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Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines
非转化细胞系中睫毛的组装/解聚测定   

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EMBO Reports
Aug 2017

 

Abstract

The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases. The in vitro serum-stimulated ciliary assembly/disassembly assay has gained popularity in addressing the functions of the protein-of-interest in ciliary dynamics. Here, we describe a well-tested protocol for transfecting human retinal pigment epithelial cells (RPE-1) and performing ciliary assembly/disassembly assays on the transfected cells.

Keywords: Primary cilium (初级纤毛), Acetylated α-tubulin (乙酰化α-微管蛋白), Ciliary assembly (纤毛组装), Ciliary disassembly (纤毛解聚), RPE-1 cells (RPE-1细胞), Short hairpin RNA (短发夹RNA)

Background

Primary cilia are hair-like sensory organelles that appear at the G0/G1 phase, and are disassembled prior to the S phase of the cell cycle (Tucker et al., 1979). Previous studies have confirmed that certain non-transformed cell types (i.e., RPE-1 cells, 3T3 fibroblasts, and mouse embryonic fibroblasts [MEFs]) can be starved to induce quiescence and ciliary formation. Subsequent re-addition of serum triggers biphasic ciliary resorption, which peaks at 2 h and 24 h following stimulation (Tucker et al., 1979; Li et al., 2011). This phenomenon lays the foundation for the serum-stimulated ciliary assembly/disassembly assay commonly used in the literature to identify proteins involved in ciliary assembly and disassembly (Pugacheva et al., 2007; Saito et al., 2017). MEFs derived from transgenic mice (in which the gene-of-interest is deleted) are often used to investigate the dynamic role of a given protein in the ciliary assembly/disassembly assays. When the specific MEF types are not accessible, one may modify the expression level of the targeted protein in naive RPE-1 cells (or 3T3 or MEFs) using transfection of cDNA or short hairpin RNA. We describe the protocol of these procedures that are routinely carried out in the lab. To unambiguously identify the cell autonomous effect on ciliary assembly or disassembly in the transfected cells, we typically ‘tag’ the transfected cells with green fluorescence via the expressed GFP or GFP-fusion protein.

Materials and Reagents

  1. 10 µl pipette tips (Corning, catalog number: 4110 , or Denville Scientific, catalog number: P1096 )
  2. 200 µl pipette tips (FUKAEKASEI and WATSON, catalog number: 110-705Y , or Denville Scientific, catalog number: P1122 )
  3. 1,000 µl pipette tips (FUKAEKASEI and WATSON, catalog number: 110-706B , or Denville Scientific, catalog number: P2103-N )
  4. 100 mm cell culture dish (AS ONE, Violamo, catalog number: 2-8590-03 , or Corning, catalog number: 430167 )
  5. 1.5 ml microcentrifuge tube (Sorenson BioScience, catalog number: 11510 , or National Scientific, catalog number: CN1700-BP )
  6. Precleaned, sterile 12-13 mm micro-glass coverslips (Matsunami Glass, catalog number: C013001 , or Thermo Fisher Scientific, catalog number: 12CIR-1.5 )
  7. 35 mm cell culture dish (Corning, Falcon®, catalog number: 353001 , or Corning, catalog number: 430165 )
  8. 15 ml centrifuge tube (FUKAEKASEI and WATSON, catalog number: 1332-015S , or Corning, catalog number: 430053 )
  9. 24-well plate (NuncTM, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 142475 , or Corning, catalog number: 3527 )
  10. Parafilm
  11. 14 x 16 cm glass (or plastic) plates
  12. Micro-slide glass (Matsunami Glass, catalog number: S024410 )
  13. 0.22 µm filter (Corning, catalog number: 431097 , or Advantec MFS, catalog number: 25CS020AS )
  14. RPE-1 cells (ATCC, catalog number: CRL-4000 )
  15. Midi-prepared, or maxi-prepared plasmid (> 1 µg/µl is preferred)
    Note: We typically have the cDNA (under the CAG or CMV promoter) or short hairpin RNA (shRNA; under the U6 promoter) inserted in the same plasmid that also expresses GFP (e.g., pCAGIG vector).
  16. 0.05% Trypsin-0.53 mM EDTA (Wako Pure Chemical Industries, catalog number: 202-16931 )
  17. Fetal bovine serum (FBS) (PAA Laboratories, catalog number: A15-701 )
  18. Dulbecco’s modified Eagle medium/Ham’s F-12 (DMEM/F12) (Wako Pure Chemical Industries, catalog number: 048-29785 )
  19. Methanol
  20. Monoclonal anti-acetylated α-tubulin (Ac-Tub) antibody, clone 6-11B-1 (Sigma-Aldrich, catalog number: T6793 )
  21. Anti-Tubulin Antibody, Detyrosinated (Merck, catalog number: AB3201 )
  22. Monoclonal anti-γ-tubulin antibody, clone GTU-88 (Sigma-Aldrich, catalog number: T5326 )
  23. Polyclonal anti-GFP tag antibody (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-6455 )
  24. Goat anti-mouse IgG (H+L) antibody, Alexa Fluor 568 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11004 )
  25. Goat anti-mouse IgG2b antibody, Alexa Fluor 568 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-21144 )
  26. Goat anti-mouse IgG1 antibody, Alexa Fluor 647 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-21240 )
  27. Goat anti-Rabbit IgG (H+L) antibody, Alexa Fluor 488 (Thermo Fisher Scientific, InvitrogenTM, catalog number: R37116 )
  28. Fluorescent mounting medium (Agilent Technologies, Dako, catalog number: S302380-2 , code number: S3023) or ProLong Gold Antifade Mountant (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36930 )
  29. Nail polish
  30. Dulbecco’s modified Eagle medium (DMEM) (NACALAI TESQUE, catalog number: 08458-16 )
  31. 100 mM pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
  32. 10x D-PBS (-) (Wako Pure Chemical Industries, catalog number: 048-29805 )
  33. Calcium chloride dihydrate (CaCl2·2H2O) (Wako Pure Chemical Industries, catalog number: 031-00435 )
  34. Magnesium chloride hexahydrate (MgCl2·6H2O) (Wako Pure Chemical Industries, catalog number: 135-00165 )
  35. 16% Paraformaldehyde (PFA) aqueous solution (Electron Microscopy Science, catalog number: 15710 )
  36. Ammonium chloride (NH4Cl) (Wako Pure Chemical Industries, catalog number: 017-02995 )
  37. Bovine serum albumin (BSA) (Wako Pure Chemical Industries, catalog number: 010-15114 )
  38. Triton X-100 (GE Healthcare, catalog number: 17-1315-01 )
  39. Sodium azide (NaN3) (Wako Pure Chemical Industries, catalog number: 190-01272 )
  40. 4’,6-Diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D1306 )
  41. Growth medium (see Recipes)
  42. PBS for cell culture (see Recipes)
  43. 2x PBSc/m (see Recipes)
  44. PBSc/m (see Recipes)
  45. 4% PFA/PBSc/m (see Recipes)
  46. 50 mM NH4Cl (see Recipes)
  47. BTPAD (see Recipes)

Equipment

  1. 5% CO2/95% air tissue culture incubator (SANYO, model: MCO-18AIC )
  2. Water bath (TOKYO RIKAKIKAI, Eyela, model: NTT-1200 )
  3. Hemocytometer chamber (Erma, catalog number: 03-303-1 )
  4. Low-speed centrifuge (TOMY SEIKO, model: LC-100 ; Eppendorf, model: 5702 )
  5. Pipette 20 µl (Pipetman P) (Gilson, catalog number: F123600 )
  6. Pipettes 200 µl (Pipetman P) (Gilson, catalog number: F123601 )
  7. Pipettes 1,000 µl (Pipetman P) (Gilson, catalog number: F123602 )
  8. For Procedure B1:
    NeonTM transfection system (Thermo Fisher Scientific, InvitrogenTM, catalog number: MPK5000 )
  9. For Procedure B2:
    1. NucleofectorTM device (Lonza, model: NucleofectorTM I )
    2. Cuvettes PlusTM Electroporation Cuvettes & transfer tips (BTX, catalog number: 45-0135 )
    3. Amaxa® Cell Line Nucleofector® Kit V (Lonza, catalog number: VCA-1003 )
  10. Forceps (Fine Scientific Tool, Dumont, model: #5/45 )
  11. Autoclave (TOMY SEIKO, model: LBS-245 )
  12. Epifluorescent microscope (Carl Zeiss, model: Axioplan 2 imaging ) equipped with objective lens (EC Plan-Neofluar 40x/0.75, Carl Zeiss, catalog number: 440350-9903-000 )
  13. Confocal microscope (ZEISS, model: LSM-780 )

Procedure

The present assay is performed by the following timeline and procedures (Figure 1). In the present protocol, we show the preparation of four samples for each transfection (e.g., before assembly, 0 h-, 2 h- and 24 h-after serum re-addition). Each researcher can prepare samples depending on the need. For example, when we focus on mechanisms of ciliary disassembly, we typically prepare three samples for each transfection (0 h-, 2 h- and 24 h-after serum re-addition). Additional sample(s) can be added for testing the transfection efficiency (on Day 3).
Day 1: Cell subculture (see Procedure A)
Day 2: Cell transfection (see Procedure B); Induction of ciliary assembly (see Procedure C)
Day 3: Checking cell transfection efficiency
Day 4: Induction of ciliary disassembly (see Procedure D); Early time point (e.g., 0 h, 2 h) cell harvest (see Procedure E)
Day 5: Late time point (e.g., 24 h) cell harvest
Immunostaining (see Procedure F) and data analysis


Figure 1. A schematic representation of the timeline of transfection and ciliary assembly/disassembly assay

  1. Cell subculture (Day 1)
    RPE-1 cells maintained in 10% FBS-containing growth medium (see Recipes) in a 5% CO2 humidified incubator at 37 °C are passaged and plated on 100 mm cell culture dishes using standard techniques. The cells are plated at a density expecting 50-80% confluence on the second day. In general, one 100 mm dish of cells (total of ~2-4 x 106 cells) is sufficient for 4-6 transfection reactions when Procedure B1 is used (3 x 105 cells for each reaction) and 2-4 transfection reactions when Procedure B2 is used (1 x 106 cells for each reaction).
    Note: We avoid using old (> 15 passage) or overgrown RPE-1 cells, which tend to have a poor cilium induction rate.

  2. Cell transfection (Day 2)
    NeonTM (Procedure B1) and NucleofectorTM (Procedure B2) transfection systems generate similar transfection efficiencies and low cell death. More than 60% of GFP plasmid-transfected RPE-1 cells express green fluorescence on the second day after transfection. In contrast, Lipofectamine 2000® or polyethylenimine (PEI) do not generate a high number of GFP-expressing cells.
    Prepare the following before conducting the transfection procedures described in Procedure B1 or B2.
    1. Prewarm growth medium in a 37 °C water bath. Place a single plasmid (or premixed multiple plasmids, when applicable) in a sterile 1.5 ml tube. For the transfection reactions of Procedures B1 and B2, ~3 µg and ~6 µg of DNA are to be used, respectively.
      If performing Procedure B2, prepare the Amaxa transfection master mixture. For each transfection reaction, mix 85.9 µl of solution I and 19.1 µl of solution II by gentle pipetting and quick spin. Prepare excess reactions to account for pipetting error.
    2. Place three or more acid-washed sterile coverslips in each 35 mm cell culture dish. Add 1 ml of growth medium and transfer to a 5% CO2 incubator at 37 °C.
    3. For each transfection reaction, prepare 1 ml of prewarmed growth medium in a 1.5 ml sterile tube and leave it in a 37 °C water bath.
    4. Use trypsin-EDTA solution to detach RPE-1 cells (0.5 ml per 100 mm plate). After ~2 min of incubation at 37 °C, collect the detached cells into a 15 ml centrifuge tube containing 9.5 ml of warmed growth medium, in which the FBS inactivates trypsin.
    5. Count the number of cells using a hemocytometer chamber.

    B1. NeonTM transfection system
    1. Centrifuge the cell suspension at 280 x g (1,200 rpm) for 2 min at room temperature.
    2. Aspirate the supernatant. Resuspend the cell pellet in 10 ml of sterile PBS (see Recipes).
    3. Centrifuge the cell suspension at 280 x g (1,200 rpm) for 2 min at room temperature.
    4. Carefully aspirate all of the supernatant. Resuspend every 1 x 106 cells with 100 µl of Resuspension Buffer R (included in NeonTM Transfection System). Thirty microliters of cell suspension (i.e., 3 x 105 cells) is required for each transfection.
    5. Gently mix 3 µg of the plasmid in 30 µl of cell suspension by pipetting.
    6. Use a 10 µl NeonTM Tip to obtain 10 µl of cell-DNA mixture and then apply an electrical pulse according to the manufacturer’s protocol using the following parameters (Pulse voltage ‘1,050 V’, Pulse width ‘35 msec’, and Pulse number ‘2 times’). Repeat until the entire volume (30 µl) of the cell-DNA mixture is ‘pulsed’.
    7. Immediately after each pulse, transfer the transfected cells into a 1.5 ml tube containing 1 ml of warmed growth medium. Use the same tube to collect all 3 x 105 transfected cells.
    8. Plate the transfected cells onto a 35 mm dish containing coverslips and immediately transfer to a 5% CO2 incubator at 37 °C for 12 h.

    B2. NucleofectorTM transfection system
    1. For each transfection reaction, transfer a volume of cell suspension equivalent to 1 x 106 trypsin-EDTA-detached RPE-1 cells into a 15 ml tube.
    2. Centrifuge the cell suspension at 280 x g (1,200 rpm) for 2-3 min at room temperature.
    3. Carefully aspirate all of the supernatant. Gently resuspend the cell pellet with 100 µl of Amaxa transfection master mixture using a P200 pipette.
      Note: Up to 4 transfection reactions may be carried out in the same batch by pooling 4 x 106 cells in 400 µl of Amaxa transfection master mixture.
    4. For each transfection reaction, transfer 100 µl of the cell suspension into a 1.5 ml tube containing DNA. Gently pipette no more than twice and avoid air bubbles.
    5. Transfer the entire cell-DNA mixture into an Electroporation Cuvette (BTX). Do not pipette out the last drop to avoid air bubbles.
    6. Place the cuvette in the NucleofectorTM device and pulse the cells with the program ‘T23’. This is a program preset in the device, which can be selected by a simple button pressing.
    7. Immediately after the pulse, collect the transfected cells in the cuvette using a fine tip dropper (included in the individually packaged Electroporation Cuvette). Transfer them into a 1.5 ml tube containing 1 ml of warmed growth medium.
    8. Plate the transfected cells onto the 35 mm dish containing coverslips and growth medium, and immediately transfer the cells to a 5% CO2 incubator at 37 °C for 12 h.

  3. Induction of ciliary assembly (Day 2)
    1. Approximately 8-12 h after cell transfection (Procedure B), the cells should have settled at the bottom. Follow Procedure E in the case that researcher wishes harvest cell samples before serum starvation (e.g., observing cilia before serum starvation or testing the transfection efficiency by immunostaining). Remove the culture medium, gently rinse the cells in 2 ml of PBS (three times), and add 2 ml of pre-warmed serum-free DMEM (or DMEM/F12). We do not notice any obvious differences between DMEM and DMEM/F12 in this step for the assembly or the subsequent disassembly of cilia.
    2. Incubate in a 5% CO2 incubator at 37 °C for 36-48 h. Optional: during this period (on Day 3), we use an inverted phase/fluorescent microscope to check the green fluorescence-positive cells to estimate the transfection efficiency. We also pay attention to the cell toxicity by observing the number of the cells detached from the coverslips/dishes.

  4. Induction of ciliary disassembly (Day 4)
    1. At the end of serum-starvation, using a pair of sterile forceps, remove one coverslip and immediately wash and fix them (0 h time-point; see Procedure E). Aspirate the serum-free medium and replace it with growth medium.
    2. Incubate the cells in a 5% CO2 incubator at 37 °C for the indicated periods; 2 h and 24 h would be good starting points.

  5. Cell harvest (Day 2, 4 and 5)
    1. Transfer one or more coverslips from the incubated 35 mm dish into an empty 24-well plate using a pair of sterile forceps with the cell-side facing up.
    2. Rinse the coverslip in 1 ml of PBSc/m (see Recipes) three times and fix in 0.5 ml of 4% PFA/PBSc/m (see Recipes) for 10 min at room temperature.
    3. Rinse the coverslip in 1 ml of PBSc/m once, and fill with 1 ml of another PBSc/m.

  6. Immunostaining
    Primary cilium can be visualized by immunostaining acetylated α-tubulin (Ac-Tub) or detyrosinated tubulin (Figure 2). Basal body can be detected by γ-tubulin (γ-Tub) immunostaining (Figure 2B). The γ-Tub staining conveniently indicates the proximal end of the cilium. It is also a good landmark particularly in the cells with a short or no detectable cilium. However, to obtain γ-Tub stained signal, the cells have to be treated with cold methanol (see Step F2).
    Each researcher can perform immunostaining procedure according to own preference, such as the procedures described previously (Phua et al., 2017; Shnitsar et al., 2015; Tomoshige et al., 2017). Below, we describe our procedures of triple staining for Ac-Tub, γ-Tub and GFP. GFP staining is used to enhance the detection of transfected cells that express GFP (or GFP-fusion proteins). So that role of the target molecule(s) in ciliary assembly/disassembly can be unambiguously identified in GFP-positive transfected cells.
    The entire procedure detailed below is performed in a light-blocking humidity chamber (homemade) at room temperature.
    1. Place a piece of Parafilm on 14 x 16 cm of glass (or plastic) plates in the chamber.
    2. Pre-treat the previously fixed coverslip with pre-chilled methanol for 1-3 min at -20 °C.
    3. Immediately rinse the cells with PBSc/m, two times.
    4. Place the coverslip on the Parafilm with the cell-side facing up.
    5. Add ~300 µl of 50 mM NH4Cl (see Recipes) from the edge of the coverslips using a P1000 pipette (or a dropper) and incubate for 10 min.
    6. Aspirate the NH4Cl from the edge of the coverslips, and rinse in 300 µl of PBSc/m once.
    7. Incubate in 100 µl of BTPAD (see Recipes) for 30 min.
    8. Incubate in 50-100 µl of primary antibody-containing BTPAD for 60 min at room temperature. Antibody dilution: anti-Ac-Tub (mouse IgG2b, 1:1,000), anti-γ-Tub (mouse IgG1, 1:1,000), anti-GFP (rabbit IgG, 1:1,000).
    9. Remove unbound primary antibodies by incubating the cells in 300 µl of PBSc/m for 5 min, three times.
    10. Incubate in 50-100 µl of secondary antibody-containing BTPAD for 45 min at room temperature. Antibody dilution: Alexa Fluor 568-conjugated goat anti-mouse IgG2b (for anti-Ac-Tub, 1:400), Alexa Fluor 647-conjugated goat anti-mouse IgG1 (for anti-γ-Tub, 1:400), and Alexa Fluor 488-conjugated goat anti-rabbit IgG (for anti-GFP, 1:400).
      Note: When the cells are labeled for Ac-Tub, but not for γ-Tub, in Step F8, the isotype specific secondary antibody is not necessary. We use Alexa Fluor 568-conjugated anti-mouse IgG (H+L) antibody instead.
    11. Rinse in 300 µl of PBSc/m for 5 min, three times.
    12. Remove the excess PBSc/m, and wipe the side of coverslips without cells.
    13. Mount the coverslip with 4 µl of mounting media on a micro-slide glass.
    14. Seal the rim of coverslip with clear nail polish.
    15. Observe samples under an epifluorescence microscope (ZEISS, Axioplan 2 imaging) or a confocal microscope (ZEISS, model: LSM-780). For an epifluorescence microscope, objective lens: 40x magnification without immersion. Ocular lens: 10x magnification. For a confocal microscope, objective lens: 63x magnification, oil immersion. Representative images taken on the confocal microscopy are shown in Figure 2.


      Figure 2. Primary cilia are assembled and disassembled by depletion and re-addition of serum, respectively. Representative images of primary cilia assembled in RPE-1 cells. For presentation purpose, images were acquired using a confocal microscope (ZEISS, LSM-780). The cells were harvested before serum starvation (before assembly), 36 h after the serum-starvation (0 h), and the subsequent serum re-addition for 2 h and 24 h, and labeled for Ac-Tub (red; A, B), GFP (green; A), and γ-Tub (cyan; B). Nucleus was stained with DAPI (blue; A). Dashed lines demarcate the cell borders. Asterisks in (A) highlight the GFP+ cells. Arrows in (A) point to the cilia in GFP+ cells. Scale bars = 10 µm (A) and 2 µm (B).

Data analysis

Because the length of the cilium is positively correlated with the number of cells exhibiting cilia, and because measuring the cilium length is labor-intensive, the number of cells exhibiting detectable cilium is counted as a surrogate index instead (Li et al., 2011).
Notes: For early-stage researchers, we recommend observing cilia with higher-magnification lens (e.g., 63x, 100x). However, we must note that researchers need to adjust Z-positions to find each cilium between cells of interest due to the higher magnification.

  1. Count the number of cells expressing Ac-Tub among GFP+-transfected cells in a double-blind fashion. More than 100 cells are counted in each experiment (two sets of 50 cells; one from the left side and the other from the right side of each coverslip). At least three independent experiments are performed for each condition. Data from independent experiments are represented as the mean percentages ± SEM. One-way analysis of variance (ANOVA) or two-way ANOVA is followed by Tukey’s test or Bonferroni’s test (as a post hoc test), respectively (Figure 3).
  2. Figure 3A shows a time-dependency of ciliary assembly after serum removal in native RPE-1 cells. Eighty percent of cells expressed cilia 36 h after the serum-starvation.
  3. Figure 3B shows the representative results of ciliary disassembly assay. An example of ciliary disassembly by knockdown of Tctex-1 is also shown. Tctex-1 is a light chain of cytoplasmic dynein complex and is an indispensable molecule for ciliary disassembly (Li et al., 2011; Yeh et al., 2013; Saito et al., 2017). Both U6 promoter and short hairpin RNA (shRNA) sequence of Tctex-1 were inserted into a pCAGIG vector that also encoded GFP (see Materials and Reagents #13) (Li et al., 2011). Cells transfected with vector (Control) or Tctex-1-shRNA (Tctex-1-sh) were serum-starved for 36 h and retreated with growth medium for 2 h and 24 h. The number of cells with cilia was counted among GFP+-transfected cells. Percentage of cells with cilia decreased from 80% (0 h) to 60% (2 h) and 40% (24 h) after serum re-addition in the cells of the control setup. Although the ciliary assembly was unaffected in Tctex-1-shRNA transfected cells, the rate of ciliary disassembly in these cells was significantly suppressed.


    Figure 3. Representative results from ciliary assembly and disassembly assays. A. Ciliary assembly assay. Naive RPE-1 cells were harvested at indicated time points after serum starvation. Y-axis showed the percentage of the cells expressing cilia. B. Ciliary disassembly assay. RPE-1 cells, transfected with control vector or Tctex-1-shRNA that also expressed GFP, were first starved and then treated with grown medium for the indicated time periods. Y-axis showed the percentage of the GFP+ cells with cilia. ##P < 0.01, ###P < 0.001; one-way ANOVA followed by Tukey’s test (comparing to the 0 h time point of each group). **P < 0.01, ***P < 0.001; two-way ANOVA followed by Bonferroni’s test (comparing between groups). n = 100 cells per experiment, three (A) and five (B) independent experiments.

Recipes

  1. Growth medium
    1. Take 500 ml of DMEM/F12
    2. Add 50 ml of FBS (final 10%)
    3. Add 5.5 ml of 100 mM pyruvate (final 1 mM)
    4. Store at 4 °C
  2. PBS for cell culture
    1. Take 180 ml of ddH2O
    2. Add 20 ml of 10x D-PBS (-)
    3. Autoclave
    4. Store at room temperature
  3. 2x PBSc/m
    1. Take 795 ml of ddH2O
    2. Add 200 ml of 10x D-PBS (-)
    3. Add 400 µl of 1 M CaCl2 (final 0.4 mM)
    4. Add 4,000 µl of 1 M MgCl2 (final 4 mM)
    5. Filter through a 0.22 µm filter
    6. Store at room temperature
  4. PBSc/m
    1. Take 500 ml of ddH2O
    2. Add 500 ml of 2x PBSc/m
    3. Filter through a 0.22 µm filter
    4. Store at room temperature
  5. 4% PFA/PBSc/m
    Prepare just before the procedure in a chemical hood
    1. Take 1 ml of ddH2O
    2. Add 2 ml of 2x PBSc/m
    3. Add 1 ml of 16% PFA
  6. 50 mM NH4Cl
    1. Take 20 ml of ddH2O
    2. Add 25 ml of 2x PBSc/m
    3. Add 133.75 mg of NH4Cl (final 50 mM)
    4. Dilute up to 50 ml with ddH2O
    5. Store at room temperature
  7. BTPAD
    1. Take 25 ml of PBSc/m
    2. Add 625 µl of 20% BSA (final 0.5%)
    3. Add 313 µl of 20% Triton X-100 (final 0.25%)
    4. Add 250 µl of 2% NaN3 (final 0.02%)
    5. Add 2.5 µl of 3 mM DAPI (final 0.3 µM)
    6. Filter through a 0.22 µm filter
    7. Store at 4 °C

Acknowledgments

This protocol was adapted from a previously published study (Saito et al., 2017). This work was supported by NIH RO1 EY11307, EY016805, Research To Prevent Blindness, Stem Innovation Award recipient (RPB), and Kaohsiung Medical University Research Foundation (105KMUOR02) (to C.-H. S.); Grant-in-Aid for Scientific Research from the Japan Society for Promotion of Science (No. 23770136 and No. 15K20856 to M.S.) and Takeda Science Foundation (to M.S.). We would like to acknowledge the Biomedical Research Core (Tohoku University Graduate School of Medicine). We would also like to thank Editage (www.editage.jp/) for English language editing. The authors declare no competing financial interests.

References

  1. Li, A., Saito, M., Chuang, J. Z., Tseng, Y. Y., Dedesma, C., Tomizawa, K., Kaitsuka, T. and Sung, C. H. (2011). Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors. Nat Cell Biol 13(4): 402-411.
  2. Phua, S.C., Chiba, S., Suzuki, M., Su, E., Roberson, E.C., Pusapati, G.V., Setou, M., Rohatgi, R., Reiter, J.F., Ikegami, K., et al. (2017). Dynamic remodeling of membrane composition drives cell cycle through primary cilia excision. Cell 168(1-2): 264-279 e215.
  3. Pugacheva, E. N., Jablonski, S. A., Hartman, T. R., Henske, E. P. and Golemis, E. A. (2007). HEF1-dependent Aurora A activation induces disassembly of the primary cilium. Cell 129(7): 1351-1363.
  4. Saito, M., Otsu, W., Hsu, K. S., Chuang, J. Z., Yanagisawa, T., Shieh, V., Kaitsuka, T., Wei, F. Y., Tomizawa, K. and Sung, C. H. (2017). Tctex-1 controls ciliary resorption by regulating branched actin polymerization and endocytosis. EMBO Rep 18(8): 1460-1472.
  5. Shnitsar, I., Bashkurov, M., Masson, G. R., Ogunjimi, A. A., Mosessian, S., Cabeza, E. A., Hirsch, C. L., Trcka, D., Gish, G., Jiao, J., Wu, H., Winklbauer, R., Williams, R. L., Pelletier, L., Wrana, J. L. and Barrios-Rodiles, M. (2015). PTEN regulates cilia through Dishevelled. Nat Commun 6: 8388.
  6. Tomoshige, S., Kobayashi, Y., Hosoba, K., Hamamoto, A., Miyamoto, T., and Saito, Y. (2017). Cytoskeleton-related regulation of primary cilia shortening mediated by melanin-concentrating hormone receptor 1. Gen Comp Endocrinol 253(1): 44-52
  7. Tucker, R. W., Pardee, A. B. and Fujiwara, K. (1979). Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells. Cell 17(3): 527-535.
  8. Yeh, C., Li, A., Chuang, J. Z., Saito, M., Caceres, A. and Sung, C. H. (2013). IGF-1 activates a cilium-localized noncanonical Gbg signaling pathway that regulates cell-cycle progression. Dev Cell 26(4): 358-368.

简介

主要纤毛是一种非运动感觉细胞器,其装配和拆卸与细胞周期进程密切相关。 初级纤毛在静止细胞中从基体拉长并随着细胞重新进入细胞周期而被吸收。 睫状动力失调与纤毛病和其他人类疾病有关。 体外血清刺激的睫状体装配/分解测定已经在解决睫状动力学中感兴趣的蛋白质的功能方面受到欢迎。 在这里,我们描述了转染人视网膜色素上皮细胞(RPE-1)和对转染细胞进行睫状体装配/分解测定的充分测试的方案。

【背景】初级纤毛是毛发样感觉细胞器,其在G 0 / G 1期出现,并且在细胞周期的S期之前分解(Tucker等, et al。,1979)。先前的研究已经证实,某些未转化的细胞类型(即,甚至是RPE-1细胞,3T3成纤维细胞和小鼠胚胎成纤维细胞[MEFs])可以被饿死以诱导静止和睫状体形成。随后的血清再次添加触发双相睫状体吸收,其在刺激后2小时和24小时达到峰值(Tucker等人,1979; Li等人,2011) 。该现象为文献中常用的血清刺激的睫状体组装/分解测定奠定了基础,以鉴定参与睫状体组装和拆卸的蛋白质(Pugacheva等人,2007; Saito等人。,2017)。通常使用源自转基因小鼠(其中感兴趣的基因被删除)的MEF来研究给定蛋白质在睫状体组装/分解测定中的动态作用。当不能获得特定的MEF类型时,可以使用cDNA或短发夹RNA的转染来修饰天然RPE-1细胞(或3T3或MEF)中靶蛋白的表达水平。我们描述了在实验室中常规进行的这些程序的协议。为了明确地鉴定细胞对转染细胞中睫状体装配或分解的自主作用,我们通常通过表达的GFP或GFP融合蛋白用绿色荧光“标记”转染的细胞。

关键字:初级纤毛, 乙酰化α-微管蛋白, 纤毛组装, 纤毛解聚, RPE-1细胞, 短发夹RNA

材料和试剂

  1. 10μl移液枪头(Corning,目录号:4110或Denville Scientific,目录号:P1096)
  2. 200μl移液枪头(FUKAEKASEI和WATSON,目录号:110-705Y,或Denville Scientific,目录号:P1122)
  3. 1,000μl移液枪头(FUKAEKASEI和WATSON,目录号:110-706B,或Denville Scientific,目录号:P2103-N)
  4. 100毫米细胞培养皿(AS ONE,Violamo,目录号:2-8590-03或Corning,目录号:430167)
  5. 1.5ml微量离心管(Sorenson BioScience,目录号:11510或National Scientific,目录号:CN1700-BP)
  6. 预清洁无菌12-13毫米微玻璃盖玻片(Matsunami Glass,产品目录号:C013001或Thermo Fisher Scientific,产品目录号:12CIR-1.5)
  7. 35mm细胞培养皿(Corning,Falcon,产品目录号:353001或Corning,目录号:430165)
  8. 15 ml离心管(FUKAEKASEI和WATSON,目录号:1332-015S或Corning,目录号:430053)
  9. 24孔板(Nunc TM,Thermo Fisher Scientific,Thermo Scientific TM,目录号:142475或Corning,目录号:3527)
  10. Parafilm

  11. 14 x 16厘米玻璃(或塑料)板
  12. 微载玻片(Matsunami Glass,产品目录号:S024410)
  13. 0.22微米过滤器(Corning,目录号:431097或Advantec MFS,目录号:25CS020AS)
  14. RPE-1细胞(ATCC,目录号:CRL-4000)
  15. Midi制备的或Maxi制备的质粒(优选>1μg/μl)
    注意:我们通常将cDNA(在CAG或CMV启动子下)或短发夹RNA(shRNA;在U6启动子下)插入同样表达GFP的质粒中(例如,pCAGIG载体)。
  16. 0.05%胰蛋白酶-0.53mM EDTA(和光纯药工业,目录号:202-16931)
  17. 胎牛血清(FBS)(PAA Laboratories,目录号:A15-701)
  18. Dulbecco改良Eagle培养基/ Ham's F-12(DMEM / F12)(Wako Pure Chemical Industries,目录号:048-29785)
  19. 甲醇
  20. 单克隆抗乙酰化α-微管蛋白(Ac-Tub)抗体,克隆6-11B-1(Sigma-Aldrich,目录号:T6793)
  21. 抗微管蛋白抗体,Detyrosinated(默克,目录号:AB3201)
  22. 单克隆抗γ-微管蛋白抗体,克隆GTU-88(Sigma-Aldrich,目录号:T5326)
  23. 多克隆抗GFP标签抗体(Thermo Fisher Scientific,Invitrogen TM,目录号:A-6455)
  24. 山羊抗小鼠IgG(H + L)抗体Alexa Fluor 568(Thermo Fisher Scientific,Invitrogen TM目录号:A-11004)
  25. 山羊抗小鼠IgG 2b抗体,Alexa Fluor 568(Thermo Fisher Scientific,Invitrogen TM,目录号:A-21144)。
  26. 山羊抗小鼠IgG1抗体,Alexa Fluor 647(Thermo Fisher Scientific,Invitrogen TM,目录号:A-21240)。
  27. 山羊抗兔IgG(H + L)抗体Alexa Fluor 488(Thermo Fisher Scientific,Invitrogen TM,目录号:R37116)
  28. 荧光固定介质(Agilent Technologies,Dako,目录号:S302380-2,代码号:S3023)或ProLong Gold Antifade Mountant(Thermo Fisher Scientific,Invitrogen TM,目录号:P36930)
  29. 指甲油
  30. 达尔伯克改良伊格尔培养基(DMEM)(NACALAI TESQUE,目录号:08458-16)
  31. 100mM丙酮酸(Thermo Fisher Scientific,Gibco TM,目录号:11360070)
  32. 10×D-PBS( - )(Wako Pure Chemical Industries,目录号:048-29805)
  33. 氯化钙二水合物(CaCl 2·2H 2 O)(Wako Pure Chemical Industries,目录号:031-00435)
  34. 氯化镁六水合物(MgCl 2·6H 2 O)(Wako Pure Chemical Industries,目录号:135-00165)
  35. 16%多聚甲醛(PFA)水溶液(电子显微镜科学,目录号:15710)
  36. 氯化铵(NH 4 Cl)(Wako Pure Chemical Industries,目录号:017-02995)
  37. 牛血清白蛋白(BSA)(Wako Pure Chemical Industries,目录号:010-15114)
  38. Triton X-100(GE Healthcare,目录号:17-1315-01)
  39. 叠氮化钠(NaN 3)(Wako Pure Chemical Industries,目录号:190-01272)
  40. 4',6-二脒基-2-苯基吲哚(DAPI)(Thermo Fisher Scientific,Invitrogen TM,目录号:D1306)
  41. 生长培养基(见食谱)
  42. 用于细胞培养的PBS(见食谱)
  43. 2x PBSc / m(见食谱)
  44. PBSc / m(见食谱)
  45. 4%PFA / PBSc / m(见食谱)
  46. 50mM NH 4 Cl(参见食谱)
  47. BTPAD(见食谱)

设备

  1. 5%CO 2/95%空气组织培养箱(SANYO,型号:MCO-18AIC)

  2. 水浴(TOKYO RIKAKIKAI,Eyela,型号:NTT-1200)
  3. 血细胞计数器室(Erma,目录号:03-303-1)
  4. 低速离心机(TOMY SEIKO,型号:LC-100; Eppendorf,型号:5702)
  5. 移液器20μl(Pipetman P)(Gilson,目录号:F123600)
  6. 移液器200μl(Pipetman P)(Gilson,目录号:F123601)
  7. 移液器1,000μl(Pipetman P)(Gilson,目录号:F123602)
  8. 对于程序B1:
    Neon TM转染系统(Thermo Fisher Scientific,Invitrogen TM,目录号:MPK5000)。
  9. 对于程序B2:
    1. Nucleofector TM装置(Lonza,型号:Nucleofector TM TM)
    2. 比色皿Plus TM电穿孔比色皿&amp;转移提示(BTX,目录号:45-0135)
    3. Amaxa®细胞系Nucleofector®Kit V(Lonza,目录号:VCA-1003)
  10. 镊子(精密科学工具,杜蒙,型号:#5/45)
  11. 高压灭菌器(TOMY SEIKO,型号:LBS-245)
  12. 配备物镜(EC Plan-Neofluar 40x / 0.75,Carl Zeiss,产品目录号:440350-9903-000)的Epifluorescent显微镜(Carl Zeiss,型号:Axioplan 2成像)
  13. 共焦显微镜(ZEISS,型号:LSM-780)

程序

本分析按照以下时间表和程序进行(图1)。在本实验方案中,我们展示了每次转染的4个样品的制备(例如,在组装之前,0小时,2小时和24小时 - 在血清重新添加之后)。每个研究人员可以根据需要准备样品。例如,当我们专注于睫状体拆卸机制时,我们通常为每次转染准备三个样品(在血清重新添加后0 h-,2 h和24 h)。可以添加额外的样本来测试转染效率(第3天)。
第1天:细胞传代培养(见程序A)
第2天:细胞转染(参见程序B);诱导睫状体组装(见程序C)
第3天:检查细胞转染效率
第4天:诱导睫状体分解(见程序D);早期时间点(例如,0小时,2小时)细胞收获(见程序E)
第5天:晚点(例如,24小时)细胞收获
免疫染色(见程序F)和数据分析


图1.转染和睫状体组装/拆卸测定的时间线示意图

  1. 细胞亚文化(第1天)
    在含有10%FBS的生长培养基中保持的RPE-1细胞(参见配方)在37℃的5%CO 2湿润培养箱中传代并使用标准技术铺板在100mm细胞培养皿上。第二天将细胞以预期50-80%融合的密度铺板。通常,当使用步骤B1(3×10 5个细胞)时,一个100mm培养皿的细胞(总共2-4×10 6个细胞)足以进行4-6次转染反应,用于每个反应的细胞)和2-4个转染反应,当使用程序B2时(每个反应1×10 6个细胞)。
    注意:我们避免使用旧的(> 15次传代)或过度生长的RPE-1细胞,这往往具有较差的纤毛诱导率。

  2. 细胞转染(第2天)
    Neon TM(程序B1)和Nucleofector TM(程序B2)转染系统产生相似的转染效率和低细胞死亡。转染后第二天,超过60%的GFP质粒转染的RPE-1细胞表达绿色荧光。相反,Lipofectamine 2000或聚乙烯亚胺(PEI)不会产生大量表达GFP的细胞。
    在 之前准备以下 进行程序B1或B2中描述的转染程序。
    1. 在37℃的水浴中预热生长培养基。将一个质粒(或预先混合的多个质粒,如果适用的话)放入一个无菌的1.5ml试管中。对于程序B1和B2的转染反应,分别使用〜3μg和〜6μg的DNA。
      如果执行程序B2,准备Amaxa转染主混合物。对于每个转染反应,通过轻轻移液和快速旋转将85.9μl溶液I和19.1μl溶液II混合。准备多余的反应来解决移液错误。
    2. 在每个35毫米的细胞培养皿中放置三个或更多的酸洗过的无菌盖玻片。加入1毫升的生长培养基,并转移到37℃的5%CO 2培养箱中。
    3. 对于每个转染反应,在1.5 ml无菌试管中准备1 ml预热生长培养基,并置于37°C水浴中。
    4. 使用胰蛋白酶-EDTA溶液分离RPE-1细胞(每100毫米板0.5毫升)。在37℃孵育〜2分钟后,将分离的细胞收集到含有9.5ml温热生长培养基的15ml离心管中,其中FBS使胰蛋白酶失活。
    5. 用血细胞计数器计数细胞的数量。

    的 B1。霓虹 TM 转染系统
    1. 在室温下将细胞悬浮液在280×g(1,200rpm)离心2分钟。
    2. 吸出上清液。
      在10毫升无菌PBS中重悬细胞沉淀(参见食谱)。
    3. 在室温下将细胞悬浮液在280×g(1,200rpm)离心2分钟。
    4. 小心地吸出所有的上清液。用100μlResuspension Buffer R(包含在Neon TM转染系统中)每1×10 6个细胞重悬一次。每次转染需要30微升细胞悬液(即,3×10 5个细胞)。

    5. 轻轻混合3微克质粒在30微升细胞悬液中
    6. 使用10μlNeon TM尖端获得10μl细胞-DNA混合物,然后根据制造商的协议使用以下参数施加电脉冲(脉冲电压'1,050 V',脉冲宽度'35毫秒“,脉冲数”2次“)。重复操作,直到细胞-DNA混合物的整个体积(30μl)被“脉冲”。
    7. 紧接着每个脉冲之后,将转染的细胞转移到含有1ml温热生长培养基的1.5ml管中。使用相同的管收集所有3×10 5个转染的细胞。
    8. 将转染的细胞铺在含盖玻片的35mm培养皿上并立即转移到37℃的5%CO 2培养箱12小时。

    的 B2。 Nucleofector TM 转染系统
    1. 对于每个转染反应,将相当于1×10 6胰蛋白酶-EDTA分离的RPE-1细胞的一定体积的细胞悬液转移到15ml管中。
    2. 在室温下将细胞悬浮液在280×g(1,200rpm)下离心2-3分钟。
    3. 小心地吸出所有的上清液。
      使用P200移液器,用100μlAmaxa转染母液轻轻悬浮细胞沉淀。
      注意:通过将400μl的4×10 6细胞与4×10 6细胞混合,可以在同一批次中进行多达4次转染反应。 Amaxa转染主混合物。
    4. 对于每个转染反应,将100μl细胞悬液转移到含有DNA的1.5ml管中。轻轻吸移不超过两次,避免气泡。
    5. 将整个细胞-DNA混合物转移到电穿孔比色杯(BTX)中。
      不要吸走最后一滴以避免气泡。
    6. 将比色杯放入Nucleofector TM 设备中,用程序'T23'对单元进行脉冲。这是在设备中预设的程序,可以通过按下一个简单的按钮来选择。
    7. 在脉冲之后立即使用精细吸头滴管(包含在独立包装的电穿孔比色杯中)将转染的细胞收集在比色杯中。将它们转移到含有1ml温热生长培养基的1.5ml试管中。
    8. 将转染的细胞铺板在含有盖玻片和生长培养基的35mm培养皿上,立即将细胞转移到37℃的5%CO 2培养箱中12小时。

  3. 诱导睫状体组装(第2天)
    1. 细胞转染后大约8-12小时(程序B),细胞应该沉降在底部。在研究人员希望在血清饥饿之前收集细胞样品(例如,在血清饥饿前观察纤毛或通过免疫染色测试转染效率)后,按照程序E进行。取出培养基,轻轻冲洗2毫升PBS中的细胞(三次),并加入2毫升预热的无血清DMEM(或DMEM / F12)。在此步骤中,我们没有注意到DMEM和DMEM / F12之间在装配或随后拆卸纤毛方面存在明显差异。
    2. 在37℃,5%CO 2培养箱中孵育36-48小时。可选:在此期间(第3天),我们使用倒置相位/荧光显微镜检查绿色荧光阳性细胞以估计转染效率。
      通过观察从盖玻片/盘子上分离的细胞数量,我们也注意细胞毒性
  4. 诱导睫状体分解(第4天)
    1. 在血清饥饿结束时,使用一对无菌镊子,取出一片盖玻片并立即清洗并固定它们(0 h时间点;参见程序E)。吸出无血清培养基并用生长培养基代替。
    2. 将细胞在37℃的5%CO 2培养箱中培养指定的时间; 2小时和24小时将是很好的起点。

  5. 细胞收获(第2,4和5天)
    1. 将一个或多个盖玻片从孵育后的35 mm培养皿转移到一个空的24孔培养板中,使用一对无菌镊子,细胞侧朝上。
    2. 用1ml PBSc / m冲洗盖玻片3次(见食谱),并在室温下固定0.5ml 4%PFA / PBSc / m(见食谱)10分钟。
    3. 用1ml PBSc / m冲洗盖玻片一次,并填充1ml另一种PBSc / m。

  6. 免疫染色
    可以通过免疫染色乙酰化α-微管蛋白(Ac-Tub)或去酪氨酸微管蛋白来显示初级纤毛(图2)。可以通过γ-微管蛋白(γ-Tub)免疫染色检测基底体(图2B)。 γ-Tub染色方便地显示了纤毛的近端。这也是一个很好的里程碑,特别是在短或无法检测到纤毛的细胞中。然而,为了获得γ-Tub染色的信号,细胞必须用冷甲醇处理(参见步骤F2)。
    每个研究人员可以根据自己的偏好进行免疫染色程序,诸如先前描述的程序(Phua et al。,2017; Shnitsar et al。,2015; Tomoshige et al。,2017)。下面,我们描述我们的Ac-Tub,γ-Tub和GFP的三重染色程序。 GFP染色用于增强对表达GFP(或GFP-融合蛋白)的转染细胞的检测。因此,目标分子在睫状体装配/分解中的作用可以在GFP阳性转染的细胞中明确地确定。
    下面详述的整个过程在室温下在遮光湿度室(自制)中进行。

    1. 在一个14 x 16厘米的玻璃(或塑料)板上放置一块石蜡膜。

    2. 在-20°C预处理预先固定的盖玻片预冷甲醇1-3分钟。
    3. 立即用PBSc / m冲洗细胞两次。
    4. 将盖玻片放在封口膜上,使细胞面朝上。
    5. 使用P1000移液管(或滴管)从盖玻片边缘加入〜300μl50mM NH 4 Cl(参见食谱)并孵育10分钟。
    6. 从盖玻片边缘吸出NH 4 4 Cl,并用300μlPBSc / m冲洗一次。

    7. 孵育100μlBTPAD(参见食谱)30分钟。
    8. 在室温下孵育50-100μl含有一抗的BTPAD 60分钟。抗体稀释液:抗Ac-Tub(小鼠IgG 2b:1:1,000),抗γ-Tub(小鼠IgG 1:1:1,000),抗GFP (兔IgG,1:1,000)。
    9. 通过将细胞以300μlPBSc / m孵育5分钟,除去未结合的一级抗体三次。
    10. 在室温下孵育50-100μl含二次抗体的BTPAD 45分钟。抗体稀释:Alexa Fluor 568偶联的山羊抗小鼠IgG 2b(对于抗Ac-Tub,1:400),Alexa Fluor 647偶联的山羊抗小鼠IgG 1 (对于抗γ-Tub,1:400)和Alexa Fluor 488缀合的山羊抗兔IgG(对于抗GFP,1:400)。
      注意:当细胞标记为Ac-Tub而不是γ-Tub时,在步骤F8中,同种型特异性二抗不是必需的。我们使用Alexa Fluor 568偶联的抗小鼠IgG(H + L)抗体。
    11. 用300μlPBSc / m冲洗5分钟,三次。
    12. 去除多余的PBSc / m,擦去盖玻片一侧的细胞。

    13. 在盖玻片上安装4μl安装介质

    14. 用清澈的指甲油密封盖玻片边缘
    15. 在落射荧光显微镜(ZEISS,Axioplan 2成像)或共焦显微镜(ZEISS,型号:LSM-780)下观察样品。对于落射荧光显微镜,物镜:放大40倍而不浸泡。目镜:放大10倍。对于共聚焦显微镜,物镜:放大63倍,油浸。图2显示了在共聚焦显微镜上拍摄的代表性图像。


      图2.初级纤毛分别通过耗尽和重新添加血清进行装配和分解。 在RPE-1细胞中组装的原代纤毛的代表性图像。为了呈现目的,使用共焦显微镜(ZEISS,LSM-780)获取图像。在血清饥饿(装配前),血清饥饿36小时后(0小时)和随后的血清再加入2小时和24小时收集细胞并标记为Ac-Tub(红色; A,B ),GFP(绿色; A)和γ-Tub(青色; B)。核用DAPI(蓝色; A)染色。虚线划分了单元格边界。 (A)中的星号突出显示GFP + 细胞。 (A)中的箭头指向GFP + 细胞中的纤毛。比例尺= 10微米(A)和2微米(B)。

数据分析

因为纤毛的长度与呈现纤毛的细胞的数量正相关,并且因为测量纤毛长度是劳动密集型的,所以显示出可检测纤毛的细胞的数量被计数为替代指标(Li等人, ,2011)。
注:对于早期阶段的研究人员,我们建议用高倍镜片观察纤毛(例如63x,100x)。然而,我们必须注意到,研究人员需要调整Z位置以找到由于更高放大倍数而感兴趣的细胞之间的每个纤毛。

  1. 以双盲方式计数在GFP + - 转染的细胞中表达Ac-Tub的细胞的数量。在每个实验中计数超过100个细胞(两组50个细胞;一个来自每个盖玻片的左侧和另一个从右侧)。每个条件至少进行三次独立实验。来自独立实验的数据以平均百分比±SEM表示。单因素方差分析(ANOVA)或双因素方差分析(Two-way ANOVA)分别接受Tukey检验或Bonferroni检验(作为事后检验)(图3)。
  2. 图3A显示天然RPE-1细胞清除血清后睫状体组件的时间依赖性。
    80%的细胞在血清饥饿后36小时表达纤毛。
  3. 图3B显示了睫状体分解测定的代表性结果。还显示了通过敲除Tctex-1的睫状体分解的实例。 Tctex-1是细胞质动力蛋白复合物的轻链,并且是用于睫状体分解的不可缺少的分子(Li等人,2011; Yeh等人,2013; Saito等人, 等。,2017)。将Tctex-1的U6启动子和短发夹RNA(shRNA)序列插入也编码GFP的pCAGIG载体(参见材料和试剂#13)(Li等人,2011)。用载体(对照)或Tctex-1-shRNA(Tctex-1-sh)转染的细胞血清饥饿36小时并用生长培养基退回2小时和24小时。在GFP + - 转染的细胞中计数具有纤毛的细胞数量。在对照装置的细胞中重新加入血清后,具有纤毛的细胞的百分比从80%(0h)降至60%(2h)和40%(24h)。尽管在Tctex-1-shRNA转染的细胞中睫状体装配未受影响,但这些细胞中的睫状体分解速率明显受到抑制。


    图3.睫状体装配和分解测定的代表性结果。 :一种。睫状体装配测定。在血清饥饿后的指定时间点收获幼稚RPE-1细胞。 Y轴显示表达纤毛的细胞的百分比。 B.睫状体分解测定。首先使用也表达GFP的对照载体或Tctex-1-shRNA转染的RPE-1细胞饥饿,然后用生长培养基处理指定的时间段。 Y轴显示了具有纤毛的GFP + / +细胞的百分比。 ## P &lt; 0.01, ### P &lt; 0.001;单因素方差分析,然后进行Tukey检验(与每组0小时时间点比较)。 ** P &lt; 0.01,*** P 0.001;双因素方差分析后接Bonferroni检验(组间比较)。每个实验n = 100个细胞,三个(A)和五个(B)独立实验。

食谱

  1. 生长培养基
    1. 取500毫升的DMEM / F12
    2. 加入50毫升FBS(最后10%)
    3. 加入5.5毫升100mM丙酮酸盐(最终1mM)
    4. 在4°C储存
  2. 用于细胞培养的PBS
    1. 取180ml ddH 2 O
    2. 加入20毫升10×D-PBS( - )
    3. 高压灭菌器
    4. 在室温下储存
  3. 2x PBSc / m
    1. 取795毫升ddH 2 O
    2. 加入200毫升的10倍D-PBS( - )
    3. 加入400μl1M CaCl 2(最终0.4mM)
    4. 加入4,000μl1M MgCl 2(最终4mM)
    5. 通过0.22μm过滤器过滤
    6. 在室温下储存
  4. PBSC /米
    1. 取500毫升ddH 2 O
    2. 添加500毫升2x PBSc / m
    3. 通过0.22μm过滤器过滤
    4. 在室温下储存
  5. 4%PFA / PBSc / m
    在化学罩内的操作之前准备
    1. 取1ml ddH 2 O
    2. 加2 ml 2x PBSc / m
    3. 加1毫升16%PFA
  6. 50mM NH 4 Cl
    1. 取20毫升ddH 2 O
    2. 添加25毫升2x PBSc / m
    3. 加入133.75mg NH 4 Cl(最终50mM)
    4. 用ddH 2 O稀释至多50ml
    5. 在室温下储存
  7. BTPAD
    1. 取25毫升的PBSc / m
    2. 加625μl20%BSA(最后0.5%)
    3. 加313μl20%Triton X-100(最后0.25%)
    4. 加入250μl2%NaN3(最终0.02%)
    5. 加入2.5μl3mM DAPI(最终0.3μM)
    6. 通过0.22μm过滤器过滤
    7. 在4°C储存

致谢

该协议是从以前发表的研究(Saito等人,2017年)改编的。这项工作得到了NIH RO1 EY11307,EY016805,防盲研究,创新奖获奖者(RPB)和高雄医科大学研究基金会(105KMUOR02)(对C.-H. S.)的支持。日本科学促进会科学研究资助计划(编号23770136,编号15K20856,M.S.)和武田科学基金会(编号M.S.)。我们要感谢生物医学研究核心(东北大学医学研究生院)。我们还要感谢Editage( www.editage.jp/ )进行英文编辑。作者声明没有竞争的财务利益。

参考

  1. Li,A.,Saito,M.,Chuang,J.Z.,Tseng,Y.Y.,Dedesma,C.,Tomizawa,K.,Kaitsuka,T。和Sung,C.H。(2011)。 磷酸化Tctex-1的纤毛过渡区激活控制睫状体吸收,S期进入和神经的命运祖细胞。 Nat Cell Biol 13(4):402-411。
  2. Phua,S.C.,Chiba,S.,Suzuki,M.,Su,E.,Roberson,E.C.,Pusapati,G.V.,Setou,M.,Rohatgi,R.,Reiter,J.F.,Ikegami,K.,et al。 (2017年)。
  3. Pugacheva,E.N.,Jablonski,S.A.,Hartman,T.R.,Henske,E.P。和Golemis,E.A。(2007)。 HEF1依赖的Aurora A激活诱导了对主要纤毛的拆解。 Cell 129(7):1351-1363。
  4. Saito,M.,Otsu,W.,Hsu,K. S.,Chuang,J.Z.,Yanagisawa,T.,Shieh,V.,Kaitsuka,T.,Wei,F.Y.,Tomizawa,K。和Sung,C.H。(2017)。 Tctex-1通过调节分支肌动蛋白聚合和内吞作用来控制睫状吸收 EMBO Rep 18(8):1460-1472。
  5. Shnitsar,I.,Bashkurov,M.,Masson,GR,Ogunjimi,AA,Mosessian,S.,Cabeza,EA,Hirsch,CL,Trcka,D.,Gish,G.,Jiao,J.,Wu,H. ,Winklbauer,R.,Williams,RL,Pelletier,L.,Wrana,JL和Barrios-Rodiles,M.(2015)。 PTEN通过Dishevelled来控制纤毛 Nat Commun 6: 8388。
  6. Tomoshige,S.,Kobayashi,Y.,Hosoba,K.,Hamamoto,A.,Miyamoto,T.和Saito,Y。(2017)。 黑色素浓集激素受体1介导的原发性纤毛缩短的细胞骨架相关调节。 Gen Comp Endocrinol 253(1):44-52
  7. Tucker,R. W.,Pardee,A. B.和Fujiwara,K.(1979)。 中央静脉系统疾病与3T3细胞中的静止和DNA合成有关 细胞 17(3):527-535。
  8. Yeh,C.,Li,A.,Chuang,J.Z.,Saito,M.,Caceres,A.and Sung,C.H。(2013)。 IGF-1激活一种调节细胞周期进程的纤毛局部非典型Gbg信号通路。 Dev Cell 26(4):358-368。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Saito, M., Sakaji, K., Otsu, W. and Sung, C. (2018). Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines. Bio-protocol 8(6): e2773. DOI: 10.21769/BioProtoc.2773.
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