


整合时间


20个μ 小号


时差


0 μ 小号


定居时间


0 毫秒


Z位置


23000


 


注(1.产品离子到步骤d ):使用一个的荧光标记的分子是在执行荧光显微镜的关键。染料的确切选择(即Alexa Fluor TM 488,Alexa Fluor TM 594等)取决于用户。


单半胱氨酸突变蛋白的荧光标记
在存在标记缓冲液的情况下,根据制造商的规程使用Alexa Fluor 488 TM C 5 马来酰亚胺进行Alexa Fluor TM 488(AF488)标记(配方3 )。
通过使蛋白质通过用平衡缓冲液平衡的7 kDa 分子量截断(MWCO)Zeba 旋转脱盐柱(2 ml)除去多余的荧光标记(配方4)。按照离心机和体积参数的制造商规程指南。
通过测量A 280 并校正AF488对吸光度的贡献来确定AF488标记的蛋白质的浓度(制造商的在线手册中提供的说明)。
 


注(1.产品离子方法E ):当执行液滴的显微镜成像,我们建议的“掺杂”未标记的蛋白质的技术用少量的荧光标记的蛋白,它可以帮助最小化由保存标记的蛋白质的成本。使用哪种显微镜取决于用户的偏好和实验室提供的访问权限;但是,使用能够激发和检测用户选择的荧光团(即AF488标记的蛋白的GFP滤光片)发射能力的显微镜至关重要。


成像牛逼echnique:荧光液滴的显微镜
使用来自过程A的未标记蛋白和来自过程D的标记蛋白以1:488-1:500 AF488标记的蛋白与未标记的蛋白的比例制备样品。步骤B 中的步骤应用于设计条件。预混合标记和未标记的蛋白质,然后添加到其余溶液成分中。彻底混合。
添加〜15 微升的蛋白质溶液至所述的表面FluoroDish中和盖盖玻片。
在BZ-X710上通过明场和GFP滤镜立方体获得图像样本。的100×油浸物镜的使用可以提供微滴的最佳分辨率,直径范围从1-5 μ 米上BZ-X710,尽管其他显微镜可以在较低放大倍数提供足够的成像。成像可以在碟子的表面进行,否则液滴会沉淀或在碟子表面上方的溶液中。不要在盖玻片的边缘附近进行成像,因为溶液的蒸发可能会导致伪影。液滴可立即成像或经过长时间(例如,通过使用延时成像)成像。通常,液滴将经历生长,沉降和融合,最终达到平衡。因此,如果要进行比较,则成像的时间跨条件应保持一致。
 


注(1.产品离子方法F ):为不同形态原子力显微镜可以提供更详细的见解相分离和聚集物种之间分配办法。当可以在液滴内或液滴周围识别出纤维状物种时,这一点最为明显- 荧光显微镜难以观察到的细节水平,而原子力显微镜则更容易观察到。因此,重要的是,AFM可以区分具有和没有纤维的液滴。但是,应注意一个固有的缺点,其中所提供的协议对在云母上干燥的物种进行成像。目前尚不清楚在干燥过程中是否会引入伪影。因此,执行互补原子力显微镜和荧光显微镜成像都是有帮助的。另外,AFM无法以这种方式评估液滴的类液体特性(有关如何执行此操作的更多信息,请参见过程G)。


成像牛逼echnique:总量和液滴的原子力显微镜
如过程AC中所述准备原纤维或液滴。
使用粘贴片将单个云母盘附着到AFM样品盘的表面。用苏格兰TM 胶带ST RIP云母表面(坚持磁带的云母和用力按压的表面。顺利取出了从磁带的云母表面)。条云母直到该表面是光滑的。
向云母表面添加约10μl 样品。盖上样品以防止灰尘进入。在环境温度下孵育样品5分钟。
漂洗云母表面用100 微升的的Milli-Q H 2 ö4-5倍。
使用约1分钟干燥云母表面一氮气的轻柔气流。
在空气模式下使用ScanAsyst 在多模式原子显微镜上进行图像采样(氮化物杠杆上具有SCANASYST-AIR硅尖端)。
推荐的成像参数:0.977-1.95 Hz扫描速率;512个样本/行; 反馈增益:5.289;0.1 V峰值力设定点; ScanAsyst 自动控制:开启。


 


注(1.产品离子到方法G ):在荧光显微镜和原子力显微镜成像可有助于从区分纤维状液滴状液滴可原纤之前可能发生内物种,成熟或聚集经由成像观察到。因此,光漂白后的荧光恢复(FRAP)是评估此中间时间段内相分离蛋白动态的理想选择。该程序提供了对液滴进行光漂白以确定其动态性质的一般方法。许多作品都采用了这种方法,但是在方法和分析上略有不同。对于可能有兴趣了解更多信息的用户,我们建议使用以下参考资料(Taylor等,2019)。


光漂白后的荧光恢复(FRAP)以监控液滴的成熟
根据制造商的指南打开Leica HyVolution SP8共聚焦显微镜。让488 nm氩气激光器预热〜30分钟。
在观察到相分离的溶液条件下制备样品。进行FRAP实验需要液滴。如步骤E1-E2 所述,将样品添加到FluoroDish 中。
初始样品成像和漂白后帧应使用不会漂白样品的激光强度进行收集。可以通过在每个FRAP实验开始时记录3-5个预漂白帧来确定是否存在漂白。
识别沉淀在显微镜皿底部的液滴,因为这些液滴将移动最少并易于进行数据分析。
使用一致的关注区域(ROI)直径,对10个连续帧进行漂白样本(2.4 mW 或类似的激光强度)。最小化每个漂白框架之间的时间。ROI大小的变化可能会影响观察到的回收率,因此应在所有样品之间保持一致。
监视恢复时间超过300秒(每5秒1帧)或在足够的时间内观察恢复。
 


数据分析


 


注(1.产品离子到数据分析):数据分析的设计主要是通过使用原子力显微镜和使用荧光显微镜液滴的强调淀粉样蛋白的鉴定。FRAP实验有助于确定液滴的动态性(如液体)还是动态性(如潜在的聚集体)更小。分别使用硫黄素-T和浊度测定法的数据进行淀粉样蛋白形成和相分离程度的定量评估。


影像分析:
原子力显微镜


在Nanoscope Analysis 1.5中打开AFM图像。应显示“高度”和“峰值力错误”图像。
对于“高度”图像,请使用以下推荐参数应用图像展平过滤器(过滤器→展平):展平顺序:1 st ; 展平Z阈值方向:无阈值;使用直方图:关闭。示例成像可以在图1A中看到。
在某些情况下,干燥液滴的高度可能太大,以至于扁平化的“高度”图像可能不是最佳的可视化效果。在这种情况下,“峰值力误差”图像还可以提供有关样品形态的信息(图1C )。
使用“分析” →“ 日记本质量导出” 导出图像(推荐参数:导出类型:TIFF;每英寸点数:300或更高)
 


荧光显微镜


在BZ-X分析仪软件中打开图像。所有成像分析将在此软件中执行。
对于明场图像,可以使用雾度降低滤镜以促进更好地观察液滴。推荐的防雾霾设置:模糊大小8;亮度3.9 ; 减少率0.75
对于荧光图像,可以使用黑色背景校正以使液滴更清晰(图1B )。
 


使用Microsoft Excel进行浊度和硫黄素-T数据分析
常规分析(浊度测量和ThT 迹线)


在Microsoft Excel中,分别平均每个技术复制集。对于特定条件,从各个实验迹线的平均值中减去空白的平均值,以计算校正值(浊度测量为A 600 ,ThT 迹线为荧光强度值)。
从原始值计算标准偏差。
为了评估NaCl浓度对LLPS倾向的影响,绘制平均浊度作为NaCl浓度的函数(可以将其应用于其他实验参数,例如温度)。
为了评估硫黄素-T测量的聚集,对于每种条件,绘制平均荧光强度作为时间的函数。如果发生了聚集,则离散的滞后和伸长阶段应该很明显(图2 )。
 


滞后时间分析(硫黄素-T测量)


滞后时间应使用未经校正且未经平均的荧光强度迹线(即单个原始ThT 迹线)计算。将每个迹线绘制为时间的函数。
使用Microsoft Excel,通过滞后阶段数据点插入一条趋势线,并通过延伸阶段数据点插入第二条趋势线,以形成单个迹线。这可以在Excel中执行,方法是将每个单独的迹线分成两个数据集,其中一个包含滞后相位数据点,而第二个包含伸长相位数据点。我们建议,如果可能的话,请尽量减少滞后和伸长阶段之间的过渡所产生的数据点数量(图2 )。不包括平稳相位数据点。
使用Excel计算通过每组数据点绘制的趋势线的斜率和y截距。计算两条线相交的x值(时间点)。这是单个轨迹的延迟时间(Nielsen 等,2001)。
计算出多条迹线后,在单个条件下平均滞后时间并计算标准偏差。
 






图2.淀粉样蛋白形成的代表性荧光强度迹线。淀粉样蛋白的形成可以描述为具有三个不同的阶段:(1)成核/滞后阶段,(2)伸长/指数生长期,和(3)平稳期。动力学差异可以通过测量滞后时间来量化。通过生成通过滞后相位数据点(蓝色)的趋势线和通过伸长相位数据点(红色)的第二条线来执行此操作。这两条线相交的时间点被定义为滞后时间(Nielsen 等,2001)。


 


使用Leica LAS X和Microsoft Excel进行FRAP 分析
完成FRAP实验后,LAS X内置软件会针对所使用的特定ROI获取荧光强度曲线。在该软件内,在未漂白的(对照)墨滴上绘制额外的ROI,以监视由于z轴漂移而引起的强度波动。强度跟踪可以导出为ASCII或Excel文件,以使用Microsoft Excel进行分析。
在Excel中,将每个漂白和对照迹线绘制成时间的函数(图3A )。如果存在明显的漂移,则可以使用从漂白液滴强度迹线中减去控制液滴强度迹线来减少漂移的影响(图3B )。
在漂白前和漂白后帧中标准化每个液滴强度迹线。
计算与恢复曲线的平均值和标准偏差(图3C )。




图3. FRAP实验分析。答:单个迹线通常包含由于z轴漂移(黑色)而导致的荧光强度波动。可以通过在未漂白的液滴(灰色)上绘制ROI进行解析。B.从漂白的ROI荧光迹线减去未漂白的ROI荧光迹线可以校正z轴漂移。C.应当对多个FRAP实验进行标准化和平均化,以评估特定条件下液滴的动态性。


 


笔记


 


影像学
孵育时间可能会极大地影响FluoroDish 表面上液滴的数量和观察到的大小。有关更多信息,请参见McGuire 等。,1995年。因此,我们建议使用恒定时间窗口在整个样品条件下成像。


 


浊度和硫黄素-T荧光测定
对于硫黄素-T聚集迹线,测得的孔数有对滞后相长度产生影响的趋势,这可能是由于不同的搅动程度导致每次读板器读取更多数量的荧光强度读数时间点。要解决此问题,我们建议将所有读板器实验标准化为测量的相同孔数(即96个孔,即使所有孔中都不存在样品)和两次读取之间的相同时间间隔(10分钟)。
将气泡或气穴引入96孔板的孔中可能会导致伪影。我们建议点击板的侧面以去除气泡。如果存在气泡,则测量不应视为有效。
 


光漂白后的荧光恢复
如果在一个框架中存在许多液滴,则可以一次漂白多个液滴,以增加实验中技术重复的次数。
在评估时间对荧光恢复的影响时,我们建议准备足够体积的反应溶液,以便在每个感兴趣的时间点将样品多次添加到新皿中。结果,对于在试管中的液滴老化,可以观察到液滴成熟对FRAP的影响,并且这使由菜盘底部的液滴滞留或与玻璃表面相互作用而导致的潜在老化影响最小化。
 


配方小号


 


100mM磷酸盐和Aceta TE库存卜ffers(10X)
在Milli-Q H 2 O中准备1 M的KH 2 PO 4 ,K 2 HPO 4 ,CH 3 COONa和CH 3 COOH 储备液。通过在Milli-Q中稀释10倍以达到最终浓度100,准备二级储备液每个毫米
要制备磷酸盐缓冲液(pH 6、7或7.3),在存在pH计的情况下,边搅拌边混合1:1 100 mM KH 2 PO 4 和100 mM K 2 HPO 4 。调整到所述通过添加额外的100mM KH所需pH 2 PO 4 (使更多的酸性)或100mMķ 2 HPO 4 (使更多的碱性)。一旦在达到期望的pH,确保pH保持恒定超过15分钟
为了制备乙酸盐缓冲液(pH 4),在pH计的存在下,在搅拌的同时将1:1 100 mM CH 3 COONa和100 mM CH 3 COOH混合。添加额外的100mM CH 3 COOH(使更多的酸性)或100mM CH 3 COONa(使更多的碱性),直到所述d 达到esired pH值
过滤器具有在使用前新鲜制备的缓冲器的Millex ® - GV过滤器单元(0.22 μ M)。储存在环境温度下
硫黄素-T库存缓冲液
准备在Milli-Q H新鲜1.5mM的硫磺素-T原液2 ○B ÿ添加一个的的Milli-Q H适当体积2 O操作硫磺素-T粉末的已知质量
与使用前过滤器的Millex ® - GV过滤器单元(0.22 μ M)。储存在4 °C
标记缓冲液
请注意,该标记缓冲液源自如前所述(Babinchak 等,2019)的1步次氮基三乙酸镍亲和纯化中使用的洗脱缓冲液。


准备1 M的Tris库存。准备500 mM TCEP库存
要准备1 L标记缓冲液(pH 8的20 mM Tris缓冲液,含8 M尿素,200 mM NaCl,250 mM咪唑和1 mM TCEP),请合并20 ml 1M Tris,480.48 g尿素,17.02 g咪唑,2毫升500 mM TCEP和40毫升5 M NaCl。将Milli-Q H 2 O 填充至1 L,并使用HCl调节pH
平衡缓冲液
准备1 M的Tris库存
要制备50 ml平衡缓冲液(pH 8的20 mM Tris缓冲液,含8 M尿素和150 mM NaCl),请合并1 ml 1 M Tris,24.02 g尿素和1.5 ml 5 M NaCl。填充Milli-Q H 2 O至50 ml,并使用HCl调节pH
 


致谢


 


这项研究最初发表在生物化学杂志:Babinchak ,WM; Haider,R; Dumm ,BK; 萨卡(P. Surewicz ,K;崔JK;Surewicz ,WK。J.Biol。化学 2019; 294(16):6306-6317。NIH赠款F30 AG059350(WMB),T32 NS077888,T32 GM 007250,P01 AI106705(WKS),R01 NS103848(WKS)和RF1 AG061797(WKS)支持了这项工作。共焦显微检查工作得到了NIH ORIP资助S10 OD024996的支持。我们感谢本杰明· 达姆(Benjamin Dumm)和拉扎·海德尔(Raza Haider)对这项工作的严格评估和校对。


 


利益争夺


 


作者宣称没有利益冲突。


 


参考文献


 


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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Babinchak, W. M. and Surewicz, W. K. (2020). Studying Protein Aggregation in the Context of Liquid-liquid Phase Separation Using Fluorescence and Atomic Force Microscopy, Fluorescence and Turbidity Assays, and FRAP. Bio-protocol 10(2): e3489. DOI: 10.21769/BioProtoc.3489.
  2. Babinchak, W. M., Haider, R., Dumm, B. K., Sarkar, P., Surewicz, K., Choi, J. K. and Surewicz, W. K. (2019). The role of liquid-liquid phase separation in aggregation of the TDP-43 low-complexity domain. J Biol Chem 294(16): 6306-6317.
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