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Aug 2020
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Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells
用RUSH蛋白分选法追踪HeLa细胞中的蛋白囊泡   

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Abstract

Monitoring vesicle trafficking is an excellent tool for the evaluation of protein dynamics in living cells. Such study is key for the understanding of protein sorting and secretion. Recent developments in microscopy, as well as new methodologies developed to study synchronized trafficking of proteins, allowed a better understanding of signaling, regulation and trafficking dynamics at the secretory pathway. One of the most helpful tools so far developed is the Retention Using Selective Hooks (RUSH) system, a methodology that facilitates the evaluation of synchronized cargo trafficking by monitoring fluorescent vesicles in cells upon biotin addition. Here we present a protocol that allows the quantitative evaluation of protein cargo trafficking at different fixed time points and an analytic approach that enables a better examination of specific cargo trafficking dynamics at the secretory pathway.


Graphic abstract:



Schematic representation of RUSH sorting assay in mammalian cells


Keywords: Protein trafficking (蛋白质运输), RUSH (RUSH), Cargo sorting (蛋白(货物)分选), Confocal microscopy (共聚焦显微镜), Vesicle tracking (囊泡跟踪)

Background

Monitoring vesicle trafficking is an excellent tool for the evaluation of secretory protein dynamics in living cells. Given that around 30% of the total amount of newly synthesized proteins in mammals follow the secretory pathway (Pfeffer, 2010; Boncompain and Weigel, 2018), the study of its trafficking dynamics is key for the understanding of protein sorting and secretion. Proteins that follow the secretory pathway go through several maturation steps, starting at the ER, they are transported along the different Golgi stacks until they reach the trans-Golgi network (TGN), a sorting station. In the TGN they are finally packed into vesicles and initiate their route to their final destination (Glick and Luini, 2011; Pantazopoulou and Glick, 2019).


Recent developments in microscopy, as well as new methodologies developed to study the synchronized trafficking of proteins allowed to better understand signaling, regulation and trafficking dynamics at the secretory pathway (Stephens and Perez, 2013). One of the most helpful tools so far developed is the Retention Using Selective Hooks (RUSH) system, a methodology that facilitates the evaluation of synchronized cargo trafficking by monitoring fluorescent vesicles in cells upon biotin addition (Boncompain et al., 2012).


The RUSH system has two main elements: a protein of interest (POI) tagged with a fluorophore and bound to a streptavidin binding peptide (SBP); and a streptavidin molecule bound to a retention signal (known as the “hook”, e.g., a KDEL sequence for retention in the ER; Figure 1). This complex, in the absence of biotin will be retained in the donor compartment, but once biotin is added to the culture medium, the POI-fluorophore-SBP complex will be released to the next compartment. This set-up allows to follow up the POI at different time points by confocal microscopy (Boncompain et al., 2012).



Figure 1. Scheme representing the RUSH system. A. Protein complex containing the protein of interest (here matrix metalloprotease 2, MMP2), a streptavidin binding peptide (SBP) and a fluorescent protein (eGFP). The complex is bound via SBP to streptavidin (Str), which is linked to a KDEL sequence for its retention in the endoplasmic reticulum (donor compartment). Without biotin addition, the complex remains in the donor compartment; however, once biotin is added to the media, it binds to streptavidin and enables the trafficking of the MMP2-SBP-eGFP protein complex to the acceptor compartment. Figure adapted from Boncompain et al. (2012). B. Immunofluorescence images showing the trafficking of MMP2 across the secretory pathway, with MMP2 localizing at the ER when biotin is absent, and later localizing at the Golgi (15 and 30 min) and in post-Golgi vesicles (45 min) upon biotin addition. TGN46: trans-Golgi marker. Figure taken from Pacheco-Fernandez et al. (2020).


Among other techniques, the RUSH system has the advantage of enabling the monitoring of cells under physiological conditions. Given that biotin is a non-toxic small molecule, the synchronization of cargo release does not lead to high stress for the cells (Boncompain and Perez, 2013). Also, compared to other techniques such as the light-triggered protein secretion system (Chen et al., 2013), it does not require a previous aggregation of the protein, which may lead to trafficking through different routes, higher protein degradation rates, and consequently, higher cell toxicity (Boncompain and Perez, 2013).


Building on the original RUSH methodology, here we present a protocol that allows to quantitatively evaluate protein cargo trafficking at different fixed time points. Importantly, in this protocol we provide a quantitative analysis to better examine intra-Golgi trafficking dynamics by evaluating specific cargo trafficking dynamics along the secretory pathway using confocal microscopy. This allows to evaluate the impact of knocking-out or silencing secretory pathway proteins on cargo trafficking (Crevenna et al., 2016; Deng et al., 2018; Pacheco-Fernandez et al., 2020).

Materials and Reagents

  1. Circle coverslips, thickness 1 (ThermoScientificTM, catalog number: CB00120RA120MNT0)

  2. 6-well cell culture plates (Corning®, Costar®, catalog number: CLS3516-1EA)

  3. SuperfrostTM Microscopy Slides (ThermoScientificTM, catalog number: AA00008332E00MNT10)

  4. Polyethylenimine, linear, MW 25,000, transfection grade (PEI 25KTM; Polysciences, catalog number: 23966-1).

  5. 60-70% confluent HeLa cells

  6. 1× Dulbecco’s Phosphate Buffered Saline (DPBS), no calcium, no magnesium (Life Technologies, Gibco, catalog number: 14190144). Storage temperature: 4 °C.

  7. pIRESneo3-Str-KDEL-MMP2-SBP-EGFP vector (Figure 2). Storage temperature: -20 °C

    Important note: This plasmid was generated by replacing the ST (ST6GAL1) sequence by MMP2 (our protein of interest) in the Addgene plasmid number 65264 (Str-KDEL_ST-SBP-EGFP).



    Figure 2. Vector map for the pIRESneo2-Str-KDEL-MMP2-SBP-eGFP construct used as an example in this protocol. The plasmid depicts the KDEL-bound streptavidin and the reporter MMP2-SBP-eGFP. An IRES element in the plasmid enables the simultaneous expression of both Str-KDEL and MMP2-SBP-eGFP in one single plasmid. For more details about the construct, please see Boncompain et al. (2012). The map was generated with SnapGene®.


  8. Opti-MEM® reduced serum media (Life Technologies, Gibco, catalog number: 31985070). Storage temperature: 4 °C

  9. 500 mM d-biotin (Merck, SUPELCO, catalog number: 47868). Storage temperature: 4 °C

  10. 4% paraformaldehyde (PFA) in 1× PBS. Prepared using the formaldehyde solution, ROTIPURAN® 37%, p.a., ACS (Roth, catalog number: 4979.1)

  11. Primary antibodies

    1. For the protein of interest

    2. Depending on the analysis, specific organelle markers can be used. For example, calnexin can be used as an ER marker, whereas, depending on the analytical evaluation, different Golgi markers could be used (e.g., GM-130 for cis-Golgi and TGN-46 for trans-Golgi network)

  12. Secondary antibodies

    Our studies were performed with Alexa-Fluor antibodies 488, 594 and 633 targeting different species (Life Technologies, ThermoScientific, catalog number varies according to species and wavelength). Storage temperature: 4 °C.

  13. ProLong Gold antifade reagent (Life Technologies, Invitrogen, catalog number: P36934)

  14. PEI solution at 1 mg/ml (see Recipes)

  15. Dulbecco’s Modified Eagles Medium (DMEM) complete medium (see Recipes)

    1. DMEM, high glucose, GlutaMAXTM Supplement, pyruvate (Life Technologies, GibcoTM, catalog number: 31966021)

    2. Fetal Bovine Serum (FBS, Life Technologies, GibcoTM, catalog number: 10270106)

    3. Penicillin / streptomycin (P/S, Life Technologies, GibcoTM, catalog number: 15140122)

  16. Permeabilizing solution (see Recipes)

    1. Triton X-100 (Carl Roth, catalog number: 3051.2)

    2. Sodium dodecyl sulphate (SDS) 10% solution

  17. Blocking solution (see Recipes)

    1. Bovine Serum Albumin (BSA) lyophilized powder (PAN Biotech, catalog number: P06-1391050)

    2. Phosphate buffered saline (PBS) powder (Sigma, catalog number: P3813-1PAK): Prepare a 1× solution according to manufacturer’s instructions

Equipment

  1. Tweezers

  2. Laminar flow hood

  3. Cell incubator set at 37 °C, 5% CO2

  4. Water bath set at 37 °C

  5. Zeiss laser scanning LSM780 confocal microscope (Carl Zeiss)

  6. Zeiss 100× (NA 1.46, oil) objective (Carl Zeiss)

  7. 488-nm laser line

Software

  1. Zeiss Zen software 2010 (Zeiss, https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html)

  2. ImageJ v.1.37 (National Institutes of Health [NIH], https://imagej.nih.gov/ij/)

  3. ImageJ macro for “RUSH-Vesicle-Analysis” (https://github.com/MehrshadPakdel/RUSH-Vesicle-Analysis)

Procedure

  1. Cell culture and plasmid transfection

    1. All steps involving cell culture must be performed in a laminar flow hood.

    2. Seed 3 × 104 HeLa cells in a 6-well plate containing 2-3 small coverslips using 2 ml DMEM complete medium per well. Prepare 1 well per time point (see Note 1).

    3. Incubate the cells at 37 °C, 5% CO2 for 24 h.

    4. After 24 h incubation, set up PEI transfection reaction. Per well:

      1. 200 µl OptiMEM

      2. 2 µg RUSH construct, e.g., pIRESneo3-SS-Str-KDEL-MMP2-SBP-EGFP

      3. 15 µl 50mM PEI solution

    5. Vortex the transfection reaction and incubate it at room temperature for 20 min.

    6. Add dropwise to the cells and incubate at 37 °C, 5% CO2 for at least 16 h. Avoid incubation periods longer than 24 h. If they are necessary, change cell medium after 24 h.


  2. RUSH experiment

    1. After incubation, prepare 2 ml of DMEM complete with d-biotin to a final concentration of 40 µM per well. Prepare enough volume according to the number of wells to analyze (for example, for 4 wells (2 ml medium per well), prepare 9 ml of DMEM complete + 7.2 µl d-biotin 500 mM). Keep it warm at 37 °C.

    2. Remove medium from cells either by aspiration with a micropipette or with a vacuum pump and wash once with 1× DPBS.

    3. Add 2 ml DMEM complete without biotin to all the time points except the latest one (see Note 2). For example, for MMP2 four time points are analyzed (0, 15, 30 and 45 min). Add medium only to the wells labeled as 0, 15 and 30 min.

    4. Add 2 ml DMEM complete + biotin (from Step B1) to the latest time point (for MMP2, to the well labeled as 45 min. Figure 3, “start of the experiment”).

    5. Incubate cells at 37 °C, 5% CO2.

      The incubation time is determined by the trafficking kinetics of each construct and in order to facilitate the experimental handling we recommend to start adding biotin first to the well that needs to be incubated the longest time (45 min for MMP2) and then continue with the earlier time points (30 and 15 min, see scheme in Figure 3).

      Important:Here is recommended to take the cells out of the incubator at least 2 min before triggering cargo trafficking in the next time point (for MMP2, the plate is taken out of the incubator 13 min after adding biotin to the previous well, namely, 2 min before starting the next time point. See note 2 for a more detailed explanation).



      Figure 3. Scheme depicting the handling times during time intervals for the RUSH experiment. The scheme depicts the handling time for an experienced person using the MMP2 construct with the described time points of 0, 15, 30 and 45 min.


    6. Remove media from the next time point to be analyzed and add 2 ml DMEM complete + biotin.

    7. Repeat Steps B2, B4 and B5 until completing the full-time cycle (for MMP2, 45 min with a last biotin addition at 15 min before fixation, Figure 3).


  3. Coverslip montage

    1. Right before incubation ends, wash the cells (all the wells) 2 times with 1× PBS.

    2. Incubate cells for 10 min in 4% PFA in PBS under low light conditions. Make sure that the coverslips are completely covered with the PFA solution.

    3. Wash 3 to 5 times with 1× PBS.

    4. If no co-staining is performed: add a small amount of PBS to the well to facilitate the handling of coverslips and mount them using the ProLong antifade Gold reagent. For this purpose:

      1. Take a microscopy glass slide.

      2. Put one drop of ProLong antifade Gold reagent on the slide.

      3. Take carefully one of the coverslips with tweezers.

      4. Place the coverslip facing down on top of the ProLong antifade Gold reagent (i.e., the side of the coverslip with cells must be in contact with the antifade reagent).

      5. Carefully store them in a dark place until imaging. It is recommended to allow the montaged slides to dry for at least 24 h, ideally for 48 h.

    5. If co-staining is performed:

      1. Permeabilize the cells with permeabilization solution for 5 min under low light conditions.

      2. Wash 3 to 5 times with 1× PBS.

      3. Incubate with blocking solution for 1 h at room temperature or overnight at 4 °C under low light conditions.

      4. Freshly prepare the primary antibody solutions by diluting the antibody in blocking solution. Depending on the antibody, this information may be recommended by the manufacturer or require previous characterization in the lab to determine the ideal time and temperature.

      5. Wash 3 to 5 times with 1× PBS.

      6. Incubate with primary antibody for 1 h at room temperature or overnight at 4 °C under low light conditions.

      7. Freshly prepare the secondary antibody solutions by diluting the amounts of antibody in blocking solutions recommended by the manufacturer.

      8. Wash 3 to 5 times with 1× PBS.

      9. Incubate with secondary antibody for 1 h at room temperature under low light conditions.

      10. Wash 3 to 5 times with 1× PBS.

      11. Mount the glass slides using the ProLong antifade Gold reagent (see Step C4).


  4. Imaging

    1. Start the microscope and warm up the required lasers. This process can take up to around 30 min depending on the lasers and the microscope.

    2. Set up a z-stack acquisition experiment. We used a 0.35 µm distance per slice with typically 10 to 15 planes to cover most of the cell volume.

    3. Focus a single cell (or several clearly defined and healthy single cells per field of view) and set up the number of stacks to be imaged using the signal from the RUSH channel.

    4. Adjust gain and intensity from the other channels, if necessary.

    5. Start imaging.

    6. Repeat Steps D3 and D4 until completing at least 15 cells per time point.

    7. Save files as *.lsm.

Data analysis

  1. Open Fiji.

  2. Open in Fiji our custom-made ImageJ macro for “RUSH-Vesicle-Analysis” available at https://github.com/MehrshadPakdel/RUSH-Vesicle-Analysis. For more details about the ImageJ functions used in this macro, see Note 7.

  3. Open the microscopy (*.lsm) file in Fiji. Other microscopy files such as *.lif can also be used.

  4. Click on run for the macro to initiate the analysis (Figure 4).



    Figure 4. Screenshot of the RUSH-Vesicle analysis macro window in Fiji


  5. Select the number of cells to analyze (Figure 5).



    Figure 5. Screenshot of step 1 from RUSH-analysis macro


  6. Selection of regions of interest (ROIs) to analyze: Identify each cell by drawing a polygon around it (Figure 6). The selected area will be saved automatically as an ROI. Click ok to select the next cell.



    Figure 6. Screenshot of step 2 from RUSH-analysis macro


  7. Generation of binary image and threshold adjustment: After selecting the last cell and clicking ok, you will see 2 windows appearing automatically in your screen (Figure 7, before): one showing the binary generated image (left window, title: MAX_Bin_Image) and another one showing the original maximal projection (right window, title: MAX_Image). In addition, two control windows will appear: one named “Threshold” that enables manual adjustment of black and white balance for the MAX_Bin_Image, and another one named “Action Required”. In order to avoid losing low-intensity vesicles in the counting, you need to visually adjust the threshold of the generated binary image in comparison to the original one (Figure 7). To do so, slide the bars in the “Threshold” window until MAX_Bin_Image resembles MAX_Image (Figure 7, after). Once both images are identical, click “Apply” in the threshold window. Importantly, once you click apply it is not possible to undo the threshold modification. After you click apply, click OK on the “Action Required” window to confirm the changes and continue with the macro (Figure 7, after).



    Figure 7. Screenshot of step 3 from RUSH-analysis macro. The figure “Before” shows the binary image generated (MAX_Bin_Image, left window), the original maximal projection (MAX_Image), the threshold control window and the “Action Required” window. To avoid losing low-intensity vesicles in the count you have to adjust manually the black and white balance of the MAX_Bin_Image having the fluorescent maximal projection as a reference (MAX_Image). Proper adjustment is illustrated in the figure named as “After”.


  8. Analyze particles: Once you click ok on the “Action Required” window, the macro will start automatically counting the number of vesicles on each one of the selected ROIs. Therefore, 3 new windows will appear: a window with the drawing (blue square, Figure 8 here named as Cell1_Example_eGFP_45min_Quanti.tif), a log window indicating the number of vesicles counted per cell (magenta square, Figure 8), and the ROI manager window showing the selected ROIs for the cells (green square, Figure 8). These numbers can be copied to an Excel file or similar spreadsheet file for further analysis.



    Figure 8. Screenshot of the final step from quantitative analysis. Three windows will appear automatically after clicking ok in step no. 7: one with a draw of the counted vesicles per cell (generated for each analyzed cell, blue square), a log window that depicts the number of vesicles counted per cell (magenta square) and a ROI window containing the information of the selected ROIs (selected cells, green square). The log window values can be copied to an excel file or statistical analysis software for further evaluation.


  9. Once the macro finishes running, all the results are saved automatically in a new results folder in the original file directory (Figure 9). The files saved are the original file (Orig_filename), the maximal projection of the original image (MAX_filename), the generated binary image (Binary_filename), one drawing file showing the number of counted vesicles for each cell selected (CellNumber_filename) and the log file containing the total number of counted vesicles per cell (log_filename).



    Figure 9. Screenshot of the files generated after running the macro


  10. The number of vesicles can be further compared performing statistical tests such as t-test, ANOVA or their corresponding non-parametric test, to evaluate differences between time points and treatments. Figure 10 shows an example of vesicle quantification for MMP2eGFP positive vesicles in HeLa cells.



    Figure 10. Final result from MMP2 RUSH vesicle analysis. Panel (A) depicts immunofluorescent images of HeLa cells expressing the RUSH MMP2eGFP construct at different time points. Scale bars: 10 µm. Panel (B) shows the accumulated number of MMP2-positive vesicles from at least 25 analyzed cells. Differences between time points were evaluated with a Kruskall-Wallis test. **** P-value < 0.0001.


  11. When using secretory pathway markers, other statistical tests could be used to evaluate co-localization, such as Pearson’s or Manders coefficients. For a more detailed description of such analysis, please refer to Pacheco-Fernandez et al. (2020). Figure 11 shows an example of MMP2 colocalization with the Golgi marker TGN 46, visualized by confocal microscopy.



    Figure 11. Immunofluorescent images of HeLa cells transfected with MMP2-RUSH and co-stained with TGN46. Cells were fixed at the indicated time points, permeabilized and incubated with GFP and anti-TGN46 antibodies (TGN-46 is a trans-Golgi marker). Green: MMP2-SBP-eGFP, magenta: trans-Golgi network. Scale bars: 10 µm.

Notes

  1. Time points are defined according to the cargo evaluated. If you are using an uncharacterized cargo, a previous experiment with a known cargo and its described time points should be performed and used as a positive control. In our experience, the soluble secretory cargo we have evaluated in the lab (such as Lysozyme C and Cathepsin D) traffics through the secretory pathway in a time span of 60 to 75 min. The unknown cargo must be firstly evaluated in different intervals to establish the kinetics of its trafficking. For example, when we assessed the trafficking dynamics of MMP2 for the first time, we evaluated 5 min intervals to determine the critical points of transport (from ER to Golgi and from Golgi to extracellular space). However, the time points to evaluate will always depend on the desired kind of analysis.

  2. This protocol refers to the latest incubation time as the last time point evaluated in the experiment (i.e., in the present protocol 45 min) and earlier incubation times as any time below such time point (i.e., 0, 15 and 30 min).

  3. Keep in mind that the process of taking the plate out of the incubator, removing medium and adding medium with biotin takes at least 2 min for experienced people. For the first time, a working time window of 5 min between time points is recommended. Therefore, take the plate out of the incubator either 2 or 5 min (according to experience in handling) before the start of the next time point. For example, with the MMP2 construct here described, once DMEM + biotin has been added to the 45 min time point, take the plate out of the incubator after 13 min incubation so you have enough time to place the plate in the hood, remove medium from the 30 min well, replace with new DMEM + biotin and place the plate back in the incubator at 37 °C exactly 15 min after the 45 min incubation has started (Figure 3).

  4. Protein trafficking is a temperature sensitive process; therefore, it is recommended to adjust your experimental conditions to avoid periods shorter than 5 min of cell incubation at 37 °C.

  5. The set up for confocal microscopy can also be done in 35-mm dishes. We recommend counting at least 15 cells for a valid statistical analysis.

  6. A control is necessary for the validation of this assay. In this case, a RUSH construct without a protein cargo (i.e., containing Str, the retention hook, SBP and the fluorophore to be analyzed) can be used as a control for the validation of any construct. Importantly, this control is suitable to evaluate the efficacy of the assay but for further kinetic dynamics comparisons, it is recommended to use a construct encoding a protein cargo with well-described trafficking dynamics as a control.

  7. The macro here used for the analysis uses 7 functions (in the following order):

    1. Background subtraction: Uses ImageJ subtract background function that is based in a rolling ball algorithm (https://imagej.net/Rolling_Ball_Background_Subtraction). This algorithm adjusts the pixel size to the radius of the largest object in the image that is not part of the background. For the present analysis, this radius was adjusted to the Golgi size.

    2. Enhance contrast: This function facilitates the visibility of small vesicle particles. It enhances the contrast by stretching the image histogram without altering the pixel values (https://imagej.net/docs/guide/146-29.html#sub:Enhance-Contrast).

    3. Z Project: Generates a 2D image with the flattened maximum intensities of the z-stack series (https://imagej.nih.gov/ij/docs/guide/146-28.html#sub:Z-Project ...).

    4. Image processing filter “median”: The filter reduces the noise in the selection. In this macro is used to smoothen the vesicles and to generate less noisy objects after thresholding (https://imagej.nih.gov/ij/docs/guide/146-29.html#sub:Median ...).

    5. Threshold: Segments grayscale images (binary image in this analysis) into feature of interest and background. Selecting threshold generates a binary image that defines which objects should not be count as background, and is required for subsequent particle quantification (https://imagej.nih.gov/ij/docs/guide/146-28.html#sub:Threshold...[T]).

    6. Analyze particles: Detects and quantifies particles within a given pixel size (https://imagej.nih.gov/ij/docs/guide/146-30.html#toc-Subsection-30.2).

Recipes

  1. PEI solution

    1. Dissolve 50 mg of Polyethylenimine in 40 ml of distilled water

    2. Mix well the solution with a magnetic stirrer on a slightly warmed plate

    3. Adjust pH to 7.4

    4. Complete volume up to 50 ml. Aliquot in 500 µl and store at -20 °C

  2. DMEM complete medium

    Prepare 500 ml of DMEM medium by adding FCS and P/S to a final concentration of 10% and 1%, respectively

  3. Permeabilizing solution

    1. Mix in 1× PBS: 0.2% Triton X-100 and 0.5% sodium dodecyl sulphate (SDS; dilute from a 20% or 10% SDS solution).

    2. Make 5 ml aliquots and store long term at -20 °C. Once thawed, store at 4 °C and warm up at room temperature for working. If some precipitate forms, briefly warm up at 37 °C until the solution becomes clear.

  4. Blocking solution

    Dissolve 20 g of bovine serum albumin (BSA, molecular biology grade) in 500 ml of 1× PBS. Aliquot in 50 ml and store long term at -20 °C. Once thawed, store at 4 °C and keep on ice while working at room temperature.

Acknowledgments

This protocol was previously documented in the publications (Deng et al., 2018) and (Pacheco-Fernandez et al., 2020). This work was supported by grants from the Deutscher Akademischer Austauschdienst (DAAD), Deutsche Forschungsgemeinschaft (DFG), National Institutes of Health (NIH), the Perspective Program (Boehringer Ingelheim Fonds) and the Max Planck Institute of Biochemistry.

Competing interests

The authors declare no competing financial interests.

References

  1. Boncompain, G., Divoux, S., Gareil, N., de Forges, H., Lescure, A., Latreche, L., Mercanti, V., Jollivet, F., Raposo, G. and Perez, F. (2012). Synchronization of secretory protein traffic in populations of cells. Nat Methods 9(5): 493-498.
  2. Boncompain, G. and Perez, F. (2013). Fluorescence-based analysis of trafficking in mammalian cells. Methods Cell Biol 118: 179-194.
  3. Boncompain, G. and Weigel, A. V. (2018). Transport and sorting in the Golgi complex: multiple mechanisms sort diverse cargo. Curr Opin Cell Biol 50: 94-101.
  4. Chen, D., Gibson, E. S. and Kennedy, M. J. (2013). A light-triggered protein secretion system. J Cell Biol 201(4): 631-640.
  5. Crevenna, A. H., Blank, B., Maiser, A., Emin, D., Prescher, J., Beck, G., Kienzle, C., Bartnik, K., Habermann, B., Pakdel, M., Leonhardt, H., Lamb, D. C. and von Blume, J. (2016). Secretory cargo sorting by Ca2+-dependent Cab45 oligomerization at the trans-Golgi network. J Cell Biol 213(3): 305-314.
  6. Deng, Y., Pakdel, M., Blank, B., Sundberg, E. L., Burd, C. G. and von Blume, J. (2018). Activity of the SPCA1 Calcium Pump Couples Sphingomyelin Synthesis to Sorting of Secretory Proteins in the Trans-Golgi Network. Dev Cell 47(4): 464-478 e468.
  7. Glick, B. S. and Luini, A. (2011). Models for Golgi traffic: a critical assessment. Cold Spring Harb Perspect Biol 3(11): a005215.
  8. Pacheco-Fernandez, N., Pakdel, M., Blank, B., Sanchez-Gonzalez, I., Weber, K., Tran, M. L., Hecht, T. K., Gautsch, R., Beck, G., Perez, F., Hausser, A., Linder, S. and von Blume, J. (2020). Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs. J Cell Biol 219(8).
  9. Pantazopoulou, A. and Glick, B. S. (2019). A Kinetic View of Membrane Traffic Pathways Can Transcend the Classical View of Golgi Compartments.Front Cell Dev Biol 7: 153.
  10. Pfeffer, S. R. (2010). How the Golgi works: a cisternal progenitor model. Proc Natl Acad Sci U S A 107(46): 19614-19618.
  11. Stephens, D. and Perez, F. (2013). Preface. In: Perez, F. and Stephens, D. (Eds.). Methods for Analysis of Golgi Complex Function (Vol. 118, pp. xix–xx). Academic Press.

简介

[摘要]监测囊泡运输是评估活细胞中蛋白质动力学的出色工具。这项研究对于理解蛋白质的分类和分泌至关重要。显微镜的最新发展,以及研究同步蛋白运输的新方法,使人们对蛋白质的理解有了更深入的了解。 分泌途径中的信号传导,调节和运输动态。迄今为止,开发的最有用的工具之一是使用选择性钩子保留(RUSH)系统,该方法可通过在添加生物素后监测细胞中的荧光囊泡来促进同步货物运输的评估。在这里,我们提出了一个协议,允许蛋白质贩卖货物在不同的固定时间点的定量评价和ANALY抽动的方法,使一个更好的检查特定货物走私力度的分泌途径。

图形摘要:

哺乳动物细胞中RUSH分选测定的示意图

[背景技术[ 0002 ]监测囊泡运输是评估活细胞中分泌蛋白动力学的极好的工具。鉴于哺乳动物新合成蛋白质总量中约有30%遵循分泌途径(Pfeffer,2010; Boncompain and Weigel,2018),因此研究其运输动力学是了解蛋白质分类和分泌的关键。遵循分泌途径的蛋白质经历了多个成熟步骤,从内质网开始,沿着不同的高尔基体堆栈运输,直到到达反式高尔基体网络(TGN),即分选站。在TGN中,它们最终被包装到囊泡中,并开始到达最终目的地的路线(Glick和Luini,2011; Pantazopoulou和Glick,2019)。

显微镜的最新发展以及研究同步蛋白运输的新方法学使人们可以更好地了解分泌途径的信号传导,调节和运输动态(Stephens和Perez,2013)。迄今为止开发的最有用的工具之一是使用选择性钩子保留(RUSH)系统,该方法可通过在添加生物素后监测细胞中的荧光囊泡来促进同步货物运输的评估(Boncompain等人,2012)。

RUSH系统具有两个主要元素:用荧光团标记并与链霉亲和素结合肽(SBP)结合的目的蛋白(POI);链霉亲和素分子与保留信号结合(称为“钩子”,例如在ER中保留的KDEL序列;图1)。在没有生物素的情况下,该复合物将保留在供体隔室中,但是一旦将生物素添加到培养基中,POI-荧光团-SBP复合物将被释放到下一个隔室中。这种设置允许通过共聚焦显微镜在不同的时间点对POI进行随访(Boncompain等,2012)。





图1 。表示RUSH系统的方案。一。P rotein复杂含有目的蛋白(这里基质金属蛋白酶2,MMP2),链亲和素结合肽(SBP)和荧光蛋白(eGFP) 。该复合物通过SBP与抗生蛋白链菌素(Str)结合,该链霉亲和素与KDEL序列连接以保留在内质网(供体区室)中。没有添加生物素,复合物保留在供体区; 但是,一旦将生物素添加到培养基中,它就会与抗生蛋白链菌素结合,并使MMP2-SBP-eGFP蛋白复合物转运到受体区室。该图改编自Boncompain等人。(2012年)。乙。免疫荧光图像显示了MMP2在整个分泌途径中的运输,当缺少生物素时MMP2定位在ER处,随后在添加生物素后在高尔基体(15和30分钟)和高尔基后囊泡中定位(45分钟)。TGN46:反高尔基标记。图取自Pacheco-Fernandez等。(2020年)。



除其他技术外,RUSH系统的优势在于能够在生理条件下监视细胞。鉴于生物素是一种无毒的小分子,货物释放的同步化不会对细胞造成高压力(Boncompain和Perez,2013)。而且,与其他技术(例如光触发的蛋白质分离系统)相比(Chen等,2013),它不需要先前的蛋白质聚集,这可能导致通过不同途径的运输,更高的蛋白质降解率,因此,细胞毒性更高(Boncompain和Perez,2013年)。

在原始的RUSH方法论的基础上,这里我们提出一种协议,该协议可以定量评估在不同固定时间点的蛋白质货物运输。重要的是,在该协议中,我们提供了定量分析,以通过使用共聚焦显微镜评估沿分泌途径的特定货物运输动态来更好地检查高尔基体内的运输动态。这允许评估的影响的敲除或货物上贩卖沉默分泌途径的蛋白(Crevenna等人,2016;邓。等人,2018;帕切科费尔南德斯等人,2020) 。

关键字:蛋白质运输, RUSH, 蛋白(货物)分选, 共聚焦显微镜, 囊泡跟踪

材料和试剂
厚1的圆形盖玻片(ThermoScientific TM ,目录号:CB00120RA120MNT0)
6孔细胞培养板(Corning ® ,Costar公司® ,目录号:CLS3516-1EA)
Superfrost TM显微镜载玻片(ThermoScientific TM ,目录号:AA00008332E00MNT10)
聚乙烯亚胺,线性,MW 25 ,000,转染级(PEI 25K TM ;自Polysciences,目录号:23966-1)。
60-70%融合的HeLa细胞
1 × Dulbecco磷酸盐缓冲盐水(DPBS),无钙,无镁(Life Technologies,Gibco,目录号:14190144 )。储存温度:4 ℃。
pIRESneo3-Str-KDEL-MMP2-SBP-EGFP载体(图2)。储存温度:-20 °C
重要说明:此质粒是通过用Addgene质粒编号65264(Str-KDEL_ST-SBP-EGFP)中的MMP2(我们的目标蛋白)替换ST(ST6GAL1)序列而产生的。


图2 。在本协议中以pIRESneo2-Str-KDEL-MMP2-SBP-eGFP构建体为载体的图谱。该质粒描述了结合KDEL的链霉亲和素和报道分子MMP2-SBP-eGFP。质粒中的IRES元件可在单个质粒中同时表达Str-KDEL和MMP2-SBP-eGFP。有关构建体的更多详细信息,请参见Boncompain等。(2012年)。地图与SnapGene产生® 。


的Opti-MEM ®减少血清培养基(Life Technologies公司,GIBCO,目录号:31985070) 。储存温度:4 °C
500 mM d生物素(Merck,SUPELCO,目录号:47868)。储存温度:4 °C
1 × PBS中的4%多聚甲醛(PFA)。使用甲醛溶液制备,ROTIPURAN ® 37%,PA,ACS(罗斯,目录号:4979.1)
一抗
对于感兴趣的蛋白质
根据分析,可以使用特定的细胞器标记。例如,钙结合蛋白可以用作ER标记,而根据分析评估,可以使用不同的Golgi标记(例如,顺式-Golgi为GM-130 ,反式-Golgi网络为TGN-46 )
二抗
我们的研究使用针对不同物种的Alexa-Fluor抗体488、594和633 (Life Technologies,ThermoScientific,目录号根据物种和波长而变化)进行。储存温度:4 °C 。


ProLong金抗褪色试剂(Life Technologies,Invitrogen,目录号:P36934)
PEI溶液的浓度为1 mg / ml (请参阅食谱)
Dulbecco的改良Eagles培养基(DMEM)完全培养基(请参阅食谱)
DMEM,高葡萄糖,GlutaMAX TM补充剂,丙酮酸(Life Technologies,Gibco TM ,目录号:31966021)
胎牛血清(FBS,Life Technologies,Gibco TM ,目录号:10270106)
青霉素/链霉素(P / S,Life Technologies,Gibco TM ,目录号:15140122)
增透溶液(请参见配方)
海卫一X-100(卡尔·罗斯,目录号3051.2)
十二烷基硫酸钠(SDS)10%溶液
阻止解决方案(请参阅食谱)
牛血清白蛋白(BSA)冻干粉(PAN B iotech,目录号:P06-1391050)
磷酸盐缓冲盐水(PBS)粉末(Sigma,目录号:P3813-1PAK):根据制造商的说明制备1 ×溶液


设备


镊子
层流罩
细胞培养箱设置在37 °C ,5 %CO 2
水浴温度设定为37 °C
蔡司激光扫描LSM780共焦显微镜(卡尔·蔡司)
蔡司100×(NA 1.46,油)物镜(卡尔·蔡司)
488-nm激光线


软件


Zeiss Zen软件2010(Zeiss,https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html)
ImageJ v.1.37(美国国立卫生研究院[ NIH ] ,https: //imagej.nih.gov/ij/ )
用于“ RUSH-Vesicle-Analysis”的ImageJ宏(https://github.com/MehrshadPakdel/RUSH-Vesicle-Analysis)


程序


细胞培养和质粒转染
所有涉及细胞培养的步骤都必须在层流罩中进行。
在每孔含2 ml的DMEM完全培养基的6孔板中接种3 × 10 4 HeLa细胞,其中含有2-3个小盖玻片。制备每个时间点1个阱(见Ñ OTE 1)。
将细胞在37 °C ,5%CO 2下孵育24小时。
孵育24小时后,建立PEI转染反应。每口井:
200 µl OptiMEM
2 µg RUSH构建体,例如pIRESneo3-SS-Str-KDEL-MMP2-SBP-EGFP
15 µl 50mM PEI解决方案
涡旋转染反应,并在室温下孵育20分钟。
滴加到细胞中,并在37 °C ,5%CO 2下孵育至少16小时。避免孵育时间超过24小时。如果有必要,请在24小时后更换细胞培养基。


RUSH实验
温育后,制备2-米升DMEM完全与d -生物素,以每孔40μM的终浓度。根据W的数量准备足够的体积厄尔来分析(例如,对于4个孔(2米升每孔培养基),制备9米升DMEM的完全+ 7 。2μ升d -生物素500毫米)。将其保持在37 °C的温度下。
用微量移液器或真空泵从细胞中去除培养基,并用1 × DPBS洗涤一次。
添加2米升DMEM完全没有生物素,除了最新一期各时间点(见ñ OTE 2)。例如,对于MMP2,分析了四个时间点(0、15、30和45分钟)。仅向标记为0、15和30分钟的孔中添加培养基。
添加2米升DMEM完全+生物素(来自小号TEP B1)到最新的时间点(MMP2,到孔标记为45分钟。图3中,“开始实验的”)。
在37 °C ,5%CO 2下孵育细胞。
孵育时间取决于每种构建体的运输动力学,为了便于进行实验处理,我们建议首先开始将生物素添加到需要孵育时间最长的孔中(对于MMP2,孵育时间为45分钟),然后继续使用的时间点(30和15分钟,参见方案˚F igure 3)。


重要的是:ħ建议ERE采取细胞从培养箱中在至少2分钟的在下一时间点(触发货物贩卖MMP2之前,将板在加入生物素到先前井,即后13分钟取出的保育箱的出,在开始下一个时间点之前2分钟。有关详细说明,请参见注释2)。




图3 。方案描述了RUSH实验的时间间隔内的处理时间。该方案描述了使用MMP2构造的有经验的人的处理时间,所述时间点为0、15、30和45分钟。


从下一个要分析的时间点取出培养基,并添加2 m l DMEM complete +生物素。
重复小号TEPS B2,B4和B5,直到完成所述专职周期(MMP2,45分钟,最后的生物素添加在15分钟固定之前,˚F igure 3)。


盖玻片蒙太奇
孵育即将结束之前,用1 × PBS洗涤细胞(所有孔)2次
在弱光条件下,将细胞在PBS中的4%PFA中孵育10分钟。确保盖玻片完全被PFA解决方案覆盖。
用1 × PBS清洗3至5次。
如果不进行共染色:向孔中加入少量PBS,以利于盖玻片的处理,并使用ProLong antifade Gold试剂将其安装。以此目的:
拿一张显微镜载玻片。
将一滴ProLong防褪色金试剂放在幻灯片上。
小心地用镊子盖好一张盖玻片。
将盖玻片面朝下放在ProLong防褪色金试剂的顶部(即,盖玻片上带有细胞的一侧必须与防褪色剂接触)。
小心地将它们存放在黑暗的地方,直到成像。建议让蒙太奇的幻灯片干燥至少24小时,理想情况下干燥48小时。
如果进行联合染色:             
在弱光条件下,用通透溶液使细胞通透5分钟。
用1 × PBS清洗3至5次。
与封闭溶液在室温下孵育1小时,或在弱光条件下于4 °C孵育过夜。
通过在封闭溶液中稀释抗体来新鲜制备一抗溶液。取决于抗体,此信息可能由制造商推荐或需要实验室中的先前表征以确定理想的时间和温度。
用1 × PBS清洗3至5次。
与一抗在室温下孵育1小时,或在弱光条件下于4 °C孵育过夜。
通过稀释制造商推荐的封闭溶液中的抗体量来新鲜制备第二抗体溶液。
用1 × PBS清洗3至5次。
与二抗在室温下于弱光条件下孵育1小时。
用1 × PBS清洗3至5次。
安装使用的ProLong抗淬灭金试剂的载玻片(参见小号TEP C4)。


影像学
启动显微镜并预热所需的激光。根据激光和显微镜的不同,此过程最多可能需要30分钟。
设置Z堆栈获取实验。我们使用每个切片0.35 µm的距离,通常使用10到15个平面来覆盖大部分细胞体积。
聚焦单个细胞(或每个视野几个清晰定义且健康的单个细胞),并使用来自RUSH通道的信号设置要成像的堆叠数量。
如有必要,请调整其他通道的增益和强度。
开始成像。
重复小号TEPS D3和D4直至完成每个时间点至少15个细胞。
将文件另存为* .lsm 。


数据分析


打开斐济。
在斐济打开用于“ RUSH-Vesicle-Analysis”的定制ImageJ宏,网址为https://github.com/MehrshadPakdel/RUSH-Vesicle-Analysis。有关此宏中使用的ImageJ函数的更多详细信息,请参见注释7。
打开斐济的显微镜(* .lsm)文件。也可以使用其他显微镜文件,例如* .lif。
单击运行运行宏以启动分析(图4)。




图4 。斐济RUSH-囊泡分析宏窗口的屏幕截图


选择要分析的细胞数量(图5)。




图5 。小号从高峰分析宏步骤1的creenshot


选择要分析的感兴趣区域(ROI):通过在每个单元周围绘制多边形来标识每个单元(图6)。所选区域将自动保存为ROI。单击确定以选择下一个单元格。




图6 。RUSH-analysis宏的第2步的屏幕截图


生成二进制图像和阈值调整:选择最后一个单元并单击ok之后,您将看到2个窗口自动出现在屏幕上(图7,之前):一个显示二进制生成的图像(左窗口,标题:MAX_Bin_Image),另一个显示一个显示原始最大投影(右窗口,标题:MAX_Image)。另外,将出现两个控制窗口:一个名为“ Threshold”的阈值,用于手动调整MAX_Bin_Image的黑白平衡,另一个名为“需要采取的措施”。为了避免在计数过程中丢失低强度囊泡,与原始图像相比,您需要在视觉上调整生成的二进制图像的阈值(图7)。为此,请在“阈值”窗口中滑动这些条,直到MAX_Bin_Image类似于MAX_Image(后图7)。一旦两个图像相同,请在阈值窗口中单击“应用”。重要的是,一旦单击“应用”,就无法撤消阈值修改。单击“应用”后,在“需要执行的操作”窗口上单击“确定”以确认所做的更改,并继续执行宏(后图7)。




图7 。RUSH-analysis宏中第3步的屏幕截图。图“之前”显示生成的二进制图像(MAX_Bin_Image,左窗口),原始最大投影(MAX_Image),阈值控制窗口和“需要操作”窗口。为了避免丢失低强度囊泡,您必须手动调整以荧光最大投影为参考的MAX_Bin_Image的黑白平衡。正确的调整在名为“ After”的图中显示。


分析颗粒:在“需要采取的措施”窗口上单击“确定”后,宏将开始自动计算每个选定ROI上囊泡的数量。因此,将出现3个新窗口:一个带有图形的窗口(蓝色正方形,图8,此处命名为Cell1_Example_eGFP_45min_Quanti.tif),一个日志窗口,指示每个单元格计数的囊泡数量(洋红色正方形,图8),以及ROI管理器该窗口显示了为单元格选择的ROI(绿色正方形,图8)。可以将这些数字复制到E xcel文件或类似的电子表格文件中以进行进一步分析。




图8 。定量分析最后一步的屏幕截图。在步骤编号中单击“确定”后,将自动出现三个窗口。7:绘制每个细胞计数的囊泡(为每个分析的细胞生成,蓝色正方形),描述每个细胞计数的囊泡数目的日志窗口(洋红色正方形)和包含所选ROI信息的ROI窗口(选定的单元格,绿色正方形)。日志窗口值可以复制到excel文件或统计分析软件中以进行进一步评估。


宏运行完毕后,所有结果将自动保存在原始文件目录中的新结果文件夹中(图9)。保存的文件是原始文件(Orig_filename),原始图像的最大投影(MAX_filename),生成的二进制图像(Binary_filename),一个图形文件,其中显示了每个选定单元格的计数囊泡数(CellNumber_filename)和日志文件包含每个细胞计数的囊泡总数(log_filename)。




图9 。运行宏后生成的文件的屏幕截图


可以进一步比较囊泡的数量以进行统计测试,例如t-检验,ANOVA或它们相应的非参数测试,以评估时间点和治疗之间的差异。图10显示了HeLa细胞中MMP2eGFP阳性囊泡的囊泡定量示例。




图10 。MMP2 RUSH囊泡分析的最终结果。小图(A)描绘了在不同时间点表达RUSH MMP2eGFP构建体的HeLa细胞的免疫荧光图像。刻度条小号:10微米。图(B)显示了来自至少25个分析细胞的MMP2阳性小泡的累积数量。使用Kruskall-Wallis检验评估时间点之间的差异。* *** P值<0.0001。


当使用分泌途径标记物时,可以使用其他统计测试来评估共定位,例如Pearson或Manders系数。有关此类分析的更详细说明,请参阅Pacheco-Fernandez等。(2020年)。图11显示了MMP2与高尔基标记TGN 46共定位的示例,通过共聚焦显微镜观察。




图11 。用MMP2-RUSH转染并与TGN46共染色的HeLa细胞的免疫荧光图像。将细胞固定在指定的时间点,进行透化,并与GFP和抗TGN46抗体(TGN-46为反式-高尔基体标记)一起孵育。绿色:MMP2-SBP-eGFP,洋红色:反-高尔基体网络。比例尺:10 µm。


笔记


时间点是根据所评估的货物定义的。如果您使用的是未表征的货物,则应执行先前对已知货物及其描述的时间点的实验,并将其用作阳性对照。根据我们的经验,我们在实验室中评估的可溶性分泌货物(例如溶菌酶C和组织蛋白酶D)在60到75分钟的时间内通过分泌途径运输。未知的货物必须先LY在不同的时间间隔评估,以确定其贩卖的动力学。例如,当我们首次评估MMP2的运输动态时,我们评估了5分钟的间隔以确定运输的关键点(从ER到高尔基体,从高尔基体到细胞外空间)。但是,评估的时间点将始终取决于所需的分析类型。
这个协议是指最新孵育时间为在实验中进行评估(最后时间点即,在本协议45分钟)和更早的温育时间为这样的时间点(下文任何时候即,0,15和30分钟)。
请记住,对于经验丰富的人员,将板从培养箱中取出,除去培养基并添加含生物素的培养基至少需要2分钟。首次建议在两个时间点之间使用5分钟的工作时间窗口。因此,在下一个时间点开始前2或5分钟(根据操作经验)将板从培养箱中取出。例如,使用此处描述的MMP2构建体,将DMEM +生物素添加到45分钟的时间点后,孵育13分钟后将板从培养箱中取出,以便您有足够的时间将板放置在通风橱中,取出培养基从30分钟的孔中移出,替换为新的DMEM +生物素,然后在开始45分钟的孵育后恰好15分钟,将板放回到37 °C的孵育器中(图3)。
蛋白质运输是一个对温度敏感的过程。因此,建议调整实验条件,以免在37 °C下细胞孵育时间少于5分钟。
共聚焦显微镜的设置也可以在35毫米培养皿中进行。我们建议至少计数15个单元格,以进行有效的统计分析。
对照对于验证该测定是必需的。在这种情况下,没有一个蛋白货物匆忙构建体(即,含有海峡,保持钩,SBP和所述荧光团待分析)可被用作用于任何构建体的验证的控制。重要的是,该对照适合评估测定的功效,但对于进一步的动力学动力学比较,建议使用编码具有良好描述的运输动力学的蛋白质货物的构建体作为对照。
此处用于分析的宏使用7个功能(按以下顺序):
背景减除:Ü是基于在一个滚球算法SES ImageJ的减背景功能(https://imagej.net/Rolling_Ball_Background_Subtraction)。此算法将像素大小调整为图像中不属于背景的最大对象的半径。对于本分析,此半径已调整为高尔基体大小。
增强对比度:牛逼他的功能方便的小囊泡颗粒的知名度。它通过拉伸图像直方图而不改变像素值(https://imagej.net/docs/guide/146-29.html#sub:Enhance-Contrast)来增强对比度。
ž项目:ģ enerates与2D图像扁平的z堆系列的最大强度(https://imagej.nih.gov/ij/docs/guide/146-28.html#sub:Z-Project ... )。
图像处理滤波器“中值”:Ť他滤波器减少了在选择的噪声。在此宏中,用于使囊泡变平滑并在阈值化后生成更少的噪点对象(https://imagej.nih.gov/ij/docs/guide/146-29.html#sub:Median ...)。
阈值:小号egments灰度图像(在此分析二值图像)进入的兴趣和背景的特征。选择阈值会生成一个二进制图像,该图像定义了哪些对象不应该算作背景,并且是后续粒子定量所必需的(https://imagej.nih.gov/ij/docs/guide/146-28.html#sub:Threshold ... [T ] )。
:分析颗粒d给定的像素大小(内etects并量化颗粒https://imagej.nih.gov/ij/docs/guide/146-30.html#toc-Subsection-30.2)。


菜谱


PEI解决方案
溶解50毫克聚乙烯亚胺在40米升的蒸馏水
用电磁搅拌器在稍微加热的板上将溶液充分混合
调节pH至7.4
完整体积可达50米升。分装成500 µl,并储存在-20 °C
DMEM完全培养基
通过添加FCS和P / S分别至终浓度分别为10%和1%来制备500 ml的DMEM培养基


增透溶液
混入1 × PBS:0.2%Triton X-100和0.5%十二烷基硫酸钠(SDS;用20%或10%SDS溶液稀释)。
使5米升等分并存储长期在-20 ℃下。解冻后,储存在4 °C并在室温下预热以进行工作。如果形成沉淀,则在37 °C短暂加热直至溶液变澄清。
阻塞解决方案
将20 g牛血清白蛋白(BSA,分子生物学级)溶解在500 ml的1 × PBS中。等分试样在50米升和存储长期在-20 ℃下。解冻后,储存在4 °C并在室温下工作时保持在冰上。


致谢


该协议先前已在出版物(Deng等人,2018)和(Pacheco-Fernandez等人,2020)中记录过。这项工作得到了Deutscher Akademischer Austauschdienst(DAAD),Deutsche Forschungsgemeinschaft(DFG),美国国立卫生研究院(NIH),透视计划(Boehringer Ingelheim Fonds)和马克斯·普朗克生物化学研究所的资助。


利益争夺


作者宣称没有相互竞争的经济利益。


参考


Boncompain,G.,Divoux,S.,Gareil,N.,de Forges,H.,Lescure,A.,Latreche,L.,Mercanti,V.,Jollivet,F.,Raposo,G.和Perez,F. (2012)。细胞群体中分泌蛋白运输的同步。Nat Methods 9(5):493-498。
Boncompain,G.和Perez,F.(2013年)。基于荧光的哺乳动物细胞运输分析。方法细胞生物学118:179-194。
Boncompain,G.和Weigel,AV(2018)。高尔基综合体中的运输和分类:多种机制对各种货物进行分类。Curr Opin Cell Biol 50:94-101。
Chen D.,ES Gibson和MJ Kennedy(2013)。光触发的蛋白质分泌系统。J Cell Biol 201(4):631-640。
克雷文纳(美国)AH,布兰克(B.),马克萨(Maiser),艾敏(Emin),D。普雷舍尔(Prescher),贝克(B.G.),肯兹勒(Kienzle),巴特尼克(K. ,H.,Lamb,DC和von Blume,J.(2016)。在反式高尔基体网络上通过Ca 2+依赖的Cab45寡聚进行分泌货物分类。J Cell Biol 213(3):305-314。
Deng,Y.,Pakdel,M.,Blank,B.,Sundberg,EL,Burd,CG和von Blume,J.(2018)。SPCA1钙泵的活动将鞘磷脂合成与反式高尔基体网络中的分泌蛋白分选相结合。Dev Cell 47(4):464-478 e468。
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Stephens D.和Perez F.(2013年)。前言。在:佩雷斯(F.)和史蒂芬斯(D.)(编辑)。高尔基复合函数分析方法(第118卷,第xix–xx页)。学术出版社。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Pacheco-Fernandez, N., Pakdel, M. and von Blume, J. (2021). Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells. Bio-protocol 11(5): e3936. DOI: 10.21769/BioProtoc.3936.
  2. Pacheco-Fernandez, N., Pakdel, M., Blank, B., Sanchez-Gonzalez, I., Weber, K., Tran, M. L., Hecht, T. K., Gautsch, R., Beck, G., Perez, F., Hausser, A., Linder, S. and von Blume, J. (2020). Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs. J Cell Biol 219(8).
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