Real-time Three-dimensional Tracking of Endocytic Vesicles

Materials and Reagents

1. 35 mm glass bottom cell culture dish (Ibidi GmbH, catalog number: 81158 )
2. Microcentrifuge tubes: 1.5 ml (Axygen Corning^®, catalog number: MCT-150-C )
3. 6-well plate (Nunc^®, catalog number: 140675 )
4. 100 mm dish (VWR^®, catalog number: 172931 )
5. SKBr3 breast carcinoma cells (ATCC^® HTB-30) 
6. Hepatocyte growth factor, HGF (Genentech, Inc.)
7. DMEM-Dulbecco's Modified Eagle Medium (Gibco^®, catalog number: 11966025 )
8. Rat tail collagen I (Roche^®, catalog number: 11179179001 )
9. Foetal Bovine Serum (FBS) (Gibco^®, catalog number: 26140079 )
10. Green fluorescent protein (GFP)-Rab4 plasmid [provided by Robert Lodge and cloned as described in Zaoui et al. (2019a)]
11. Cell line nucleofector Kit V (Lonza^®, catalog number: VCA-1003 )
12. Phosphate-buffered saline (PBS) (Wisent Inc, catalog number: 311-012-LL )
13. Trypsin-EDTA (Gibco^®, catalog number: 25200056 )
    

Equipment

1. Cell incubator Steri-Cycle CO₂ (Thermo Fisher Scientific^®, catalog number: 51030303 )
2. Centrifuge (Eppendorf^®, catalog number: 5424/5424R )
3. Nucleofector 2b device (Lonza^®, catalog number: AAB-1001 )
4. Motorized Inverted microscope Axiovert 200 M (Carl Zeiss, Inc.)
5. Stage top chamber with 5% (v/v) CO₂ in air at 37 °C (Climabox, Carl Zeiss, Inc.).
6. Objective 100x plan Apochromat NA 1.4 objective
7. Hemocytometer (Sigma-Aldrich^®, catalog number: Z359629-1EA )
   

Software

1. AxioVision software (Carl Zeiss, Inc.)
2. Imaris software (Bitplane Inc.)
3. Microsoft Excel^®
   

Procedure

1. Collagen I coating
   
   1. Coat the 35 mm glass bottom cell culture dish with 1 ml rat-tail collagen I diluted in PBS (25 µg/ml).
      
   2. Incubate the 35 mm glass bottom cell culture dish coated with 1 ml collagen I at 37 °C, for 1 h.
      
   3. Wash the 35 mm glass bottom cell culture dish twice with 1 ml pre-warm PBS and aspirate off PBS buffer.
   Note: Collagen I must be kept on ice to avoid polymerization and PBS must be kept at 37 °C.
   
   
2. Cell transfection and seeding
   
   1. Retrieve culture plate from incubator, aspirate off media, and wash twice cells with 1 ml pre-warm PBS.
      
   2. Detach SKBr3 cells (80-90% confluency) by adding 1 ml Trypsin-EDTA and incubate the cells at 37 °C, for 5 min.
      
   3. Neutralize trypsinization reaction with supplemented culture medium (10% FBS) when most of the cells (> 90%) are detached.
      
   4. Determine cell density with hemocytometer.
      
   5. Transfer (1 x 10⁶ cells per sample) into the microcentrifuge tubes.
      
   6. Centrifuge the cells at 200 x g for 10 min at room temperature.
      
   7. Gently resuspend cell pellet in 100 μl nucleofector solution V at room temperature. The solution V is provided with the nucleofector Kit V.
      
   8. Combine 100 μl of cell suspension with 3 μg DNA of GFP-Rab4.
      
   9. Transfer cell/DNA suspension into certified cuvette provided with nucleofector Kit V (sample must cover the bottom of the cuvette without air bubbles). Close the cuvette with the cap.
      
   10. Select the appropriate Nucleofector^® Program X-005 on the Nucleofector^® 2b device.
       
   11. Insert the cuvette with cell/DNA suspension into the Nucleofector^® Cuvette Holder and apply the selected program by pressing the X-button.
       
   12. Take the cuvette out of the holder once the program is finished.
       
   13. Immediately add 500 μl of the culture medium (10% FBS) to the cuvette.
       
   14. Use the supplied pipettes to transfer the sample (600 µl final volume) into the collagen I pre-coated 35 mm glass bottom cell culture dish prepared in Procedure A.
       
   15. Add 2 ml supplemented culture medium with 10% FBS per 35 mm glass bottom cell culture dish and incubate at 37 °C, for 24 h.
   Note: Avoid repeated aspiration of the sample in Step B14.
   
   
3. Cell washing and stimulation
   
   1. Retrieve the 35 mm glass bottom cell culture dish from incubator, aspirate off media, and wash twice cells with 1 ml pre-warm PBS.
      
   2. Add 2 ml pre-warm culture medium (10% serum)
      
   3. Add HGF at 0.5 nM (or any other growth factor) for 20 min to stimulate the cells. An unstimulated condition is needed as a negative control, to evaluate the effect of Met receptor tyrosine kinase activation with HGF.
   Note: HGF must be kept on ice and PBS and supplemented culture medium at 37 °C.
   
   
4. Microscope setup for time-lapse imaging in 3D (Figure 1 and Video 1)
   
   1. Microscope setup
      
      1. Turn on the motorized Inverted microscope Axiovert 200 M and the connected computer.
         
      2. Select the appropriate stage holder and the 100x plan Apochromat NA 1.4 objective.
         
      3. Turn on the temperature unit and the CO₂ control module, set temperature at 37 °C, humidity > 80% and CO₂ at 5%. Warm up for approximately 2 h.
         
   2. Live-cell imaging in 3D
      
      1. Open the AxioVision software and choose Multidimensional Acquisition module.
         
      2. Place the 35 mm glass bottom cell culture dish on the plate holder. Focus on the selected cells.
         
      3. Set up the cell duration of acquisition for 2 min with a capturing interval of 4 s and 10 of Z sections with 0.5 μm spacing.
         
      4. Acquire single cell images in 3D (X, Y, Z) and analyze them in real-time.
   Note: The exposure time parameter varied slightly between experiments and depends on the transfection efficiency. Depending of the size of the cell additional Z sections may be required.
   
   
   Figure 1. Microscope configuration for live cell imaging. Example of a complete setup for cell tracking using Axiovert 200 M motorized inverted microscope equipped with a Perspex environmental chamber, CO₂ enrichment module, and heating controller. Please refer to the McGill University Advanced Bioimaging Facility for additional details.
   
   
   
   Video 1. Visualization of GFP-Rab4 protein by time-lapse video microscopy.  Movie of SKBr3 cells expressing GFP-Rab4 stimulated with HGF 0.5 nM for 20 min.

Data analysis

In the following section, we will demonstrate procedures for the analyses of Rab4-positive vesicles mobility in 3D using spots tracking algorithm of Imaris^® software.

1. Visualization and segmentation (Figure 2)
   1. Transfer the measurements to Imaris^® software and create a volume reconstruction of the data set select using the “Surpass mode”.
   2. Click on “Edit”, open “Display Adjustment” window, select as source channel the Channel 1 (green for GFP) and adjust the minimum and the maximum.
   3. Click on the left tab “Spot Object” button and a new object spot is now added in the object list.
      
      
      Figure 2. Three-dimensional analysis of Rab4-GFP movement by live cell imaging. A. Representative image of spot assignment of the Rab4-GFP vesicle using the Spots tracking algorithm of Imaris^® software. Stack image of SKBr3 cells transfected with GFP-Rab4 and treated or not (CTL) with HGF 0.5 nM for 20 min. Insets show enlargement from the cell cortex. B-E. Individual Rab4-positive vesicles were tracked over time (2 min) after 20 min stimulation with HGF 0.5 nM. The percentage of directed movement of vesicles to leading edge (B), to center (C) and, the speed of Rab4-positive vesicles (D) were analyzed. E) Cell velocity was determined by tracking cells every 5 min for 420 min. Ninety cells were tracked for CTL and HGF treated cells. Scale bars = 10 μm. Graphed data represent the average values ± SEM from at least three independent experiments. Statistical significance was assessed using a two-tailed Student’s t test. *** P < 0.001. Representative movie of SKBr3 cells expressing GFP-Rab4 stimulated with HGF 0.5 nM for 20 min (Video 1).
      
      
2. Tracking algorithm
   
   1. Select “Track Spots (over time)” in create properties. Click on “Next”.
      
   2. Select as Source Channel the Channel 1 (green for GFP); set the “Estimated Diameter” to 0.5 µm. Add “Background Subtraction” option. Click on “Next”.
   Note: Imaris^® software detects spots automatically you can either accept the automatic values or change them by selecting the manual option.
   
   
3. (Optional) Selecting the manual option
   
   1. Click on “Manual” option and select a subset of the detected spots by modifying the lower and higher threshold of the green fluorescence. Put the Lower Threshold value at 10 and to verify spots detection click on play button. Click on “Next”.
      
   2. Add or delete spots at this step (if needed). Click on “Next”.
      
   3. Select “Brownian Motion” algorithm. Set Maximum Distance at 10 µm and Maximum Gap Size at 3 µm. Click on “Next”.
      Note: The “Brownian Motion” algorithm is appropriated for the tracking of random movements without discernable pattern and frequent directional changes. Here, this algorithm has been used to track the Rab4-GFP vesicles because in absence of HGF the Rab4-GFP vesicles move slowly, in a random manner, without any directional persistence. However, the Imaris^® software provides “Autoregressive Option”, this algorithm tolerates high rate of directional change during the displacement and can be used to track objects with continuous motion.
      
   4. Sort and filter the result tracks by selecting “Track Speed Average” in “Filter Type menu”. Click on Finish.
   5. Export the data to Microsoft Excel^® software for further analysis. The tracking measurements are stored in the “Detailed Tab” found under “Tab Statistics”.
      
   6. Summarize vesicle speed of at least 500 vesicles from 30 cells as desired (i.e., mean ± SEM) and perform statistical analysis as desired (such as unpaired t-test).
      
   7. Plot summarized information using preferred graph (such as bar graph).
      
   Note: Under “Specific Values Tab”, a second pull-down list at the top of the window displays available specific statistical values. Select the variable and a list of values will be displayed. Statistical variables associated with objects (speed) have values at every time point. Therefore, for these variables the “Average values” are displayed per time point. The changing the time points, statistical variable values and images are consequently changed.
   

Acknowledgments

This work was performed as a part of the publication of Zaoui et al. (2019a), Traffic and was supported by a Susan G. Komen for the Cure Postdoctoral fellowship (KG111467) to K. Zaoui.

Competing interests

The authors declare no competing financial interests.

References

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