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Biochemical Analysis

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Total cell lysate

Tetrahymena cell from 5 ml culture of the density 2.5 x 105cells/ ml were pelleted at 1700 xg, cell pellet was resuspended in 100 μl of 10 mM Tris-HCl, pH = 7.4. To lysate cells, an equal volume of hot 2.5-fold concentrated Laemmli buffer (0.156 M Tris-HCl pH = 6.8, 5% SDS, 25% glycerol, 12.5% 2-mercaptoethanol, 0.005% bromophenol blue) was added to the cell suspension (Laemmli, 1970) for 4 minutes at 95 °C, cooled on ice, centrifuged for 2 minutes at 16000 xg at 4 °C.

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Isolation of cytoskeletal fraction

Tetrahymena cells from 150 ml culture of the density 2x105cells/ml were harvested at 1700 xg, washed with Tris-HCl pH =7.5, pelleted again and cooled on ice. To lyse the cells, cold lysis buffer (20 mM Tris-HCl pH = 9.0, 1.4 M glucose, 4 mM EGTA, 4 mM EDTA, 2% Triton X-100) with protease inhibitors (cOmplete Protease Inhibitor Cocktail, Roche) was added and the mixture was incubated on ice for 1 minute. The lysate was centrifuged at 21100 xg for 10 minutes at 4 °C. The pellet containing the cytoskeletal fraction was washed in 10 mM Tris-HCl pH = 7.4 with protease inhibitors, pelleted and resuspended in the same buffer.

Cilia purification for biochemical analyses

Tetrahymena cells from 600 ml of culture of the density of 2.5-3 x105cells/ml were centrifuged at 1700 xg, and washed with 10 mM Tris-HCl pH = 7.4. Cell suspension was recentrifuged under the same conditions, the cell pellet was resuspended in 40 ml of the deciliation buffer (10 mM Tris-HCl, pH = 7.4, 10 mM CaCl2 and 50 mM glucose supplemented with protease inhibitor mixture). To induce deciliation 840 µl of 0.5 M acetic acid was added to the cell suspension (pH shock) and swirled for 60-80 seconds in 250 ml flask. To neutralize the medium, 720 μl of 0.6 M KOH was added. Ciliary fraction was extracted from the cells by two centrifugations for 5 minutes at 1700 xg at 4 °C. The cilia in the supernatant were centrifuged for 30 minutes at 21100 xg at 4 °C.

The ciliary pellet was resuspended in 10 mM Tris-HCl pH 7.4 with protease inhibitors, the ciliary fraction was either directly analyzed or further fractioned. Salt stripped axonemes were extracted by first demembraning the cilia by 2% NP-40, then the dynein arms were detached by adding an equal volume of the high salt 80mM Tris solution containing 1.2 M NaCl with protease inhibtors and incubated on ice for 30 minutes. The suspension was passed 10 times through BD microfine plus syringe and was recentrifuged at 20,000 xg for 30 minutes at 4 °C, then the radial spokes were detached from the demembraned cilia by resuspending the pellet in 200 ml KI buffer (5mM MgSO4, 1mM DTT, 10mM HEPES, 0.5mM EDTA, 30mM NaCl, 0.5 mM KI). The suspension was incubated on ice for 45 minutes then centrifuged at 20,000 xg at 4⁰C for 20 minutes. The resultant suspension contained the radial spokes fraction in the supernatant while the pellet contained the axonemal fraction.

BioID-based identification of ciliary proteins located in close proximity to BirA

The Tetrahymena cells expressing the HA-BirA* tagged either FAP69 or FAP246 or Spef2A and wild-type cells were grown in 200 ml SPP medium to a density of 2 × 105cells/ml, washed and starved for 14-20 hours in Tris-HCl pH 7.4. After adding biotin to the medium (final concentration 50 µM) cells were cultured for additional 4 hours at 30 °C. BirA* is a ligase capable of attaching biotin to any amino acids of proteins within 10 nm from the BirA* ligase. Cilia were isolated from cells as described in (2.3) and effectively demembraned by suspension twice in 10% NP-40 supplemented 490 μl of axoneme stabilizing buffer with protease inhibitors (20 mM KAc, 5 mM MgSO4, 20 mM HEPES, 0.5mM EDTA) (Suryavanshi et al., 2010), the samples were incubated for 5 minutes on ice and centrifuged for 10 minutes at 21100 xg at 4 °C. The obtained axonemal pellet was lysed for 1 hour in a lysis buffer (50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.4% SDS, 1 mM DTT) at room temperature followed by centrifugation for 11 minutes at 8000 xg at 4 °C.

An equal volume of 50 mM Tris-HCl, pH = 7.4 was added to the collected supernatant to dilute concentrated NaCl and SDS. Suspended proteins (250 μg) were incubated with 100 μl of streptavidin-coated Dynabeads (Dyanbeads M-280, Streptavidin, Invitrogen) overnight at 4 °C on the laboratory shaker. To remove unbound protein, the samples were washed 6 times for 5 minutes with a buffer of the following composition: 15 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 0.3 mM DTT at 4 °C on the laboratory shaker. Proteins bound beads (15 μl) were suspended in 2x Laemmli buffer and then separated as described in (2.7) followed by western blot as described in (2.8). Biotinylated proteins were visualized using Streptavidin conjugated with horseradish peroxidase (Thermo Scientific) in dilution 1:40,000. By the use of Pierce™ ECL Plus Western Blotting Substrate on the nitrocellulose membranes, proteins bands were visualized using chemi-luminescent CCD-based system in Sygene G:BOX. The remaining amount of the beads was washed 3x with 10mM Tris pH 7.4 at 4°C on laboratory shaker to remove protease inhibitor and was analyzed by mass spectrometry in (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland)

Cilia proteome analysis

In order to analyze the cilia proteome, Tetrahymena WT and mutant cells were deciliated as described previously in (2.3), cilia were centrifuged and washed with 10 mM Tris pH 7.4 and an equal amount (300-600 µg) of ciliary protein was run on SDS polyacrylamide gel, protein bands were separated and analyzed by mass spectrometry in (Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland)

Bicinchoninic Assay

Protein concentration was determined using Bicinchoninic assay (BCA) which is a simpler form of Lowry assay (Smith et al (1985) using Pierce BCA Protein Assay Kit by ThermoFisher as instructed. The protein concentrations are determined and reported with reference to standards of a bovine serum albumin (BSA) incubated at 37 ⁰C in 96 well plate for 30 minutes. After cooling to room temperature, sample concentration was measured using the spectrophotometer (Perkin Elmer “Enspire” multimode plate reader).

Polyacrylamide Gel-Electrophoresis

Equal amounts of proteins from different samples were diluted with 5x concentrated Laemmli buffer (10 % SDS, 0.312 M Tris pH 6.8, 50% glycerol, 25% 2-mercaptoethanol and 0.01% bromophenol blue the protein was denatured for 5 minutes at 95 °C and resolved on polyacrylamide SDS-PAGE gel (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) under denaturing conditions in Tris-glycine buffer (25 mM Tris-HCl pH 7.4, 192 mM glycine, 0.1% SDS, pH 8.3). The protein extracts were separated in 0.7 or 1 mm thick 10% (w/v) polyacrylamide gel using a Mini-Protean II apparatus (Bio-Rad) at 120V. The molecular masses of the proteins were determined by comparing their electrophoretic mobility with that of PAGE PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (ThermoFisher).

Western Blot

Proteins were electrotransferred to the nitrocellulose membrane in transfer buffer (25 mM Tris-HCl pH = 8.3, 192 mM glycine, 20% methanol) according to the method of (Towbin et al. 1979) in the Mini Trans-Blot (Bio Rad) for 1.5 – 2 hours at 10 °C with a constant intensity of 160 mA. For rapid detection of protein, blots were stained for 2 minutes with 2% Ponceau S (Sigma), then washed several times with distilled water. To prevent the non-specific binding of antibodies, the nitrocellulose membrane was blocked by incubating for 1 hour with 5% non-fat milk in TBST (10 mM Tris-HCl pH = 8.0, 150 mM NaCl, 0.1% Tween 20) at room temperature. Afterwards, membrane was incubated at 4 °C with the primary antibody overnight (Table 3) diluted in 5% non-fat milk in TBS Tween 20 buffer. The nitrocellulose membrane was washed 4 times for 10 minutes in TBST buffer and incubated for one hour with an appropriate secondary antibody conjugated with horseradish peroxidase (Table 4), suspended in a 1% non-fat milk solution in TBST buffer. The excess antibodies were removed by 3 successive washes with TBST buffer and once with TBS buffer. Detection of the immunochemical reaction was carried out using the Thermofisher ECL Plus chemi-luminescence kit according to the manufacturer’s instructions. The visualization was carried out in the G: Box apparatus (Syngene) with the GenSys software.

Phenotypic studies on live ciliary mutants

Proliferation rate

Tetrahymena cell cultures in the logarithmic growth phase were diluted to a density of 2.5 x 104 cells/ ml by adding SPP medium and grown at 30°C. Density of the culture was measured at 3 hours interval by counting cells fixed in 20% TCA in the Bürker chamber. The experiment was repeated 3 times.

Cell motility

The rate of cell motility was measured by capturing the paths of WT and FAP69 KO cells using the video microscopy (Hennessey and Lampert, 2012) and length of the paths was analyzed using imageJ software.

Phagocytosis rate

To test the functionality of the oral cilia in mutant cells, the rate of uptake of the food particles with India ink by phagocytosis was determined by adding 1% India ink to SPPA media and allowing Tetrahymena cells to form food vacuoles for 10 and 30 minutes at 30⁰C. Next, cells were fixed by mixing 1:1 with 2% paraformaldehyde in PHEM buffer. The number of dark vacuoles filled with India ink per cell was determined in a total of 100 cells from one sample. The experiment was repeated 3 times using a microscope with low magnification on a bright field

Cilia number and length measurement:

Tetrahymena cells in midlog phase were fixed, and using immunoflourescence, cilia were labeled by 12G10 anti-a-tubulin primary antibody, followed by secondary antibody, anti-mouse IgG as described in (3.7) and the recorded confocal images were processed using ImageJ.

Deciliation and cilia regeneration for microscopic analyses

Tetrahymena cells from 20 ml of (2×10^5 cells/ml) culture were harvested at 1700 ×g, washed with Tris HCl, pH 7.4 and resuspended in 900 µl of deciliation medium (10% Ficoll 400 (Merck), 10 mM sodium acetate, 10 mM EDTA, 10 mM CaCl2, pH = 4.2). Cilia detachment was enhanced mechanically by passing the cells through a 1mm diameter needle 8 times during 80-100 seconds. The cell suspension was diluted with 5 volumes of SPPA and cells were fixed just after deciliation and next every 30 min till 120 minutes. Fixed cells were stained with anti-a-tubulin antibodies as described in (3.7). Modified Calzone and Gorovsky method is used for this experiment (Calzone and Gorovsky, 1982).

Comparative immunofluorescence

To compare the intensity of the cilia staining WT and mutant cells, were grown to the density 2x 105 cells. Before fixation WT cells were cultured for 10 minutes in medium supplemented with India ink to form dark food vacuoles via phagocytosis (as explained in 3.3). An excess of India ink was washed out with 10 mM Tris-HCl pH 7.5 by centrifugation (3 min 1700 × g). An equal number of wild-type and mutant cells (10 µl total) was mixed on cover slip and immediately fixed for immunofluorescence as described below.

Immunofluorescence

To fix Tetrahymena cells, 10 µl of culture in midlog phase was mixed with 15 µl of 1% NP-40 (Merck) + 2% PFA in PHEM buffer (60 mM PIPES, pH = 6.9, 25 mM HEPES, 10mM EGTA, 2mM MgCl2) in 1:1 ratio v/v. Air dried cells were blocked with 3% BSA in PBS buffer pH of 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate). Next, fixed cells were incubated with suitable primary antibody in a cold room overnight. Next day, the cover slips were washed with PBS 4 times at 5 min intervals and were incubated with secondary antibody at RT for 1 hour, after which the washing was repeated under the same conditions. The coverslips were mounted onto 8 µl drop of Fluoromount-G™ on a microscope slide, and the edges were sealed with a thin layer of nail polish.

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