Monitoring the Recruitment and Fusion of Autophagosomes to Phagosomes During the Clearance of Apoptotic Cells in the Nematode Caenorhabditis elegans

Materials and Reagents

1. In vivo reporters

   1. LGG-1 reporter construct: P_ced-1mCherry::lgg-1 (for labeling autophagosomes)

   2. LGG-2 reporter construct: P_ced-1mCherry::lgg-2 (for labeling autophagosomes)

   3. Pseudopod and phagosomal surface reporter construct: P_ced-1ced-1::gfp (for labeling nascent phagosomes).

      
      

2. The Caenorhabditis elegans strains that carry both reporters

   1. ZH2919, genotype: enIs82 [pUNC-76(+)(20 ng/μL), P_ced-1ced-1::gfp (5 ng/mL), P_ced-1mCherry::lgg-1 (5 ng/μL)] II; unc-76(e911) V

   2. ZH2898, genotype: enIs83[pUNC-76(+)(20 ng/μL), P_ced-1ced-1::gfp (5 ng/mL), P_ced-1 mCherry::lgg-2 (5 ng/μL)] II; unc-76(e911) V

   In these strains, the recruitment and subsequent fusion of autophagosomes to phagosomes can be monitored by recording the dynamic localization patterns of both the mCherry and GFP reporters.

   
   

3. Consumable reagents

   1. Microscope slides (Premiere, catalog number: 9101)

   2. Coverslips (22 × 22 mm) (Fisher Scientific, catalog number: 12-542B)

   3. DeltaVision immersion oil, N = 1.516 (Cytiva, catalog number: 29162940)

   4. Handmade worm pick, which is a platinum wire (Alfa Aesar, catalog number: 10287) mounted on a Pasteur pipette (Fisher Scientific, catalog number: 13-678-20B) (Figure 3)

   5. High vacuum grease (Fisher Scientific, catalog number: 14-635)

   6. Agarose (Fisher Scientific, catalog number: BP160-500)

   7. KH₂PO₄ (MilliporeSigma, catalog number: PX1565)

   8. Na₂HPO₄ (MilliporeSigma, catalog number: 567550)

   9. NaCl (Fisher Scientific, catalog number: S671)

   10. 4% agarose solution (see Recipes)

   11. M9 buffer (see Recipes)

       
       

   
   Figure 3. A handmade worm pick

Equipment

1. Stereomicroscope (Nikon, catalog number: SMZ645)

2. DeltaVision Elite Deconvolution imaging system (GE Healthcare, Inc.), including AP1 F1- DeltaVision microscope (Olympus) equipped with 20×, 63×, and 100× Uplan Apo objectives, excitation/emission filter sets, fluorescent light source, differential interface contrast (DIC) microscopy accessories, motorized stage (X, Y, and Z-axis), and a Coolsnap HQ2 digital camera (Photometrics). For fluorescent imaging, two sets of fluorescence filters (Chroma Inc.) are used, including the GFP filter (excitation wavelength 475/28 nm; emission wavelength 525/50 nm) and the mCherry filter (excitation wavelength 575/25 nm; emission wavelength 632/60 nm). The DeltaVision microscope is kept in a room where the temperature is maintained at 20 °C.

3. Computer for image processing

Software

1. SoftWoRx 5.5 software (for the deconvolution and processing of images) (GE Healthcare, Inc.)

2. Microsoft Excel (Microsoft, Inc.)

3. Prism GraphPad (Dotmatics, Inc.)

Procedure

1. Mounting embryos on an agar pad

   1. Melt 4% agarose solution in a microwave oven.

   2. Dispense between 100 and 300 μL of the melted agarose solution in the center of the slide. Be cautious not to generate air bubbles. Immediately, flatten the drop by placing a glass slide perpendicular to the one holding the drop and press gently (Figure 4A). Let it stand for 1–2 min to allow the agarose to solidify.

   3. Gently separate the two slides by sliding one against the other. Using a clean glass slide as a blade, trim the flattened agar pad into an approximately 12 × 12 mm square (Figure 4B).

   4. Place 3 μL of M9 buffer at the center of the agar pad. The buffer is used to wash and maintain the embryos alive during the procedure.

   5. Under the stereotype microscope, collect 50–80 embryos with the worm pick and transfer them to the drop of M9 buffer on the agarose pad. When transferring embryos from a Petri dish to the agar pad on the slide, carry as little bacteria as possible. If too many bacteria are transferred, they will consume oxygen and cause the embryos to die.

   6. Gently squeeze a thin line of high vacuum grease around the agarose path (Figure 4C). Place a cover slip over the vacuum grease and press the corners gently to seal the slide (Figure 4D).

      
      

   
   Figure 4. Construction of an agarose pad. (A) Agar flattened by two cover slips. (B) Agar pad cut into a square, marked by the black arrow. (C) High vacuum grease (black arrow) placed around the agar pad. (D) Agar pad covered with a glass cover slip (yellow arrow) by pressing gently pressing the corners with a piper tip (black arrow).

   
   

2. Time-lapse recording of C1, C2, and C3 phagosomes

   1. Set up microscope parameters: for DIC images, an exposure time of 0.2 s with a 10% neutral density filter is enough. The percentage of a neutral density filter represents the fraction of light that passes through the filter and illuminates the sample. For recording the mCherry::LGG-1/2 reporters, the exposure parameter for the mCherry channel is 0.1 s with 5% neutral density filter. For recording the engulfment marker ced-1::gfp, the parameter is 0.15 s with a 5% neutral density filter.

   2. For optimal imaging, align the DIC light path according to the manufacturer's instructions.

   3. Under the 20× objective, find embryos. Under the 100× objective, identify embryos at the age between 280 and 300 min post their first cell division and with their ventral side facing up (Figure 5). Mark these embryos utilizing the “point marking” and “position visiting” functions of the SoftWoRx software. For each strain, we image 15 embryos.

   4. Define the position where the z-section recording should start. The serial z-section recording is performed from the embryo's ventral surface and proceeds to the center of the embryo. Set up 12–16 z-sections at 0.5 μm thickness for each section, which is sufficient to cover the entire ventral region.

   5. Set up the time-lapse recording parameters. Start recording when the embryo is approximately 320 min post the first cell division; a moment later, the hypodermal cells will engulf apoptotic cells C1, C2, and C3 (Figure 1A). A clue hinting that the engulfment of C1, C2, and C3 is about to happen is the event when the middle part of the embryo slightly contracts towards its center, as shown in Figure 5. The recording time interval is set at every 2 mins for 60 to 120 min or until the embryos reach the 1.5-fold developmental stage, which is at 420 min post the first embryonic division.

   6. Keep observing the images during the acquisition process. Adjust the starting focal plane if necessary. Abort the recording if the embryos slow down or arrest their development. Examples of time-lapse images of LGG⁺ vesicles are recruited to the phagosomal surfaces and subsequently fuse to phagosomes are shown in Figure 1B.

      
      

   
   Figure 5. DIC image of a C. elegans embryo at 300 min post first cell division. Scale bar is 5 µm.

Data analysis

Measuring the signal intensity inside the phagosomal lumen

To measure the fluorescence signal intensity inside the phagosomal lumen over time in embryos expressing P_ced-1mCherry::lgg-1 or -2, the boundary of a phagosome is identified at the 0 min time point when a nascent phagosome forms by the CED-1::GFP marker, which labels the surfaces of the extending pseudopods and nascent phagosomes. At each time point, the total mCherry::LGG-1 or -2 signal intensity within a fixed area (4 × 4 pixels) in the center of a phagosome (Int_phagosome) is recorded (Figure 6B), as is the intensity of an area of the same size (4 × 4 pixels) outside the embryo as the background image intensity (Int_background). The relative image intensity (RInt) at a particular time point (Tn) comparing to the start point (T0) is calculated as RInt_Tn = (Int_phagosome - Int_background)_Tn / (Int_phagosome - Int_background)_T0. The RInt_Tn value of 1.0 indicates no entry of LGG-1- or LGG-2-labeled autophagosomes into the phagosomal lumen. The data collected are plotted in a graph with the X-axis indicating the time points and RInt_Tn in the Y-axis. In addition, for each strain to be characterized, the RInt_T50 (at the +50 min time point) of 15 phagosomes is measured. Standard statistical analysis is performed (Figure 6D). Whether the RInt_T50 in a particular mutant strain is significantly different from that in the wild-type is determined by Student’s t-test.

Measuring the signal intensity on the surface of a phagosome

To evaluate the efficiency of recruitment of autophagosomes to phagosomes, the intensity of mCherry::LGG-1 or -2 is measured on the surfaces of phagosomes. First, the boundary of a phagosome at 0 min is identified by the CED-1::GFP reporter, which labels the surfaces of the extending pseudopods and nascent phagosomes. At a particular time point (Tn), the surface of a phagosome is outlined by two closed polygons (Figure 6A). The total signal intensities, as well as the areas of the polygons, are recorded. The unit signal intensity of the donut-shaped area between the two polygons is calculated as follows:

  Unit Intensity (UI_phagosome) = (Intensity_{external polygon} - Intensity_{internal polygon}) / (Area_{external polygon} - Area_{internal polygon}). The Unit Background Intensity (UI_background) was measured from a polygon outside the embryo and calculated as follows: UI_background = Intensity_background / Area_background. At the time point Tn, the relative signal intensity on phagosomal surfaces (Rsurface_Tn) = (UI_phagosome - UI_background)_Tn / (UI_phagosome - UI_background)_T0. The Rsurface_Tn value 1.0 indicates no recruitment of the LGG-1- or LGG-2-labeled autophagosomes on phagosomal surfaces compared to the 0 min time point. The data collected are plotted in a graph with the X-axis indicating the time points and Y-axis indicating Rsurface_Tn. In addition, for each strain to be characterized, the Rsurface_T12 (at the +12 min time point) of 15 phagosomes is measured (Figure 6C). Standard statistical analysis will be performed. Whether the Rsurface_T50 in a particular mutant strain is significantly different from that of the wild-type will be determined by the Student’s t-test.

Figure 6. Two diagrams depicting the areas where the LGG signal intensity is measured in the C3 phagosome. (A) Diagram illustrating where the mCherry signal on the surface of a phagosome is measured over time. The area is delimited by the two black-dotted polygons. (B) Diagram illustrating where the relative mCherry signal in the center of a phagosome is measured over time. (C) Box and whisker plots of the relative mNeonGreen (mNG) signal intensities measured on the surfaces of ten C3 phagosomes 12 min post the formation of nascent phagosomes from wild-type and ced-1(e1735) mutant embryos. This graph is adapted from Peña-Ramos et al. (2022). (D) Box and whiskers plots of the relative mNG signal intensities measured in the center of fifteen C3 phagosomes 50 min post the formation of nascent phagosomes from wild-type and ced-1(e1735) mutant embryos. (C and D) Red dashed lines indicate the position of value 1.0, which represents no signal enrichment relative to the background signal. ***, p < 0.001, Student’s t-test of the ced-1(e1735) mutants compared to the wild-type value.

Notes

1. The reporter constructs are available upon request.

2. The strains mentioned in this protocol are available upon request.

3. Although this protocol specifically describes the usage of the DeltaVision Elite Deconvolution imaging system for the real-time recording of autophagosome recruitment and fusion events, it can be easily adapted to other real-time imaging systems such as the spinning disk confocal microscope. In addition, in C. elegans, programmed cell death events also occur in larvae and in the gonad of adult hermaphrodites in addition to embryos. Our protocol can also be applied to study the incorporation of autophagosomes into phagosomes in larvae and adults. Furthermore, besides C. elegans embryos, any experimental system in which real-time imaging can be conducted and that allows the expression of transgenic reporter constructs can adopt this protocol as well.

Recipes

1. 4% agarose solution

   1. Place 2.0 g of agarose in 50 mL of deionized water and heat up in a microwave until the agarose is completely dissolved.

   2. After usage, cap the flask with aluminum foil and store it at room temperature.

   3. To reuse the agar solution, melt the solidified solution for 35 s in the microwave.

2. M9 Buffer (1 L)

   1. Dissolve 5.8 g of Na₂HPO₄ (40.9 mM), 0.5 g of NaCl (8.6 mM), 1.0 g of NH₄Cl (18.7mM), and 3.0 g KH₂PO₄ (22.0 mM) in 400 mL of deionized water.

   2. Bring the volume to 1 L and autoclave for 40 min.

Acknowledgments

This work is supported by NIH grant GM067648. This protocol is adapted from Peña-Ramos et al. (2022).

Competing interests

There are no conflicts of interest or competing interests.

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