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Ciliary and Flagellar Membrane Vesicle (Ectosome) Purification
纤毛和鞭毛囊泡(外皮层)的纯化   

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Abstract

Eukaryotic cilia/flagella are ideal organelles for the analysis of membrane trafficking, membrane assembly, and the functions of a variety of signal transduction molecules. Cilia are peninsular organelles and the membrane lipids, membrane proteins, and microtubular-associated components are selectively transported into cilia through the region formed by the basal body/transition region and tightly associated ciliary membrane. Cilia can be isolated from many organisms without disrupting cells and many will rapidly regenerate cilia (with the ciliary membrane lipids and proteins) to replace those that are released. Despite their ease of isolation, we have relatively little understanding of the mechanisms that regulate lipid and protein transport into ciliary membranes (Pazour and Bloodgood, 2008; Bloodgood, 2009; Bloodgood, 2012).
Chlamydomonas flagella shed membrane vesicles, also called ectosomes (Wood et al., 2013) from flagellar tips and these vesicles can be purified from the culture medium without damaging or deflagellating cells (McLean et al., 1974; Bergman et al., 1975; Snell, 1976; Kalshoven et al., 1990). Based on a comparison of biotinylated proteins on the shed vesicles with biotinylated proteins isolated from purified flagella and cell bodies, the ectosomes contain most, but not all, flagellar surface proteins and none of the major cell body proteins (Dentler, 2013). Although ectosomes have only been purified from Chlamydomonas cells, preliminary evidence indicates that similar vesicles are released from Tetrahymena cilia (Dentler, unpublished).
Flagellar (and ciliary) membranes or membrane proteins also can be released from purified flagella/cilia. Most membrane proteins can be solubilized by extracting purified cilia with nonionic detergent [Triton X-100 or X-114 or Nonidet P-40 (NP-40)] and pelleting the microtubules (axonemes). However, not all membranes are released by detergent (Dentler, 1980) and the supernatant also contains all of the flagellar proteins that are not attached to the microtubules.
Intact membrane vesicles can be released from flagella by agitation of flagella, often with low concentrations of nonionic detergents or freeze-thawing (Witman et al., 1972; Snell, 1976; Dentler, 1980; Dentler, 1995; Bloodgood and May, 1982; Pasquale and Goodenough, 1987; Iomini et al., 2006; Huang et al., 2007). Once released, they can be purified from axonemes by differential centrifugation.
Each of these methods may enrich for different populations of axonemal and membrane proteins and lipids. The different solubility of membranes may reveal local differences in lipid or protein composition (Bloodgood, 2009). The ectosomes contain most but not all surface proteins found on purified Chlamydomonas flagella (Dentler, 2013). The ectosomes vesicles may be enriched in different soluble flagellar proteins than those trapped as vesicles are released from purified flagella. The detergent-solubilized “membrane+matrix” will contain all soluble membrane proteins as well as all of the soluble proteins in the flagellar compartment.
In this paper, a method to purify ectosomes vesicles released from the tips of living Chlamydomonas cells is presented as are two methods to release flagellar membrane vesicles and proteins from purified flagella.


Figure 1. A: Purified flagella (phase contrast); B: Purified flagella (TEM); C: Shed membrane vesicles (TEM). Bars = 500 nm

Materials and Reagents

  1. Cells
    Chlamydomonas cells can be obtained from a variety of sources and pure strains can be obtained from the Chlamydomonas Resource Center (http://chlamycollection.org/contact-us/).
  2. Media and cell culture (see Notes)
  3. Culture media for washing cells (2-4 L)
  4. 0.5 N acetic acid
  5. 0.5 N KOH
  6. Sucrose
  7. Triton X-100 or Nonidet P-40
  8. Cilia wash buffer (CWB) (see Recipes)

Equipment

  1. Pellicon tangential flow microfiltration cassettes (Millipore, www.millipore.com)
    Note: Cells can be harvested using 450 ml centrifuge bottles and large rotor but, for 8-16 L of cells, harvesting is more rapid and fewer cells are deflagellated using a Pelicon.
  2. Preparative centrifuge with rotors for 450 ml bottles and for 40 ml tubes
  3. 450 ml centrifuge bottles
  4. 40 ml centrifuge tubes
  5. 12 ml centrifuge tubes
  6. Medium speed centrifuge with swinging bucket rotor and angle rotor
  7. Fernbach flasks (2,800 ml) or large flasks
  8. Ultracentrifuges and rotors
    Note: For 750 ml, I use Beckman 35 or Ty45Ti rotors and two Beckman ultracentrifuges.
  9. Phase contrast microscope
  10. Orbital shaker
  11. Bright fluorescent light - generally 4-6 F20/40 PL/AQ lamps
  12. Transmission electron microscope
  13. Magnetic stirrer
  14. pH electrode

Procedure

Method 1. Purification of ectosomes
Ectosomes are released by flagella during the incubation period and contain most of the surface-exposed (biotinylated) proteins found on purified flagella (Dentler, 2013).

  1. Harvest 8-16 liters of cells using a Pellicon cassette system. Further concentrate cells by centrifugation (1,500 x g, 4 min) if necessary.  
  2. Gently suspend the cell pellets in fresh culture medium.
    1. To avoid deflagellating cells, suspend pellets by gentle swirling or by agitation with a large bore glass or plastic pipette. The total suspension volume depends on the availability of centrifuges.  
    2. Each Beckman 35 or 45Ti rotor can hold six 70 ml tubes, for a total volume of 490 ml/centrifuge, so suspend cells in a total volume of 700-900 ml of M medium. If there is access to larger capacity centrifuges or additional centrifuges and rotors, it is best to increase the volume of cells to avoid crowing.
    3. Examine cells by phase contrast microscopy to insure that there are no dividing cells and that nearly 100% of the cells are fully flagellated and motile. If cells are sick or not fully flagellated, the preparations will be contaminated by cell debris or released flagella and it’s best not to proceed with these cells.
  3. Incubate suspended cells in constant light for 6 h or longer.  
    1. Cells can be aerated with a sintered glass bubbler in 1 L bottles or can be gently swirled in 2,700 ml Fernbach flasks using an orbital shaker under bright fluorescent light.
  4. At the end of the incubation period, check cells by microscopy to insure that all are intact, flagellated, and motile. At the end of the incubation period, pellet the cells by centrifugation for 1,500 x g, 4 min. This can be done at 4 °C or at room temperature (~23 °C), the temperature at which the cells were incubated.
    1. Carefully decant the supernatant (containing shed vesicles) to avoid disturbing the cell pellet or aspirate the supernatant from the bottles.
    2. Examine the supernatant by phase contrast microscopy to insure that no flagella are present. If flagella or cells are present, centrifuge the supernatant at 11,000 x g for 10 min. The cell pellets can be gently suspended in M medium and deflagellated to purify flagella (see below).
  5. Pellet the shed membranes by centrifuging the supernatant for 60 min, 125,000 x g, 4 °C.
  6. Remove the supernatant by aspiration. The vesicle-containing pellets are nearly transparent and are easily dislodged. Often, there is a tight green pellet containing cell bodies or chloroplasts that is overlain by a more transparent pellet. Try to suspend the membrane-containing transparent material without disturbing the green pellet. Suspend in a small volume of CWB, keeping the combined volume of all suspended pellets less than 2 ml.  
  7. Layer the suspended pellets over ~ 8 ml of CWB with 25% sucrose and centrifuge 3,000 x g, 10 min, 4 °C, in a swinging bucket rotor.
  8. Remove the layer (containing vesicles) above the 25% sucrose/CWB and centrifuge at 46,000 x g for 30 min, 4 °C to pellet membrane vesicles. Suspend the pellet in a small volume of CWB and check for flagella or cell bodies by phase contrast and by negatively staining and examining by transmission electron microscopy.

Method 2. Purification of vesicles from isolated flagella

  1. Flagellar purification - pH shock
    Flagella can be isolated using dibucaine or pH shock but I’ve found the membrane to be more intact in pH shocked flagella. For other methods to isolate and fractionate cilia and flagella from various organisms, see Methods in Cell Biology, Vol 47, 1995.
    1. Harvest cells using a pelicon harvester and by centrifugation (see above) and suspend cells in fresh M medium to a final volume of 100-200 ml. The cells used to isolate released membrane vesicles (above) also can be used to isolate flagella.
    2. Using a magnetic stirrer and calibrated pH electrode, deflagellate cells by lowering the pH to 4.0 by dropwise addition of 0.5 N acetic acid. Do not leave cells at pH 4 for longer than 1-1.5 min. Raise the pH to 7.0 by dropwise addition of 0.5 N KOH. For 100 ml of cells in M medium, use approximately 4.5 ml of 0.5 N acetic acid and 5.2 ml of 0.5 M KOH. For most purposes, it is best to monitor the pH change but, for mass-deflagellations in microtitre plates, adding defined quantities of acetic acid and KOH is useful.
    3. Pellet cells by centrifugation at 1,100 x g, 3 min.
    4. Aspirate the supernatant (containing flagella) with a large bore pipette and layer over CWB with 25% sucrose.
      1. Centrifuge in a swinging bucket rotor at 2,500 x g for 10 min, 4 °C.  
      2. Flagella will collect at the interface above the 25% sucrose.
    5. Aspirate the white flagella layer, dilute with CWB, and pellet flagella at 17,000 x g, 20 min, 4 °C.
    6. Suspend flagella in CWB using a large bore pipette to avoid breaking flagella and check with phase contrast microscopy to insure the preparation is free of cell bodies or other debris.

  2. For detergent-solubilized membrane + matrix.
    Detergent-extraction of cilia and flagella solubilizes most, but not all, of the membrane but also releases soluble ciliary proteins.  Bonafide flagellar trans-membrane proteins can be unambiguously identified if they are biotinylated on intact cells (see Reference 7).
    1. Suspend the flagella in CWB, add 10% Triton X-100 or Nonidet P-40 to a final concentration of 1%, incubate 10-20 min, 4 °C.
    2. Centrifuge for 15 min at 27,000 x g, 4 °C to pellet the detergent-insoluble axonemes and detergent-insoluble membranes. The solubilized membrane and matrix proteins will be in the supernatant.

  3. Vesicle release and purification
    Purification of membrane vesicles should insure that all of the proteins and lipids are components of the membrane but not the soluble flagellar matrix proteins. However, contamination of the vesicles with soluble flagellar proteins is possible as the membranes are released from flagella and vesiculate, trapping flagellar matrix proteins. Additionally, rubbing membranes off axonemes or freeze-thawing them leads to considerable breakage of the axonemal microtubules and release of axonemal components. Detergent extraction can be done with no significant axonemal breakage.
    This is a modification of the method used to purify Tetrahymena ciliary membranes (Dentler, 1980; Dentler, 1995). Other methods to release membranes from purified flagella include freeze-thawing and rubbing off membranes (Wang and Snell, 2003; Huang et al., 2007; Pasquale and Goodenough, 1987; Iomini et al., 2006; Snell, 1976) or treatment with low concentrations of detergents (Witman et al., 1972; Bloodgood and May, 1982).
    1. Start with purified flagella (above) suspended in CWB.  
    2. Agitate the flagella by shearing with a glass pipette or by vortexing for 1 min, incubate on ice 1 min, and vortex again. Repeat 5-10 times.  
    3. Examine by phase contrast microscopy to assay for the formation of small membrane vesicles (will appear as dots) and the membrane-free axonemes (which will be thin and less refractile than intact cilia).  
    4. If membranes cannot easily be released, add Nonidet P-40 or Triton X-100 to a final concentration of 0.01-0.02% during vortexing.
    5. Layer the sheared or vortexed flagella over sucrose step gradients composed of 1 ml 20% sucrose (w/v) in CWB, 1 ml of 30% sucrose (w/v) in CWB, and 1.2 ml of 40% sucrose (w/v) in CWB.  
    6. Centrifuge in a swinging bucket rotor.  Centrifuge samples at 200,000 x g, 2 h and collect membranes from the interface between the 30% and 40% sucrose layers (Dentler, 1995).  
      Note: Alternatively, vesicles may be separated from axonemes by layering the sheared flagella over ~8 ml of CWB with 25% sucrose and centrifuging 3,000 x g, 10 min, 4 °C as was done for the released membranes (above). The axonemes with attached membranes will be in the pellets.
    7. Carefully remove membranes from the interfaces, dilute with cold CWB, and pellet vesicles by centrifugation at 48,000 x g for 1 h.
    8. Suspend the vesicles in a small amount of CWB and examine by phase contrast and/or negative staining and transmission electron microscopy to insure that the vesicles are intact and are not contaminated with cell debris or axonemes. 

Notes

  1. Notes about media and cell culture
    1. Culture media recipes are available at http://www.chlamy.org/media.html. I prefer to culture cells in Sager and Granick minimal (M) medium (http://www.chlamy.org/SG.html) but TAP medium is widely used (http://www.chlamy.org/TAP.html). Media preparation and culture conditions also are described in References 8 and 15.
    2. Autoclave media in 4 L Pyrex bottles. Aerate cells with a sintered glass filter and house air. Culture in constant light or, to obtain a uniform population of synchronized cells, culture in M medium on a 12 h light/dark cycle. 
    3. For most preparations, I use 8-16 liters of cells grown to ~ 5 x 105 cells/ml.

Recipes

  1. Cilia wash buffer (CWB)
    50 mM Tris-HCl (pH 7.4)
    3 mM MgSO4
    100 mM EGTA (pH 7.4)
    250 mM sucrose

Acknowledgments

This protocol was adapted from Dentler (2013).

References

  1. Bergman, K., Goodenough, U. W., Goodenough, D. A., Jawitz, J. and Martin, H. (1975). Gametic differentiation in Chlamydomonas reinhardtii. II. Flagellar membranes and the agglutination reaction. J Cell Biol 67(3): 606-622.
  2. Bloodgood, R. A. (2009). The Chlamydomonas flagellar membrane and its dynamic properties. The Chlamydomonas Sourcebook 2nd edition. 309-368.
  3. Bloodgood, R. A. (2012). The future of ciliary and flagellar membrane research. Mol Biol Cell 23(13): 2407-2411.
  4. Bloodgood, R. A. and May, G. S. (1982). Functional modification of the Chlamydomonas flagellar surface. J Cell Biol 93(1): 88-96.
  5. Dentler, W. L. (1980). Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis. J Cell Biol 84(2): 364-380.
  6. Dentler, W. L. (1995). Isolation and fractionation of ciliary membranes from Tetrahymena. Methods Cell Biol 47: 397-400.
  7. *Dentler, W. (2013). A role for the membrane in regulating Chlamydomonas flagellar length. PLoS One 8(1): e53366.
  8. Harris, E. H. (1989). A comprehensive guide to biology and laboratory use. The Chlamydomonas Sourcebook. Academic Press.
  9. Huang, K., Diener, D. R., Mitchell, A., Pazour, G. J., Witman, G. B. and Rosenbaum, J. L. (2007). Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella. J Cell Biol 179(3): 501-514.
  10. Iomini, C., Li, L., Mo, W., Dutcher, S. K. and Piperno, G. (2006). Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas. Curr Biol 16(11): 1147-1153.
  11. Kalshoven, H., Musgrave, A. and Van den Ende, H. (1990). Mating receptor complex in the flagellar membrane of Chlamydomonas eugametos gametes. Sexual Plant Reproduction 3(2): 77-87.
  12. McLean, R.J., Laurendi, C. J. and Brown, R. M., Jr. (1974). The relationship of gamone to the mating reaction in Chlamydomonas moewusii. Proc Natl Acad Sci U S A 71(7): 2610-2613.
  13. Pazour, G. J. and Bloodgood, R. A. (2008). Targeting proteins to the ciliary membrane. Curr Top Dev Biol 85: 115-149.
  14. Pasquale, S. M. and Goodenough, U. W. (1987). Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii. J Cell Biol 105(5): 2279-2292.
  15. Sager, R. and Granick, S. (1953). Nutritional studies with Chlamydomonas reinhardi. Ann N Y Acad Sci 56(5): 831-838.
  16. Snell, W. J. (1976). Mating in Chlamydomonas: a system for the study of specific cell adhesion. I. Ultrastructural and electrophoretic analyses of flagellar surface components involved in adhesion. J Cell Biol 68(1): 48-69.
  17. Wang, Q. and Snell, W. J. (2003). Flagellar adhesion between mating type plus and mating type minus gametes activates a flagellar protein-tyrosine kinase during fertilization in Chlamydomonas. J Biol Chem 278(35): 32936-32942.
  18. Witman, G. B., Carlson, K., Berliner, J. and Rosenbaum, J. L. (1972). Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes. J Cell Biol 54(3): 507-539.
  19. Wood, C. R., Huang, K., Diener, D. R. and Rosenbaum, J. L. (2013). The cilium secretes bioactive ectosomes. Curr Biol 23(10): 906-911.

简介

真核纤毛/鞭毛是分析膜运输,膜组装和各种信号转导分子的功能的理想细胞器。纤毛是半岛细胞器,并且膜脂质,膜蛋白和微管相关组分通过由基底体/过渡区和紧密相关的睫状膜形成的区域选择性地转运到纤毛中。纤毛可以从许多生物体中分离而不破坏细胞,并且许多将快速再生纤毛(具有睫状膜脂质和蛋白质)以替代释放的纤毛。尽管它们容易分离,但我们对调节脂质和蛋白质转运到睫状膜的机制的理解相对较少(Pazour和Bloodgood,2008; Bloodgood,2009; Bloodgood,2012)。
< >鞭毛脱落的膜囊泡,也称为来自鞭毛尖端的卵母细胞(Wood等人,2013),并且可以从培养基中纯化这些囊泡而没有损伤或去鞭毛细胞(McLean等人, ,1974; Bergman et al。,1975; Snell,1976; Kalshoven et al。,1990)。基于脱落囊泡上的生物素化蛋白质与从纯化鞭毛和细胞体分离的生物素化蛋白质的比较,外周体含有大多数但不是全部的鞭毛表面蛋白质,并且没有主要的细胞体蛋白质(Dentler,2013)。虽然ectosomes只从衣原体细胞中纯化,但初步证据表明类似的囊泡从四膜虫纤毛释放(Dentler,未发表)。
鞭毛(和睫状体)膜或膜蛋白也可以从纯化的鞭毛/纤毛释放。大多数膜蛋白可以通过用非离子去污剂[Triton X-100或X-114或Nonidet P-40(NP-40)]提取纯化的纤毛并将微管(轴突)造粒来溶解。然而,不是所有的膜都被去污剂释放(Dentler,1980),并且上清液还包含所有未附着到微管的鞭毛蛋白。通过搅动鞭毛,通常使用低浓度的非离子型洗涤剂或冷冻融化,可以从鞭毛中释放完整的膜囊泡(Witman等人,1972; Snell,1976; Dentler,1980 ; Dentler,1995; Bloodgood和May,1982; Pasquale和Goodenough,1987; Iomini等人,2006; Huang等人,2007)。一旦释放,可以通过差速离心从轴突中纯化它们。
这些方法中的每一种可以富集轴突和膜蛋白和脂质的不同群体。膜的不同溶解度可以揭示脂质或蛋白质组成的局部差异(Bloodgood,2009)。外来体含有在纯化的衣藻鞭毛上发现的大多数但不是全部表面蛋白(Dentler,2013)。外来体囊泡可以富集在不同的可溶性鞭毛蛋白中,而不是当从纯化的鞭毛中释放囊泡时捕获的那些。洗涤剂增溶的"膜+基质"将包含鞭毛区室中的所有可溶性膜蛋白以及所有可溶性蛋白。
在本文中,提出了从活的衣藻细胞的末端释放的外来体囊泡的方法,以提供从纯化的鞭毛中释放鞭毛膜囊泡和蛋白质的两种方法。


图1。 A:纯化鞭毛(相差); B:纯化鞭毛(TEM); C:脱膜膜囊泡(TEM)。 条= 500nm

材料和试剂

  1. 单元格
    衣藻细胞可以从多种来源获得,并且纯菌株可以从衣藻资源中心获得( http://chlamycollection.org/contact-us/)。
  2. 培养基和细胞培养(见注释)
  3. 用于洗涤细胞的培养基(2-4L)
  4. 0.5 N乙酸
  5. 0.5 N KOH
  6. 蔗糖
  7. Triton X-100或Nonidet P-40
  8. 纤毛洗涤缓冲液(CWB)(参见配方)

设备

  1. Pellicon切向流微滤盒(Millipore, www.millipore.com
    注意:可以使用450ml离心瓶和大转子收获细胞,但是对于8-16L细胞,收获更快,并且使用Pelicon进行更少的细胞絮凝。
  2. 带有用于450ml瓶和40ml管的转子的制备型离心机
  3. 450 ml离心瓶
  4. 40 ml离心管
  5. 12ml离心管
  6. 带有摆动斗式转子和角度转子的中速离心机
  7. Fernbach烧瓶(2,800ml)或大烧瓶
  8. 超速离心机和转子
    注意:对于750 ml,我使用Beckman 35或Ty45Ti转子和两个Beckman超速离心机。
  9. 相差显微镜
  10. 轨道振动器
  11. 明亮的荧光灯 - 通常为4-6 F20/40 PL/AQ灯
  12. 透射电子显微镜
  13. 磁力搅拌器
  14. pH电极

程序

方法1.纯化ectosome
外膜体在孵育期间由鞭毛释放并且包含在纯化鞭毛上发现的大多数表面暴露的(生物素化的)蛋白质(Dentler,2013)。

  1. 使用Pellicon盒系统收获8-16升的细胞。 如果需要,通过离心进一步浓缩细胞(1,500×g,4分钟)。  
  2. 轻轻悬浮细胞沉淀在新鲜培养基中。
    1. 为了避免絮凝细胞,通过温和的旋动或通过悬浮颗粒 用大口径玻璃或塑料移液管搅拌。 总数 悬浮液体积取决于离心机的可用性。  
    2. 每个Beckman 35或45Ti转子可以容纳六个70毫升管,总共 体积为490 ml /离心机,使细胞悬浮在总体积中 700-900ml的M培养基。 如果有访问更大的容量 离心机或附加离心机和转子,最好增加   避免淹没的细胞体积。
    3. 通过相检查细胞 对比显微镜,以确保没有分裂细胞和那 几乎100%的细胞完全鞭毛和活动。 如果单元格 生病或不完全鞭打,制剂将受到污染 细胞碎片或释放的鞭毛,最好不要继续这些   细胞。
  3. 孵育悬浮细胞在恒定光照6小时或更长时间。  
    1. 可以在1L瓶或罐中用烧结玻璃鼓泡器对小室充气   使用轨道摇床在2,700ml Fernbach烧瓶中轻轻涡旋 在明亮的荧光灯下。
  4. 在孵育期结束时,通过显微镜检查细胞以确保所有是完整的,鞭毛和运动。 在孵育期结束时,通过离心1500×g,4分钟沉淀细胞。 这可以在4℃或在室温(〜23℃),细胞孵育的温度下进行。
    1. 小心倾析上清液(包含脱落的囊泡)以避免 干扰细胞沉淀或从瓶中吸出上清液。
    2. 通过相差显微镜检查上清液 确保不存在鞭毛。 如果存在鞭毛或细胞, 在11,000×g离心上清液10分钟。 细胞沉淀 可以轻轻地悬浮在M培养基中并且絮凝以纯化鞭毛   (见下文)。
  5. 通过将上清液离心分离60分钟,125,000×g,4℃来制备脱落的膜。
  6. 通过抽吸除去上清液。 含囊泡的丸粒几乎是透明的,并且容易脱落。 通常,存在包含细胞体或叶绿体的紧密绿色沉淀物,其被更多的上覆 透明丸。尝试悬浮含膜透明材料而不干扰生球团矿。悬浮在小体积的CWB中,保持所有悬浮颗粒的组合体积小于2ml。  
  7. 将悬浮的小球在含有25%蔗糖的〜8ml的CWB上并在摇摆转子中离心3,000×g,10分钟,4℃。
  8. 除去25%蔗糖/CWB上的层(含囊泡),并在46,000×g离心30分钟,4℃,以沉淀膜囊泡。将小丸悬浮在小体积的CWB中,并通过相差和通过负染和通过透射电子显微镜检查检查鞭毛或细胞体。

方法2.从分离的鞭毛中纯化囊泡

  1. 鞭毛净化 - pH休克
    鞭毛可以使用地布卡因或pH休克分离,但我发现膜在pH震荡鞭毛中更完整。对于从各种生物体分离和分离纤毛和鞭毛的其它方法,参见Methods in Infect Cell Biology,Vol 47,1995。
    1. 收获细胞使用pelicon收获机和离心(见 并将细胞悬浮在新鲜M培养基中至终体积100-200 ml。 用于分离释放的膜囊泡(上面)的细胞也 可用于分离鞭毛。
    2. 使用磁力搅拌器和 校准pH电极,通过将pH降低至4.0来使鞭毛细胞生长   滴加0.5N乙酸。 不要将细胞置于pH 4下 超过1-1.5分钟。 通过滴加0.5N将pH升至7.0   KOH。 对于100ml培养基中的细胞,使用约4.5ml的0.5N   乙酸和5.2ml的0.5M KOH。 对于大多数用途,最好是 监测pH变化,但对于微量滴定中的质量减少 板,加入规定量的乙酸和KOH是有用的
    3. 通过在1,100×g离心3分钟沉淀细胞。
    4. 用大口径移液管吸出上清液(含鞭毛),并在含有25%蔗糖的CWB上铺层。
      1. 在2,500×g离心10分钟,4℃的摆动转子中。  
      2. 鞭毛将在25%蔗糖以上的界面处聚集。
    5. 吸出白鞭毛层,用CWB稀释,并以17,000×g /小时,20分钟,4℃沉淀鞭毛。
    6. 使用大口径移液管悬挂鞭毛在CWB,以避免打破 鞭毛和检查与相差显微镜以确保 制备无细胞体或其他碎片。

  2. 对于去污剂溶解的膜+基质。
    洗涤剂提取的纤毛和鞭毛溶解大多数,但不是所有的膜,但也释放可溶性睫状蛋白。 如果Bonafide鞭毛跨膜蛋白在完整细胞上生物素化,可以被明确鉴定(参见参考文献7)。
    1. 将鞭毛悬浮于CWB中,加入10%Triton X-100或Nonidet P-40至终浓度为1%,在4℃孵育10-20分钟。
    2. 在27,000×g离心15分钟,4℃,沉淀 洗涤剂不溶性轴突和去污剂不溶性膜。 的 溶解的膜和基质蛋白将在上清液中。

  3. 囊泡释放和纯化
    膜囊泡的纯化应当确保所有的蛋白质和脂质是膜的组分,但不是可溶性鞭毛基质蛋白。然而,囊泡与可溶性鞭毛蛋白的污染是可能的,因为膜从鞭毛和囊泡释放,捕获鞭毛基质蛋白。另外,从轴突摩擦膜或冻融它们导致轴突微管的相当破坏和轴突组分的释放。洗涤剂提取可以在没有显着的轴突破裂的情况下进行 这是用于纯化四膜虫睫状膜的方法的改进(Dentler,1980; Dentler,1995)。从纯化的鞭毛中释放膜的其它方法包括冻融和摩擦膜(Wang和Snell,2003; Huang等人,2007; Pasquale和Goodenough,1987; Iomini等人, ,2006; Snell,1976)或用低浓度的去污剂处理(Witman等人,1972; Bloodgood和May,1982)。
    1. 从纯化的鞭毛(上面)开始悬浮在CWB。  
    2. 通过用玻璃吸管剪切或通过涡旋搅动鞭毛 1分钟,在冰上孵育1分钟,再次涡旋。重复5-10次。  
    3. 通过相差显微镜检查以检测形成 小膜囊泡(将显示为点)和无膜 轴突(其将是薄的并且比完整纤毛更少的折射)。  
    4. 如果膜不容易释放,添加Nonidet P-40或Triton X-100,在涡旋期间最终浓度为0.01-0.02%
    5. 在蔗糖梯度梯度上层剪切或涡旋鞭毛 由在CWB中的1ml 20%蔗糖(w/v),1ml 30%蔗糖(w/v) CWB和1.2ml 40%蔗糖(w/v)的CWB溶液。  
    6. 离心机中 摆动转子。 以200,000×g,2小时和2小时离心样品 从30%和40%蔗糖之间的界面收集膜 层(Dentler,1995)。  
      注意:或者,囊泡可以 通过将剪切的鞭毛分层超过〜8ml从轴突分离 CWB与25%蔗糖并且以3,000×g离心10分钟,4℃ 对释放的膜(上)做。 附着的轴突 膜将在颗粒中。
    7. 小心取出膜 从界面,用冷CWB稀释,和颗粒囊泡 在48,000×g离心1小时。
    8. 暂停囊泡在   少量的CWB并通过相差和/或负检查 染色和透射电子显微镜以确保 囊泡是完整的,并且不被细胞碎片或 轴突。

笔记

  1. 关于媒体和细胞培养的注意事项
    1. 您可以在 http://www.chlamy.org/media.html 上查看文化媒体食谱。 我更喜欢在Sager和Granick minimal(M)培养基中培养细胞( http://www.chlamy.org/SG.html ),但广泛使用TAP媒体( http://www.chlamy.org/TAP.html )。 培养基制备和培养条件也在参考文献8和15中描述
    2. 高压灭菌媒体在4 L Pyrex瓶中。 充气电池与烧结 玻璃过滤器和室内空气。 文化在恒光或者,获得 均匀细胞群,在M培养基中培养12 h 明/暗循环。
    3. 对于大多数制备,我使用8-16升生长至〜5×10 5个细胞/ml的细胞。

食谱

  1. 纤毛洗涤缓冲液(CWB)
    50mM Tris-HCl(pH7.4) 3mM MgSO 4 100mM EGTA(pH7.4) 250mM蔗糖

致谢

该协议改编自Dentler(2013)。

参考文献

  1. Bergman,K.,Goodenough,U.W.,Goodenough,D.A.,Jawitz,J.and Martin,H。(1975)。 在莱茵衣藻中的配子分化。 II。鞭毛膜和凝集反应。细胞生物学67(3):606-622。
  2. Bloodgood,R.A。(2009)。衣藻鞭毛膜及其动力学性质。 The Chlamydomonas Sourcebook 2 。 309-368。
  3. Bloodgood,R. A.(2012)。 纤毛和鞭毛膜研究的未来。 em> 23(13):2407-2411。
  4. Bloodgood,R.A。和May,G.S。(1982)。 衣藻表面的功能修饰 > J Cell Biol 93(1):88-
  5. Dentler,W.L。(1980)。 纤毛中的微管 - 膜相互作用。 I.来自四膜虫的睫状膜的分离和表征 J Cell Biol 84(2):364-380。
  6. Dentler,W.L。(1995)。 从四膜虫分离和分离睫状膜。 < em> Methods Cell Biol 47:397-400。
  7. * Dentler,W。(2013)。 膜在调节衣藻类睫毛长度方面的作用。 PLoS One 8(1):e53366。
  8. Harris,E.H。(1989)。生物学和实验室使用的综合指南。 The Chlamydomonas Sourcebook 。学术出版社
  9. Huang,K.,Diener,D.R.,Mitchell,A.,Pazour,G.J.,Witman,G.B。和Rosenbaum,J.L。(2007)。 羽毛衣藻(Chlamydomonas reinhardtii)中的PKD2的功能和动态。 flagella。 J Cell Biol 179(3):501-514
  10. Iomini,C.,Li,L.,Mo,W.,Dutcher,S.K.and Piperno,G。(2006)。 两个鞭毛基因,AGG2和AGG3,介导衣藻中的光照方向 。 Curr Biol 16(11):1147-1153。
  11. Kalshoven,H.,Musgrave,A。和Van den Ende,H。(1990)。 在衣藻属eugametos 配子的鞭毛膜中的配合受体复合物。 性植物繁殖 3(2):77-87
  12. McLean,R.J.,Laurendi,C.J。和Brown,R.M.,Jr。(1974)。 gamone与衣藻的交配反应的关系 Proc Natl Acad Sci USA 71(7):2610-2613。
  13. Pazour,G.J.and Bloodgood,R.A。(2008)。 将蛋白质靶向睫状膜。 Curr Top Dev Biol 85:115-149。
  14. Pasquale,S.M.and Goodenough,U.W。(1987)。 环AMP作为莱茵衣藻配子中的主要性信号。 J Cell Biol 105(5):2279-2292
  15. Sager,R。和Granick,S。(1953)。 使用衣藻属的营养研究。 Ann NY Acad Sci 56(5):831-838。
  16. Snell,W.J。(1976)。 在衣藻中的配合:用于研究特定细胞粘附的系统。 I.参考粘附的鞭毛表面组分的超微结构和电泳分析。细胞生物学杂志68(1):48-69。
  17. Wang,Q.and Snell,W.J。(2003)。 嫁接型加上和交配型之间的鞭毛粘附减少配子在受精期间激活鞭毛蛋白酪氨酸激酶
  18. Witman,G.B.,Carlson,K.,Berliner,J.and Rosenbaum,J.L。(1972)。 Chlamydomonas flagella 。 I.微管,基质,膜和mastigonemes的分离和电泳分析。细胞生物学54(3):507-539。
  19. Wood,C.R.,Huang,K.,Diener,D.R。和Rosenbaum,J.L。(2013)。 纤毛分泌生物活性蛋白。 Curr Biol 23 10):906-911。
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引用:Dentler, W. (2014). Ciliary and Flagellar Membrane Vesicle (Ectosome) Purification. Bio-protocol 4(12): e1156. DOI: 10.21769/BioProtoc.1156.
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