Isolation of Intraflagellar Transport Particle Proteins from Chlamydomonas reinhardtii

CHAPTER ONE

Isolation of Intraflagellar Transport Particle Proteins from Chlamydomonas reinhardtii Elizabeth Richey, Hongmin Qin1 Department of Biology, Texas A&M University, College Station, Texas, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Media 2.1 Tips on making media 2.2 Media choices 3. Isolating Flagella 3.1 Recipe of stock solutions (the chemicals are from Sigma) 3.2 Preparing the culture 3.3 Isolating flagella 4. Preparing Soluble Flagellar Proteins 5. Isolating IFT Particle Proteins by Fractioning Soluble Flagellar Proteins Through Sucrose Density Gradient 5.1 Prepare low- and high-density sucrose solutions 5.2 Making the gradient 5.3 Gradient centrifugation 5.4 Collecting the gradient fractions 5.5 Analyzing the fractions by gel electrophoresis 6. Isolating IFT Particle Proteins by Immunoprecipitation from Soluble Flagellar Proteins 7. Conclusions Acknowledgments References

2 4 4 5 5 5 6 7 9 9 9 11 12 13 14 14 15 15 16

Abstract Cilia, the hair-like protrusions found on most eukaryotic cells, were once considered vestigial organelles. The recent renaissance of research in cilia arose from the discoveries of intraflagellar transport (IFT) and the involvement of IFT particle proteins in human diseases. Many IFT particle proteins have since been identified, and research on IFT particle complexes and their protein components continues to provide insight into the mechanism of IFT and the etiology of ciliopathies. In this chapter, we describe the methods of

Methods in Enzymology, Volume 524 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-397945-2.00001-9

#

2013 Elsevier Inc. All rights reserved.

1

2

Elizabeth Richey and Hongmin Qin

isolating IFT particles from the flagella of Chlamydomonas reinhardtii. Two methods, sucrose density gradient fractionation and immunoprecipitation, are explained in detail. Troubleshooting information is presented to illustrate the critical steps of the procedure to ensure successful implementation of these methods in individual labs.

1. INTRODUCTION Although tiny, eukaryotic cilia are important cellular organelles, performing a variety of signaling and motility-based functions. However, the critical function of the primary cilium, which is present in almost all differentiated cells in the human body, had long been under-appreciated. A new chapter in cilia biology was opened in the year 2000 when a protein essential for cilia assembly, IFT88, was found to be mutated in a polycystic kidney disease (PKD) mouse model (Murcia et al., 2000; Pazour et al., 2000). Since then, many other human diseases are found to be due to perturbation of ciliary proteins or ciliary function (Hildebrandt, Benzing, & Katsanis, 2011). The protein IFT88 was originally isolated as a subunit of intraflagellar transport (IFT) particles from Chlamydomonas flagella (Cole et al., 1998). By trafficking bidirectionally between the ciliary base and tip, the linear trains of IFT particles carry protein precursors synthesized in the cell body to support ciliogenesis and cilia maintenance (Ahmed, Gao, Lucker, Cole, & Mitchell, 2008; Hou et al., 2007; Ishikawa & Marshall, 2011; Mukhopadhyay et al., 2010; Qin, Diener, Geimer, Cole, & Rosenbaum, 2004). After 12 years of extensive research, ciliary defects, including those caused by IFT deficiencies, are linked to a large class of ciliopathies such as PKD and Bardet–Biedl syndrome (Hildebrandt et al., 2011). Chlamydomonas reinhardtii is a single-cell green alga with two long flagella (structurally identical to cilia), which has long served as a model organism for basic research on cilia. The ease of observing flagella under the microscope and the ability to isolate unlimited amounts of pure flagella played decisive roles in the discovery of IFT and purification of IFT particles (Cole et al., 1998). In addition, over the past 50 years, the Chlamydomonas community has accumulated a large collection of flagellar mutants, making this model organism even more powerful for studying flagella. The temperature-sensitive mutant f la10ts, which harbors a mutation in the anterograde motor subunit FLA10 (Cole et al., 1998; Walther, Vashishtha, & Hall, 1994), is instrumental to the successful isolation of IFT particle proteins. This mutant has normal flagella and IFT at the permissive temperature. However, within the first hour of shifting the mutant cells to the

IFT Particle Protein Isolation

3

nonpermissive temperature, the structure of the flagella still remains intact, but neither the anterograde nor the retrograde IFT is observable under light or electron microscopes (Iomini, Babaev-Khaimov, Sassaroli, & Piperno, 2001; Kozminski, Beech, & Rosenbaum, 1995). Although in the absence of IFT, the flagella of the fla10ts mutant will gradually shorten and eventually disappear completely, the first hour after the temperature shift provides a sufficient time window allowing isolation of flagella without IFT particles (Cole et al., 1998). Taking these experimental advantages, Cole in the Rosenbaum lab (Cole et al., 1998) and the Piperno lab (Piperno & Mead, 1997) found that a group of flagellar proteins that sediment at 16S fractions on the sucrose density gradient disappeared when IFT was inactivated in fla10ts cells (Cole et al., 1998). This group of proteins is known as IFT particle proteins (Cole et al., 1998). Obviously, the identification of IFT particle proteins in Chlamydomonas has relied primarily on biochemical purifications (Cole et al., 1998; Piperno & Mead, 1997). Peptide sequences used to clone the IFT genes were obtained by microsequencing using the proteins excised from 1D and 2D gels of sucrose gradient fractions containing 16S IFT particles (Cole et al., 1998). The original purification done by the Rosenbaum lab revealed 4 polypeptides in IFT complex A and 11 in complex B (Cole et al., 1998). Recently, we modified the flagella isolation method to increase the yield of IFT particle proteins. In addition, we found an electrophoresis system to resolve closely migrating bands (Wang, Fan, Williamson, & Qin, 2009). Through these efforts, we identified three additional IFT proteins, IFT121 (A), IFT25(B), and IFT22(B). IFT25 and IFT27 form a heterodimer, which can associate and dissociate with the main complex B (Wang et al., 2009). IFT22 is a small GTPase similar to RABL5 (Silva, Huang, Behal, Cole, & Qin, 2012). IFT22 stays with the complex B core, but its direct binding partner is still unknown. IFT121 interacts with IFT43 and is in the complex A core (Behal et al., 2012). The Caenorhabditis elegans ciliary proteins DYF-1, DYF-3, DYF-11, and DYF-13 have functions comparable to the known IFT particle subunits and display IFT-like motility (Ou, Blacque, Snow, Leroux, & Scholey, 2005; Ou et al., 2007; Pathak, Obara, Mangos, Liu, & Drummond, 2007). The homologues of these proteins in other species also show similar functions in ciliogenesis. However, they are not among the originally identified IFT particle subunits (Cole, 2003; Cole et al., 1998). Through rigorous biochemical assays, we confirmed that DYF-1 is a bona fide IFT complex B protein. Chlamydomonas DYF-1 (CrDYF-1) directly interacts with

4

Elizabeth Richey and Hongmin Qin

IFT46 and is a complex B core protein. We renamed CrDYF-1 as IFT70 based on its molecular weight (Fan et al., 2010). Similar methods used to confirm the IFT particle protein identity of DYF-1 are readily applicable for DYF-3, DYF-11, and DYF-13. These three proteins, which have been identified as IFT complex B-associated proteins (Ishikawa & Marshall, 2011), are likely true IFT complex B subunits of Chlamydomonas. This chapter describes the challenge and the detailed troubleshooting information in isolating IFT particle proteins.

2. MEDIA Chlamydomonas cells can be cultured both on solid media and in liquid. Many labs keep the Chlamydomonas strain stocks on slants under illumination and streak the cells on fresh plates when needed. The strains on solid slants require passage on new slants every 3 months or even shorter for some mutants. For long-term storage, Chlamydomonas strains can be frozen in liquid nitrogen (Piasecki, Diller, & Brand, 2009).

2.1. Tips on making media There are two most commonly used media, Tris–acetate–phosphate (TAP) and the minimal 1 (M1). The protocols on making TAP and M1 media are detailed on the Chlamydomonas resource Web site (http://www.chlamy. org/). Both TAP and M1 media are completely made of synthetic chemicals, so many chemicals are required in the media. “Hunter’s trace elements,” an important stock solution for making the TAP medium, is difficult to prepare. Again, the Chlamydomonas resource Web site offers a detailed step-by-step method on how to correctly make the Hunter’s trace elements. One way to check if the media are correctly made is to check the final pH; TAP media should be around 7.0–7.4 and M1 media are 6.9. If the pH is off, the media are not properly prepared. The quality of water used in the medium is very important for the Chlamydomonas culture. The deionized (DI) water equipped in many biology labs does not always meet the requirement for culturing healthy Chlamydomonas cells. In the past, we found that the wild-type cc125 cells cultured in both TAP and M1 media made with the DI water either were in clumps or had short and defective flagella. These issues were rectified by switching to double-distilled water.

IFT Particle Protein Isolation

5

2.2. Media choices The TAP medium provides a carbon source, which allows the Chlamydomonas cells to grow and reach high density quickly. The M1 medium does not contain a carbon source. The cells grown in M1 media are entirely dependent on photosynthesis; therefore, it takes longer for the cell to grow and the culture rarely reaches more than 107 cells/ml. However, the cells cultured in M1 are thought to be healthier and may have advantages in certain applications. Regarding flagella isolation, the TAP medium allows the growth of large amount of cells in a shorter period of time. So, if the flagella form well in TAP media, as they do in wild-type cells, it is much more efficient to use TAP than M1 media for growing the cells. However, some strains, including IFT particle complex A mutants fla15 and fla17 as well as IFT particle complex B mutant fla11, form clumps when cultured in TAP media but flagellate well in M1 media. In those cases, M1 is the preferred media.

3. ISOLATING FLAGELLA Witman, Carlson, Berliner, and Rosenbaum (1972) first described the sucrose underlay flagella isolation method, and it has since been implemented in many labs which use biochemical methods to study flagella. The method described here is modified to enrich the IFT particle proteins in the flagella.

3.1. Recipe of stock solutions (the chemicals are from Sigma) 1 M HEPES (store at 4  C) 119.16 g HEPES Add double-distilled H2O to 500 ml, use KOH to adjust pH to 7.4 50% sucrose (store at 4  C) 250 g sucrose ddH2O to 500 ml 25% sucrose in HMDE (store at 4  C) 10 mM HEPES (10 ml of 1 M HEPES for 1 l) 5 mM MgSO4 (5 ml of 1 M MgSO4 for 1 l) 1 mM DTT (add 1000 stock solution right before use) 0.25 mM EGTA (0.5 ml of 0.5 M EGTA, pH 8.5 for 1 l) 25% sucrose (250 g sucrose for 1 l)

6

Elizabeth Richey and Hongmin Qin

10 HMDEK (store at 4  C) 10 mM HEPES (10 ml of 1 M HEPES for 100 ml) 5 mM MgSO4 (5 ml of 1 M MgSO4 for 100 ml) 1 mM DTT (add 1000 stock solution right before use) 5 mM EDTA (10 ml of 0.5 M EDTA, pH 8.0 for 100 ml) 25 mM KCl (1.86 g for 100 ml) 100 mM PMSF (store at room temperature) 1740 mg PMSF Isopropanol to 100 ml

3.2. Preparing the culture We use a three-step method to prepare the culture for flagellar isolation. All three steps require sterile operation. The quality and density of the culture are checked under a compact microscope. Make sure that the cells are flagellated, healthy, and uncontaminated (steps 2 and 3). 1. The first step is to grow the cells on a plate. It is important to start with healthy cells. After streaking the cells onto a fresh TAP plate, it takes about a week under light at room temperature for the cells to grow into thick dark green lawns. 2. Second, prepare the starter culture. Once the plate is ready, inoculate one or two loops of fresh culture from the plate to 250 ml liquid media. Culture the cells for 3–5 days with air bubbling until the solution turns dark green (5  107 cells/ml). 3. Lastly, prepare the production culture. Use the starter culture to inoculate big bottles of media. We use 50 or 100 ml of the starter culture to inoculate a 4- or 8-l Kimble glass bottle. The cells are cultured for 3–5 days with air bubbling until the culture turns dark green (5  107 cells/ml). Note: Tips on isolating flagella from mutants with cells clumping together: Flagella are important for the daughter cells to swim away from the mother cell wall once cell division is completed. Thus flagella defective mutants including many IFT mutants and motility defective mutants are often in clumps. Treating the cell clumps with autolysin is very effective to help the daughter cells escape from the mother’s cell wall. However, the amount of autolysin should be carefully calculated. Currently, autolysin has to be house made in individual labs since no commercial product is available. The strength of autolysin from batch to batch varies greatly. If the autolysin treatment removes not only the mother but also the daughter cell wall, it will lead

IFT Particle Protein Isolation

7

to cell lysis and contaminate the purification. To prevent autolysin from damaging the daughter cell wall, once individual cells are released, we quickly remove autolysin by centrifugation the cells at 1400 rpm for 4 min at the room temperature with a swing-bucket rotor and then resuspend the cell pellets in large volume of 10 mM HEPES, pH 7.5.

3.3. Isolating flagella 1. Steps 1–3 concentrate the cells so that the volume is reduced to a workable amount. Harvest cells by spinning them down at 1400 rpm for 4 min at the temperature that was used to culture the cells. We use 1-l bottles and the Beckman J6 centrifuge equipped with a swingbucket Rotor JS-4.0. This step is time consuming if the volume of the culture is big. Some labs use the Pellicon Flow Filtration system from Millipore, Inc. We find that the cells concentrated by flow filtration are not healthy, and a high fraction of cells lose their flagella due to the harsh process. We therefore recommend the low-speed centrifugation method. 2. Discard the media and resuspend the cells in 10 mM HEPES, pH 7.5 (1 l of 10 mM HEPES for 8 l culture). 3. Allow flagellar regeneration in 10 mM HEPES with bubbling for 2 h. 4. Check under a compact microscope to make sure that cells have fulllength flagella. 5. Spin cells down in 250-ml conical tubes at 1400 rpm for 4 min using the Beckman J6 centrifuge equipped with a JS-4.0 Rotor. Quickly decant the supernatant and resuspend the cells in 10 mM HEPES, pH 7.5 (100–150 ml for 8 l original culture). 6. If your aim is just to isolate IFT particles from wild-type cells, we strongly recommend treating the cells with 5% sucrose for 30 min. This treatment increases the flagellar concentration at least two- to threefolds (Lucker et al., 2005). Otherwise, skip this step and go to step 7 directly. 7. Deflagellate the cells by adding 0.5 M acetic acid to drop pH to 4.5. Check cells under the microscope to make sure that deflagellation is complete and then bring the pH up to 7.0 by adding drops of 0.5 M KOH. This step should take no more than 2 min. If the cells do not deflagellate, add a few drops of 1 M CaCl2. A pH meter is essential for monitoring the pH change. Avoid letting the pH drop below 4 and increase over 7.5. Such extreme pH levels cause the cells to die,

8

Elizabeth Richey and Hongmin Qin

which leads to the releasing of the cytoplasm, which is impossible to remove by the sucrose underlay purification. 8. Keep cells on ice immediately after deflagellation and orderly add the following stock solutions to yield the respective final concentration.

9.

10. 11.

12.

13. 14.

Stock solution

Final concentration

1 M MgSO4 (200 ) 1 M DTT (1000 ) 50% sucrose (10 ) 0.5 M EGTA (2000 ) 100 mM PMSF (100 )

5 mM 1 mM 5% sucrose 0.25 mM 1 mM

If the cells have already been treated with 5% sucrose in step 6, skip adding sucrose in this step. Perform the sucrose underlay purification. Aliquot 150 ml of cells in 250-ml conical tubes. Use an electronic transfer pipette to gently underlay 50 ml of 25% sucrose in HMDE to the bottom of the conical tube. Two clear layers should be readily seen after the transfer: the clear sucrose layer at the bottom and the cells on the top. The separation of these two layers should be distinct. Centrifuge at 1800 rpm for 5 min at 4  C in the Beckman J6 centrifuge equipped with a JS-4.0 Rotor. After the centrifugation, the cell bodies are pelleted to the bottom, while flagella are in the top 25% sucrose layer. Collect and combine the supernatant and discard the pellet. The supernatant contains flagella, while the pellet contains cell bodies. If the supernatant is greenish, aliquot 30 ml of supernatant in 50-ml conical tubes and underlay with 8 ml of 25% sucrose in HMDE. Then centrifuge at 1800 rpm for 10 min at 4  C in Eppendorf 5810R equipped with a swing-bucket rotor A-4-44. Transfer the supernatant to 50-ml round-bottom tubes and collect flagella by centrifuge at 10,000 rpm for 10 min at 4  C in Beckman J2 equipped with a fixed angle rotor JA20. Resuspend the flagella in 1  HMDEK plus the protease inhibitors (1 mM PMSF, 50 mg/ml soybean trypsin inhibitor, 1 mg/ml pepstatin A, 2 mg/ml aprotinin, and 1 mg/ml leupeptin). Then, combine all the samples and pellet the flagella by centrifugation in a microcentrifuge

IFT Particle Protein Isolation

9

at the top speed for 10 min at 4  C. Resuspend the flagella in equal volume of 1  HMDEK plus the protease inhibitors. The stock solutions of protease inhibitors (all from Sigma) are made in 1000 concentration and stored at 4  C except leupeptin, which should be stored at 20  C. Make 1000 leupeptin stock solution and store singleuse small aliquots to avoid frequent freeze and thaw. 15. Check the isolated flagella under the microscope. The isolated flagella should be milky white. If not used right away, freeze flagella at 80  C.

4. PREPARING SOLUBLE FLAGELLAR PROTEINS Soluble flagellar proteins can be extracted from the purified flagella by the freeze/thaw method in 1  HMDEK or by treatment with low concentration of a nonionic detergent. The commonly used detergent is 0.1% NP-40. The most critical factor for the success of both sucrose density gradient centrifugation and the immunoprecipitation methods is the protein concentration of the flagellar extracts. We usually use very concentrated extracts. For example, we use 64 l of cultured cells at around 5  107cells/ ml density for a single isolation of flagellar IFT particle proteins by sucrose density gradient centrifugation. The protein concentration of the flagella extract should be no less than 5 mg/ml. Moreover, before the flagellar extracts are loaded on gradients or used for immunoprecipitation, it is very important to remove all insoluble proteins. Insoluble proteins interfere with gradient centrifugation and immunoprecipitation. We usually centrifuge the extracts at least twice at 100,000  g for 10 min to be sure that the extracts are clear of aggregates.

5. ISOLATING IFT PARTICLE PROTEINS BY FRACTIONING SOLUBLE FLAGELLAR PROTEINS THROUGH SUCROSE DENSITY GRADIENT Figure 1.1 provides an overview of the steps described in Sections 5.1–5.5.

5.1. Prepare low- and high-density sucrose solutions Make a solution that is 1  HMDEK with the desired sucrose concentration with double-distilled water. You need a low-density and a high-density sucrose solution. A common gradient is 10–25%, but can also be others such

10

Elizabeth Richey and Hongmin Qin

Isolate flagella, extract soluble proteins Load onto sucrose density gradient 10–25% sucrose Spin

Collect fractions

25%

10%

Figure 1.1 The flowchart of the method using sucrose density gradient centrifugation to isolated IFT particle proteins.

as 5–20%. In addition, sometimes the concentration of salt in the gradient is altered to disrupt complex B. When the flagellar extracts are treated with high concentration of NaCl (300 mM ), the peripheral proteins of complex B dissociate from complex B core, while complex A stays intact (Lucker et al., 2005). In this case, complex A and B core are further separated apart on the gradient: complex A proteins cosediment in the 16S fractions, whereas complex B core proteins sediment at 11S. Moreover, this treatment also allows assigning a complex B protein to be a peripheral or a core protein. Using this method, IFT88, IFT81, IFT74, IFT70, IFT52, IFT46, IFT27, IFT25, and IFT22 are found to be complex B core proteins (Cole, 2005; Fan et al., 2010), IFT172, IFT80, IFT57, and IFT20 to be peripheral proteins (Lucker et al., 2005). Example of low sucrose solution (10%):

10 HMDEK 50% sucrose Distilled water

2 ml 4 ml 14 ml 20 ml

11

IFT Particle Protein Isolation

Example of high sucrose solution (25%): 10  HMDEK 50% sucrose Distilled water

2 ml 10 ml 8 ml 20 ml

Keep these two solutions on ice until ready to use.

5.2. Making the gradient 1. Linear sucrose density gradients are used for IFT particle protein isolation. The essential apparatus to make a gradient includes a J17 Jule gradient maker (http://www.precastgels.com/formers1.htm), a pump (multistaltic pump from Buchler instruments, or similar models), a magnetic stirrer, connecting tubing, a centrifuge tube (Thinwall, Ultra-Clear™, 14 ml, 14  95 mm from Beckman Coulter), and a glass capillary micropipette (25 ml, 5-in. long from Corning). Before making the gradient, wash tubing and apparatus in distilled water. To do this, place one end of the tubing with the glass capillary micropipette and hook the other end up to the gradient apparatus and around the pump wheel. Place the end with the glass capillary micropipette in a waste beaker. Pour distilled water into both gradient chambers and turn pump on forward to allow water to flow through. At the end of the wash, make sure all the water is out of the tubing and the chambers of the gradient maker. Insert a long glass pipette to the hole in the bottom of the chambers to remove water from the connector tube which connects two chambers on the back of the gradient maker. 2. When finished washing, place the glass capillary micropipette on the end of the tubing into the Thinwall, Ultra-Clear™ centrifuge tube. Be sure that the centrifuge tube is secured in a straight upright position and the capillary micropipette reaches the bottom of the centrifuge tube. Tape the tubing connected to the capillary micropipette to the table so that the micropipette is stable and will not move or fall out of the collection tube. 3. There are two chambers of the gradient maker. The high sucrose chamber is the one that is not directly connected to the pump. It is for holding the high concentration sucrose solution. The other chamber connects to the pump, which holds the low sucrose solution. A connector tubing in the back of the gradient maker connects these two chambers. To

12

4.

5.

6.

7. 8.

Elizabeth Richey and Hongmin Qin

prevent bubbles to form during making gradient, we remove the air from the connector tube by squeezing low concentration of sucrose solution into the connector tube. Then close the connector tube with the supplied plastic clamp. Remove any extra solution from high sucrose chamber. Prevent bubbles from forming in the tubing connected to the low sucrose chamber. First, add 1 ml low sucrose solution to the low sucrose chamber. Then turn pump on in the forward direction and allow solution to run into the tubing. Then turn the pump on in the reverse direction to allow solution to flow back into low sucrose chamber, pushing any bubbles that have formed out of the tubing. When the solution has almost all made back to the low sucrose chamber but still fills about 0.5 cm long of the initial part of the tubing, stop the pump. Grease all connecting points to make sure they are sealed. Transfer 6.4 ml of the low-density sucrose solution into the low sucrose chamber and 6.4 ml of the high-density sucrose solution into the high sucrose chamber (for 14-ml tube used in SW41 rotor). Place piston inside the chambers until both black O-ring ends are touching the sucrose solutions. Close the air-releasing tubes on top of the piston and open the connector tube which connects the two chambers. Place the gradient maker on the magnetic stirrer and turn on the stirrer. Make sure the stir bar in the low-density sucrose chamber rotates in a smooth and fast fashion. Then set the pump to forward at a proper low speed. Turn on and allow the pump to slowly pump mixed sucrose solutions into the centrifuge tube. Stop the pump when centrifuge tube fills up almost completely, leaving just enough space for the sample (500–750 ml) at the top. Too much space on top will mess with the vacuum and will disrupt the centrifugation. In addition, during making the gradient, make sure that there are no bubbles running into the gradient. Any significant bubbles will disrupt your gradient and you will need to start over. Also be sure that the magnet is spinning smoothly on high in the low sucrose chamber during the entire process. When finished making the gradient, gently pull the capillary micropipette straight up as not to disturb the gradient. Flush the gradient chambers and tubing with water again as in step 1 and then disassemble apparatus.

5.3. Gradient centrifugation 1. Cut the end of a 200-ml pipette tip and gently pipette about 500–750 ml (no more than 750 ml) of the prepared soluble flagellar proteins onto the top of the gradient by placing the tip on the side of the tube without

IFT Particle Protein Isolation

2.

3.

4.

5.

13

touching the sucrose gradient and without allowing the sample to mix with the sucrose solution. Create balance tubes. If the tubes are not balanced, the centrifuge will stop which destroys the gradient. The SW41 rotor has six swinging buckets. These buckets are secured on a metal rack and are labeled 1–6. Bucket 1 has to balance with 4, 2 with 5, and 3 with 6. To balance, put a tube rack on the scale and zero. Then place your sucrose gradient in tube rack to weigh. Place the tube in bucket 1 on metal rack. To make a balance tube, put an empty tube on tube rack on scale and use sucrose solution to add until it weighs the same as the tube with the flagellar extracts. Do not use water to balance since it is much lighter than sucrose solution. Repeat until all tubes are balanced. Screw to close the tops of the buckets firmly. Place the buckets in the centrifuge rotor in the correct order so that balanced tubes are across from each other. Place the rotor into the centrifuge. We use the Beckman L8-M Ultracentrifuge. Spin the gradients at 37,000 rpm at 5  C for 18 h. To start the machine, push vacuum first and then set the temperature and speed and push run. Make sure that the vacuum works well; the temperature drops and the speed reaches 37,000 rpm before leaving the centrifuge unattended. When the centrifugation is finished, turn off the vacuum and remove the rotor right away from the centrifuge. It is important to remove the rotor immediately after the centrifuge stops; otherwise, the gradient can be disrupted causing the separated proteins spread into more fractions. Place the tubes from the buckets on ice in straight upright positions. To avoid moisture buildup, turn off the power of the centrifuge only when the centrifuge temperature goes back to room temperature.

5.4. Collecting the gradient fractions 1. We use the Fraction Collector Frac-100 to collect 0.5-ml fractions. This fraction collector is connected to the same pump as used in making the gradient. The speed of the pump is set at 1.8. At this speed, it takes 1 min to collect one gradient fraction. Before collecting the samples, always use water to test if the settings are correct. 2. The gradient is collected from the bottom to the top. To collect the fractions, gently set the glass capillary micropipette (25 ml) into the gradient until it reaches the bottom of the centrifuge tube. Then turn on the pump. When the first drop falls into the first collecting tube, start the fraction collector and let it run until all the fractions are collected. 3. Label tubes 1, 2, 3, 4. . .n, starting with the first tube that is collected until the last one. If the gradient is from 10% to 25% sucrose, the first tube

14

Elizabeth Richey and Hongmin Qin

16S 1 2

3

4

5

6

7 8

9 10 11 12 13 14

15 16 17 18 19 20 21 22 23 24

205 116 97 66 45

IFT27 IFT25 IFT22 IFT20

IFT144 IFT140+IFT139 IFT122 IFT121

Figure 1.2 The profile of fractions of a sucrose density gradient. The soluble flagellar proteins isolated from flagella of the wild-type cells were fractionated on a 12 ml of 10–25% sucrose gradient. The Coomassie blue-stained 10% SDS-PAGE gel of gradient fractions 1 (25% sucrose) to 24 (10% sucrose) is shown. IFT particle proteins peaked at the 16S fractions. The proteins highlighted in gray-lined rectangles are IFT particle complex A proteins with higher molecular weights. IFT27, IFT25, and IFT 22 polypeptides are highlighted with arrows. This figure is reproduced from Wang et al. (2009).

should contain 25% sucrose and the last tube should contain 10% sucrose. The total number of tubes should be around 24–28. Freeze fractions at 20  C for short-term or 80  C for long-term storage.

5.5. Analyzing the fractions by gel electrophoresis Sodium dodecyl sulfate (SDS) affects the migration rates of the proteins on the SDS-polyacrylamide gel. We prefer to use the SDS from the EMD Company. Figure 1.2 is a Coomassie blue-stained 10% SDS-PAGE gel of fractions of a 10–25% sucrose gradient of flagella extracts. On this gel, IFT22 is well separated from IFT20.

6. ISOLATING IFT PARTICLE PROTEINS BY IMMUNOPRECIPITATION FROM SOLUBLE FLAGELLAR PROTEINS IFT particle proteins also can be isolated by coimmunoprecipitation by certain antibodies against IFT particle proteins. IFT complexes A and B can be coprecipitated separately or together depending on the antibodies used (Cole et al., 1998; Qin et al., 2004; Silva et al., 2012). To obtain the best

IFT Particle Protein Isolation

15

results of coimmunoprecipitation, it is important to use highly concentrated flagellar extracts and strong specific antibodies based on our experiences. Here is the protocol: 1. To carry out an immunoprecipitation experiment, we usually use a total of 200 ml flagellar pellet and resuspend the pellet in 1  HMDEK to 500 ml total volume. 2. The flagella suspension is subjected to freeze–thaw to release soluble proteins. The insoluble flagellar axoneme fraction is removed by centrifugation at 14,000 rpm for 10 min at 4  C in a table-top centrifuge. 3. Prior to the immunoprecipitation experiments, protein A–Sepharose beads (GE Healthcare, NJ) should be washed three times for 10 min at room temperature with 1  HMDEK buffer. Then the beads are incubated in 1  HMDEK buffer plus 3% BSA for 1 h at room temperature to block nonspecific bindings. 4. Flagellar extracts are clarified by centrifugation twice at 100,000  g for 10 min. Sixty microliters of flagellar extracts are mixed with 60 ml of purified anti-IFT22 antibody or 60 ml of rabbit IgG control. The preparation is then incubated with antibodies for 1–2 h on ice. 5. Add 30 ml of the pretreated protein A–Sepharose beads and rotate the mixtures for 8 h at 4  C. Immune complexes recovered on the beads are then washed three times with 1 ml of 1 HMDEK plus 0.05% NP-40 at room temperature. Each wash takes 10 min. 6. Proteins are eluted from the beads by boiling in 1  Laemmli loading buffer and analyzed by SDS-PAGE and immunoblotting.

7. CONCLUSIONS The model organism Chlamydomonas has provided excellent experimental advantages for flagella and IFT particle protein isolation. The protocols described here detail the steps involved in flagellar isolation and IFT particle protein purification. We hope these protocols will not only be useful for other labs to implement these methods but also for developing modified methods that are useful for other applications, such as isolating native IFT particles for high-resolution electronic microscopic analysis.

ACKNOWLEDGMENTS The authors would like to thank Xiaomeng Huang for her input in the flagellar isolation protocol and Garrison Mathis for critical reading of the manuscript. The research in the Qin lab was supported by the National Science Foundation Grant MCB-0923835 to H. Q.

16

Elizabeth Richey and Hongmin Qin

REFERENCES Ahmed, N. T., Gao, C., Lucker, B. F., Cole, D. G., & Mitchell, D. R. (2008). ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery. The Journal of Cell Biology, 183, 313–322. Behal, R. H., Miller, M. S., Qin, H., Lucker, B. F., Jones, A., & Cole, D. G. (2012). Subunit interactions and organization of the Chlamydomonas reinhardtii intraflagellar transport complex A proteins. The Journal of Biological Chemistry, 287, 11689–11703. Cole, D. G. (2003). The intraflagellar transport machinery of Chlamydomonas reinhardtii. Traffic, 4, 435–442. Cole, D. G. (2005). Intraflagellar transport: Keeping the motors coordinated. Current Biology, 15, R798–R801. Cole, D. G., Diener, D. R., Himelblau, A. L., Beech, P. L., Fuster, J. C., & Rosenbaum, J. L. (1998). Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. The Journal of Cell Biology, 141, 993–1008. Fan, Z. C., Behal, R. H., Geimer, S., Wang, Z. H., Williamson, S. M., Zhang, H. L., et al. (2010). Chlamydomonas IFT70/CrDYF-1 is a core component of IFT particle complex B and is required for flagellar assembly. Molecular Biology of the Cell, 21, 2696–2706. Hildebrandt, F., Benzing, T., & Katsanis, N. (2011). Ciliopathies. The New England Journal of Medicine, 364, 1533–1543. Hou, Y., Qin, H., Follit, J. A., Pazour, G. J., Rosenbaum, J. L., & Witman, G. B. (2007). Functional analysis of an individual IFT protein: IFT46 is required for transport of outer dynein arms into flagella. The Journal of Cell Biology, 176, 653–665. Iomini, C., Babaev-Khaimov, V., Sassaroli, M., & Piperno, G. (2001). Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases. The Journal of Cell Biology, 153, 13–24. Ishikawa, H., & Marshall, W. F. (2011). Ciliogenesis: Building the cell’s antenna. Nature Reviews. Molecular Cell Biology, 12, 222–234. Kozminski, K. G., Beech, P. L., & Rosenbaum, J. L. (1995). The Chlamydomonas kinesinlike protein FLA10 is involved in motility associated with the flagellar membrane. The Journal of Cell Biology, 131, 1517–1527. Lucker, B. F., Behal, R. H., Qin, H., Siron, L. C., Taggart, W. D., Rosenbaum, J. L., et al. (2005). Characterization of the intraflagellar transport complex B core: Direct interaction of the IFT81 and IFT74/72 subunits. The Journal of Biological Chemistry, 280, 27688–27696. Mukhopadhyay, S., Wen, X., Chih, B., Nelson, C. D., Lane, W. S., Scales, S. J., et al. (2010). TULP3 bridges the IFT-A complex and membrane phosphoinositides to promote trafficking of G protein-coupled receptors into primary cilia. Genes & Development, 24, 2180–2193. Murcia, N. S., Richards, W. G., Yoder, B. K., Mucenski, M. L., Dunlap, J. R., & Woychik, R. P. (2000). The Oak Ridge Polycystic Kidney (orpk) disease gene is required for left-right axis determination. Development, 127, 2347–2355. Ou, G., Blacque, O. E., Snow, J. J., Leroux, M. R., & Scholey, J. M. (2005). Functional coordination of intraflagellar transport motors. Nature, 436, 583–587. Ou, G., Koga, M., Blacque, O. E., Murayama, T., Ohshima, Y., Schafer, J. C., et al. (2007). Sensory ciliogenesis in Caenorhabditis elegans: Assignment of IFT components into distinct modules based on transport and phenotypic profiles. Molecular Biology of the Cell, 18, 1554–1569. Pathak, N., Obara, T., Mangos, S., Liu, Y., & Drummond, I. A. (2007). The zebrafish fleer gene encodes an essential regulator of cilia tubulin polyglutamylation. Molecular Biology of the Cell, 18, 4353–4364.

IFT Particle Protein Isolation

17

Pazour, G. J., Dickert, B. L., Vucica, Y., Seeley, E. S., Rosenbaum, J. L., Witman, G. B., et al. (2000). Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. The Journal of Cell Biology, 151, 709–718. Piasecki, B. P., Diller, K. R., & Brand, J. J. (2009). Cryopreservation of Chlamydomonas reinhardtii: A cause of low viability at high cell density. Cryobiology, 58, 103–109. Piperno, G., & Mead, K. (1997). Transport of a novel complex in the cytoplasmic matrix of Chlamydomonas flagella. Proceedings of the National Academy of Sciences of the United States of America, 94, 4457–4462. Qin, H., Diener, D. R., Geimer, S., Cole, D. G., & Rosenbaum, J. L. (2004). Intraflagellar transport (IFT) cargo: IFT transports flagellar precursors to the tip and turnover products to the cell body. The Journal of Cell Biology, 164, 255–266. Silva, D. A., Huang, X., Behal, R. H., Cole, D. G., & Qin, H. (2012). The RABL5 homolog IFT22 regulates the cellular pool size and the amount of IFT particles partitioned to the flagellar compartment in Chlamydomonas reinhardtii. Cytoskeleton (Hoboken), 69, 33–48. Walther, Z., Vashishtha, M., & Hall, J. L. (1994). The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein. The Journal of Cell Biology, 126, 175–188. Wang, Z., Fan, Z. C., Williamson, S. M., & Qin, H. (2009). Intraflagellar transport (IFT) protein IFT25 is a phosphoprotein component of IFT complex B and physically interacts with IFT27 in Chlamydomonas. PLoS One, 4, e5384. Witman, G. B., Carlson, K., Berliner, J., & Rosenbaum, J. L. (1972). Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes. The Journal of Cell Biology, 54, 507–539.