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Dec 2018
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This protocol has been amended. See the updated notice

Lipid-exchange Rate Assay for Lipid Droplet Fusion in Live Cells
活体细胞中脂滴融合的脂类交换速率检测   

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Abstract

Lipid droplets (LDs) are central organelles in maintaining lipid homeostasis. Defective LD growth often results in the development of metabolic disorders. LD fusion and growth mediated by cell death–inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) family proteins are crucial for various biological processes including unilocular LD formation in the adipocytes, lipid storage in the liver, milk lipid secretion in the mammary epithelia cells, and lipid secretion in the skin sebocytes. Previous methodology by Gong et al. (2011) first reported a lipid-exchange rate assay to evaluate the fusion ability of each LD pair in the cells mediated by CIDE family proteins and their regulators, but photobleaching issue remains a problem and a detailed procedure was not provided. Here, we provide an improved and detailed protocol for the lipid-exchange rate measurement. The three key steps for this assay are cell preparation, image acquisition, and data analysis. The images of the fluorescence recovery are acquired after photobleaching followed by the measurement of the intensity changes in the LD pair. The difference in fluorescent intensity is used to obtain the lipid exchange rate between the LDs. The accuracy and repetitiveness of the calculated exchange rates are assured with three-cycle of photobleaching process and the linear criteria in data fitting. With this quantitative assay, we are able to identify the functional roles of the key proteins and the effects of their mutants on LD fusion.

Keywords: Lipid metabolism (脂代谢), Lipid droplet fusion (脂滴融合), Lipid-exchange rate (脂交换速率), Fluorescence recovery after photobleaching (光脱色荧光恢复技术), Lipid droplet size (脂滴大小)

Background

Lipid droplets (LDs) play a key role in maintaining lipid homeostasis (Farese and Walther, 2009; Yang et al., 2012). Defective LD maturation and growth are closely associated with the development of metabolic diseases such as obesity, fatty liver disease, cardiovascular disease, and type II diabetes (Krahmer et al., 2013; Rosen and Spiegelman, 2014; Gluchowski et al., 2017). LDs are dynamic organelles which budded from the endoplasmic reticulum (ER) (Gross et al., 2011; Choudhary et al., 2015) and continue to grow via triglyceride synthesis and lipid transfer from the ER (Fujimoto et al., 2007; Wilfling et al., 2013; Xu et al., 2018) or LD fusion (Gong et al., 2011; Somwar et al., 2011). LD-associated cell death–inducing DNA fragmentation factor alpha-like effector (CIDE) family proteins including CIDEA, CIDEB, and CIDEC/Fsp27 (Gao et al., 2017) are crucial regulators in the lipid homeostasis by governing atypical LD fusion and growth for lipid storage. Previously, we reported that CIDEC mediates LD fusion through directional lipid transfer from small (donor) to large (acceptor) LDs (Gong et al., 2011; Somwar et al.). The enrichment of CIDEC at the LD-LD contact site (LDCS) and the formation of the fusion pore are two essential steps for lipid exchange and transfer to occur. To evaluate the fusion ability of each LD pair in cells mediated by the CIDE family proteins and their regulators such as Perilipin1 (Sun et al., 2013a) or Rab8a (Wu et al., 2014), lipid-exchange rate assay was first proposed and performed as previously described (Gong et al., 2011; Sun et al., 2013b). However, the analytic process reported in the previous methodology neither eliminated the photobleaching effect upon laser exposure nor provided a detailed procedure to ensure experimental reproducibility and accuracy.

Here, we detailed the protocol of our renewed lipid-exchange rate assay used in Wang et al. (2018). The three key steps in the assay including cell preparation, fluorescence recovery after photobleaching (FRAP) image acquisition, and data analysis using mean of intensities (MOI) and BODIPY-C12-stained size measurement of LDs were reported. Upon performing a three-cycle photobleaching process and the pre-estimation of exchange rate based on the linear criteria in the data analysis, we can ensure the accuracy and repetitiveness of the fitted exchange rates by using our proposed equation underlying molecular thermodynamics in the Theory section. This lipid-exchange rate assay is also applicable for the evaluation of a nanometer size channel connecting two vesicles when the micrometer volumes of the vesicles are measured in advance.

Materials and Reagents

  1. Materials
    1. Pipette tips (Corning, Axygen, catalog numbers: T-1000-B, T-200-Y, T-300)
    2. 100 mm culture dish (Thermo Fisher, Nunc, catalog number: 150462)
    3. 35 mm glass bottom culture dish (Thermo Fisher, catalog number: 150682)
    4. 1.5 ml Eppendorf tube (Corning, Axygen, catalog number: MCT-150-C)
    5. 50 ml, 15 ml centrifuge tubes (Thermo Fisher, catalog numbers: 339652, 339650)
    6. Gene Pulser (Bio-Rad, catalog number: 165-2086)

  2. Biological materials
    1. 3T3-L1 pre-adipocyte (ATCC, catalog number: CL-173)
    2. Cidec-GFPN1 plasmid (Wang et al., 2018; available from the corresponding author upon request)
    Note: The full-length Cidec gene (NCBI accession number: NM_178373) was inserted into pEGFPN1 plasmid at the Xhol and EcoRI restriction enzyme sites.

  3. Reagents
    1. BODIPY 558/568 C12 (Thermo Fisher, Molecular Probes, catalog number: D3835)
    2. Sodium oleate (Sigma-Aldrich, catalog number: O7501)
    3. Dulbecco’s modified Eagle’s medium (DMEM), high glucose, with L-Glutamine and Phenol Red (Gibco, catalog number: 11965084)
    4. Fetal bovine serum (FBS) (Gibco, catalog number: 16140071)
    5. Penicillin-streptomycin Mixed Solution (P/S) (Gibco, catalog number: 15140122)
    6. Electroporation buffer (Bio-Rad, catalog number: 1652676)
    7. Trypsin-EDTA (Thermo Fisher, Life Technologies, catalog number: 25200-072)
    8. NaCl (Sigma-Aldrich, catalog number: S3014)
    9. KCl (Sigma-Aldrich, catalog number: P9541)
    10. Na2HPO4 (Sigma-Aldrich, catalog number: S5136)
    11. KH2PO4 (Sigma-Aldrich, catalog number: P9791)
    12. CaCl2 (Sigma-Aldrich, catalog number: C5670)
    13. MgCl2 (Sigma-Aldrich, catalog number: M4880)
    14. Phosphate-buffered saline (PBS) (see Recipes)

Equipment

  1. Pipettes (Gilson, models: PIPETMAN P10, P20, P200, P1000)
  2. Cell counting chamber (Easybio, catalog number: BE6138)
  3. 37 °C, 5% CO2 cell culture incubator (NuAire, model: NU-49SOE)
  4. Biosafety cabinet (NuAire, model: NU-425-400E)
  5. Centrifuge (Cence, China, model: TDZ5-WS)
  6. Electroporation (Lonza, model: Amaxa Nucleofactor II)
  7. Confocal fluorescent microscope (Nikon Instruments, model: Nikon A1+ Confocal Microscope)
  8. Live cell station (Oko laboratory, model: A1 Confocal)
  9. Computer (Lenovo, model: ThinkStation P510)

Software

  1. NIS-element analysis (Nikon, https://www.microscope.healthcare.nikon.com/products/software)
  2. Fiji (NIH software, http://fiji.sc/Fiji)
  3. Open-source plugin-based image analysis software based on ImageJ (https://imagej.nih.gov/ij/)
  4. LabVIEW 8.5 with a plug-in installation of NI Vision 8.6 module (National Instruments, http://www.ni.com/en-us/support/downloads/software-products/download.labview.html)
  5. Custom-made LabVIEW modules:
    1. 1_Exchange rate assay.llb (applied in Step B of Data analysis)
    2. 2_Check a fitting region.vi (applied in Step C of Data analysis)
    3. 3_Calculation of exchange rate.vi (applied in Step D of Data analysis)
  6. Prism 5 (GraphPad Inc.)

Procedure

Theory: For the estimation of LD fusion ability, we measure a lipid-exchange rate as an alternative indicator of potential pore size between two contacted LDs. During LD fusion, the lipids constantly exchange between the two LDs. For the measurement of the lipid-exchange rates between the LDs in live cells, the small (donor) LD of each LD pair pre-stained with BODIPY-C12 was photobleached. The fluorescence-labeled lipids in the large (acceptor) LD diffused into the small LD, and the photobleached lipids went into the large LD. The images of the small and large (acceptor) LDs, before and after photobleaching, were recorded and their fluorescent intensities were normalized to the initial fluorescent intensity of the large LD at time 0 s after photobleaching, giving rise to the initial intensity ratio of 1 for the large LD. The ratio of fluorescent intensity of the photobleached small LD to the large LD at time 0 s is denoted as G0. At any time after photobleaching, the time-dependent ratios of the fluorescent intensities of the large and small LDs are denoted as G1 (t) and G2 (t), respectively. Therefore, a time-invariable exchange rate (∅e) of the neutral lipid molecules between an LD pair is calculated from the measurement of the fluorescence recovering rate in the LD pair according to the ordinary differential equation as follows:



where V1 and V2 represent the volumes of the large and small LDs, respectively. The unit of the lipid-exchange rate is μm3/s. The detailed equation derivation is shown in Supplementary Information.


  1. Overall procedure
    The procedure for the lipid-exchange rate assay of LDs in live cells is shown in Figure 1.


    Figure 1. Schematics of LD lipid-exchange rate measurement in live cells

  2. Cell preparation
    1. Culture 3T3-L1 pre-adipocytes in 10 cm plastic dish in 10 ml DMEM supplemented with 10% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin.
    2. At 80% confluence, trypsinize the cells using 2 ml 0.25% trypsin solution. Terminate the process using 5 ml DMEM. Transfer the cells with DMEM into a 15 ml tube and centrifuge at 150 x g for 3 min, discard the supernatant.
    3. Resuspend the cell using 1 ml PBS.
    4. Count cells using a cell counting chamber.
    5. Centrifuge at 150 x g for 3 min, discard the supernatant.
    6. Resuspend the cells with appropriate electroporation buffer to reach a cell density of 2-3 x 106/ml.
    7. Aspirate 100 μl of cells, mix with 0.5-1 μg plasmid (Cidec-GFP) in a 1.5 ml Eppendorf tube.
    8. Transfer the cell-DNA mix into a genepulser, place the genepulser into Nucleofector II, choose the program A-033, which is optimized for 3T3-L1 pre-adipocytes transfection, execute the electroporation step.
    9. Add 100 μl of DMEM into the genepulser, aspirate out the mixture into a 15 ml centrifuge tube with 2 ml DMEM medium.
    10. Add BODIPY-C12 558/568 into the medium to a final concentration of 1 μg/ml.
    11. Add sodium oleate solution into the medium to a final concentration of 200 μM to provide lipids for LD formation and growth in cells.
    12. After the addition of the above reagents, aspirate the mixture into a 35 mm glass bottom culture dish and incubate for 18-20 h in an incubator at 37 °C, 5% CO2.
    13. Change the culture medium with 2 ml fresh DMEM for two reasons: 1) for the removal of BODIPY-C12 dye from the medium to eliminate background fluorescence; 2) for the removal of sodium oleate to reduce the influence of triglycerides synthesis on LD fusion. Next, place the culture dish with the cells in the incubator for 1 h before FRAP experiments to help the cells accommodate to the changed medium.

  3. Fluorescence recovery after photobleaching (FRAP) image acquisition
    The module of FRAP is conventional in most commercial confocal microscopes. Here, we used a Nikon A1 confocal microscope to perform FRAP measurements as an example. This following procedures can also be used for FRAP experiments when using the other confocal microscopes.
    1. Turn on the live cell station, set the temperature to 37 °C, switch on the 5% CO2, add an appropriate amount of double distilled water in a heating trough.
    2. Turn on the confocal microscope, laser generator, microscope controller, mercury lamp, electric automatic stage, z-axis piezoelectric stage, and computer workstation. Turn on the perfect focus system (PFS). Launch the Nis-element software. Choose the 100x oil-immersion objective (Numerical aperture 1.45). Add objective oil dropwise onto the objective, place the 35 mm glass bottom culture dish onto the sample holder, and adjust the height of the objective to immerse the culture dish in the oil. Allow the cells to accommodate for 1 h if possible, or at least 0.5 h to ensure the stability of the following measurement.
    3. Set the parameters of the microscope as shown in Figure 2. Click on the channel button, select “Fluorescein isothiocyanate (FITC)” and “Tetramethylrhodamine (TRITC)” channels. Choose “none” in Channel series. To capture the fluorescent images, fine settings of the optimized laser power and the gain and sensitivity of detector are critical. There are several commercial microscopes but their definitions of the setting parameters are different. Here, we used Nikon A1 confocal microscope as an example to show our settings, in which “Laser power” indicates the level of real laser power, “HV” indicates the gain of detector (brightness of images), and “Offset” indicates the sensitivity of detector (contrast of images). Thus, set the FITC laser power to “6.0”, HV “60-120”, off set “0”; the TRITC laser “0.3”, HV “60-120”, off set “0”. Set pinhole as home under the wavelength of 561 nm. Set scan size to “512 x 512 pixels”, scan speed “1 frame/s”, zoom “3.0”, and line average “none”.


      Figure 2. NIS-element settings of images acquisition. The image shows the settings of the laser channel, laser power, HV, Offset, pinhole size, scan size, scan speed, and other detailed parameters for image acquisition.

    4. Set the photobleaching parameters:
      1. Switch on the photoactivation interface as shown in Figure 3. Set the photobleaching laser as the wavelength of 561 nm, power of 100%. Set the stimulation scanning speed as 0.25 sec/frame.


        Figure 3. NIS-element settings of photobleaching. The image shows the settings of laser power and scan speed for photobleaching.

      2. Go to the N-dimensional (ND) Stimulation Interface as shown in Figure 4, in which we could set the XYZ locations of a few images captured and the time schedule options. Create three phases corresponding to the three-steps in time-sequential operations. In Phase 1, choose “Acquisition” in Acq/Stim, “No delay” in Interval, a duration of 1.06 s and 1 loop to capture one image, which shows the initial fluorescent intensities of an LD pair before photobleaching. In Phase 2 and Phase 3, set 3x in Group option. In addition, in Phase 2, set “Stimulation” in Acq/Sim, “S1” in ROIs and 1 loop to perform photobleaching. The stimulation duration will be automatically set according to the setting of stimulation scanning speed in Figure 3. In Phase 3, choose “Acquisition” in Acq/Stim, “1.06 s” in time Interval, and 30 (fast exchange rate) or 50 (slow exchange rate) loops to capture time-lapse images with a time interval of 1.06 s, which record the change in the fluorescent intensities of the LD pair after photobleaching. Subsequently, the duration is automatically calculated. Choose “Perform Time Measurement”. Check “Save to File”, input the path and name the file.


        Figure 4. NIS-element settings of ND Stimulation. The image shows three phases corresponding to the three-step time-sequential operations in the ND stimulation setting.

      3. Click on the button “Time measurement” in the menu to stick the time measurement interface in front of all interfaces. The interface is used to monitor the change in fluorescent signals of the selected regions-of-interest over time.

    5. Find target LD pairs:
      1. Click on the FITC or GFP button in the interface of the NIS-element software and switch to the microscope control mode. Using the mercury lamp illumination, select an LD pair with GFP signal enriched at the LD-LD contact site (LDCS) as shown in Figure 5. Adjust the focus plane of the objective to get a clear image of the LD pair. The diameters of the selected donor LDs are preferred to be in the range of 3-6 μm to reduce variation.


        Figure 5. An example image of a suitable LDCS. The image shows the red signal of BODIPY-C12 stained LD pairs and the green signal of Cidec-GFP enriched at the LDCS. Scale bar, 5 μm.

      2. Switch to the computer control mode by clicking on the button “A1”. Click on the button “Laser interlocked” to stimulate the laser. Click on the button “Live” to preview the LD pair. Move the cursor on the interface of the software to the LDCS, right click on the mouse, choose “move the point to the center of the image” to place the LD pair at the center of the vision. Scroll the mouse to adjust the focus plane of the objective once the LD pair went out of focus. Adjust HV in GFP and TRITC channels to achieve the highest fluorescent intensity without overexposure as the photograph taken by detector. Press the button “PFS” on the microscope front panel to lock the imaging plane during the acquisition process.
      3. Click on the ROI button as shown in Figure 6, select the round circle, draw a circle covering about 70% area of the donor LD at its center, and denote as S1 (stimulation ROI). The reason for photobleaching donor LDs is described in the Notes section below. Next, draw another circle at the center of the donor LD that covers about 70% area of the donor LD; Draw a third circle at the center of the acceptor LD that covers about 70% area of the acceptor LD. The reason for selecting 70% area is dependent on our experience with the optimal HV value and bleaching laser power used. It is possible to adjust the percentage area on case by case basis. In addition, the ROIs selection here was only used to preview the real-time fluorescent intensities of the BODIPY-C12 dyes in LDs and to pre-check the fluorescence recovery data.


        Figure 6. ROI settings. The image shows the ROIs and the schematic diagram of circles on LDs. S1, a photobleaching region on the donor LD of an LD pair; Circle 2, the monitoring ROI on the photobleaching donor LD; Circle 3, the monitoring ROI on the acceptor LD.

    6. Execute the photobleaching process. Before the process, make sure the LD pair is ready on the imaging plane and all the parameters are set correctly. Click on the button “run now” in the ND stimulation interface and allow the program to complete.
    7. Look for next LD pairs and repeat Steps C5 and C6 to acquire FRAP data for a series of LD pairs.

Data analysis

Below, we will demonstrate two alternative methods to analyze the FRAP image data. Specifically, the first method is a partial manual measurement, using Fuji software. The second method is a totally automatic measurement, using custom-built codes in LabVIEW program. After testing on cases of LD pairs ranged from 3 to 6 μm in diameter, the final FRAP results measured using both the above methods are almost alike. But, in view of the other to-be-measured LD pairs maybe of different sizes and fluorescent intensities, as well as the potential limitation of the custom-built LabVIEW program to handle any condition of FRAP data, we also provide the manual method to process the most difficult cases. For example, for donor LDs with a diameter smaller than 1 μm. However, if possible, we recommend the automatic method.


  1. Mean of intensity (MOI) and size measurement of BODIPY-C12 stained LDs by manual
    1. Open the Fiji software.
    2. Go to “File” → “Import” → “Image Sequence”, to open a series of time-sequential images (8-bit TIF format files, the data in Figure 7 is used as an example).


      Figure 7. Import a series of 8-bit TIF format images. A. The menu of the Fiji software. B. The sequence options of the imported image sequence. C. A representative set of time-sequential images input. D. The scale of the input images.

    3. To set the scale bar of the images, go to “Analysis” → “Set Scale”. Here the calibrated scale is 12.107 pixels/μm as shown in Figure 7D.
    4. To obtain a series of “Mean gray values (Mean)” information of a large LD over time.
      1. Select at least 70% of large LD area with “Oval” tool.
      2. Go to “Analyze” → “Measure”, to get “Area”, “Mean gray value (Mean)”, “Min gray value (Min)”, and “Max gray value (Max)” information of the large LD.
      3. Press on the button “►” as shown in the lower right of Figure 7C or “>” on the keyboard to go to the next frame of the sequential images.
      4. Click the center of the selected circle and move the circle to the center of the large LD in the next frame with drag-and-drop.
      5. Repeat steps b-d to obtain a series of information of the large LD to the last frame (Figure 8).


        Figure 8. “Mean gray values (Mean)” data of a large LD in a series of time-lapse fluorescent images quantified by Fiji. Here, the total number of frames analyzed in this image is 91 with a time interval of 1.12 s

    5. Save the data set of the large LD as an Excel file.
    6. To obtain a series of “Mean” information of a small LD over time.
      1. Press on the back button “◄” as shown in the lower left of Figure 9A or “<” on the keyboard to return to the first frame of the sequential images (for example press 90 times for the sequential set of 91 images).
      2. Re-select at least 70% of the small LD area with “Oval” tool.
      3. Go to “Analyze” → “Measure”, to get “Area”, “Mean gray value (Mean)”, “Min gray value (Min)”, and “Max gray value (Max)” information of the small LD.
      4. Press on the button “►” or “>” to go to the next frame of the sequential images.
      5. Click the center of the selected circle and move the circle to the center of the small LD in the next frame with drag-and-drop.
      6. Repeat steps c-e to get a series of information of the small LD to the last frame (Figure 9).


        Figure 9. “Mean gray values (Mean)” data of a small LD in a series of time-lapse fluorescent images quantified by Fiji

    7. Save the data set of the small LD as an Excel file.
    8. To obtain a series of “Size” information in the region of the large LD over time.
      1. Press on the back button “◄” or “<” to return to the first frame of the sequential images (for example press 90 times for the sequential set of 91 images).
      2. Measure the size of the large LD with the “Line” tool.
      3. Go to “Analyze” → “Measure”, to get “Area”, “Mean gray value (Mean)”, “Min gray value (Min)”, “Max gray value (Max)”, “Angle”, and “Length” information of the large LD. For the following analytical process, only “Length” information is required.
      4. Press on the button “►” or “>” to go to the next frame of the sequential images.
      5. Repeat steps b-d to get a series of the size information of the large LD to the last frame (Figure 10).


        Figure 10. “Size” data of a large LD in a series of time-lapse fluorescent images quantified by Fiji

    9. Save the data set of the large LD as an Excel file.
    10. To obtain a series of “Size” information of the small LD over time.
      1. Press on the back button “◄” or “<” to return to the first frame of the sequential images (for example press 90 times for the sequential set of 91 images).
      2. Measure the size of the small LD with the “Line” tool to obtain a series of the size information of the small LD until the last frame by repeating the same steps 8b-8e for the large LD measurement (Figure 11).


        Figure 11. “Size” data of a small LD in a series of time-lapse fluorescent images quantified by Fiji

    11. Save the data set of the small LD as an Excel file.
    12. To get the plot of time-sequential MOIs over time, only “Mean” value from the intensity measurement and the “Length” values from the size measurement are required. Next, reformat the data sets as a new Excel file as shown in Figure 12. The data is now ready for the subsequent analysis in Step C of Data analysis.


      Figure 12. Merge and reformat the data sets in a new Excel file

    13. Plot the sequential MOIs of the two regions-of-interest of the LD pair over time in the Excel software (Figure 13).


      Figure 13. Plot of sequential MOIs over time

  2. MOI and size measurements of BODIPY-C12 stained LDs using an automatic identification program
    1. Open the LabVIEW software (here using the version of LabVIEW 8.5 with a plug-in installation of NI Vision 8.6 module).
    2. Double-click the LabVIEW sub-llb file “1_Exchange rate assay.llb” to open the main module “Main program.vi” as shown in Figure 14. The interface of the sub-VI program is shown in Figure 15.


      Figure 14. The dialog box of LLB manager


      Figure 15. The interface of the main module “Main program.vi”. Red asterisks indicate a set of regular steps in a standard procedure. Follow the steps to obtain the series of information of each LD pair over time.

    3. To obtain a series of “Mean” information of the large and small LDs over time.
      1. Set the scale of input images marked by “Scale” in Figure 15 (here using the value of 12.107 pixels/μm).
      2. Run the sub-VI program by pressing on the button marked by “Step 1” in Figure 15 or alternatively press on a combination keyboard button “Ctrl + R.”
      3. Press on “Load file” marked by “Step 2” in Figure 15 to open a series of time-sequential images (8-bit TIF format files, here using the data of Figure 16A as example).
      4. Select the whole large LD area with a default “Square” tool by drag-and-drop directly on the image as shown in Figure 16B and press on “Learn Large Template” marked by “Step 3” in Figure 15 to record the template image of the large LD.
      5. Repeat step d for the small LD: select the whole small LD area with “Square” tool as shown in Figure 16C and press on “Learn Small Template” marked by “Step 4” in Figure 15 to record the template image of the small LD.


        Figure 16. Loading an image set and recording the template images of large and small LDs. A. A set of time-sequential images of an LD pair were loaded into the computer memory and showed accordingly on the interface of the main program. B. A template image of a large LD was selected with “Square” tool and recorded into the computer memory as a reference for the subsequent identification of the large LD in the next frames. C. A template image of a small LD was selected with “Square” tool and recorded into the computer memory as a reference for the subsequent identification of the small LD in the next frames. Scale bars, 5 μm.

      6. Select “Large” or “Small” in the Search Test menu and press on the button “Search” marked by “Step 5” in Figure 15 to confirm the accurate identification of the selected large or small LDs (Here showing Figure 17 as an example).


        Figure 17. Confirmation of the identified LD size. Select “Large” (A) or “Small” (C) to check that the identification of the selected large (B) or small (D) LD is accurate. Scale bars, 5 μm.

      7. Select “Large” in the Search Test menu and press on the button “Analysis” marked by “Step 6” in Figure 15 to get a series of “Frame”, “Mean gray value (Mean)”, “Min gray value (Min)”, and “Size” information of the large LD over time automatically.
      8. Select “Small” in the Search Test menu and press on the button “Analysis” again to get a series of “Frame”, “Mean gray value (Mean)”, “Min gray value (Min)”, and “Size” information of the small LD over time automatically.
      9. Press on the button “Load file” marked by “Step 2” in Figure 15 to input the next set of time-sequential images and repeat steps d-h to get the series of information of the next LD pairs over time. Or press on the button “Return” marked by “Finish” in Figure 15 after the completion of the MOI measurement of the LD pairs.
        Note: After pressing either the “Load file” or “Return” button, the series of Mean and Size information of the larger and smaller LDs will be saved as an Excel file automatically. The generated Excel file is in ready format for the subsequent analysis in Step C of Data analysis.
    4. Check the data sets of the LD pairs saved previously as an Excel file (here using the two sets of data as example).
    5. Plot the sequential MOIs of the two regions-of-interest in the individual LD pairs over time in the Excel software (Figure 18).
    6. Create a folder name “MOI&Size” and copy the Excel data set into the folder for the subsequent exchange rate analysis. Transfer the folder with these data into the same folder where there are LabVIEW sub-VIs “2_Check a fitting region.vi” and “3_Calculation of exchange rates.vi” files.


      Figure 18. Plot of sequential MOIs over time. Data shown in (A) are the same data shown in Figure 13. B. In contrast, the MOIs were obtained using another set of time-sequential images as mentioned in Step B3i of Data analysis.

  3. Pre-estimation of the exchange rate underlying linear criteria
    After the imaging process and obtaining “MOI” and “Length” information generated in Step A or B of Data analysis, the Excel files are now ready for the following analysis. Here, we show the pre-estimation process of the exchange rate.
    1. Open the LabVIEW software.
    2. Open the LabVIEW sub-VI “2_Check a fitting region.vi” file. The interface of the sub-VI program is shown in Figure 19.


      Figure 19. The interface of sub-VI “2_Check a fitting region.vi”. Here using the data from Figure 18B as an example.

    3. Obtain the optimal fitting range of an LD pair’s exchange rate.
      1. Run the sub-VI program by pressing on the button marked by “Periodic running” in Figure 19.
      2. Adjust and choose the maximum value of the fitting range marked by “Fitting range” in Figure 19 to ensure the linearity (MSE) and repetitiveness (%) of the adjusted fitted lines satisfy the experienced optimal values (MSE < 0.01 and % < 30%). We define the two upper limit values as linear criteria.
      3. Record each optimal fitting range for each set of LD pair measured.

  4. Calculation of the exchange rates between individual fused LD pairs
    1. Open the LabVIEW sub-VI “3_Calculation of exchange rates.vi” file. The interface of the sub-VI program is shown in Figure 20.


      Figure 20. The interface of sub-VI “3_Calculation of exchange rates.vi”

    2. Input all the optimal fitting ranges in the table marked by “Optimal fitting range” as shown in Figure 21A.
    3. Run the sub-VI program by pressing on “Ctrl + R” to obtain the mean ± SD values of the exchange rates. Exchange rates were plotted against large or small LD sizes as shown in Figure 21B.


      Figure 21. Results of running the sub-VI “3_Calculation of exchange rates.vi”. A. Table of optimal fitting range. B. A plot showing the exchange rates against the sizes of large and small LDs.

Notes

  1. The photobleaching process has to be performed on the smaller (namely donor) LD of an LD pair three times for statistical analyses as described in our original publication (Wang et al., 2018). Briefly, because there are more fluorescent molecules in the larger LDs, photobleaching these large LDs more than twice will result in the rapid decline of the fluorescent intensities in the LD pair and pose difficulty in the fluorescence recovery detection.
  2. The most critical step in this protocol is to obtain the optimal linearity (mse) and repetitiveness (%) of three fitted lines as described in “Procedure C”. If either the evaluated linearity or the repetitiveness of the fitted lines in the three cycles measured is out of the linear criteria, the data set should be discarded as these indicate that the exchange rate event might be unstable during the acquisition interval. These unstable exchange rates possibly resulted from the physical size change of the LD pair, the height of imaging plane, deformation of LD fusion complex, and serious photobleaching et al. We recommend 50 sets or more of the lipid-exchange rates to be measured. In general, 30-40 events of lipid-exchange is sufficient to satisfy the criteria if acquired under an optimal environmental control condition.

Recipes

  1. Phosphate-buffered saline (PBS)
    1. Mix 1.37 M NaCl, 25 mM KCl, 8 mM Na2HPO4, 14.7 mM KH2PO4, 9 mM CaCl2, and 5 mM MgCl2 in 900 ml deionized H2O
    2. Adjust to a final pH of 7.4
    3. Add deionized H2O to 1,000 ml
    4. Autoclave to store (This is 10x PBS stock)
    5. Dilute PBS stock with deionized H2O to 1x PBS

Acknowledgments

We thank the members of P. Li Laboratory at Tsinghua University for their helpful discussions and Jinyu Wang at SLSTU-Nikon Biological Imaging Center for imaging support. This work was supported by grants from National Basic Research Program Grants 2018YFA0506900 (to P.L.) and National Natural Science Foundation of China Grants 91857103 (to F.J.C), 31430040, 31690103, and 31621063 (to P.L.). This protocol was adapted from our previous publication in the Journal of Biological Chemistry (Wang et al., 2018).

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. Choudhary, V., Ojha, N., Golden, A. and Prinz, W. A. (2015). A conserved family of proteins facilitates nascent lipid droplet budding from the ER. J Cell Biol 211(2): 261-271.
  2. Farese, R. V., Jr. and Walther, T. C. (2009). Lipid droplets finally get a little R-E-S-P-E-C-T. Cell 139(5): 855-860.
  3. Fujimoto, Y., Itabe, H., Kinoshita, T., Homma, K. J., Onoduka, J., Mori, M., Yamaguchi, S., Makita, M., Higashi, Y., Yamashita, A. and Takano, T. (2007). Involvement of ACSL in local synthesis of neutral lipids in cytoplasmic lipid droplets in human hepatocyte HuH7. J Lipid Res 48(6): 1280-1292.
  4. Gao, G., Chen, F. J., Zhou, L., Su, L., Xu, D., Xu, L. and Li, P. (2017). Control of lipid droplet fusion and growth by CIDE family proteins. Biochim Biophys Acta Mol Cell Biol Lipids 1862(10 Pt B): 1197-1204.
  5. Gluchowski, N. L., Becuwe, M., Walther, T. C. and Farese, R. V., Jr. (2017). Lipid droplets and liver disease: from basic biology to clinical implications. Nat Rev Gastroenterol Hepatol 14(6): 343-355.
  6. Gong, J., Sun, Z., Wu, L., Xu, W., Schieber, N., Xu, D., Shui, G., Yang, H., Parton, R. G. and Li, P. (2011). Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites. J Cell Biol 195(6): 953-963.
  7. Gross, D. A., Zhan, C. and Silver, D. L. (2011). Direct binding of triglyceride to fat storage-inducing transmembrane proteins 1 and 2 is important for lipid droplet formation. Proc Natl Acad Sci U S A 108(49): 19581-19586.
  8. Krahmer, N., Farese, R. V., Jr. and Walther, T. C. (2013). Balancing the fat: lipid droplets and human disease. EMBO Mol Med 5(7): 973-983.
  9. Rosen, E.D. and Spiegelman, B.M. (2014). What we talk about when we talk about fat. Cell 156(1-2): 20-44.
  10. Sun, Z., Gong, J., Wu, H., Xu, W., Wu, L., Xu, D., Gao, J., Wu, J. W., Yang, H., Yang, M. and Li, P. (2013a). Perilipin1 promotes unilocular lipid droplet formation through the activation of Fsp27 in adipocytes. Nat Commun 4: 1594.
  11. Sun, Z., Gong, J., Wu, L. and Li, P. (2013b). Imaging lipid droplet fusion and growth. Methods Cell Biol 116: 253-268.
  12. Wang, J., Yan, C., Xu, C., Chua, B. T., Li, P. and Chen, F. J. (2018). Polybasic RKKR motif in the linker region of lipid droplet (LD)-associated protein CIDEC inhibits LD fusion activity by interacting with acidic phospholipids. J Biol Chem 293(50): 19330-19343.
  13. Wilfling, F., Wang, H., Haas, J. T., Krahmer, N., Gould, T. J., Uchida, A., Cheng, J. X., Graham, M., Christiano, R., Frohlich, F., Liu, X., Buhman, K. K., Coleman, R. A., Bewersdorf, J., Farese, R. V., Jr. and Walther, T. C. (2013). Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets. Dev Cell 24(4): 384-399.
  14. Wu, L., Xu, D., Zhou, L., Xie, B., Yu, L., Yang, H., Huang, L., Ye, J., Deng, H., Yuan, Y. A., Chen, S. and Li, P. (2014). Rab8a-AS160-MSS4 regulatory circuit controls lipid droplet fusion and growth. Dev Cell 30(4): 378-393.
  15. Xu, D., Li, Y., Wu, L., Li, Y., Zhao, D., Yu, J., Huang, T., Ferguson, C., Parton, R. G., Yang, H. and Li, P. (2018). Rab18 promotes lipid droplet (LD) growth by tethering the ER to LDs through SNARE and NRZ interactions. J Cell Biol 217(3): 975-995.
  16. Yang, H., Galea, A., Sytnyk, V. and Crossley, M. (2012). Controlling the size of lipid droplets: lipid and protein factors. Curr Opin Cell Biol 24(4): 509-516.
  17. Somwar, R., Roberts Jr., C. T. and Varlamov, O. (2011). Live-cell imaging demonstrates rapid cargo exchange between lipid droplets in adipocytes. FEBS Lett 585(12):1946-1950.

简介

LD生长缺陷通常会导致代谢紊乱的发生.LD融合和DNA片段化因子α(DFF)介导的生长Gong 等人的先前方法。 >(2011)首先报道了脂质交换速率测定,以评估由CIDE家族蛋白及其调节剂介导的细胞中每个LD对的融合能力,但仍存在问题的问题,并且未提供详细的程序。该测定的三个关键步骤是细胞制备,图像采集和数据分析通过光漂白后获得荧光恢复程度,然后测量LD对中的强度变化。荧光强度的差异程度用于获得LD之间的泄漏程度。精确度和重复性质的性质通过这种定量分析,我们能够确定关键蛋白的功能作用及其突变体对LD融合的影响。
【背景】脂质滴(LDs)在维持脂质稳态中起着关键作用(Farese和Walther,2009; Yang et al。,2012)。缺乏LD成熟和生长与代谢疾病的发展密切相关,如作为肥胖症,脂肪肝病,心血管疾病和II型糖尿病(Krahmer 等人,<2013; Rosen和Spiegelman,2014; Gluchowski等人,2017)。 LD是从内质网(ER)发芽的动态细胞器(Gross et al。,2011; Choudhary et al。,2015),并通过甘油三酯合成继续生长。来自ER的脂质转移(Fujimoto et al。,2007; Wilfling et al。,2013; Xu et al。,2018)或LD融合(Gong et al。,2011).LD相关细胞死亡诱导DNA片段化因子α样效应蛋白(CIDE)家族蛋白,包括CIDEA,CIDEB和CIDEC / Fsp27(Gao et al。,2017)是控制非典型L的脂质体内平衡的关键调节剂以前,我们已经报道了CIDEC通过从小(供体)到大(受体)LD的定向脂质转移来介导LD融合(Gong et al。,2011)。评估LD-LD接触部位(LDCS)的融合能力和融合孔的形成是脂质交换和转移发生的两个必要步骤。评估每种LD对在CIDE家族蛋白和它们介导的细胞中的融合能力。调节剂如Perilipin 1(Sun et al。,2013a)或Rab 8a(Wu et al。,2014),脂质交换率测定首先被提出并如前所述进行(Gong et al。,2011; Sun et al。,2013 b)。然而,之前的方法报告的分析过程消除了激光曝光的照片效果确保实验重现性和准确性的详细程序。

细胞分析中的三个关键步骤,包括细胞回收,光漂白后的荧光恢复(FRAP)图像在使用LD的强度平均值(MOI)和BODIPY-C12染色尺寸测量值进行数据分析中的线性标准时,执行三循环光漂白过程和预测汇率。这种脂质交换速率测定也适用于纳米弹性体的评估,因为它用于评估拟合的电子交换率,使用我们在理论部分中进行分子热力学的方程式。预先测量囊泡的数量。

关键字:脂代谢, 脂滴融合, 脂交换速率, 光脱色荧光恢复技术, 脂滴大小

材料和试剂

  1. 材料
    1. 移液器吸头(Corning,Axygen,目录号:T-1000-B,T-200-Y,T-300)
    2. 100毫米培养皿(Thermo Fisher,Nunc,目录号:150462)
    3. 35毫米玻璃底培养皿(Thermo Fisher,目录号:150682)
    4. 1.5毫升Eppendorf管(Corning,Axygen,目录号:MCT-150-C)
    5. 50毫升,15毫升离心管(Thermo Fisher,目录号:339652,339650)
    6. Gene Pulser(Bio-Rad,目录号:165-2086)

  2. 生物材料
    1. 3T3-L1前体细胞(ATCC,目录号:CL-173)
    2. Cidec-GFPN1质粒(Wang et al。。,2018;根据要求可从相应作者处获得)
    注意:将全长Cidec基因(NCBI登录号:NM_178373)插入XhoI和EcoRI限制酶位点的pEGFPN1质粒中。

  3. 试剂
    1. BODIPY 558/568 C12(Thermo Fisher,Molecular Probes,目录号:D3835)
    2. 油酸钠(Sigma-Aldrich,目录号:O7501)
    3. Dulbecco改良的Eagle's培养基(DMEM),高葡萄糖,含L-谷氨酰胺和酚红(Gibco,目录号:11965084)
    4. 胎牛血清(FBS)(Gibco,目录号:16140071)
    5. 青霉素 - 链霉素混合溶液(P / S)(Gibco,目录号:15140122)
    6. 电穿孔缓冲液(Bio-Rad,目录号:1652676)
    7. 胰蛋白酶-EDTA(Thermo Fisher,Life Technologies,目录号:25200-072)
    8. NaCl(Sigma-Aldrich,目录号:S3014)
    9. KCl(Sigma-Aldrich,目录号:P9541)
    10. Na 2 HPO 4 (Sigma-Aldrich,目录号:S5136)
    11. KH 2 PO 4 (Sigma-Aldrich,目录号:P9791)
    12. CaCl 2 (Sigma-Aldrich,目录号:C5670)
    13. MgCl 2 (Sigma-Aldrich,目录号:M4880)
    14. 磷酸盐缓冲盐水(PBS)(见食谱)

设备

  1. 移液器(Gilson,型号:PIPETMAN P10,P20,P200,P1000)
  2. 细胞计数室(Easybio,目录号:BE6138)
  3. 37°C,5%CO 2 细胞培养箱(NuAire,型号:NU-49SOE)
  4. 生物安全柜(NuAire,型号:NU-425-400E)
  5. 离心机(Cence,中国,型号:TDZ5-WS)
  6. 电穿孔(Lonza,型号:Amaxa Nucleofactor II)
  7. 共聚焦荧光显微镜(尼康仪器,型号:尼康A1 +共聚焦显微镜)
  8. 活细胞站(Oko实验室,型号:A1 Confocal)
  9. 电脑(联想,型号:ThinkStation P510)

软件

  1. NIS元素分析(尼康, https://www.microscope.healthcare.nikon.com/产品/软件)
  2. 斐济(NIH软件, http://fiji.sc/Fiji )
  3. 基于ImageJ的基于开源插件的图像分析软件( https://imagej.nih.gov/ij/ < / a>)
  4. LabVIEW 8.5带有NI Vision 8.6模块的插件安装(National Instruments, http://www.ni.com/en-us/support/downloads/software-products/download.labview.html )
  5. 定制的LabVIEW模块:
    1. 1_Exchange rate assay.llb(应用于数据分析的步骤B)
    2. 2_检查拟合region.vi(在数据分析的步骤C中应用)
    3. 3_兑换rate.vi(应用于数据分析的步骤D)
  6. Prism 5(GraphPad Inc.)

程序

理论:为了估计LD融合能力,我们测量脂质交换率作为两个接触的LD之间潜在孔径的替代指标。为了测量活细胞中LDs之间的脂质交换速率,用BODIPY-C12预染色的每个LD对的小(供体)LD被光漂白。荧光标记的脂质在大(受体)中记录光漂白之前和之后的小和大(受体)LD的图像,并将它们的荧光强度归一化为大LD的初始荧光强度。光漂白后0秒,大LD的初始强度比为1,光漂白小LD的荧光强度与0时的大LD的比率表示为 G > 0 。之后的任何时间光漂白,大强度小LD的时间相关比率表示为 G 1 ( t )和G 2 ( t ),可观的是,中性脂质分子的时间不变的汇率(∅ e )根据常规微分方程,根据LD对中荧光恢复率的测量计算LD对之间的关系:



其中 V 1 和 V 2 分别代表小型和大型LD的体积。详细的等式推导显示在补充信息。


  1. 整体程序
    活细胞中LDs的脂质交换率测定程序如图1所示。


    图1.活细胞中LD脂质交换率测量的示意图

  2. 细胞制备
    1. 在10ml塑料培养皿中在10ml补充有10%FBS,100μg/ ml链霉素和100U / ml青霉素的DMEM中培养3T3-L1前脂肪细胞。
    2. 使用5ml DMEM终止该过程。将细胞用DMEM转移到15ml管中并在150μl离心机中离心3分钟,在80%汇合时,用2ml 0.25%胰蛋白酶溶液对细胞进行胰蛋白酶消化。丢弃上清液。
    3. 使用1ml PBS重悬细胞。
    4. 使用细胞计数室计数细胞。
    5. 在150 x g 下离心3分钟,弃去上清液。
    6. 用合适的电穿孔缓冲液重悬细胞,使细胞密度达到2-3×10 6 sup / ml。
    7. 吸出100μl细胞,在1.5ml Eppendorf管中与0.5-1μg质粒(Cidec-GFP)混合。
    8. 将细胞DNA混合物转移到genepulser中,将genepulser置于Nucleofector II中,选择针对3T3-L1前脂肪细胞转染进行优化的程序A-033,执行电穿孔步骤。
    9. 向基因推进器中加入100μlDMEM,将混合物吸出到带有2ml DMEM培养基的15ml离心管中。
    10. 将BODIPY-C12 558/568加入培养基中至终浓度为1μg/ ml。
    11. 将油酸钠溶液加入培养基中至终浓度为200μM,以提供LD形成和细胞生长的脂质。
    12. 加入上述试剂后,将混合物吸入35mm玻璃底培养皿中,在37℃,5%CO 2 的培养箱中孵育18-20小时。
    13. 用2ml新鲜DMEM更换培养基有两个原因:1)从培养基中除去BODIPY-C12染料以消除背景荧光; 2)除去油酸钠以减少甘油三酯合成对LD融合的影响。接下来,在FRAP实验之前将培养物与细胞一起放置在培养箱中1小时,以帮助细胞具有改变的培养基。

  3. 光漂白(FRAP)图像采集后的荧光恢复
    在这里,我们使用尼康A1共焦显微镜进行FRAP测量作为一个例子。当使用其他配置示波器时,以下程序也可用于FRAP实验。
    1. 打开活细胞站,将温度设置为37°C,打开5%CO 2,在加热槽中加入适量的双蒸水。
    2. 选择100x机油软件。启动Nis-element软件。在共聚焦显微镜,激光发生器,显微镜灯,电动自动平台,z轴压电平台和计算机工作站上选择100x油轮。让细胞在物镜上滴加浸油,将35毫米玻璃底培养皿放在样品架上,调节高度至物镜以浸没培养皿。如果可能,可容纳1小时,或至少0.5小时,以确保以下测量的稳定性。
    3. 设置显微镜的参数,如图2所示。单击通道按钮,选择“异硫氰酸荧光素(FITC)”和“四甲基罗丹明(TRITC)”通道。在通道系列中选择“无”。要捕获荧光图像,可以有几种商用显微镜,但它们对设置参数的定义不同。这里,我们以尼康A1闭合显微镜为例,显示优化激光功率的设置和灵敏度的增益。激光功率“表示实际激光功率的水平,”HV“表示检测器的增益(图像的亮度),”偏移“表示检测器的灵敏度(图像的对比度)。 TRITC激光器“0.3”,HV“60-120”,关闭设置为“0”。在561 nm波长下将针孔设置为原位。将扫描尺寸设置为“512 x 512像素”,扫描速度“1 f rame / s“,缩放”3.0“,行平均”无“。


      图2.图像采集的NIS元素设置。图像显示激光通道的设置,激光功率,HV,偏移,针孔尺寸,扫描尺寸,扫描速度以及图像的其他详细参数收购。

    4. 设置光漂白参数:
      1. 打开光活化界面,如图3所示。将刺激扫描速度设置为0.25秒/帧。


        图3.光漂白的NIS元素设置。图像显示了激光功率和光漂白扫描速度的设置。

      2. 转到N维(ND)刺激界面,如图4所示,我们可以设置捕获的一些图像的XYZ位置和时间表选项。创建与时序操作中的三个步骤相对应的三个阶段在阶段1中,在Acq / Stim中选择“采集”,在间隔中选择“无延迟”,持续1.06秒和1个循环以捕获一个图像,其显示在光漂白之前LD对的初始荧光强度。另外,在阶段2中,在Acq / Sim中设置“Stimulation”,在ROI中设置“S1”,在1个循环中设置进行光漂白。刺激持续时间将根据设置自动设置图3中的刺激扫描速度。在阶段3中,选择Acq / Stim中的“Acquisition”,时间间隔为“1.06 s”,30(快速汇率)或50(慢速汇率)循环以捕获时间推移图像时间间隔为1.06秒,记录荧光的变化随后,自动计算持续时间。选择“执行时间测量”。选中“保存到文件”,输入路径并命名文件。


        图4. ND刺激的NIS元素设置。图像显示了与ND刺激设置中的三步时序操作相对应的三个阶段。

      3. 该界面用于监测所选区域的荧光信号随时间的变化。

    5. 找到目标LD对:
      1. 使用水银灯照明,选择LD对,以及LD-LD接触点(LDCS)富集的GFP信号,如图所示LD的直径优选在3至6μm的范围内选择以减少变化。


        图5.合适的LDCS的示例图像。该图像显示BODIPY-C12染色的LD对的红色信号和在LDCS富集的Cidec-GFP的绿色信号。

      2. 单击“A1”按钮切换到计算机控制模式。单击按钮&nbsp;
      3. 单击“实时”按钮预览LD对。将界面上的光标移动到LDCS,右键单击鼠标,选择“将点移动到中心位置”。调整GFP和TRITC通道中的HV以获得最高荧光强度而不会过度曝光图像“将LD对置于视觉中心。一旦LD对失焦,滚动鼠标以调整物镜的焦平面。按下显微镜前面板上的“PFS”按钮,在采集过程中锁定成像平面。
      4. 单击ROI按钮,如图6所示,选择圆形圆圈,在其中心绘制一个覆盖供体LD约70%面积的圆圈,并表示为S1(刺激ROI)。光致LDs的原因描述于接下来,在供体LD的中心绘制约70%的供体LD面积的另一个圆;在受体LD的中心绘制约70%的受体LD的第三个圆。选择70%面积的原因取决于我们使用最佳HV值和漂白激光功率的经验。可以根据具体情况调整百分比面积。此外,此处的ROI选择仅用于预览论文LD中BODIPY-C12染料的实时荧光强度,并预先检查荧光恢复数据。


        图6. ROI设置。图像显示了LD上的圆的ROI和圆形示意图.S1,LD对的供体LD上的光漂白区域;圆2,光漂白的监测ROI Donor LD; Circle 3,受体LD上的监测ROI。

    6. 在此过程之前,确保LD对已在成像平面上准备就绪并且所有参数都已正确设置。单击ND刺激界面中的“立即运行”按钮并允许程序完成。
    7. 寻找下一个LD对并重复步骤C5和C6以获取一系列LD对的FRAP数据。

数据分析

具体来说,第一种方法是使用Fuji软件进行部分手动测量。第二种方法是使用LabVIEW程序中的定制代码进行全自动测量。下面,我们将演示两种可供选择的方法来分析FRAP图像数据。但是,在LD对的情况下,使用上述两种方法测量的最终FRAP结果几乎相似。例如,对于直径小于1μm的LD。但是,如何定制LabVIEW程序的潜在限制来处理FRAP数据的任何条件,我们还提供了处理最困难情况的手动方法。如果可能,我们建议使用自动方法。


  1. BODIPY-C12染色LDs的强度平均值(MOI)和大小测量值手册
    1. 打开斐济软件。
    2. 转到“文件”→“导入”→“图像序列”,打开一系列时序图像(8位 TIF格式文件,图7中的数据用作示例)


      图7.导入一系列8位TIF格式图像。 A.斐济软件菜单.B。导入图像序列的序列选项C.代表时间序列集图像输入.D。输入图像的比例。

    3. 校准后的刻度为12.107像素/μm,如图7D所示。要设置图像的比例尺,请转到“分析”→“设置比例”。
    4. 获得一系列随时间变化的大LD的“平均灰度值(平均值)”信息。
      1. 使用“椭圆”工具选择至少70%的大LD区域。
      2. 转到“分析”→“测量”,得到大LD的“面积”,“平均灰度值(平均值)”,“最小灰度值(最小值)”和“最大灰度值(最大值)”信息。
      3. 按下图7C右下方的“►”按钮或键盘上的“>”按钮,转到顺序图像的下一帧。
      4. 单击所选圆的中心,然后通过拖放将圆移动到下一帧中大LD的中心。
      5. 重复步骤b到d以获得大LD到最后一帧的一系列信息(图8)。


        图8.由斐济量化的一系列延时荧光图像中大LD的“平均灰度值(平均值)”数据。此处,此图像中分析的帧总数为91时间间隔为1.12秒

    5. 将大型LD的数据集保存为Excel文件。
    6. 随着时间的推移获得一小部分LD的“平均”信息。
      1. 按下后退按钮“◄”,如图9A左下方所示,或按下键盘上的“<”按钮返回顺序图像的第一帧(例如,按顺序设置的91张图像按90次)。
      2. 使用“椭圆”工具重新选择至少70%的小LD区域。
      3. 转到“分析”→“测量”,得到小LD的“面积”,“平均灰度值(平均值)”,“最小灰度值(最小值)”和“最大灰度值(最大值)”信息。
      4. 按“►”或“>”按钮转到顺序图像的下一帧。
      5. 单击所选圆的中心,然后通过拖放将圆移动到下一帧中小LD的中心。
      6. 重复步骤c-e,得到小LD到最后一帧的一系列信息(图9)。


        图9.斐济量化的一系列延时荧光图像中小LD的“平均灰度值(平均值)”数据

    7. 将小文件的数据集保存为Excel文件。
    8. 随着时间的推移,在大LD的区域内获得一系列“尺寸”信息。
      1. 按后退按钮“◄”或“<”返回顺序图像的第一帧(例如,按顺序设置的91张图像按90次)。
      2. 使用“Line”工具测量大LD的大小。
      3. 转到“分析”→“测量”,得到“面积”,“平均灰度值(平均值)”,“最小灰度值(最小值)”,“最大灰度值(最大值)”,“角度”和“长度”对于以下分析过程,仅需要“长度”信息。
      4. 按“►”或“>”按钮转到顺序图像的下一帧。
      5. 重复步骤bd以获得大LD到最后一帧的一系列大小信息(图10)。


        图10.斐济量化的一系列延时荧光图像中大型LD的“尺寸”数据

    9. 将大型LD的数据集保存为Excel文件。
    10. 随着时间的推移获得一小部分LD的“尺寸”信息。
      1. 按后退按钮“◄”或“<”返回顺序图像的第一帧(例如,按顺序设置的91张图像按90次)。
      2. 使用“线”工具测量LD的大小,通过对大LD测量重复相同的步骤8b-8e,获得具有最后帧的小LD的一系列信息(图11)。 >

        图11.斐济量化的一系列延时荧光图像中小LD的“尺寸”数据

    11. 将小文件的数据集保存为Excel文件。
    12. 接下来,将数据集重新格式化为新的Excel文件,如图12所示:随时间变化的MOQ,随时间的连续MOI,仅需要来自强度测量的“平均”值和来自尺寸测量的“长度”值。现在,数据已准备好用于数据分析的步骤C中的辅助分析。


      图12.在新的Excel文件中合并和重新格式化数据集

      13.在Excel软件中绘制LD对的两个感兴趣区域的连续MOI(图13)。


      图13.连续MOI随时间的变化

  2. 使用自动识别程序测量BODIPY-C12染色LD的MOI和尺寸
    1. 打开LabVIEW软件(此处使用带有NI Vision 8.6模块插件安装的LabVIEW 8.5版本)。
    2. 双击LabVIEW sub-llb文件“1_Exchange rate assay.llb”打开主模块“Main program.Vi”,如图14所示。子VI程序的界面如图15所示。


      图14. LLB管理器的对话框


      图15.主模块“主程序.vi”的界面。红色星号表示标准程序中的一组常规步骤。按照步骤获取每对信息的一系列信息中的一系列信息时间。

    3. 随着时间的推移获得大小LD的一系列“均值”信息。
      1. 设置图15中标有“Scale”的输入图像的比例(此处使用12.107像素/μm的值)。
      2. 通过按下图15中“步骤1”标记的按钮运行子VI程序,或者按下组合键盘按钮“Ctrl + R”。
      3. 按下图15中“步骤2”标记的“加载文件”,打开一系列时序图像(8位TIF格式文件,此处使用图16A的数据作为示例)。
      4. 通过拖放直接在图像上选择具有默认“方形”工具的整个大LD区域,如图16B所示,并按下图15中“步骤3”标记的“学习大模板”以记录模板图像大LD的。
      5. 对小LD重复步骤d:用“方形”工具选择整个小LD区域,如图16C所示,并按下图15中“步骤4”标记的“学习小模板”,记录小LD的模板图像


        A.根据计算机存储器将一对LD对的时间顺序图像加载到计算机中并相应地在界面上显示B.用“Square”工具选择的大LD的模板图像,并记录到计算机存储器中作为下一帧中的大LD的后续识别的参考C.模板图像比例尺,5μm。
        使用“Square”工具选择小LD并将其记录到计算机中作为参考,用于下一帧中的小LD的后续识别。
      6. 在搜索测试菜单中选择“大”或“小”,并按下图15中标记为“步骤5”的“搜索”按钮,以确认所选大或小LD的准确识别。 )。


        图17.确认已识别的LD大小。选择“大”(A)或“小”(C)以检查所选大(B)或小(D)LD的标识是比例尺,5微米。

      7. 在搜索测试菜单中选择“大”并按下图15中“步骤6”标记的“分析”,得到一系列“帧”,“平均灰度值(平均值)”,“最小灰度值(最小值)” ),以及大LD随时间的“大小”信息自动。
      8. 在“搜索测试”菜单中选择“小”并再次按“分析”按钮以获得一系列“帧”,“平均灰度值(平均值)”,“最小灰度值(最小值)”和“大小”信息随着时间的推移,小LD自动。
      9. 按由“步骤2”,在图15中输入标记下一组时间顺序图像并重复步骤dh以获取下一系列信息完成LD对的MOI测量后。
        注意:按“加载文件”或“返回”按钮后,较大和较小LD的一系列均值和大小信息将自动保存为Excel文件。生成的Excel文件已准备好格式数据分析的步骤C中的辅助分析。
    4. 检查先前保存为Excel文件的LD对的数据集(此处使用两组数据作为示例)。
    5. 在Excel软件中绘制各个LD对中两个感兴趣区域的连续MOI随时间的变化(图18)。
    6. 将具有相同数据的文件夹传输到LabVIEW子VI的相同文件夹中“2_Check a fitting region.vi”和“3_Calculation of exchange rates.vi”文件。


      图18.连续MOI随时间变化的情节。(A)中显示的数据与图13中显示的数据相同.B。相比之下,使用另一组时间序列图像获得MOI在数据分析的步骤B3i中提到。

  3. 预测汇率强调线性标准
    在成像过程并获得在数据分析的步骤A或B中生成的“MOI”和“长度”信息之后,Excel文件现在可用于以下分析。 br />
    1. 打开LabVIEW软件。
    2. 打开LabVIEW子VI“2_检查拟合区域.Vi”文件。子VI程序的界面如图19所示。


      图19.子VI的界面“2_Check a fitting region.vi”。这里使用图18B中的数据作为示例。

    3. 获得LD对汇率的最佳拟合范围。
      1. 按下图19中“Periodic running”标记的按钮,运行sub-VI程序。
      2. 调整以通过图19中的“拟合范围”选择拟合范围的最大值,以确保调整的拟合线的线性度(MSE)和重复性(%)满足最佳值(MSE <0.01且%<&lt;&lt; 30%)。我们将两个上限值定义为线性标准。
      3. 记录每组测量的LD对的最佳拟合范围。

  4. 计算各个融合LD对之间的汇率
    1. 打开LabVIEW子VI“3_Calculation of exchange rate.Vi”文件。子VI程序的界面如图20所示。


      图20.子VI的界面“3_Calculation of exchange rates.vi”

    2. 在“最佳拟合范围”标记的表格中输入所有最佳拟合范围,如图21A所示。
    3. 如图21B所示,针对大或小LD尺寸绘制汇率。
      按“Ctrl + R”运行子VI程序以获得汇率的平均值±SD值。

      图21.运行子VI“3_计算汇率.vi”的结果。 A.最佳拟合范围表B.显示汇率与大型和小型LD的大小的关系图。 。

笔记

  1. 光漂白过程的简要描述必须在三个LD的较小(即供体)LD上进行三次以进行统计分析,如我们的原始出版物中所述(Wang et al。,2018)。简短,因为那里在较大的LD中,荧光分子更多,将这些大的LD光漂白两倍以上将导致LD对中荧光强度的快速下降,并且导致荧光恢复检测的困难。
  2. 该协议中最关键的步骤是获得三个拟合线的最佳线性度(mse)和重复性(%),如“程序C”中所述。如果三个循环中的拟合线的评估线性或重复性为mes。这些不稳定的汇率可能在采集间隔期间发生。这些不稳定的汇率可能由LD对的物理尺寸变化,成像平面的高度引起。我们建议测量50组或更多的脂质交换率。一般来说,30-40次脂质交换事件足以满足标准,如果获得的话。最佳环境控制条件。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    1. 混合1.37M NaCl,25mM KCl,8mM Na 2 HPO 4 ,14.7mM KH 2 PO ,9毫升CaCl 2 和5 mM MgCl 2 在900毫升去离子H 2 O中
    2. 调整至最终pH 7.4
    3. 将去离子H 2 O加入1,000ml
    4. 高压灭菌器存储(这是10倍PBS库存)
    5. 用去离子H 2 O稀释PBS原液至1×PBS

致谢

这项工作得到了国家研究计划拨款2018 YFA 0506900(PL)和国家自然财富的资助,清华大学P. Li实验室成员中国科学基金会的资助91857103(到FJC),31430040,31690103和31621063(以PL),该协议是从我们以前的出版物适用于在生物化学杂志(王的等的,2018 )。

竞争利益

作者声明没有利益冲突或竞争利益。

参考

  1. Choudhary,V.,Ojha,N.,Golden,A。和Prinz,WA(2015)。一个保守的蛋白质家族有助于新生脂质从ER中出芽。 J Cell Biol 211(2):261-27。
  2. Farese,RV,Jr。和Walther,TC(2009)。脂质水滴终于得到了一点尊重 Cell 139(5):855-860。
  3. Fujimoto,Y.,Itabe,H.,Kinoshita,T.,Homma,KJ,Onoduka,J.,Mori,M.,Yamaguchi,S.,Makita,M.,Higashi,Y.,Yamashita,A。和Takano ,T。(2007)。 ACSL参与局部合成中性脂质的细胞静态脂质滴人肝细胞HuH7。 J Lipid Res 48 (6):1280-1292。
  4. Gao,G.,Chen,FJ,Zhou,L.,Su,L.,Xu,D.,Xu,L。和Li,P。(2017)。通过CIDE家族蛋白控制脂滴融合和生长。 Biochim Biophys Acta Mol Cell Biol Lipids 1862(10 Pt(10 Pt) B):1197-1204。
  5. Gluchowski,NL,Becuwe,M.,Walther,TC和Farese,RV,Jr。(2017)。脂滴和肝脏疾病:从基础生物学到临床意义。 Nat Rev Gastroenterol Hepatol 14(6):343-355。
  6. Gong,J.,Sun,Z.,Wu,L.,Xu,W.,Schieber,N.,Xu,D.,Shui,G.,Yang,H.,Parton,RG and Li,P。(2011 )。 Fsp27通过脂质交换和脂滴接触部位转移促进脂滴生长。 J Cell Biol 195(6):953-963。
  7. Gross,DA,Zhan,C。和Silver,DL(2011)。将甘油三酯直接结合到诱导脂肪储存的跨膜蛋白1和2对于脂滴形成是重要的。 Proc Natl Acad Sci USA 108(49):19581-19586。
  8. Krahmer,N。,Farese,RV,Jr。和Walther,TC(2013)。平衡脂肪:脂滴和人类疾病。 EMBO Mol Med 5(7):973-983。
  9. Rosen,ED和Spiegelman,BM(2014)。当我们谈论脂肪时我们谈论的是什么。 Cell 156(1-2):20-44。
  10. Sun,Z.,Gong,J.,Wu,H.,Xu,W.,Wu,L.,Xu,D.,Gao,J.,Wu,JW,Yang,H.,Yang,M。和Li Perilipin 1通过激活脂肪细胞中的Fsp27促进独特的脂滴形成。 Nat Commun 4:1594。
  11. Sun,Z.,Gong,J.,Wu,L。和Li,P。(2013 b)。成像脂质液滴融合和生长。 Methods Cell Biol 116:253-268。
  12. Wang,J.,Yan,C.,Xu,C.,Chua,BT,Li,P。和Chen,FJ(2018)。多肽RKKR基序在脂滴(LD)的连接区域 - 相关蛋白CIDEC抑制剂LD融合活性通过相互作用的磷脂。 J Biol Chem 293(50):19330-19343。
  13. Wilfling,F.,Wang,H.,Haas,JT,Krahmer,N.,Gould,TJ,Uchida,A.,Cheng,JX,Graham,M.,Christiano,R.,Frohlich,F.,Liu,X ,Buhman,KK,Coleman,RA,Bewersdorf,J.,Farese,RV,Jr。和Walther,TC(2013)。三酰基甘油合成酶通过从ER重新定位到脂滴来脂质化脂滴生长。 Dev Cell 24(4):384-399。
  14. Wu,L.,Xu,D.,Zhou,L.,Xie,B.,Yu,L.,Yang,H.,Huang,L.,Ye,J.,Deng,H.,Yuan,YA,Chen ,S。和Li,P。(2014)。 Rab8a-AS160-MSS4调节电路控制脂质液滴融合和生长。 Dev Cell 30(4):378-393。
  15. Xu,D.,Li,Y.,Wu,L.,Li,Y.,Zhao,D.,Yu,J.,Huang,T.,Ferguson,C.,Parton,RG,Yang,H。和Li ,P.(2018)。 Rab18通过将ER束缚于LD来促进脂滴(LD)生长通过SNARE和NRZ的相互作用。 J Cell Biol 217(3):975-995。
  16. Yang,H.,Galea,A.,Sytnyk,V。和Crossley,M。(2012)。控制脂滴的大小:脂质和蛋白质因子。 Curr Opin Cell Biol 24(4):509-516。
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Copyright: © 2019 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. Wang, J., Chua, B. T., Li, P. and Chen, F. (2019). Lipid-exchange Rate Assay for Lipid Droplet Fusion in Live Cells. Bio-protocol 9(14): e3309. DOI: 10.21769/BioProtoc.3309.
  2. Wang, J., Yan, C., Xu, C., Chua, B. T., Li, P. and Chen, F. J. (2018). Polybasic RKKR motif in the linker region of lipid droplet (LD)-associated protein CIDEC inhibits LD fusion activity by interacting with acidic phospholipids. J Biol Chem 293(50): 19330-19343.
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