This method can help to answer key question in the centriole field, such as localizing protein to a specific region of the centriole. The main advantage of this techniques that we provide the means to concentrate centriole to get them in multiple orientations. Though this method can provide insights into Chlamydomonas centriole biology, it can also be applied to other system, such as isolated human and paramecium centrioles.
Demonstrating the procedure will be PhD student Nikolai Klena, another PhD student Maeva Le Guennec, and the postdoc in the laboratory Davide Gambarotto. To begin this procedure, first prepare all media and culture the C.Reinhardtii cells as outlined in the text protocol. Transfer the prepared cells to 50 milliliter conical tubes and centrifuge them at 600 times g for 10 minutes.
Wash the pellet once with 50 milliliters of 1x PBS and spin the cells again at 600 times g for 10 minutes. Use a pipette to resuspend the pellet in 100 milliliters of room temperature deflagellation buffer. Place the beaker on a magnetic stirrer, and slowly add 0.5 molar acetic acid to a final pH of 4.5 to 4.7.
Let this mixture incubate at room temperature for two minutes. After this slowly add drops of one normal potassium hydroxide to restore the pH to 7.0, transfer the cells to 50 milliliter tubes, and centrifuge them at 600 times g for 10 minutes, to remove any detached flagella. Discard the supernatant and store the pellet on ice until ready to wash.
When ready, wash the pellet twice using 50 milliliters of 1x PBS at four degrees Celsius per wash. Spin the washed pellet at 600 times g and four degrees Celsius for 10 minutes. Resuspend the pellet in 30 milliliters of 1x PBS.
Then, slowly load the suspension onto a 20 milliliter cushion of 25%sucrose, without mixing. Spin at 600 times g and four degrees Celsius for 15 minutes to remove any remaining flagella and spread the cells in the sucrose. Using an aspirator, carefully aspirate the supernatant keeping only the bottom-most 20 milliliters.
Wash the remaining sucrose by adding 20 milliliters of cold 1x PBS. Centrifuge at 600 times g and four degrees Celsius for 10 minutes. Resuspend the pellet in 10 milliliters of 1x PBS at four degrees Celsius, making sure that there are no clumps in the solution.
Transfer the resuspended pellet to a new 250 milliliter bottle, add the lysis buffer, supplemented with 5, 000 units of DNase, to the bottle containing the cells. Incubate at four degrees Celsius for one hour, while mixing every 15 minutes by carefully inverting the bottle. Transfer the lysed cells to a 50 milliliter conical tube and centrifuge at 600 times g and four degrees Celsius for 10 minutes.
Using a pipette, collect the supernatant and load it into a 30 milliliter round-bottom tube on ice containing two milliliters of 60%sucrose cushion. Centrifuge this at 10, 000 times g for 30 minutes at four degrees Celsius. After this, aspirate the supernatant up to one milliliter above the sucrose cushion.
Note the yellow interface between the one milliliter of remaining supernatant, and the two milliliters of sucrose cushion. Using a cut P1000 tip, gently mix the remaining supernatant with the sucrose. Pool all of the sucrose cushions and store them on ice.
Next, prepare a 40%to 70%sucrose gradient in a thin-walled 38.5 milliliter polypropylene tube as outlined in the text protocol. Slowly load the pooled interfaces into the gradient. Balance the tubes with the 10 millimolar K-PIPES buffer, and centrifuge at 68, 320 times g and four degrees Celsius for 75 minutes.
After this, use a 0.8 millimeter needle to make a hole in the bottom of the tube, making sure not to disturb the different sucrose layers. Use this hole to collect 12 500 microliter fractions at four degrees Celsius. Using a cut P200 tip, prepare an additional 10 microliter aliquot from each fraction to be used during immunofluorescence.
Then, snap-freeze the fractions in liquid nitrogen. To begin, place a sterile 12 millimeter coverslip onto the recessed bottom end of the concentrator, making sure to keep the PDL-coated side down. Place the adaptor directly on top, to cap the coverslip.
Then, invert the round-bottom tube and place it over the concentrator, coverslip, and adaptor. Using tweezers, gently push the ensemble until it reaches the bottom of the tube, and then invert the tube. Add 10 millimolar of K-PIPES buffer until it reaches the top of the concentrator.
Ensure that there are no bubbles in the central cylinder of the concentrator. Gently add 100 microliters of 10 millimolar K-PIPES buffer to one of the aliquots of the enriched centriolar fraction and thoroughly mix using a P200 pipette. Next, remove 100 microliters of K-PIPES buffer from the hollow center of the concentrator, and add 100 microliters of the buffer fraction mixture, making sure that the contents remain in the hollow center of the concentrator.
Spin at 10, 000 times g for 10 minutes in a centrifuge that is pre-cooled to four degrees Celsius. After this, use tweezers to remove the concentrator. Insert a handmade-hooked device into the hole present in the slotted edge of the adaptor and lift up gently to recover the coverslip.
Once it reaches the top of the tube, use a gloved finger to trap the edge of the adaptor and use tweezers to remove the coverslip, being mindful of which side of the coverslip contains centrioles. Then, perform fixation and immunofluorescence staining of the concentrated centrioles. To begin, incubate the coverslips with the centrioles in the prepared crystal polystyrene transmission lab box filled with 100%methanol at minus 20 degrees Celsius for 5 minutes.
Using tweezers, transfer the coverslips to a transparent laboratory box filled with 50 milliliters of 1x PBS. Let the slides wash in the PBS for five minutes at room temperature. Then, pipette 60 microliters of primary antibody mix onto a piece of laboratory sealing wrap in a humidified chamber.
Carefully lay the coverslips on top of the antibody mix with the centrioles directly facing the drop. Let the coverslips incubate for 45 minutes. After this, remove the coverslips and wash them in a box containing PBS for five minutes.
Incubate the washed coverslips for 45 minutes, with secondary antibodies in PBS 1%BSA and 0.05%Tween-20. Remove the coverslips and wash them for five minutes in a box containing PBS. Using a regular anti-fading mounting medium, mount the coverslip onto a slide.
Then, image the isolated centrioles on a confocal microscope at a 63X oil objective with an N.A.of 1.4, while applying deconvolution. Set up the top and bottom position of the Z-stack above and below the centriole signal, respectively, and acquire a large stack of images comprising the total centriole signal. Project the stack and perform single-particle averaging as outlined in the text protocol.
Representative immunofluorescence reveals that six fractions are enriched for isolated centrioles, with a peak for fraction number three, while the last two fractions are not, indicating that the purification is successful. Only about 30 centrioles are seen per field of view without the concentrator, while 183 are seen with the concentrator. This demonstrates that the concentrator step is successful, and allows for a six-fold enrichment of centrioles in a defined region, making it easier to detect and image them.
Super-resolution microscopy is then used to image centrioles stained for monoglutamylated tubulin, and software is used to generate a near-perfect average for each chosen orientation. After five iterations, two classes of averages were generated of monoglutamylated centrioles:a top view from 63 objects and a side view from 98 particles. The length of the side-view class average is 260 nanometers with a diameter of 250 nanometers, comparable to the measured monoglutamylated tubulin signal that is seen to localize within the core of the centriole.
While performing this procedure, it's important to remember to keep the isolated centriole on ice and snap-freeze them after isolation. Following this procedure, other method, like cryo-electron microscopy and mass spectrometry can be performed in order to answer additional question, such as the native architecture of the centriole or the centriole protein composition.
