参见作者原研究论文

本实验方案简略版
Apr 2021
Advertisement

本文章节


 

Live-cell Imaging and Analysis of Germline Stem Cell Mitosis in Caenorhabditis elegans
秀丽隐杆线虫生殖干细胞有丝分裂的活细胞成像和分析   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Model organisms offer the opportunity to decipher the dynamic and complex behavior of stem cells in their native environment; however, imaging stem cells in situ remains technically challenging. C. elegans germline stem cells (GSCs) are distinctly accessible for in situ live imaging but relatively few studies have taken advantage of this potential. Here we provide our protocol for mounting and live imaging dividing C. elegans GSCs, as well as analysis tools to facilitate the processing of large datasets. While the present protocol was optimized for imaging and analyzing mitotic GSCs, it can easily be adapted to visualize dividing cells or other subcellular processes in C. elegans at multiple developmental stages. Our image analysis pipeline can also be used to analyze mitosis in other cell types and model organisms.


Keywords: Live cell and tissue imaging (活细胞和组织成像), Stem cells (干细胞), Mitosis (有丝分裂), Spindle dynamics (Spindle dynamics), C. elegans (秀丽隐杆线虫), Germline (种系)

Background

While permitting the visualization of various tissue-resident stem cells in several model systems, recent advances in intravital imaging often rely on invasive surgery or sophisticated and expensive imaging modalities ( Yoshida et al., 2007; Rompolas et al., 2012; Ritsma et al., 2014; Barbosa et al., 2015; Park et al., 2016; Martin et al., 2018; Nguyen and Currie, 2018). C. elegans GSCs are an established stem cell model that has yielded generalizable insights into many aspects of stem cell biology (Kimble and White, 1981; Baugh and Sternberg, 2006; Fielenbach and Antebi, 2008; Angelo and Van Gilst, 2009). In addition, C. elegans GSCs can be imaged in living animals using standard fluorescence microscopy techniques and without surgical manipulation. In particular, live imaging of GSC mitosis provides an opportunity to investigate the dynamics of cell division and how they are influenced by in vivo factors such as tissue organization, niche signaling, and organismal physiology. Here we describe a simple, fast, and reproducible method to immobilize C. elegans and to image and analyze GSC mitosis, while preserving animal viability, fertility, and seemingly normal GSC divisions.


Adult C. elegans hermaphrodites house two populations of GSCs located at the distal ends of the two tube-shaped gonad arms (Figure 1A). Like other germlines, the C. elegans gonad is organized as a syncytium. GSCs form a rough circumferential monolayer around a shared inner core of cytoplasm named the rachis (Hirsh et al., 1976) (Figure 1A and 1C). Each GSC is connected to the rachis by a stable actomyosin ring, which forms a cytoplasmic bridge (Maddox et al., 2005; Zhou et al., 2013; Amini et al., 2014; Priti et al., 2018). Like other stem cells, C. elegans GSCs are kept in a stem-like state by signaling from a somatic niche (the distal tip cell, Kimble and White, 1981; Figure 1A and 1E). Like several types of mammalian stem cells, the size of the C. elegans GSC pool is maintained according to a population model, wherein differentiation due to displacement from the niche is balanced by symmetrical divisions to maintain a relatively constant number of stem cells. according to a population model, wherein differentiation due to displacement from the niche is balanced by symmetrical divisions, thus maintaining a relatively constant number of stem cells (Morrison and Kimble, 2006; Joshi et al., 2010).


The majority of studies in C. elegans GSCs rely on dissected, fixed, and stained gonads, or single-timepoint imaging of gonads bearing fluorescently tagged proteins in living worms. Long-term imaging of GSCs has been accomplished using “catch and release” approaches that permit periodic visualization of GSCs over hours or days (Wong et al., 2013; Rosu and Cohen-Fix, 2017). Moreover, a recently developed microfluidics device may permit continuous observation on a similar time scale (Berger et al., 2018, Berger and Spiri, 2021). Fewer studies have reported live cell imaging of GSCs under conditions suitable for documenting dynamic subcellular events such as mitosis (Gerhold et al., 2015; Gordon et al., 2020; Zellag et al., 2021).


To achieve high temporal and spatial resolution during live imaging, animals must be immobilized and the overall impact of mounting on animal and GSC physiology must be considered. Typical mounting methods immobilize animals using a combination of paralytic drugs and physical compression (Sulston and Horvitz, 1977; Chai et al., 2012; Kim et al., 2013; Hwang et al., 2014; Luke et al., 2014; Burnett et al., 2018; Fabig et al., 2020a and 2020b; Gordon et al., 2020). We use an etched silicon wafer to pattern grooves on an agarose pad, which constrains the animals in a straight position (Figure 1B); this prevents the sinusoidal movements required for worm locomotion (Gray and Lissmann, 1964) and reduces the requirement for physical compression and anesthetics.



Figure 1. A mounting method allowing for live-imaging of dividing GSCs in intact C. elegans.

A. Schematic showing a top view of a late L4 (top) and an adult (bottom) hermaphrodite worm mounted in grooves. The position and overall organization of the two gonad arms are shown in white, and key germline features are highlighted, such as the rachis (green), and the distal tip cell (DTC) or niche (magenta). The position of the GSCs is indicated by purple circles. B. Schematic showing a cross-sectional view of animals mounted in v-shaped (top) and square-shaped (bottom) grooves that match the width of the animals. (C-E) Maximum intensity projections of the distal gonad from adult animals expressing (C) mNG::ANI-1 (green) and mCH::β-tubulin (magenta; strain = UM679), D. GFP::β-tubulin (green) and mCH::Histone H2B (magenta; strain = JDU19), and (E) LAG-2::GFP (green) and mCH::β-tubulin (magenta; strain = UM211). A dividing GSC in each gonad is boxed in yellow and an enlarged image is shown below. Fluorescently labelled β-tubulin marks the mitotic spindle in all three strains, with mNG::ANI-1 marking the rachis, mCH::Histone H2B marking nuclei and LAG-2::GFP marking the DTC/niche. Scale bar = 10 µm (top, gonad view) and 5 µm (bottom, single cell view).


We have used this mounting method to provide the first characterization of GSC mitosis by live imaging (Gerhold et al., 2015). Recently, we investigated the technical factors that impact GSC mitosis during live imaging, which allowed us to define optimal mounting and imaging conditions, and to determine that animal starvation during mounting/imaging has the most deleterious effect on GSC mitosis (Zellag et al., 2021). Our data suggest that GSC mitosis can be imaged under near physiological conditions within an approximately 40 min window, which starts from the moment worms are removed from food. Although some GSCs enter mitosis after 40 min, we recommend caution when interpreting their behavior. In addition, under optimal conditions, the number of mitoses per germline and the duration of these mitoses in wild-type worms are relatively constant, and offer a reproducible baseline by which others may benchmark their results (see Videos 1 and 2 and Figure 4I-4K).


A parallel challenge that arises when using live imaging to study GSC mitosis is how to monitor mitotic progression. GSCs divide within the tube-shaped gonad and can divide in any orientation relative to the imaging plane, and at a range of depths relative to the gonad surface. Therefore, accurate monitoring of GSC mitosis requires a tracking method that accounts for the three-dimensional (3D) nature of their division. A good method will need to be sufficiently high-throughput to generate large data sets with minimal user time. In addition, fluorescently labelled proteins are often expressed at low levels in the germline (Merritt and Seydoux, 2010), which limits the availability of suitable markers.


We have shown that fluorescently tagged β-tubulin provides a robust marker for GSC mitotic centrosomes and that we can use centrosome-to-centrosome distance as a reliable readout for mitotic progression by tracking centrosome pairs in 3D (Gerhold et al., 2015; Zellag et al., 2021). In addition, tracking centrosome pairs can provide information on mitotic features, such as spindle dynamics and orientation (Zellag et al., 2021). To make this approach amenable to large-scale studies, we recently developed CentTracker, a largely automated image analysis pipeline that allows for fast extraction of mitotic parameters in any genetic background and in other cell types and organisms, provided that centrosomes are trackable (Zellag et al., 2021). Here, we describe the basic imaging processing steps required for using CentTracker. The relevant code and detailed instructions are freely available for download.

Materials and Reagents

  1. 60 mm Petri dish with cams (Sarstedt, catalog number: 82.1194.500)

  2. 1.5 mL microcentrifuge tube (Axygen, catalog number: MCT-150-C)

  3. 15 mL conical centrifuge tube (Fisher Scientific, catalog number: 14-959-53A)

  4. Aspirator tube assembly for calibrated microcapillary pipettes (Sigma, catalog number: A5177-5EA)

  5. 50 µL glass micropipette (VWR, catalog number: 53432-783)

  6. 1.7 mL plastic transfer pipette (Fisher Scientific, catalog number: 13-711-41)

  7. 200 µL pipet tips (Diamed, catalog number: DIATEC520-6752)

  8. 1,250 µL pipet tips (Diamed, catalog number: DIATEC520-6501)

  9. Single edge razorblade

  10. Fine tipped forceps/tweezers

  11. Lab tape (Fisher Scientific, catalog number: 15-901-10R)

  12. 18 × 18 mm #1.5 (0.16-0.19 mm) square cover glasses (VWR, catalog number: CA48366-205-1)

  13. Microscope slides (Fisher Scientific, catalog number: 12-55-15)

  14. Whatman 3 MM Chr blotting paper (VWR, catalog number: 21427-411), cut into ~3 cm strips

  15. KIMTECK Kimwipes 11.2 × 21.3 cm

  16. 1-well glass depression slide (VWR, catalog number: 470235-728)

  17. Silicon wafer micro-patterned by lithography, to give a series of parallel raised ridges with defined depth and width (Figure 2B-2C; see Note 1; hereafter “silicon mold”)

  18. Worm pick [99.95% Platinum, 0.05% Iridium wire, 0.01 inches diameter (Tritech, catalog number: PT-9901), flattened like a spatula at one end, and mounted on a glass Pasteur pipette (Fisher Scientific, catalog number: 13-678-20A), or worm pick handle (Tritech, catalog number: TWPH1)]

  19. Worm eyelash pick (see Note 2)

  20. C. elegans strain(s) of preferred genotype bearing a fluorescent protein (FP) tagged centrosomal marker (if using CentTracker), with or without additional FP-tagged proteins (Figure 1C-1D; strains used in this protocol are listed in Table 1).

    Note: We find that single-copy transgenes inserted by Mos1-mediated single copy insertion (MosSCI; Zeiser et al., 2011), or CRISPR-Cas9 with a single guide RNA (sgRNA) targeting a region near to established Mos1 insertion sites (Dickinson et al., 2013), and expressing FP-tagged proteins under the mex-5 promoter, and tbb-2 3’ UTR regulatory sequences produce the best results for germline-specific expression suitable for live-imaging.


    Table 1. C. elegans strains used in this protocol.

    StrainGenotype
    UM679ltSi567[pOD1517/pSW222; Pmex-5::mCherry::tbb-2::tbb-2_3′UTR; cb-unc-119(+)] I; ani-1(mon7[mNeonGreen^3xFlag::ani-1]) III
    UM211qls56[Plag2::GFP] V; wels21[Pja138(Ppie-1::mCherry::tubulin::pie-1_3’UTR)] ?
    JDU19ijmSi7[pJD348/pSW077; mosI_5′mex-5_GFP::tbb-2; mCherry::his-11; cb-unc-119(+)] I; unc-119(ed3) III
    ARG16ltSi567[pOD1517/pSW222; Pmex-5::mCherry::tbb-2::tbb-2_3′UTR; cb-unc-119(+)] I; ccm-3(mon9[ccm-3::mNeonGreen^3xFlag]) II


  21. E. coli strain OP50 (Brenner, 1974)

  22. E. coli strain HT115 (Timmons and Fire, 1998)

  23. Nematode Growth Medium (NGM) (Stiernagle, 2006; with the following modifications: 20 g agar and 3 g peptone per liter media)

  24. Distilled water (dH2O)

  25. 10 M NaOH (Bioshop, catalog number: SHY700.1; powder dissolved in dH2O)

  26. Commercial bleach (Bioshop, catalog number: SYP001.4; Sodium Hypochlorite, 12% Solution)

  27. Vaseline® 100% pure petroleum jelly

  28. Lanolin (Sigma, catalog number: L7387-250G)

  29. Paraffin wax (Bernardin Parowax Canning Wax, 454g)

  30. Tetramisole (Sigma, catalog number: L9756-5G)

  31. Agarose powder (Sigma, catalog number: A9539-500G)

  32. M9 buffer (see Recipes)

  33. Bleaching solution (see Recipes)

  34. Valap (see Recipes)

  35. 2% w/v Tetramisole solution (see Recipes)

  36. 3% w/v volume Agarose powder solution (see Recipes)

Equipment

  1. Stereomicroscope with transmitted light stand (Leica, model: S6 E, Nikon, model: SMZ 745 or equivalent)

    Note: We prefer stereomicroscopes with an external or LED light source, to avoid heating our worms during mounting.

  2. Two heat blocks, one set to 95°C, and the other to 65°C (VWR, model: Standard Dry Block Heater or equivalent)

  3. Benchtop microcentrifuge with rotor for 1.5-2 mL tubes (Eppendorf, model: 5415 D 24-tube with rotor F45-24-11, or equivalent)

  4. Vortex mixer (Scientific Industries, model: Vortex-Genie 2, or equivalent)

  5. Incubator set to 20°C (Sanyo, model: MIR-553, or equivalent)

  6. Test tube rocker (Thermo Scientific, model: Vari-Mix M48725Q, or equivalent)

  7. Single channel pipettes for 10-1,000 µL volumes (Gilson, models: Pipetman Classic P20, P200 and P1000, or equivalent)

  8. Microwave

  9. Spinning disc confocal microscope (Zeiss, model: Cell Observer, Quorum, model: WaveFX-X1, or equivalent) with excitation wavelengths and emission filters suitable for GFP and/or mNeonGreen and mCherry fluorescent proteins (e.g., 35-50 mW 488 nm or 491 nm, and 561 nm or 568 nm diode lasers, with ET525/50 and FF593/40 single band pass, or 466/523/600/677 quad band pass emission filters), and a high numerical aperture (NA) 63× oil objective (e.g., Leica, model: 63×/1.40-0.60 oil HCX PL APO, or Zeiss, model: 63× Plan-Apochromat DIC UV VIS-IR).

    Note: If available, a high NA water immersion objective (e.g., Leica, model: HC PL APO 63×/1.20 W CORR CS2) may be preferable depending on the sample.

  10. Spinning disk scanner (Yokogawa, model: CSU-X1)

  11. Scientific CMOS camera (Zeiss, model: AxioCam 506 Mono, Photometrics, model: Prime BSI, or equivalent)

Software

  1. Conda 4.9.0 or ulterior release (Anaconda, https://anaconda.org/anaconda/conda)

  2. CentTracker Github repository: https://github.com/yifnzhao/CentTracker

  3. Fiji 1.52v or ulterior release that includes TrackMate (Schindelin et al., 2012; Tinevez et al., 2017)

  4. MATLAB 2020b or ulterior release (MathWorks, https://www.mathworks.com/products/matlab.html)

Procedure

  1. Culture of C. elegans strains in preparation for GSC live imaging

    1. Maintain C. elegans animals on nematode growth medium (NGM) inoculated with Escherichia coli strain OP50, according to standard protocols (Brenner, 1974).

    2. Transfer worms regularly, to ensure a healthy and relatively synchronous population. We use a worm pick (Figure 2A) to transfer several small aggregates of ~L1 larvae (~50 animals in total) weekly (~every 2 generations) from plates maintained at 20°C, just as the bacterial lawn is becoming depleted. However, the optimal maintenance method will depend on the animals’ genotype and temperature at which they are grown (Stiernagle, 2006).

    3. Table 1 lists the strains used in this study. To use CentTracker for analysis, animals must carry a centrosome marker suitable for fluorescence microscopy. We use β-tubulin tagged with either GFP or mCherry, which marks the mitotic spindle and is enriched at centrosomes. If a more specific centrosomal marker is needed, FP-tagged centriolar or pericentriolar components (e.g., SAS-4 or SPD-2; Magescas et al., 2019) may be more suitable.


  2. Synchronization of larvae at the L1 stage

    1. Three days in advance, set up plates by transferring 2-3 larger aggregates of recently starved L1 larvae from a stock plate to fresh NGM/OP50 plates, using a worm pick. This should ensure plates full of healthy, well-fed, and gravid adults when performing the synchronization procedure.

      Note: Three days is appropriate timing if strains are maintained at 20°C; if strains require maintenance at another temperature, adjust this timing accordingly (Stiernagle, 2006).

    2. Collect gravid adults by adding ~1 mL of sterile M9 to each plate. Once most animals are floating, use a P1000 pipetman to transfer the M9 and worms to a 1.5 mL microcentrifuge tube.

    3. Spin tubes for 1 min in a benchtop microcentrifuge set to 4,000 × g, to collect animals into a loose pellet without damaging them.

    4. Remove the M9 supernatant and discard.

    5. Add 1 mL of bleaching solution (see Recipes).

    6. Agitate the animals using a vortex mixer for approximately 6-7 min. This reaction should not be prolonged for more than 10 min. Check the condition of the animals using a stereomicroscope every ~1-2 min and stop the reaction as soon as the worms have started to lyse/break apart.

    7. Immediately return tubes to the microcentrifuge and spin for 1 min at 4,000 × g.

    8. Remove the bleaching solution from the worm carcasses and embryo pellet and add 1 mL of sterile M9.

    9. Mix tubes on a vortex mixer for ~10 s and centrifuge for 1 min at 4,000 × g.

    10. Remove the M9 and repeat this washing procedure two more times.

    11. After the last spin, add 750 µL of sterile M9 to the worm pellet.

    12. Incubate tubes at 15°C on a tube rocker for ~24 h to allow for L1 hatching. L1s hatching in the absence of food will remain in developmental diapause, thereby creating a synchronous population, which can be maintained at 15°C for 5-7 days.

    13. On the day of use, determine the approximate concentration (L1s per µL) of surviving and arrested L1s in each tube, by counting the number of thrashing animals present in 10 µL of the M9 and starved L1 solution after inverting the tube several times to ensure an even distribution.

    14. Use a P20 or P200 pipetman to transfer 10-15 synchronized L1s in M9 to a 35 mm NGM plate inoculated with HT115, using the number of L1s per µL from Step B13 to select the appropriate volume. To image late L4 larvae, incubate plates at 20°C for 40-48 h.

      Note: We find that plating on HT115, rather than OP50, ensures a more synchronous population and a more consistent number of mitotic GSCs at the time of imaging.


  3. Preparation of grooved agarose pads

    1. In advance, prepare ~1 mL aliquots of 3% agarose gel, by dissolving 3 g agarose powder into 100 mL of dH2O, microwaving on high until fully melted, and dispensing into 1.5 mL microcentrifuge tubes. Agarose aliquots stored at room temperature are good to use for up to 1 month.

    2. Pre-warm a heat block to 95°C. When the heat block reaches the set temperature, add a tube of 3% agarose. After 7-9 min, check the agarose regularly (every ~1-2 min) to see if it has melted.

      Note: Overheating the agarose, either by exposing tubes to temperatures >95°C, or leaving them at 95°C for longer than 10-15 min, produces poor quality pads.

    3. Clean a glass slide and the silicon mold using water or 70% ethanol, and compressed air and/or a Kimwipe, as needed, to remove excess liquid and dust or debris.

    4. Tape two coverslips to the cleaned glass slide, such that they are separated by a distance shorter than the width of the silicon mold (Figure 2B).

    5. Using a clean plastic transfer pipette, add a drop of melted agarose onto the slide between the two taped coverslips, avoiding bubbles.

    6. Quickly lower the silicon mold onto the drop, such that it is balanced between the two taped coverslips. Press down gently on both edges of the silicon mold where they overlap with the coverslips (Figure 2C). Let the agarose solidify for ~1 min.

      Note: Doing this step quickly ensures well-patterned grooves. Do not push down on the silicon mold where it is not supported by the coverslips. Any lateral movement of the silicon mold after it has made contact with the surface of the agarose will result in broken, skewed, and generally poor-quality grooves.

    7. As soon as the agarose solidifies, gently lift the silicon mold off the agarose pad, and use a stereomicroscope to check that the grooves are straight and well-defined.

    8. Using a razorblade, cut the molded agarose into slices of at least 0.5 by 1 cm. Immediately cover with dH2O to prevent drying out (Figure 2C).

    9. Molded agarose pads can be stored in dH2O at 4°C for up to 1 week.



      Figure 2. Overview of the materials required and the mounting method.

      A. Worm pick made of a glass Pasteur pipette and a platinum wire. B. Materials for agarose patterning: 1. 3% agarose solution; 2. Silicon mold micro-patterned with ridges by lithography; 3. Plastic transfer pipet with cut tip; 4. Microscope slide with two coverslips taped to its surface to act as spacers; 5. Petri dish filled with dH2O for storing molded agarose pads. C. Method for preparing agarose pads patterned with grooves. An image of the silicon mold, balanced between two coverslips and pressed against the molten agarose is shown in top (left) and underside (middle) views. The patterned agarose pad is then cut into slices with a single-edge razorblade and the slices are transferred into a Petri dish filled with water (right). D. Materials for mounting worms: 1. Mouth pipette assembled with a microcapillary pipette; 2. M9 buffer; 3. 0.04% Tetramisole in M9; 4. 75% EtOH (for cleaning the microcapillary on the mouth pipette); 5. Blotting paper and Kimwipes cut into ~3 cm strips; 6. Worm eyelash pick; 7. Petri dish filled with water containing the agarose pad slices; 8. 1-well glass depression slide; 9. Forceps; 10. Microscope slide and square coverslip; 11. Valap; 12. Plastic transfer pipet with cut tip and assembled with a 200 µL pipette tip; E. Top view of the final preparation – the agarose pad is centered under the coverslip with the grooves facing up (i.e., towards the coverslip), the corners of the coverslip are stabilized by Valap, and the area around the agarose pad under the coverslip is backfilled with 0.04% Tetramisole in M9. F. Close up of three well-mounted L4 larvae, as viewed through a stereomicroscope.


  4. Mounting worms for live imaging

    Note: When performing the following steps, every effort should be made to avoid physical contact with the animals and to work quickly, such that imaging can begin within ~5 min of removing worms from feeding plates. These steps have been optimized for late L4 stage larvae, but also work well for L3 larvae and adult animals.

    1. Make a fresh solution of 0.04% tetramisole in M9.

      Note: While this mounting approach can immobilize worms without anesthetics, we find that residual body movements, such as muscle twitching and pharyngeal pumping, can obscure subcellular dynamics, and a low dose of anesthetic (0.04% tetramisole) produces more reliable results.

    2. Prepare a glass micropipette by heating its middle over a Bunsen burner flame and pulling gently from both ends once the glass becomes soft. Break the pipette at a length of ~6 cm and gently heat the broken end to smoothen the glass. An ideal pipette will be narrow enough to ensure precise volume control, but wide enough to transfer late L4 animals with minimal contact (i.e., wide enough for them to freely “thrash”) (Figures 2D #1 and 3A).

    3. Melt a tube of Valap (see Recipes) on a heat block set to 65°C.

    4. Transfer 100 μL of 0.04% tetramisole in M9 into the well of a glass depression slide.

    5. Under a stereomicroscope and using a mouth pipet, gently float 2-5 late L4 larvae off the surface of the plate using ~5 μL of M9, and transfer them via mouth pipette into the tetramisole on the depression slide (Figure 3A).

    6. Using the mouth pipette, blow air on the surface of the tetramisole solution to create a “mini-vortex” for ~10 s. Worm thrashing should reduce noticeably (Figure 3B).

    7. Using a razorblade or forceps, place a 3% agarose pad on a clean glass slide with the groove-molded side facing up.


    8. Note: Perform the following steps quickly to prevent the agarose pad and worms from drying out.
    9. Remove excess water from the agarose pad using Whatman paper or a Kimwipe and the mouth pipette.

    10. Using a mouth pipette, gently transfer the worms, one at a time, from the depression slide to the agarose pad, as close to the grooves as possible and using as little liquid as possible (Figure 3C).

    11. With the mouth pipette, gently blow air to move the worms until they fall into the desired groove(s). If the worms remain floating rather than falling into the grooves, then too much liquid is present, so remove excess liquid using the mouth pipette and repeat (Figure 3D).

    12. (Optional) If precise placement of worms is desired (e.g., to capture 2-3 larvae within a single field of view), use a worm eyelash pick to gently sweep the worms into position. Minimize physical contact between the worms and eyelash pick, as this can stimulate the touch receptors and cause the worms to move.

    13. Remove any excess liquid using Whatman paper or a Kimwipe (Figure 3E).

    14. Carefully lower a coverslip onto the agarose pad (Figure 3F).

      Note: Dropping the coverslip too quickly may damage the worms.

    15. Trim a 200 μL pipette tip, by cutting ~1 cm off the end using a single edge razorblade, and place it on the end of a plastic transfer pipette. Use this to add a small drop of melted Valap to each corner of the coverslip (Figures 2E and 3G).

      Note: Avoid adding too much Valap, as this may heat the coverslip. When adding the Valap, avoid lifting the coverslip.

    16. Using a P200 pipetman, slowly backfill the space under the coverslip with ~100 μL of 0.04% tetramisole in M9 (Figure 3H).

      Note: Do not overfill the chamber or fill it too fast, as this can cause the coverslip to lift and displace the worms. Check mounted worms under a stereomicroscope to ensure that they are in the grooves and not moving (Figure 2F).



      Figure 3. Schematic overview of the mounting procedure.

      A. C. elegans worms are transferred using a small volume of M9 buffer and a mouth pipette, from a feeding plate to a depression slide filled with 0.04% tetramisole in M9. B. The liquid and worms are then mixed by blowing air on the surface of the liquid using the mouth pipette, to create a “mini-vortex”. C. Individual worms are then transferred by mouth pipette in a small volume of 0.04% tetramisole in M9 to an agarose pad molded with grooves. D. Worms are moved towards the grooves and excess liquid is dispersed using the mouth pipette to blow air across the surface of the liquid in the desired direction. E. Once worms are positioned in the grooves, excess liquid is removed using Whatman paper or a Kimwipe, with the help of the mouth pipette if needed. F. A coverslip is then carefully placed on top of the agarose pad. G. The coverslip is fixed in place by adding a small drop of melted Valap at each corner. H. The area under the coverslip is then backfilled with ~100 μL of the 0.04% tetramisole in M9 remaining in the depression slide.


  5. Imaging

    Typical imaging parameters used by our lab to image GSC mitosis are as follows:

    1. Place mounted worms on inverted spinning disk confocal microscope [Zeiss Cell Observer with Yokogawa CSU-X1, using a high NA 63× oil immersion objective (Zeiss 63× Plan-Apochromat DIC (UV) VIS-IR)].

    2. Set laser intensity to 10% of maximum (35 mW 488 nm and 50 mW 561 nm lasers) and exposure time to 200 ms. Set camera (Zeiss Axio Cam 506 Mono) binning to 3 × 3, for a final pixel size of 0.1802 μm.

    3. Set the z-stack step size to 0.5 μm, with sufficient range to encompass the height of the distal germline (typically 20 μm).

    4. Image every 30 s for up to 40 min. After ~40 min of starvation, which is an unavoidable consequence of our mounting method, we observe a significant decrease in the number of GSCs entering mitosis, suggesting that cell and/or organismal physiology changes past this point (Videos 1 and 2; Zellag et al., 2021). As a result, we do not recommend imaging for longer durations.

      Note: These imaging parameters were adjusted to minimize the adverse effects of laser exposure and are specific to our microscope. We recommend that users optimize their imaging parameters using the following criteria as benchmarks: (1) A fairly consistent number of cells entering mitosis throughout a 30-40 min acquisition (Figure 4K); (2) An average of 13 mitotic entries per germline (Figure 4I), within a 40 min acquisition; and (3) An average duration of mitosis (as defined below) of ~5 min (Figure 4J).


    Video 1. Mitotic C. elegans GSCs in a late L4 larval germline over a 90-min image acquisition with centrosomes and rachis marked.

    Time-lapse movie of the distal end (left) of a gonad from a late L4 worm expressing mNG::ANI-1 (green; rachis) and mCH::β-tubulin (magenta; centrosomes) in the germline. Images were acquired every 30 s for 90 min, with an AxioCam 506 Mono camera (Zeiss) mounted on an inverted Cell Observer spinning-disk confocal microscope (Zeiss; Yokogawa), using a 60x Plan Apochromat DIC (UV) VIS-IR oil immersion objective (Zeiss), controlled by Zen software (Zeiss). A maximum intensity projection of 38 z-sections (0.5 µm sections) is shown. Images were adjusted for brightness and contrast using Fiji (Schindelin et al., 2012). Scale bar = 10 µm. Time stamp is in hour:min. Movie plays at 10 frames per second.


    Video 2. Mitotic C. elegans GSCs in a late L4 larval germline over a 40-min image acquisition with centrosomes and chromatin marked.

    Time-lapse movie of the distal end (left) of a gonad from a late L4 worm expressing GFP::β-tubulin (green; centrosomes) and mCH::Histone H2B (magenta; chromatin) in the germline. Acquisition settings were the same as for Video 1, except for acquisition duration, which was 40 min. A maximum intensity projection of 38 z-sections (0.5 µm sections) is shown. Images were adjusted for brightness and contrast using Fiji (Schindelin et al., 2012). Scale bar = 10 µm. Time stamp is in min:sec. Movie plays at 10 frames per second.


  6. Image processing

    The following steps are further detailed in the ReadMe file of our GitHub repository and can be applied to a single movie or several movies in batch mode, if grouped in the same folder.


  7. Registration

    1. Use the ImageJ macro automatedregistrationtool to manually track the spindle midpoint of one or a series of mitotic cells over the duration of the movie (Figure 4A).

      Note: Users may track objects other than the spindle midpoint, if other marked structures that follow global sample movement are visible.

    2. Use the Jupyter notebook batchmode movie registration module to generate an x-y translation matrix and to correct the movie accordingly (Figure 4B).

      Kymographs in Figure 4B show the correction of sample movement after x-y registration.


  8. Centrosome tracking

    Use the ImageJ macro automatedfixhyperstack to restore pixel scaling and frame rate in the registered movie, to crop the borders generated by registration, and perform spot detection and track creation using TrackMate (Tinevez et al., 2017) (Figure 4C). For reference, our TrackMate parameters are as follows: blob diameter = 2.5 μm; linking max distance = 2.7 μm; max frame gap = 2.

    Note: TrackMate can identify and track centrosomes marked by fluorescent proteins other than β-tubulin, in cell types other than GSCs, and in organisms other than C. elegans. TrackMate parameters should be optimized by the user based on their sample and imaging parameters, to detect the largest number of true centrosome tracks possible. Spurious tracks are generally removed by the next step.


  9. Centrosome track pairing

    Use the Jupyter notebook batchmode track pair classification module to pair spot tracks into likely centrosome track pairs (i.e., centrosomes within the same cell) (Figure 4D).

    Note: Our track pair classifier was trained on a large dataset of wild-type C. elegans GSCs. Users who wish to pair centrosomes from other cell types and/or other organisms may need to train their own model. Instructions on how to do this can be found on our GitHub repository.


  10. Scoring mitosis

    1. Use the Matlab script Step1_import_textfile_and_align_cent_tracks to import the xyzt coordinates for paired tracks and to generate a file containing the corresponding midpoint coordinates for each pair (Figure 4E).

    2. Use the ImageJ macro Step2_CropCells to generate cropped maximum intensity projections centered on the midpoint coordinates calculated in Step1 (Figure 4F).

    3. Use the Matlab script Step3_score_mitosis to plot spot-to-spot distance (hereafter “spindle length”) for each predicted pair of centrosomes. This step allows users to exclude false-positive pairs and to score mitotic events [e.g., nuclear envelop breakdown (NEBD), which occurs concomitantly with a rapid decrease in spindle length, and anaphase onset (AO), when spindle length starts to increase rapidly], by clicking on the plot. If the graph is unclear, an option allows the user to view the corresponding cropped tif (Figure 4G).

    4. Use the Matlab script Step4_calc_fits to calculate the duration of mitosis and to extract various mitotic features, including spindle length and anaphase elongation rate. Other features, such as spindle angular displacement or length fluctuations, can readily be calculated from the resulting data. We define “mitosis” as the period of time post-NEBD, once spindle length has reached a ~constant minimum, until AO (Figure 4G); this will encompass most of prometaphase and all of metaphase.



      Figure 4. Overview of image processing for analyzing GSCmitotic spindle dynamics in 3D in late L4 larvae.

      A. To correct for sample movement in x-y, the spindle midpoint of one or more cells is manually tracked over the duration of a maximum intensity projection of the raw/original movie, using the ImageJ macro automatedtegistrationtool. Scale bar = 10 µm. B. Running the movie registration module in the Jupyter notebook batchmode creates an x-y translation matrix from the coordinates of the tracked spindle midpoints and registers the raw/original movie accordingly. Generated using the KymographBuilder plugin in ImageJ (Mary et al., 2016), a kymograph along a ~8 µm-wide line scan through the spindle midpoint of a single GSC over 30 min is shown to illustrate the effectiveness of this correction, before (left) and after (right) x-y registration. Scale bar = 5 µm. C. The ImageJ macro automatedfixhyperstack restores pixel scaling and frame rate, crops the borders generated by registration, and allows the user to perform spot detection and track creation on the registered image using TrackMate (Tinevez et al., 2017). Tracks are shown as colored lines, including bona fide centrosome tracks and spurious tracks. D. TrackMate tracks are then paired by running the track pair classifier module in the Jupyter notebook batchmode. An optimal outcome is shown on the left, where four centrosome tracks from two neighboring cells have been correctly paired (green and magenta tracks) over the course of both divisions. E. The xyzt coordinates for paired spots are imported into Matlab and the midpoint for all pairs is calculated using the script Step1_import_textfile_and_align_cent_tracks. F. The midpoint coordinates are then used by the ImageJ macro Step2_CropCells to generate cropped maximum intensity projections for each pair, with the frame number shown at the upper right-hand corner G. Users can then exclude false positive pairs and score mitotic events, by either clicking on a displayed plot of spot-to-spot distance (i.e., spindle length) over time (left), or by entering timepoints manually after viewing the cropped image generated in F (right) using the Matlab script Step3_score_mitosis. NEBD = nuclear envelop breakdown. AO = anaphase onset. H. The Matlab script Step4_calc_fits calculates the duration of ‘mitosis’ (which we define as illustrated by the shaded box in G) and extracts different mitotic features. I. Number of GSCs entering mitosis (undergoing NEBD) per gonad during the first 40 min of image acquisition from 61 animals [mean ± standard deviation (SD) = 13.5082 ± 6.2786 mitotic entries]. J. Duration of mitosis for 649 cells from 74 animals (mean ± SD = 5.2512 ± 2.0175 min). K. Mitotic entries as a percent of the total number of mitoses during 40 min image acquisition, binned by 5-min intervals relative to acquisition start (n = 61 and 875 mitotic entries). L. Average spindle length during mitosis for 649 cells from 74 animals (mean ± SD = 3.0246 ± 0.3678 µm). M. Spindle elongation rate during early anaphase (First 2 min following anaphase onset) for 819 cells from 74 animals (mean ± SD = 0.0245 ± 0.0059 µm/sec). In I-M, black bars show the mean and error bars represent the SD. Data from three strains (UM679, JDU19, and ARG16, see Table 1) were combined.

Data analysis

The CentTracker image registration module corrects x-y sample movement. However, large and abrupt sample movements in z can lead to untrackable frames and to spot duplications, which can significantly impact the automated pairing module. Therefore, we exclude movies that exhibit severe z-movement. This does not include normal z-drift, which is progressive and has a minimal impact on spot detection and track creation. Typically, severe z-movement occurs when animals are not well situated in their grooves and can be avoided almost entirely by good mounting.

    The cell scoring step (J) is crucial for detecting false-positive centrosome pairs when using CentTracker. Spot-to-spot distance plots for true positive centrosome pairs have a consistent pattern (Figure 4G), so false-positive plots should be readily identifiable. However, we recommend viewing the corresponding cropped tif for any unusual plots, especially if the GSCs under analysis are not wild-type, and/or are expected to have spindle defects.

    The mitotic parameters displayed in Figure 4 are for GSCs from late L4 larvae and were calculated as described in Zellag et al. (2021), where details for the extraction of additional mitotic features can also be found. The total number of mitotic entries per gonad was determined using manual track pairing, not the track pair classifier in CentTracker, which has a discovery rate of ~80% in wild-type animals (Zellag et al., 2021).

Notes

  1. The silicon mold used in this protocol is etched to form positive bands 26 μm high and 50 μm wide and generates v-shaped grooves of the same proportions, with angled walls (Figure 1B). A simple photomask design can be created using computer-aided design (CAD) software, and the photomask production and photolithography can be achieved at most microfabrication facilities. We have subsequently found that square-shaped grooves (Figure 1B) produce similar and even potentially better results, and may be less challenging/costly for the photolithography step. We have tested the following designs and all of them are adequate for mounting:

    1. For mid to late L3 worms: Square-shaped grooves 20 μm deep and 20 μm wide.

    2. For Late L4 worms: Square-shaped grooves 35 μm deep and 38-43 μm wide.

    3. For 1 day-old adult worms: Square-shaped grooves 55 μm deep and 60-65 μm wide.

    If microfabrication facilities are not available, using a vinyl Long Play (LP) record as a mold (Zhang et al., 2008; Rivera Gomez and Schvarzstein, 2018) can yield reasonably good results (Zellag et al., 2021).

  2. Make a worm eyelash pick using a human eyelash or a cat whisker. Hold the eyelash/whisker with a pair of fine-tipped tweezers and insert the root end into the end of a 200 μL pipette tip, which is filled with molten Valap. Once the Valap solidifies, the eyelash/whisker should stay in place. Alternatively, the end of ~1,000 μL pipet tip can be melted, and the eyelash/whisker inserted while the plastic is still malleable (Figure 2D).

Recipes

  1. M9 buffer (1,000 mL)

    3 g KH2PO4

    6 g Na2HPO4

    5 g NaCl

    1 mL of 1 M MgSO4

    dH2O to bring final volume to 1,000 mL

    Combine all ingredients until dissolved and sterilize by filtration using a 0.2 μm pore size.

  2. Valap

    1:1:1 by weight Vaseline, lanolin, and paraffin

    Combine all ingredients in a glass beaker on a hot plate set at a low temperature. Stir occasionally until melted and homogenous. Store at room temperature as ~1 mL aliquots.

  3. Bleaching solution (5 mL)

    1 mL of commercial bleach (~10% available chlorine)

    250 μL of 10M NaOH

    3.75 mL of dH2O

    Add bleach and NaOH to water in a 15 mL conical tube. Makes enough to bleach five 60 mm plates of gravid adults. Make fresh every time, ideally within 1 h of use. Do not store.

    Note: As bleach degrades over time, we recommend using bottles of commercial bleach with a known and fairly recent (within 1 year) purchase date.

  4. 2% w/v Tetramisole solution

    Make a 2% w/v solution in dH2O, store at 4°C and dilute to 0.04% in M9 buffer before use.

  5. 3% w/v volume Agarose powder solution

    Make a 3% w/v volume solution in dH2O.

Acknowledgments

We thank Dr. Jean-Claude Labbé (JCL) of IRIC for advice and critical reading of this manuscript. We also thank Drs. Amy Maddox (UNC Chapel Hill), Arshad Desai (UC, San Diego), Benjamin Lacroix (Institut Jacques Monod) for sharing strains, IRIC's Bio-imaging facility and McGill’s Advanced BioImaging Facility (ABIF) for technical assistance, and members of the Labbé and Gerhold labs for help and advice. RMZ held an Alexander Graham Bell Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC) and an IRIC Doctoral Scholarship. YZ was partially supported by the Sheila Ann MacInnis Grant Science Undergraduate Research Award (SURA). This work was funded by grants from the Canadian Institutes of Health Research (PJT-153283 to JCL and ARG and PJT-159523 to ARG).

This protocol was part of and derived from Zellag et al. (2021).

Competing interests

There are no conflicts of interest or competing interests.

References


  1. Angelo, G. and Van Gilst, M. R. (2009). Starvation protects germline stem cells and extends reproductive longevity in C. elegans. Science 326(5955): 954-958.
  2. Amini, R., Goupil, E., Labella, S., Zetka, M., Maddox, A. S., Labbé, J. C. and Chartier, N. T. (2014). C. elegans Anillin proteins regulate intercellular bridge stability and germline syncytial organization. J Cell Biol 206(1): 129-143.
  3. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77(1): 71-94.
  4. Baugh, L. R. and Sternberg, P. W. (2006). DAF-16/FOXO regulates transcription of cki-1/Cip/Kip and repression of lin-4 during C. elegans L1 arrest. Curr Biol 16(8): 780-785.
  5. Barbosa, J. S., Sanchez-Gonzalez, R., Di Giaimo, R., Baumgart, E. V., Theis, F. J., Götz, M. and Ninkovic, J. (2015). Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain. Science 348(6236): 789-793.
  6. Berger, S., Lattmann, E., Aegerter-Wilmsen, T., Hengartner, M., Hajnal, A., deMello, A. and Casadevall I Solvas, X. (2018). Long-term C. elegans immobilization enables high resolution developmental studies in vivo. Lab Chip 18(9): 1359-1368.
  7. Burnett, K., Edsinger, E. and Albrecht, D. R. (2018). Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours. Commun. Biol 1: 73.
  8. Berger, S. and Spiri, S. (2021). Microfluidic-based imaging of complete Caenorhabditis elegans larval development. Development 148(18).
  9. Chai, Y., Li, W., Feng, G., Yang, Y., Wang, X. and Ou, G. (2012). Live imaging of cellular dynamics during Caenorhabditis elegans postembryonic development. Nat Protoc 7(12): 2090-2102.
  10. Dickinson, D. J., Ward, J. D., Reiner, D. J. and Goldstein, B. (2013). Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nature Methods 10(10):1028-1034.
  11. Fielenbach, N. and Antebi, A. (2008). C. elegans dauer formation and the molecular basis of plasticity. Genes Dev 22(16): 2149-2165.
  12. Fabig, G., Löffler, F., Götze, C. and Müller-Reichert, T. (2020a). Live-cell Imaging and Quantitative Analysis of Meiotic Divisions in Caenorhabditis elegans Males. Bio-protocol 10(20): e3785.
  13. Fabig, G., Kiewisz, R., Lindow, N., Powers, J. A., Cota, V., Quintanilla, L. J., Brugués, J., Prohaska, S., Chu, D. S., and Müller-Reichert, T. (2020b). Male meiotic spindle features that efficiently segregate paired and lagging chromosomes.eLife 9: e50988.
  14. Gray, J. and Lissmann, H. W. (1964). THE LOCOMOTION OF NEMATODES. J Exp Biol 41: 135-154.
  15. Gerhold, A. R., Ryan, J., Vallée-Trudeau, J. N., Dorn, J. F., Labbé, J. C. and Maddox, P. S. (2015). Investigating the regulation of stem and progenitor cell mitotic progression by in situ imaging. Curr Biol 25(9): 1123-1134.
  16. Gordon, K. L., Zussman, J. W., Li, X., Miller, C. and Sherwood, D. R. (2020). Stem cell niche exit in C. elegans via orientation and segregation of daughter cells by a cryptic cell outside the niche. eLife 9: e56383.
  17. Hirsh, D., Oppenheim, D. and Klass, M. (1976). Development of the reproductive system of Caenorhabditis elegans. Dev Biol 49(1): 200-219.
  18. Hwang, H., Krajniak, J., Matsunaga, Y., Benian, G. M. and Lu, H. (2014). On-demand optical immobilization of Caenorhabditis elegans for high-resolution imaging and microinjection. Lab Chip 14(18): 3498-3501.
  19. Joshi, P. M., Riddle, M. R., Djabrayan, N. J. and Rothman, J. H. (2010). Caenorhabditis elegans as a model for stem cell biology.Dev Dyn Off Publ Am Assoc Anat 239(5): 1539-1554.
  20. Kimble, J. E. and White, J. G. (1981). On the control of germ cell development in Caenorhabditis elegans. Dev Biol 81(2): 208-219.
  21. Kim, E., Sun, L., Gabel, C. V. and Fang-Yen, C. (2013). Long-term imaging of Caenorhabditiselegans using nanoparticle-mediated immobilization. PLoS One 8(1): e53419.
  22. Luke, C. J., Niehaus, J. Z., O'Reilly, L. P. and Watkins, S. C. (2014). Non-microfluidic methods for imaging live C. elegans. Methods 68(3): 542-547.
  23. Maddox, A. S., Habermann, B., Desai, A. and Oegema, K. (2005). Distinct roles for two C.elegans anillins in the gonad and early embryo. Development 132(12): 2837-2848.
  24. Morrison, S. J. and Kimble, J. (2006). Asymmetric and symmetric stem-cell divisions in development and cancer. Nature 441(7097): 1068-1074.
  25. Magescas, J., Zonka, J. C. and Feldman, J. L. (2019). A two-step mechanism for the inactivation of microtubule organizing center function at the centrosome. eLife 8: e47867.
  26. Martin, J. L., Sanders, E. N., Moreno-Roman, P., Jaramillo Koyama, L. A., Balachandra, S., Du, X. and O'Brien, L. E. (2018). Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of division, differentiation and loss. eLife 7: e36248.
  27. Mary, H., Rueden, C., and Ferreira, T. (2016). KymographBuilder: Release 1.2.4. Zenodo.
  28. Merritt, C., and Seydoux, G. (2010). Transgenic solutions for the germline. WormBook : The Online Review of C. elegans Biology. 1-21. doi: 10.1895/wormbook.1.148.1.
  29. Nguyen, P. D. and Currie, P. D. (2018). In vivo imaging: shining a light on stem cells in the living animal. Development 145(7).
  30. Park, S., Greco, V. and Cockburn, K. (2016). Live imaging of stem cells: answering old questions and raising new ones. Curr Opin Cell Biol 43: 30-37.
  31. Priti, A., Ong, H. T., Toyama, Y., Padmanabhan, A., Dasgupta, S., Krajnc, M., and Zaidel-Bar, R. (2018). Syncytial germline architecture is actively maintained by contraction of an internal actomyosin corset. Nat Commun 9(1): 4694.
  32. Rompolas, P., Deschene, E. R., Zito, G., Gonzalez, D. G., Saotome, I., Haberman, A. M. and Greco, V. (2012). Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration. Nature 487(7408): 496-499.
  33. Ritsma, L., Ellenbroek, S. I. J., Zomer, A., Snippert, H. J., de Sauvage, F. J., Simons, B. D., Clevers, H. and van Rheenen, J. (2014). Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging. Nature 507(7492): 362-365.
  34. Rivera Gomez, K. A., and Schvarzstein, M. (2018). Immobilization of nematodes for live imaging using an agarose pad produced with a Vinyl Record. MicroPublication Biology 2018(08).
  35. Rosu, S. and Cohen-Fix, O. (2017). Live-imaging analysis of germ cell proliferation in the C. elegans adult supports a stochastic model for stem cell proliferation. Dev Biol 423(2): 93-100.
  36. Sulston, J. E. and Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev Biol 56(1): 110-156.
  37. Stiernagle, T. (2006). Maintenance of C. elegans. WormBook. 1-11.
  38. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  39. Timmons, L. and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395(6705): 854.
  40. Tinevez, J. Y., Perry, N., Schindelin, J., Hoopes, G. M., Reynolds, G. D., Laplantine, E., Bednarek, S. Y., Shorte, S. L. and Eliceiri, K. W. (2017). TrackMate: An open and extensible platform for single-particle tracking. Methods 115: 80-90.
  41. Wong, B. G., Paz, A., Corrado, M. A., Ramos, B. R., Cinquin, A., Cinquin, O. and Hui, E. E. (2013). Live imaging reveals active infiltration of mitotic zone by its stem cell niche. Integr Biol (Camb) 5(7): 976-982.
  42. Yoshida, S., Sukeno, M. and Nabeshima, Y. (2007). A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis. Science 317(5845): 1722-1726.
  43. Zhou, K., Rolls, M. M. and Hanna-Rose, W. (2013). A postmitotic function and distinct localization mechanism for centralspindlin at a stable intercellular bridge. Dev Biol 376(1): 13-22.
  44. Zeiser, E., Frøkjær-Jensen, C., Jorgensen, E., and Ahringer, J. (2011). MosSCI and Gateway Compatible Plasmid Toolkit for Constitutive and Inducible Expression of Transgenes in the C. elegans Germline. PloS One 6(5): e20082.
  45. Zhang, M., Chung, S. H., Fang-Yen, C., Craig, C., Kerr, R. A., Suzuki, H., Samuel, A. D., Mazur, E. and Schafer, W. R. (2008). A self-regulating feed-forward circuit controlling C. elegans egg-laying behavior. Curr Biol 18(19): 1445-1455.
  46. Zellag, R. M., Zhao, Y., Poupart, V., Singh, R., Labbé, J. C. and Gerhold, A. R. (2021). CentTracker: a trainable, machine-learning-based tool for large-scale analyses of Caenorhabditis elegans germline stem cell mitosis. Mol Biol Cell 32(9): 915-930.


简介

[摘要]模式生物提供了破译干细胞在其原生环境中的动态和复杂行为的机会; 然而,原位成像干细胞在技术上仍然具有挑战性。 C. elegans 生殖干细胞 (GSC) 明显可用于原位实时成像,但相对较少的研究利用了这种潜力。 在这里,我们提供了用于安装和实时成像划分线虫 GSC 的协议,以及用于促进大型数据集处理的分析工具。 虽然本协议针对有丝分裂 GSC 的成像和分析进行了优化,但它可以很容易地适应于在多个发育阶段可视化线虫中的分裂细胞或其他亚细胞过程。 我们的图像分析管道还可用于分析其他细胞类型和模型生物的有丝分裂。

[背景]虽然允许在几个模型系统中可视化各种组织驻留干细胞,但活体成像的最新进展通常依赖于侵入性手术或复杂且昂贵的成像方式(Yoshida 等人,2007;Rompolas 等人,2012;Ritsma 等人) .,2014 年;Barbosa 等人,2015 年;Park 等人,2016 年;Martin 等人,2018 年;Nguyen 和 Currie,2018 年)。秀丽隐杆线虫 GSC 是一种已建立的干细胞模型,它对干细胞生物学的许多方面产生了普遍的见解(Kimble 和 White,1981;Baugh 和 Sternberg,2006;Fielenbach 和 Antebi,2008;Angelo 和 Van Gilst,2009)。此外,C. elegans GSCs 可以使用标准荧光显微镜技术在活体动物中成像,无需手术操作。特别是,GSC 有丝分裂的实时成像提供了一个机会来研究细胞分裂的动态以及它们如何受到组织组织、生态位信号和有机体生理学等体内因素的影响。在这里,我们描述了一种简单、快速和可重复的方法来固定线虫并成像和分析 GSC 有丝分裂,同时保留动物的生存能力、生育能力和看似正常的 GSC 分裂。


成人 C. elegans 雌雄同体容纳两个 GSC 群,位于两个管状性腺臂的远端(图 1A)。与其他种系一样,秀丽隐杆线虫性腺被组织为合胞体。 GSCs 在称为轴的共享内核周围形成一个粗糙的环形单层(Hirsh 等人,1976)(图 1A 和 1C)。每个 GSC 通过一个稳定的肌动球蛋白环与轴相连,形成一个细胞质桥(Maddox 等人,2005;Zhou 等人,2013;Amini 等人,2014;Priti 等人,2018)。像其他干细胞一样,C. elegans GSCs 通过来自体细胞生态位的信号保持在类似干细胞的状态(远端尖端细胞,Kimble 和 White,1981;图 1A 和 1E)。与几种类型的哺乳动物干细胞一样,C. elegans GSC 池的大小根据种群模型维持,其中由于从生态位移位而导致的分化通过对称分裂来平衡,以维持相对恒定的干细胞数量。根据种群模型,其中由于从生态位移位而引起的分化通过对称分裂来平衡,从而保持相对恒定的干细胞数量(Morrison 和 Kimble,2006;Joshi 等,2010)。


大多数对秀丽隐杆线虫 GSC 的研究依赖于解剖、固定和染色的性腺,或对活蠕虫中带有荧光标记蛋白的性腺进行单时间点成像。 GSC 的长期成像已使用“捕获和释放”方法完成,该方法允许在数小时或数天内定期可视化 GSC(Wong 等人,2013;Rosu 和 Cohen-Fix,2017)。此外,最近开发的一种微流体装置可能允许在类似的时间尺度上进行连续观察(Berger 等人,2018 年,Berger 和 Spiri,2021 年)。很少有研究报道在适合记录有丝分裂等动态亚细胞事件的条件下对 GSC 进行活细胞成像(Gerhold 等人,2015;Gordon 等人,2020;Zellag 等人,2021)。


为了在实时成像期间实现高时间和空间分辨率,必须固定动物,并且必须考虑安装对动物和 GSC 生理学的整体影响。典型的安装方法结合使用麻痹药物和物理压迫来固定动物(Sulston 和 Horvitz,1977;Chai 等人,2012;Kim 等人,2013;Hwang 等人,2014;Luke 等人,2014;Burnett等人,2018;Fabig 等人,2020a 和 2020b;Gordon 等人,2020)。我们使用蚀刻的硅晶片在琼脂糖垫上形成凹槽,将动物限制在笔直的位置(图 1B);这可以防止蠕虫运动所需的正弦运动(Gray 和 Lissmann,1964 年),并减少对物理压缩和麻醉剂的要求。

图 1. 一种安装方法,允许对完整的线虫中的 GSC 进行实时成像。
A. 示意图显示了安装在凹槽中的晚期 L4(顶部)和成年(底部)雌雄同体蠕虫的俯视图。两个性腺臂的位置和整体组织以白色显示,并突出显示关键的种系特征,例如轴(绿色)和远端尖端细胞(DTC)或利基(洋红色)。 GSC 的位置由紫色圆圈表示。 B. 示意图显示了安装在与动物宽度相匹配的 v 形(顶部)和方形(底部)凹槽中的动物的横截面视图。 (CE) 表达 (C) mNG::ANI-1(绿色)和 mCH::β-微管蛋白(洋红色;应变 = UM679)、D. GFP::β-微管蛋白的成年动物远端性腺的最大强度预测(绿色)和 mCH::Histone H2B(洋红色;应变 = JDU19)和(E)LAG-2::GFP(绿色)和 mCH::β-微管蛋白(洋红色;应变 = UM211)。每个性腺中的分割 GSC 用黄色框起来,放大的图像如下所示。荧光标记的 β-微管蛋白标记所有三种菌株的有丝分裂纺锤体,mNG::ANI-1 标记轴,mCH::Histone H2B 标记细胞核,LAG-2::GFP 标记 DTC/生态位。比例尺 = 10 µm(顶部,性腺视图)和 5 µm(底部,单细胞视图)。


我们已经使用这种安装方法通过实时成像提供了 GSC 有丝分裂的第一个特征(Gerhold 等人,2015)。最近,我们研究了在实时成像期间影响 GSC 有丝分裂的技术因素,这使我们能够定义最佳的安装和成像条件,并确定在安装/成像期间动物饥饿对 GSC 有丝分裂的最有害影响(Zellag 等人, 2021)。我们的数据表明,GSC 有丝分裂可以在大约 40 分钟的窗口内接近生理条件下成像,这从蠕虫从食物中取出的那一刻开始。虽然一些 GSC 在 40 分钟后进入有丝分裂,但我们建议在解释它们的行为时要小心。此外,在最佳条件下,野生型蠕虫中每个种系的有丝分裂数量和这些有丝分裂的持续时间相对恒定,并提供了一个可重复的基线,其他人可以通过该基线对其结果进行基准测试(参见视频 1 和 2 以及图 4I- 4K)。


使用实时成像研究 GSC 有丝分裂时出现的一个平行挑战是如何监测有丝分裂进程。 GSC 在管状性腺内分裂,可以相对于成像平面的任何方向以及相对于性腺表面的深度范围内分裂。因此,准确监测 GSC 有丝分裂需要一种跟踪方法,以考虑其分裂的三维 (3D) 特性。一个好的方法需要足够高的吞吐量才能以最少的用户时间生成大型数据集。此外,荧光标记的蛋白质通常在种系中以低水平表达(Merritt 和 Seydoux,2010),这限制了合适标记的可用性。


我们已经证明,荧光标记的 β-微管蛋白为 GSC 有丝分裂中心体提供了强大的标记,并且我们可以通过在 3D 中跟踪中心体对,使用中心体到中心体的距离作为有丝分裂进展的可靠读数(Gerhold 等人,2015;Zellag等人,2021)。此外,跟踪中心体对可以提供有关有丝分裂特征的信息,例如纺锤体动力学和方向(Zellag 等,2021)。为了使这种方法适用于大规模研究,我们最近开发了 CentTracker,这是一种高度自动化的图像分析管道,可以在任何遗传背景和其他细胞类型和生物体中快速提取有丝分裂参数,前提是中心体是可追踪的 (Zellag et等人,2021)。在这里,我们描述了使用 CentTracker 所需的基本成像处理步骤。相关代码和详细说明可免费下载。

关键字:活细胞和组织成像, 干细胞, 有丝分裂, Spindle dynamics, 秀丽隐杆线虫, 种系

材料和试剂


1. 带凸轮的60毫米培养皿(Sarstedt,目录号:82.1194.500

2. 1.5 mL微量离心管(Axygen,目录号:MCT-150-C

3. 15 mL锥形离心管(Fisher Scientific,目录号:14-959-53A

4. 用于校准的微毛细管移液器的吸气管组件(Sigma,目录号:A5177-5EA

5. 50 µL 玻璃微量移液器(VWR,目录号:53432-783

6. 1.7 mL塑料移液管(Fisher Scientific,目录号:13-711-41

7. 200 µL 移液器吸头(带直径,目录号:DIATEC520-6752

8. 1,250 µL 移液器吸头(带直径,目录号:DIATEC520-6501

9. 单刃剃须刀片

10. 细尖镊子/镊子

11. 实验室胶带(Fisher Scientific,目录号:15-901-10R

12. 18 × 18 mm #1.50.16-0.19 mm)方形盖玻片(VWR,目录号:CA48366-205-1

13. 显微镜载玻片(Fisher Scientific,目录号:12-55-15

14. Whatman 3 MM Chr 吸墨纸(VWR,目录号:21427-411),切成约 3 cm

15. KIMTECK Kimwipes 11.2 × 21.3 厘米

16. 1孔玻璃凹陷载玻片(VWR,目录号:470235-728

17. 通过光刻对硅晶片进行微图案化,形成一系列具有规定深度和宽度的平行凸起脊(图 2B-2C;见注 1;以下称为硅模具

18. 蜗杆 [99.95% 铂,0.05% 铱丝,0.01 英寸直径(Tritech,目录号:PT-9901),一端像抹刀一样扁平,并安装在玻璃巴斯德移液器(Fisher Scientific,目录号:13- 678-20A),或蜗杆镐柄(Tritech,目录号:TWPH1]

19. 蠕虫睫毛夹(见注2

20. 带有荧光蛋白 (FP) 标记的中心体标记 (如果使用 CentTracker) 的首选基因型线虫菌株, 有或没有额外的 FP 标记蛋白 (1C-1D;本协议中使用的菌株列于表 1 )。

注意:我们发现通过 Mos1 介导的单拷贝插入(MosSCI Zeiser et al., 2011) CRISPR-Cas9 与单个导向 RNA (sgRNA) 靶向靠近已建立 Mos1 插入位点的区域(Dickinson et al., 2013) ,并在 mex-5 启动子下表达 FP 标记的蛋白质, tbb-2 3' UTR 调控序列为适合实时成像的种系特异性表达产生最佳结果。 

 

1.本协议中使用的线虫菌株

 

21. 大肠杆菌菌株 OP50 (Brenner, 1974)

22. 大肠杆菌菌株 HT115 (Timmons and Fire, 1998)

23. 线虫生长培养基(NGM) (Stiernagle, 2006 ; 进行以下修改:每升培养基 20 g 琼脂和 3 g 蛋白胨)

24. 蒸馏水 (dH 2 O)

25. 10 M NaOHBioshop,目录号:SHY700.1;溶解在 dH 2 O 中的粉末)

26. 商业漂白剂(Bioshop,目录号:SYP001.4;次氯酸钠,12% 溶液)

27. Vaseline ® 100% 纯凡士林

28. 羊毛脂(Sigma,目录号:L7387-250G

29. 石蜡(Bernardin Parowax Canning Wax454g

30. 四咪唑(Sigma,目录号:L9756-5G

31. 琼脂糖粉末(Sigma,目录号:A9539-500G

32. M9 缓冲器(见配方)

33. 漂白溶液(见食谱)

34. Valap(见食谱)

35. 2% w/v Tetramisole 溶液(见配方)

36. 3% w/v 体积琼脂糖粉末溶液(见配方)

 

设备

 

1. 带透射光支架的体视显微镜(徕卡,型号:S6 E,尼康,型号:SMZ 745 或同等产品)

注意:我们更喜欢带有外部或 LED 光源的立体显微镜,以避免在安装过程中加热我们的蠕虫。

2. 两个加热块,一个设置为 95 °C ,另一个设置 65 °C VWR,型号:标准干块加热器或同等产品)

3. 用于 1.5-2 mL管的带转子的台式微量离心机(Eppendorf型号: 5415 D 24 管,带转子 F45-24-11,或同等产品)

4. 涡流混合器(Scientific Industries ,型号: Vortex-Genie 2,或同等产品)

5. 培养箱设置为 20°C(三洋,型号: MIR-553,或同等产品)

6. 试管摇杆(Thermo Scientific ,型号: Vari-Mix M48725Q,或同等产品)

7. 用于 10-1,000 µL 体积的单通道移液器(Gilson,型号:Pipetman Classic P20P200 P1000,或同等产品)

8. 微波

9. S针盘共聚焦显微镜(Zeiss,型号:Cell ObserverQuorum,型号:WaveFX-X1,或同等产品),具有适用于 GFP /mNeonGreen mCherry 荧光蛋白(例如35-50 mW 488 )的激发波长和发射滤光片nm 491 nm,以及 561 nm 568 nm 二极管激光器,带 ET525/50 FF593/40 单带通,或 466/523/600/677 四带通发射滤光片)和高数值孔径 (NA) 63 ×油镜(例如Leica,型号:63 × /1.40-0.60 HCX PL APO,或 Zeiss,型号:63 × Plan-Apochromat DIC UV VIS-IR)。

根据样品的不同,高 NA 水浸物镜(例如,徕卡,型号:HC PL APO 63 × /1.20 W CORR CS2)可能更可取。

10. 转盘扫描仪(横河电机,型号:CSU-X1

11. 科学 CMOS 相机(Zeiss型号:AxioCam 506 MonoPhotometrics,型号:Prime BSI,或同等产品)

 

软件

 

1. Conda 4.9.0 或别有用心的版本(Anacondahttps: //anaconda.org/anaconda/conda

2. CentTracker Github 仓库: https ://github.com/yifnzhao/CentTracker

3. 斐济 1.52v 或包含 TrackMate 的其他版本Schindelin等人2012Tinevez等人2017

4. MATLAB 2020b 或其他版本(MathWorks https://www.mathworks.com/products/matlab.html

 

程序

 

A.  GSC 实时成像做准备的C. elegans菌株的培养

1. 根据标准方案(Brenner, 1974) ,在接种了大肠杆菌菌株 OP50 的线虫生长培养基 (NGM) 维持秀丽隐杆线虫动物

2. 定期转移蠕虫,以确保健康且相对同步的种群。我们使用蠕虫挑选(图 2A)每周(约每 2 代)从保持在 20 °C 的盘子中转移几个小聚集体 ~L1 幼虫(总共约 50 只动物),就像细菌草坪正在枯竭一样。然而,最佳维持方法将取决于动物的基因型和它们生长的温度Stiernagle2006

3.  1 列出了本研究中使用的菌株。要使用 CentTracker 进行分析,动物必须携带适用于荧光显微镜的中心体标记。我们使用带有 GFP mCherry 标记的β-微管蛋白,它标记有丝分裂纺锤体并在中心体富集。如果需要更特异的中心体标记,FP 标记的中心体或中心体周围成分(例如SAS-4 SPD-2 Magescas等人。 , 2019)可能更合适。

 

B. L1 阶段幼虫的同步

1. 提前三天,通过将 2-3 个最近饥饿的 L1 幼虫的较大聚集体从库存盘转移到新鲜的 NGM/OP50 盘中,使用蠕虫挑选来设置盘子。这应确保在执行同步程序时,盘子里装满了健康、喂养良好和怀孕的成年人。

°C 三天是合适的时间;如果菌株需要在另一个温度下维持,则相应地调整此时间Stiernagle2006

2. mL的无菌 M9 来收集妊娠成人。一旦大多数动物漂浮,使用 P1000 移液器将 M9 和蠕虫转移到 1.5 mL微量离心管中。

3. 在设置为 4,000 ×的台式微量离心机中旋转试管 1 分钟 g将动物收集成松散的颗粒而不损坏它们。

4. 取出 M9 上清液并丢弃。

5. 添加 1 mL的漂白溶液(参见食谱)。

6. 使用涡流混合器搅拌动物约 6-7 分钟。该反应时间不应超过 10 分钟。每约 1-2 分钟使用立体显微镜检查动物的状况,并在蠕虫开始裂解/破裂后立即停止反应。

7. ×离心 1 分钟

8. 从蠕虫尸体和胚胎颗粒中取出漂白溶液,加入 1 mL无菌 M9

9. ×离心 1 分钟

10. 取下 M9 并再重复此清洗程序两次。

11. 最后一次旋转后,将 750 μL 的无菌 M9 添加到蠕虫颗粒中。

12.  15 °C 下孵育管子 24 小时,以允许 L1 孵化。在没有食物的情况下孵化的 L1s 将保持发育滞育,从而产生同步种群,可在 15 °C保持 5-7 天。

13. 在使用当天,通过计算 M9 10 μL 中存在的颠簸动物的数量和在倒置管数次后饥饿的 L1 溶液中的数量,确定每个管中存活和被捕 L1s 的近似浓度(L1s/μL),以确保均匀分布。

14. 使用 P20 P200 移液器将 M9 中的 10-15 个同步 L1 转移到接种 HT115 35 mm NGM 板上,使用步骤B13中每 µL L1 数量来选择合适的体积。为了成像晚期 L4 幼虫,在 20 °C下孵育板40-48 小时。

注意:我们发现在 HT115 而不是 OP50 上电镀可确保在成像时更同步的种群和更一致的有丝分裂 GSC 数量。

 

C. 凹槽琼脂糖垫的制备

1. 提前准备约 1 mL 3% 琼脂糖凝胶,将 3 g 琼脂糖粉溶解到 100 mL dH 2 O 中,在高处微波直至完全融化,然后分配到 1.5 mL微量离心管中。在室温下储存的琼脂糖等分试样可以使用长达 1 个月。

2. 将加热块预热至 95 °C 。当加热块达到设定温度时,加入一管 3% 琼脂糖。 7-9 分钟后,定期检查琼脂糖(每 ~ 1-2 分钟),看它是否已经融化。

注意:过热琼脂糖,无论是将管暴露在 >95 °C 的温度下,还是将它们在 95 °C 放置超过 10-15 分钟,都会产生质量差的垫。

3. 根据需要使用水或 70% 乙醇、压缩空气和/Kimwipe 清洁载玻片和硅模具,以去除多余的液体和灰尘或碎屑。

4. 将两个盖玻片粘贴到清洁的载玻片上,使它们之间的距离短于硅模具的宽度(图 2B)。

5. 使用干净的塑料移液管,将一滴融化的琼脂糖添加到两个带胶带的盖玻片之间的载玻片上,避免气泡。

6. 快速将硅模具降低到液滴上,使其在两个胶带盖玻片之间保持平衡。轻轻按下硅模具的两个边缘,它们与盖玻片重叠(图 2C)。让琼脂糖凝固约 1 分钟 

注意:快速执行此步骤可确保图案良好的凹槽。不要在没有盖玻片支撑的硅模具上向下推。硅模具与琼脂糖表面接触后的任何横向移动都会导致凹槽破裂、歪斜且通常质量差。

7. 一旦琼脂糖凝固,轻轻地将硅模具从琼脂糖垫上提起,并使用立体显微镜检查凹槽是否笔直且轮廓清晰。

8. 使用剃刀刀片,将模制的琼脂糖切成至少 0.5 x 1 厘米的切片。立即用 dH 2 O覆盖以防止干燥(图 2C)。

9. 成型的琼脂糖垫可在 4 °CdH 2 O 中储存长达 1 周。

 

 

2. 所需材料和安装方法概述。 

A. 由玻璃巴斯德吸管和铂丝制成的蠕虫镐。 B. 琼脂糖图案化材料: 1. 3% 琼脂糖溶液; 2. 硅模具通过光刻微纹脊; 3. 带切割吸头的塑料移液管; 4. 显微镜载玻片表面贴有两片盖玻片作为垫片; 5.装有dH 2 O培养皿,用于储存成型的琼脂糖垫。 C. 制备带有凹槽图案的琼脂糖垫的方法。硅模具的图像,在两个盖玻片之间平衡并压在熔融的琼脂糖上,显示在顶部(左)和底部(中)视图中。然后用单刃剃须刀片将带图案的琼脂糖垫切成薄片,然后将切片转移到装满水的培养皿中(右)。 D. 安装蜗杆的材料: 1 。嘴部移液器与微毛细管移液器组装在一起; 2. M9缓冲器; 3. M90.04%四咪唑; 4. 75% EtOH(用于清洁吸管上的微毛细管); 5.吸墨纸和Kimwipes切成~3厘米的条; 6.蠕虫睫毛夹; 7. 装有琼脂糖垫切片的水的培养皿; 8. 1孔玻璃凹陷载玻片; 9. 镊子; 10.显微镜载玻片和方形盖玻片; 11. 瓦拉普; 12.带切割吸头的塑料移液管,并与 200 µL移液器吸头组装在一起; E. 最终制备的顶视图 - 琼脂糖垫位于盖玻片下方的中心,凹槽朝上(,朝向盖玻片),盖玻片的角由 Valap 稳定,琼脂糖垫周围的区域位于盖玻片下方盖玻片用 M9 中的 0.0 4% Tetramisole 回填。 F. 通过立体显微镜观察三个安装良好的 L4 幼虫的特写。

 

D. 安装蠕虫进行实时成像

注意:执行以下步骤时,应尽一切努力避免与动物发生身体接触并快速工作,以便在从喂食盘中取出蠕虫后约 5 分钟内开始成像。这些步骤已针对 L4 后期幼虫进行了优化,但也适用于 L3 幼虫和成年动物。

1.  M9 中制作 0.04% 四咪唑的新鲜溶液。

注意:虽然这种安装方法可以在没有麻醉剂的情况下固定蠕虫,但我们发现残留的身体运动,如肌肉抽搐和咽部抽吸,会掩盖亚细胞动力学,低剂量的麻醉剂(0.04% tetramisole)会产生更可靠的结果。

2. 通过在本生灯火焰上加热其中间并在玻璃变软后从两端轻轻拉动来制备玻璃微量移液器。以约 6 厘米的长度折断移液器,轻轻加热折断端以使玻璃光滑。理想的移液器应足够窄,以确保精确的音量控制,但又足够宽,可以以最小的接触转移 L4 晚期动物(,足够宽,让它们自由地捶打)(图 2D #1 3A)。

3. °C的加热块上熔化一管 Valap(参见食谱) 。

4.  100 μl M9 中的 0.04% 四咪唑转移到玻璃凹陷载玻片的孔中。

5. 在立体显微镜下并使用嘴吸管,使用约 5 μl M9 2-5 L4 幼虫从板表面轻轻漂浮,并通过嘴吸管将它们转移到凹陷幻灯片上的四咪唑中(图 3A)。

6. 使用嘴吸管,在 tetramisole 溶液表面吹气,以产生约 10 秒的迷你涡流。蠕虫抖动应显着减少(图 3B)。

7. 使用剃须刀或镊子,将 3% 的琼脂糖垫放在干净的载玻片上,凹槽成型的一面朝上。

 

注意:快速执行以下步骤,以防止琼脂糖垫和蠕虫变干。

8. 使用 Whatman 纸或 Kimwipe 和吸管从琼脂糖垫中去除多余的水。

9. 使用嘴吸管轻轻地将蠕虫从凹陷滑道转移到琼脂糖垫,尽可能靠近凹槽并使用尽可能少的液体(图 3C)。

10. 用嘴吸管轻轻吹气以移动蠕虫,直到它们落入所需的凹槽中。如果蠕虫保持漂浮而不是落入凹槽中,则存在过多的液体,因此请使用口吸管去除多余的液体并重复(图 3D)。

11. (可选)如果需要精确放置蠕虫(例如,在单个视野内捕获 2-3 只幼虫),请使用蠕虫睫毛镐轻轻地将蠕虫扫入到位尽量减少蠕虫和睫毛夹之间的物理接触,因为这会刺激触觉感受器并导致蠕虫移动。

12. 使用 Whatman 纸或 Kimwipe 去除任何多余的液体(图 3E)。

13. 小心地将盖玻片降低到琼脂糖垫上(图 3F)。

注意:过快地放下盖玻片可能会损坏蠕虫。

14. 修剪 200 μl 移液器吸头,使用单刃剃须刀片从末端切割约 1 厘米,并将其放在塑料移液管的末端。使用它在盖玻片的每个角落添加一小滴融化的 Valap(图 2E 3G)。

注意:避免添加过多的 Valap,因为这可能会加热盖玻片。添加 Valap 时,避免提起盖玻片。

15. 使用 P200 移液器,在 M9 中用 ~ 100 μl 0.04% 四咪唑慢慢回填盖玻片下的空间(图 3H)。

注意:不要过度填充腔室或填充过快,因为这会导致盖玻片抬起并取代蠕虫。检查立体显微镜下安装的蠕虫,以确保它们在凹槽中并且不会移动(图 2F)。

 

 

3. 安装过程的示意图 

使用少量 M9 缓冲液和吸管将A. C. elegans蠕虫从喂食板转移到 M9 中填充有 0.04% tetramisole 的凹陷幻灯片。 B. 然后用吸管在液体表面吹气,将液体和蠕虫混合,形成微型涡流C. 然后将单个蠕虫通过嘴吸管转移到 M9 中的小体积 0.04% 四咪唑中,并将其转移到模制有凹槽的琼脂糖垫上。 D. 将蠕虫移向凹槽,并使用吸管以所需方向将空气吹过液体表面来分散多余的液体。 E. 一旦蠕虫被定位在凹槽中,使用 Whatman 纸或 Kimwipe 去除多余的液体,如果需要,在嘴吸管的帮助下。 F. 然后将盖玻片小心地放在琼脂糖垫的顶部。 G. 通过在每个角落添加一小滴融化的 Valap 将盖玻片固定到位。 H. 然后用M9 中剩余的约 100 μl 0.04% 四咪唑回填盖玻片下方的区域。

 

E. 成像

我们实验室用于对 GSC 有丝分裂进行成像的典型成像参数如下:

1. 将安装的 wor ms 放置在倒置旋转圆盘共聚焦显微镜 [Zeiss Cell Observer with Yokogawa CSU-X1,使用高 NA 63 ×油浸物镜(Zeiss 63 × Plan-Apochromat DIC (UV) VIS-IR]

2. 将激光强度设置为最大值的 10%35 mW 488 nm 50 mW 561 nm 激光器),并将曝光时间设置为 200 ms。将相机 (Zeiss Axio Cam 506 Mono) 分档设置为 3 × 3,最终像素大小为 0.1802 μm

3.  z堆栈步长设置为 0.5 μm,具有足够的范围以包含远端种系的高度(通常为 20 μm)。

4.  30 秒成像一次,最长可达 40 分钟。在饥饿约 40 分钟后,这是我们安装方法不可避免的结果,我们观察到进入有丝分裂的 GSC 数量显着减少,这表明细胞和/或有机体生理学在这一点之后发生了变化视频 1 2 Zellag等等2021。因此,我们不建议长时间成像。

注意:这些成像参数进行了调整,以尽量减少激光曝光的不利影响,并且特定于我们的显微镜。我们建议用户使用以下标准作为基准优化其成像参数:(1)在 30-40 分钟采集过程中进入有丝分裂的细胞数量相当一致(图 4K); (2)每个生殖系平均有 13 个有丝分裂条目(图 4I),在 40 分钟内采集; (3) 有丝分裂的平均持续时间(定义见下文)约 5 分钟(图 4J)。

 

 

 

 

视频 1 。在 90 分钟的图像采集中,L4 晚期幼虫种系中的有丝分裂线虫GSCs 具有中心体和轴标记。 

来自晚期 L4 蠕虫的性腺远端(左)的延时电影,在生殖系中表达 mNG::ANI-1(绿色;轴)和 mCH::β-微管蛋白(洋红色;中心体)。每 30 秒采集一次图像,持续 90 分钟,使用 60 倍平面复消色差 DIC (UV) VIS-IR 油浸式安装在倒置 Cell Observer 旋转盘共聚焦显微镜 (Zeiss; Yokogawa) 上的 AxioCam 506 Mono 相机 (Zeiss)物镜 (Zeiss),由 Zen 软件 (Zeiss) 控制。显示了 38 z 截面(0.5 µm 截面)的最大强度投影。使用 Fiji (Schindelin et al ., 2012) 调整图像的亮度和对比度。比例尺 = 10 µm。时间戳以小时:分钟为单位。电影以每秒 10 帧的速度播放。

 

 

视频 2  40 分钟的图像采集中,L4 晚期幼虫种系中的有丝分裂线虫GSCs 具有中心体和染色质标记 

来自晚期 L4 蠕虫的性腺远端(左)的延时电影,在生殖系中表达 GFP::β-微管蛋白(绿色;中心体)和 mCH::组蛋白 H2B(洋红色;染色质)。采集设置与视频 1 相同,但采集持续时间为 40 分钟。显示了 38 z 截面(0.5 µm 截面)的最大强度投影。使用 Fiji (Schindelin et al ., 2012) 调整图像的亮度和对比度。比例尺 = 10 µm。时间戳以分钟:秒为单位。电影以每秒 10 帧的速度播放。

 

F. 图像处理

以下步骤在我们的 GitHub 存储库的自述文件中进一步详细说明,如果分组在同一个文件夹中,则可以以批处理模式应用于单个电影或多个电影。

 

G. 登记

1. 使用 ImageJ 自动注册工具在电影期间手动跟踪一个或一系列有丝分裂细胞的主轴中点(图 4A)。

注意:如果跟随全局样本移动的其他标记结构可见,则用户可以跟踪主轴中点以外的对象。

2. 使用 Jupyter notebook批处理模式电影注册 模块生成 xy 平移矩阵并相应地校正电影(图 4B)。

4B 中的 Kymographs 显示了 xy 配准后样本运动的校正。

 

H. 中心体追踪

使用 ImageJ 自动修复超堆栈来恢复注册电影中的像素缩放和帧速率,裁剪注册生成的边界,并使用 TrackMate Tinevez等人2017执行点检测和轨道创建(图 4C)。作为参考,我们的 TrackMate 参数如下:blob 直径 = 2.5 μm ;链接最大距离 = 2.7 μm最大帧间隙 = 2

注意:TrackMate 可以识别和跟踪由除β-微管蛋白之外的荧光蛋白、除 GSC 之外的细胞类型以及除秀丽隐杆线虫之外的生物体中标记的中心体。 TrackMate 参数应由用户根据其样本和成像参数进行优化,以检测尽可能多的真实中心体轨迹。虚假轨道通常在下一步中被移除。

 

I. 中心体轨道配对

使用 Jupyter 笔记本批处理模式 轨道对分类模块将点轨道配对成可能的中心体轨道对(,同一细胞内的中心体)(图 4D)。

注意:我们的轨道对分类器是在一个大型数据集上训练的 野生型 C. elegans GSC。希望将中心体与其他细胞类型和/或其他生物体配对的用户可能需要训练自己的模型。可以在我们的 GitHub 存储库中找到有关如何执行此操作的说明。

 

J. 有丝分裂评分

1. 使用 Matlab 脚本Step1_import_textfile_and_align_cent_tracks导入 xyzt 坐标 f或配对轨道,并生成包含每对对应中点坐标的文件(图 4E)。

2. 使用 ImageJ Step2_CropCells生成以Step1中计算的中点坐标为中心的裁剪最大强度投影(图 4F)。

3. 使用 Matlab 脚本Step3_score_mitosis绘制每个预测的中心体对的点到点距离(以下简称主轴长度)。此步骤允许用户排除假阳性对并对有丝分裂事件进行评分[例如,核包膜破裂 (NEBD),伴随着纺锤体长度的快速减少和后期开始 (AO),当纺锤体长度开始迅速增加时]通过单击图。如果图表不清楚,用户可以使用一个选项查看相应的裁剪 tif(图 4G)。

4. 使用 Matlab 脚本Step4_calc_fits 计算有丝分裂的持续时间并提取各种有丝分裂特征,包括纺锤体长度和后期伸长率。其他特征,如主轴角位移或长度波动,可以很容易地从结果数据中计算出来。我们将有丝分裂定义为 NEBD 后的一段时间,一旦纺锤体长度达到恒定最小值,直到 AO(图 4G);这将包括大部分前中期和所有中期。

 

 

4. 图像处理概述 分析晚期 L4 幼虫的 3D 有丝分裂纺锤体动力学。 

A.为了校正 xy 中的样本移动,使用 ImageJ 自动化测试工具在原始/原始电影的最大强度投影的持续时间内手动跟踪一个或多个细胞的纺锤体中点。比例尺 = 10 µmB. Jupyter notebook 批处理模式中运行电影注册模块,从跟踪的主轴中点的坐标创建一个 xy 平移矩阵,并相应地注册原始/原始电影。使用 ImageJ (Mary et al ., 2016)中的 KymographBuilder 插件生成的 kymograph ADDIN EN.CITE Hadrien2016122(Hadrien et al., 2016)12212217Hadrien, MaryRueden, CurtisFerreire, TiagoKymographBuilber: Release 1.2.4NZenodoNZenodo201610.5281/zenodo.56702 30 分钟内通过单个 GSC 的主轴中点沿约 8 µm 宽的线扫描显示,以说明此校正的有效性,之前 (左)和后(右)xy 注册。比例尺 = 5 µmC. ImageJ 自动修复超堆栈恢复像素缩放和帧速率,裁剪配准生成的边界,并允许用户使用 TrackMate 对配准图像执行点检测和跟踪创建Tinevez等人2017。轨迹显示为彩色线,包括真正的中心体轨迹和虚假轨迹。 D. TrackMate 轨道然后通过在 Jupyter 笔记本批处理模式中运行轨道对分类器模块进行配对。左侧显示了最佳结果,其中来自两个相邻细胞的四个中心体轨迹在两个分裂过程中已正确配对(绿色和洋红色轨迹)。 E. 成对点的 xyzt 坐标被导入 Matlab 并使用脚本Step1_import_textfile_and_align_cent_tracks计算所有对的中点 F. 然后 ImageJ Step2_CropCells使用中点坐标为每对生成裁剪的最大强度投影,帧号显示在右上角 G. 然后用户可以排除假阳性对并对有丝分裂事件进行评分,通过单击显示的点到点距离(即主轴长度)随时间变化的图(左),或者在使用 Matlab 脚本Step3_score_mitosis查看 F(右)中生成的裁剪图像后手动输入时间点。 NEBD = 核包膜破裂。 AO =后期开始。 H. Matlab 脚本Step4_calc_fits计算有丝分裂的持续时间(我们定义为 G 中的阴影框所示)并提取不同的有丝分裂特征。 I.在从 61 只动物获取图像的前 40 分钟内,每个性腺进入有丝分裂(进行 NEBD)的 GSC 数量 [平均值±标准 (SD) = 13.5082 ± 6.2786 有丝分裂条目]J. 来自 74 只动物的 649 个细胞的有丝分裂持续时间(平均值± SD = 5.2512 ± 2.0175 分钟)。 K. 有丝分裂条目占 40 分钟图像采集期间有丝分裂总数的百分比,相对于采集开始(n = 61 875 有丝分裂条目)按 5 分钟间隔进行分类。 L. 来自 74 只动物的 649 个细胞在有丝分裂期间的平均纺锤体长度(平均值± SD = 3.0246 ± 0.3678 µm)。 M. 74 只动物的 819 个细胞在后期早期(后期开始后的前 2 分钟)的纺锤体伸长率(平均值± SD = 0.0245 ± 0.0059 µm/秒)。在 IM 中,黑条显示平均值,误差条代表 SD。合并了来自三个菌株(UM679JDU19 ARG16,见表 1)的数据。

 

数据分析

 

CentTracker 图像配准模块校正 xy 样本移动。然而,z 中大而突然的样本移动可能导致无法跟踪的帧和点重复,这可能会显着影响自动配对模块。因此,我们排除了表现出严重 z 运动的电影。这不包括正常的 z 漂移,它是渐进的,对点检测和轨迹创建的影响最小。通常,当动物不能很好地位于它们的凹槽中时会发生严重的 z 运动,并且可以通过良好的安装几乎完全避免。

细胞评分步骤 (J) 使用CentTracker时,对于检测假阳性中心体对至关重要。真阳性中心体对的点到点距离图具有一致的模式(图 4G),因此假阳性图应该很容易识别。但是,我们建议查看任何异常图的相应裁剪 tif,尤其是在分析的 GSC 不是野生型/或预计有纺锤体缺陷的情况下。

4 中显示的有丝分裂参数适用于来自晚期 L4 幼虫的 GSC,并按照Zellag等人的描述进行计算 (2021) ,其中还可以找到提取其他有丝分裂特征的详细信息。每个性腺的有丝分裂总数是使用手动轨迹配对确定的,而不是 CentTracker 中的轨迹配对分类器,后者在野生型动物中的发现率约为 80% Zellag等人2021

 

笔记

 

1. 该协议中使用的硅模具被蚀刻以形成 26 μm高和 50 μm宽的正带,并生成具有相同比例的 V 形凹槽,具有倾斜的壁(图 1B)。可以使用计算机辅助设计 (CAD) 软件创建简单的光掩模设计,并且可以在大多数微细加工设施中实现光掩模生产和光刻。我们随后发现,方形凹槽(图 1B产生相似甚至可能更好的结果,并且对于光刻步骤来说可能更具挑战性/成本更高。我们已经测试了以下设计,它们都足以安装:

a. μm 深和20 μm 的方形凹槽。

b. 对于晚期 L4 蠕虫:方形凹槽,深35 μm 38-43 μm

c. 对于 1 天大的成虫:方形凹槽 55 μm深和 60-65 μm宽。

如果没有微细加工设备,使用乙烯基 Long Play (LP) 唱片作为模具Zhang等人2008 年;Rivera Gomez Schvarzstein2018 年)可以产生相当好的结果Zellag等人2021 年)

2. 用人的睫毛或猫的胡须制作蠕虫睫毛。用一对细尖镊子夹住睫毛/晶须,将根端插入 200 μl移液器尖端的末端,该尖端充满熔融的 Valap。一旦 Valap 凝固,睫毛/晶须应该留在原位。或者,可以熔化约 1,000 μl移液器尖端的末端,并在塑料仍具有延展性时插入睫毛/晶须(图 2D)。

 

食谱

 

1. M9 缓冲液(1,000 毫升)

3KH 2 PO 4

6 Na 2 HPO 4

5克氯化钠

1 mL 1 M MgSO 4

dH 2 O 使最终体积达到 1,000 mL

混合所有成分直至溶解,并使用 0.2 μm 孔径过滤灭菌。

2. 瓦拉普

凡士林、羊毛脂和石蜡重量比为 1:1:1

将玻璃烧杯中的所有成分混合在设置为低温的热板上。偶尔搅拌直到融化和均匀。在室温下储存为 ±1 mL 等分试样。

3. 漂白溶液 (5 毫升)

1 mL 商业漂白剂( 10% 有效氯)

250 μL 10M NaOH

3.75毫升dH 2 O

mL锥形管中将漂白剂和 NaOH 添加到水中。足以漂白5 60 毫米的妊娠成人平板。每次新鲜,最好在使用后 1 小时内。不要储存。

注意:由于漂白剂会随着时间的推移而降解,我们建议使用已知且相当近期(1 年内)购买日期的商用漂白剂瓶。

4. 2% w/v 四咪唑溶液

dH 2 O 中制备 2% w/v 溶液,在 4°C 下储存,使用前在 M9 缓冲液中稀释至 0.04%

5.  3% w/v 体积琼脂糖粉末溶液

2 O中制作 3% w/v 体积溶液。

 

致谢


我们感谢 IRIC Jean-Claude Labbé (JCL) 博士对这份手稿的建议和批判性阅读。我们也感谢 DrsAmy Maddox (UNC Chapel Hill)Arshad Desai (UC, San Diego)Benjamin Lacroix (Institut Jacques Monod) 用于共享菌株、IRIC 的生物成像设施和 McGill 的高级生物成像设施 (ABIF) 用于技术援助,以及 Labbé 的成员和 Gerhold 实验室寻求帮助和建议。 RMZ 获得了加拿大自然科学与工程研究委员会 (NSERC) Alexander Graham Bell 加拿大研究生奖学金和 IRIC 博士奖学金。 YZ 得到了 Sheila Ann MacInnis Grant 科学本科生研究奖 (SURA) 的部分支持。这项工作由加拿大卫生研究院 (PJT-153283 JCL ARG 以及 PJT-159523 ARG) 资助。

该协议是 Zellag 等人的一部分并衍生自Zellag等人2021 年)


利益争夺


不存在利益冲突或竞争利益。

 

参考

1. Angelo, G. Van Gilst, MR (2009)饥饿可保护生殖系干细胞并延长秀丽隐杆线虫的生殖寿命 科学3265955):954-958

2. Amini, R., Goupil, E., Labella, S., Zetka, M., Maddox, AS, Labbé, JC Chartier, NT (2014)C. elegans Anillin 蛋白调节细胞间桥稳定性和种系合胞组织。 细胞生物学杂志 2061):129-143

3. 布伦纳,S.1974 年)。秀丽隐杆线虫的遗传学。 遗传学771):71-94

4. Baugh, LR Sternberg, PW (2006)DAF-16/FOXO 秀丽隐杆线虫L1 停滞期间调节 cki-1/Cip/Kip 的转录和 lin-4 的抑制 Curr Biol 168):780-785

5. Barbosa, JS, Sanchez-Gonzalez, R., Di Giaimo, R., Baumgart, EV, Theis, FJ, Götz, M. Ninkovic, J. (2015)神经发育。完整和受伤斑马鱼大脑中成体神经干细胞行为的实时成像 科学3486236):789-793

6. Berger, S.Lattmann, E.Aegerter-Wilmsen, T.Hengartner, M.Hajnal, A.deMello, A. Casadevall I Solvas, X. (2018)长期C. elegans固定化使高分辨率的体内发育研究成为可能 实验室芯片18(9)1359-1368

7. Burnett, K.Edsinger, E. Albrecht, DR (2018)对活生物体进行快速温和的水凝胶封装可以在多个小时内进行长期显微镜检查 交流。生物学173

8. Berger, S. Spiri, S. (2021)。完整秀丽隐杆线虫幼虫发育的基于微流体的成像 发展148(18)

9. Chai, Y.Li, W.Feng, G.Yang, Y.Wang, X. Ou, G. (2012)秀丽隐杆线虫胚胎后发育过程中细胞动力学的实时成像。 国家协议712):2090-2102

10. Dickinson, DJ, Ward, JD, Reiner, DJ Goldstein, B. (2013)使用 Cas9 触发的同源重组设计秀丽隐杆线虫基因组。 自然方法 10 (10):1028 - 1034

11. Fielenbach, N. Antebi, A. (2008)C. elegans dauer 的形成和可塑性的分子基础。 基因开发2216):2149-2165

12. Fabig, G.Löffler, F.Götze, C. Müller-Reichert, T. (2020 a )雄性秀丽隐杆线虫减数分裂的活细胞成像和定量分析。 生物协议10(20)e3785

13. Fabig, G., Kiewisz, R., Lindow, N., Powers, JA, Cota, V., Quintanilla, LJ, Brugués, J., Prohaska, S., Chu, DS Müller-Reichert, T. ( 2020b)雄性减数分裂纺锤体具有有效分离成对和滞后染色体的特征 188bet体育电竞9e50988

14. Gray, J. Lissmann, HW (1964)线虫的运动 J Exp Biol 41135-154

15. Gerhold, AR, Ryan, J., Vallée-Trudeau, JN, Dorn, JF, Labbé, JC Maddox, PS (2015)。通过原位成像研究干细胞和祖细胞有丝分裂进程的调节。 Curr Biol 259):1123-1134

16. Gordon, KL, Zussman, JW, Li, X., Miller, C. Sherwood, DR (2020)。干细胞壁龛通过壁龛外的隐蔽细胞定向和分离子细胞而退出秀丽隐杆线虫 188bet体育电竞9e56383

17. Hirsh, D.Oppenheim, D. Klass, M. (1976)秀丽隐杆线虫生殖系统的发育 开发生物学491):200-219

18. Hwang, H.Krajniak, J.Matsunaga, Y.Benian, GM Lu, H. (2014)用于高分辨率成像和显微注射的秀丽隐杆线虫的按需光学固定 实验室芯片14(18)3498-3501

19. Joshi, PM, Riddle, MR, Djabrayan, NJ Rothman, JH (2010)秀丽隐杆线虫作为干细胞生物学模型。 Dev Dyn Off Publ Am Assoc Anat 2395):1539-1554

20. Kimble, JE White, JG (1981)关于控制秀丽隐杆线虫的生殖细胞发育 线虫_ 开发生物学812):208-219

21. Kim, E.Sun, L.Gabel, CV Fang-Yen, C. (2013)秀丽隐杆线虫的长期成像 使用纳米粒子介导的固定的线虫。 公共科学图书馆一号81):e53419

22. Luke, CJ, Niehaus, JZ, O'Reilly, LP Watkins, SC (2014)。用于成像活秀丽隐杆线虫的非微流体方法 方法683):542-547

23. Maddox, ASHabermann, B.Desai, A. Oegema, K. (2005)两个C的不同角色。 线虫性腺和早期胚胎中的苯胺 发展13212):2837-2848

24. Morrison, SJ Kimble, J. (2006)发育和癌症中的不对称和对称干细胞分裂。 自然4417097):1068-1074

25. Magescas, J.Zonka, JC Feldman, JL (2019)中心体微管组织中心功能失活的两步机制。 188bet体育电竞8e47867

26. Martin, JL, Sanders, EN, Moreno-Roman, P., Jaramillo Koyama, LA, Balachandra, S., Du, X. O'Brien, LE (2018)果蝇成年中肠的长期实时成像揭示了分裂、分化和损失的实时动态 188bet体育电竞7e36248

27. Mary, H.Rueden, C. Ferreira, T. (2016)KymographBuilder:发布 1.2.4 泽诺多

28. Merritt, C. Seydoux, G. (2010)种系的转基因解决方案。 WormBookC. elegans 生物学的在线评论 1-21doi10.1895/wormbook.1.148.1

29. NguyenPD CurriePD2018 年)。体内成像:照亮生活中的干细胞 动物。 发展145(7)

30. Park, S.Greco, V. Cockburn, K. (2016)干细胞的实时成像:回答老问题并提出新问题 Curr Opin 细胞生物学4330-37

31. Priti, A.Ong, HTToyama, Y.Padmanabhan, A.Dasgupta, S.Krajnc, M. Zaidel-Bar, R. (2018)合胞体种系结构通过内部肌动球蛋白紧身胸衣的收缩来积极维持。 国家通讯91):4694

32. Rompolas, P., Deschene, ER, Zito, G., Gonzalez, DG, Saotome, I., Haberman, AM Greco, V. (2012)生理性毛囊再生中干细胞和后代行为的实时成像。 自然4877408):496-499

33. Ritsma, L., Ellenbroek, SIJ, Zomer, A., Snippert, HJ, de Sauvage, FJ, Simons, BD, Clevers, H. van Rheenen, J. (2014)。通过体内活体成像在单干细胞水平显示肠隐窝稳态。 自然5077492):362-365

34. Rivera Gomez, KA Schvarzstein, M. (2018)使用乙烯基记录生产的琼脂糖垫固定线虫以进行实时成像。 微出版生物学 2018 (08)

35. Rosu, S. Cohen-Fix, O. (2017)C中生殖细胞增殖的实时成像分析。 成年线虫支持干细胞增殖的随机模型 开发生物学4232):93-100

36. Sulston, JE Horvitz, HR (1977)。线虫,秀丽隐杆线虫的胚胎后细胞谱系。 开发生物学561):110-156

37. Stiernagle, T. (2006)秀丽隐杆线虫的维护。 蠕虫书 1-11

38. Schindelin, J.Arganda-Carreras, I.Frise, E.Kaynig, V.Longair, M.Pietzsch, T.Preibisch, S.Rueden, C.Saalfeld, S.Schmid, B .等人2012)。斐济:生物图像分析的开源平台 Nat 方法97):676-682

39. Timmons, L. Fire, A. (1998)摄入的 dsRNA 的特异性干扰 自然3956705):854

40. Tinevez, JY, Perry, N., Schindelin, J., Hoopes, GM, Reynolds, GD, Laplantine, E., Bednarek, SY, Shorte, SL Eliceiri, KW (2017)TrackMate:一个开放且可扩展的单粒子跟踪平台。 方法11580-90

41. Wong, BG, Paz, A., Corrado, MA, Ramos, BR, Cinquin, A., Cinquin, O. Hui, EE (2013)实时成像显示其干细胞生态位对有丝分裂区的主动浸润。 Integr Biol (Camb) 5(7): 976-982

42. Yoshida, S.Sukeno, M. Nabeshima, Y. (2007)小鼠睾丸中未分化精原细胞的脉管系统相关生态位。 科学3175845):1722-1726

43. Zhou, K.Rolls, MM Hanna-Rose, W. (2013)稳定的细胞间桥上中枢轴的有丝分裂后功能和独特的定位机制 开发生物学3761):13-22

44. Zeiser, E.Frøkjær-Jensen, C.Jorgensen, E. Ahringer, J. (2011) MosSCI Gateway 兼容的质粒工具包,用于在C. 线虫种系。 公共科学图书馆一号 6 5):e20082

45. Zhang, M., Chung, SH, Fang-Yen, C., Craig, C., Kerr, RA, Suzuki, H., Samuel, AD, Mazur, E. Schafer, WR (2008)。控制秀丽隐杆线虫产卵行为的自调节前馈电路。 Curr Biol 1819):1445-1455

46. Zellag, RM, Zhao, Y., Poupart, V., Singh, R., Labbé, JC Gerhold, AR (2021)CentTracker:一种可训练的、基于机器学习的工具,用于大规模分析秀丽隐杆线虫生殖干细胞有丝分裂。 摩尔生物细胞329):915-930




登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2022 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. Zellag, R. M., Zhao, Y. and Gerhold, A. R. (2022). Live-cell Imaging and Analysis of Germline Stem Cell Mitosis in Caenorhabditis elegans. Bio-protocol 12(1): e4272. DOI: 10.21769/BioProtoc.4272.
  2. Zellag, R. M., Zhao, Y., Poupart, V., Singh, R., Labbé, J. C. and Gerhold, A. R. (2021). CentTracker: a trainable, machine-learning-based tool for large-scale analyses of Caenorhabditis elegans germline stem cell mitosis. Mol Biol Cell 32(9): 915-930.
提问与回复

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。