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Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays
分离盘基网柄菌的乳胶珠吞噬体用于体外功能分析   

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Abstract

We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction for in vitro motility. These assays allow us to probe how lipids on the phagosome membrane recruit and organize motors, and also measure the motion and force generation resulting from underlying lipid-motor interactions. This provides a unique opportunity to interrogate native-like organelles using biophysical and biochemical assays, and understand the role of motor proteins in phagosome maturation and pathogen clearance.

Keywords: Phagosome maturation(吞噬体成熟), Dynein(动力蛋白), Kinesin(驱动蛋白), in vitro motility(体外运动), Dictyostelium(盘基网柄菌)

Background

In vitro reconstitution of biological processes is important to understand the molecular components and mechanisms underlying them. One such process is phagosome maturation, which is involved in degradation of pathogens taken up by macrophage cells of the immune system, and is also used as a process of nutrition in lower eukaryotes (Vieira et al., 2002). The transport of phagosomes on microtubules is intimately connected to their maturation (Blocker et al., 1997; Vieira et al., 2002). Notably, several intracellular pathogens disrupt phagosome transport to survive in a latent form inside cells (Harrison et al., 2004; Harrison and Grinstein, 2002; Rai et al., 2016; Sun et al., 2007). Therefore, reconstitution of phagosome transport might help to understand the strategy used by pathogens for immune evasion. Here, we describe a detailed protocol to prepare LBPs from cell extracts of the social amoeba Dictyostelium discoideum. This protocol has been adapted and modified from the work by Gotthard et al. (2006). Briefly, the cells are pulsed with latex beads and chased for different time durations to enable either early or late phagosome formation. Such phagosomes are buoyant in nature and float away from other endogenous vesicles when spun at high speeds. These phagosomes, collected along with the cytosol, show robust motion on in vitro polymerized microtubules. A detailed version of this protocol has also been published elsewhere (Barak et al., 2014). Using this method, we have recently shown cholesterol as a key regulator of phagosome transport and maturation (Rai et al., 2016). Furthermore, this assay has helped us to elucidate the mechanism of disruption of phagosome transport by lipophosphoglycan (LPG) from the parasite Leishmania donovani. A description of the phagosome extract preparation from Dictyostelium cells is detailed below. This protocol describes only the purification of LBPs. The in vitro motility assay has been described elsewhere (Barak et al., 2014).

Materials and Reagents

  1. Glass coverslip
  2. 1.5 ml microfuge tube
  3. Preassembled Acrodisc® syringe filters for lysing cells (5 μm pore size, Supor® membrane 32 mm diameter) (Pall, catalog number: 4650 )
  4. Syringes of 1-2 ml capacity for lysis
  5. 1 ml syringe with needle (26 G) for collection of LBPs
  6. Dictyostelium discoideum AX-2 strain cells (dictyBase, catalog number: DBS0238585 ) (see Note 1)
  7. HL-5 medium for cell culture: HL-5 medium with glucose (ForMediumTM, catalog number: HLG0102 ) prepared according to manufacturer’s specifications (see Note 2)
  8. Polystyrene beads: carboxylated polystyrene beads of 750 nm diameter (Polysciences, catalog number: 07759-15 ) (see Note 4)
  9. Penicillin-streptomycin (Penstrep) (10,000 μg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  10. Protease inhibitor cocktail (cOmplete EDTA-free) (Roche Diagnostics, catalog number: 11836145001 )
  11. Liquid nitrogen for snap freezing
  12. Pepstatin A (MP Biomedical, catalog number: 2195368 )
  13. Methanol
  14. KH2PO4
  15. Na2HPO4
  16. Tris
  17. EGTA
  18. Sucrose
  19. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43819 )
  20. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 78830 )
  21. Benzamidine hydrochloride (Sigma-Aldrich, catalog number: 434760 )
  22. Sorensen’s buffer (see Recipes)
  23. Cell lysis buffer (see Recipes)
  24. Centrifugation cushion buffer (see Recipes)

Equipment

  1. Rotatory shaker
  2. Differential Interference Contrast (DIC) microscope (Nikon Instruments, model: TE2000U or similar)
  3. Cell culture microscope with 10x and 20x objective for observing and counting cells
  4. Water bath sonicator (Branson 1510MT ultrasonic cleaner, frequency 40 kHz, 10 min)
    Note: This product has been discontinued.
  5. Clinical centrifuge for pelleting cells
  6. Shaking incubator at 22 °C
  7. Autoclaved 500 ml conical flasks for shaking suspension culture
  8. Beckman Coulter centrifuge with JA-10 rotor 
    1. JA-10 rotor (Beckman Coulter, catalog number: 369687 )
    2. Jars (Beckman Coulter, catalog number: 355605 )
  9. Beckman Coulter table top ultracentrifuge with MLS-50 rotor
    1. MLS-50 rotor (Beckman Coulter, catalog number: 367280 )
    2. Ultra-Clear centrifuge tubes (Beckman Coulter, catalog number: 344057 )

Procedure

Note: Steps mentioned in this protocol should be performed on ice in a walk-in cold room at 4 °C (unless stated otherwise). In case of unavailability of such a room, all steps should be performed strictly on ice.

  1. Culturing of Dictyostelium cells
    Dictyostelium AX-2 cells are cultured in HL-5 suspension media with Penstrep (100 μg/ml working concentration) at 22 °C and 150 rpm in a shaking incubator (see Note 6). The optimal cell density for phagosome extract preparation is between 4-8 x 106 cells/ml. A 100 ml suspension culture (or 4-8 x 108 cells) is usually sufficient for one preparation (see Note 7).
  2. Before each preparation a small aliquot of cells from the culture (50 µl) is put on a glass coverslip to observe motility of organelles inside cells under a 100x objective of a differential interference contrast (DIC) microscope. A video for the intracellular motility is shown (Video 1). Cells with poor intracellular motility and/or excess vacuoles are under stress, and should not be used. Vacuoles are easily observable as large membranous structures inside the cells (Figure 1B).
    If cells appear healthy, before proceeding for the extraction procedure, it is required to perform the preparatory tasks as outlined in Note 3.

    Video 1. Intracellular motility of organelles in Dictyostelium. Cell suspension was placed on a clean glass coverslip, allowed to adhere to the surface for 3-5 min and then imaged under 100x objective of a DIC microscope. The movie was acquired at 30 frames/sec using a custom program written on LabVIEW and later reduced to 10 frames/sec. Movie runs in real-time. Organelles appear to move in a directed manner inside the cell. Robust in vivo motion of organelles (as observed in the video) is imperative for isolating highly motile LBPs.


    Figure 1. Comparison of healthy versus unhealthy Dictyostelium cells. Cell suspension from either an optimally dense (A) or highly dense (B) culture was placed on a clean glass coverslip, allowed to adhere to the surface for 3-5 min and then imaged under 100x objective of a DIC microscope. Stressed cells rarely adhere to the coverslip, appear spherical and show large vacuoles as opposed to healthy cells. Such cells also show highly diffusive intracellular motility and hence cannot be used for in vitro reconstitution experiments.

  3. Bead preparation
    The 750 nm bead stock solution mentioned in the materials section has a bead density of 1.08 x 1011 particles/ml. 200 µl of the bead stock solution is taken in a 1.5 ml microfuge tube and beads are pelleted by centrifuging at 10,000 x g for 5 min at 4 °C. The supernatant is discarded and the bead pellet is resuspended in 1 ml of HL-5 medium (see Note 8). This washing step is repeated once more and the final bead pellet is resuspended in 500 µl of Sorensen’s buffer. In order to avoid clumping of beads, they are sonicated in a sonicating water bath for 10 min and kept on ice until further use.
  4. Cells are collected by centrifuging the suspension culture twice in a 50 ml Falcon tube at 900 x g for 3 min at room temperature. The cell pellet is immediately stored on ice and resuspended in 5 ml of ice-cold Sorensen’s buffer.
  5. Synchronization
    The washed bead solution (500 µl) is added to the cells and the bead-cell suspension is incubated at 4 °C for 20 min with gentle shaking on a rotatory shaker (see Note 9).
  6. Pulse
    After synchronization, the bead-cell suspension is added to 100 ml of HL-5 medium kept in a 500 ml conical flask at 22 °C to initiate bead uptake. The incubation is done at 22 °C and 150 rpm in a shaking incubator. To isolate early phagosomes, a pulse duration of 5 min (see Note 10) is used. To isolate late phagosomes, the cells are pulsed with beads for 15 min.
  7. To stop the pulse, the 100 ml cell suspension is directly added to 330 ml of ice-cold Sorensen’s buffer kept in JA-10 centrifuge bottles. The cells are then pelleted by centrifuging them at 900 x g for 5 min at 4 °C in a Beckman JA-10 rotor.
  8. Chase
    The cell pellet is resuspended in 5 ml of ice-cold HL-5 medium and then added to 100 ml of HL-5 medium kept in a 500 ml conical flask at 22 °C to initiate the chase. For chase, the cells are incubated at 22 °C and 150 rpm in a shaking incubator. To isolate early phagosomes, no chase is needed and one can proceed directly to step 10 whereas to obtain late phagosomes, the cells are chased for 45 min to complete the process of phagosome maturation. Before chase period ends, we generally take 50 µl of cells on a coverslip to check whether the cells have phagocytosed beads and to ensure that LBPs are motile in vivo (Video 2).

    Video 2. Motility of LBPs in Dictyostelium. 50 µl of cell suspension after the chase step was placed on a clean glass coverslip, allowed to adhere to the surface for 3-5 min and then imaged under 100x objective of a DIC microscope. The movie was acquired at 30 frames/sec using a custom program written on LabVIEW and later reduced to 10 frames/sec. Movie runs in real time. Latex beads (now LBPs), being highly refractile, can be easily distinguished from other endogenous organelles and show robust motion in vivo.

  9. To stop the chase, the cell suspension is directly added to 330 ml of ice-cold Sorensen’s buffer kept in JA-10 centrifuge bottles. The cells are then pelleted by centrifuging them at 900 x g for 5 min at 4 °C in a Beckman JA-10 rotor.
  10. Removal of non-phagocytosed beads
    After chase (or for early phagosomes after pulse), the cell pellet is washed thrice with ice-cold Sorensen’s buffer to remove any non-phagocytosed beads. During each washing step, the cell pellet is resuspended in 50 ml of Sorensen’s buffer and centrifuged at 900 x g for 5 min at 4 °C (see Note 11).
  11. Cell lysis
    After the final washing step, the cell pellet is weighed and then resuspended in a 1:1 (w/v) ratio of lysis buffer (LB/30% sucrose with inhibitors added and kept on ice). The cell suspension is then lysed by one passage through a preassembled syringe filter containing a 5 µm pore size membrane. The cell lysate is collected in a microfuge tube and kept on ice. Lysis efficiency is examined on a coverslip using a 20x objective of the cell culture microscope (Figure 2). For optimal yield, 70-80% lysis of cells is desirable (see Note 12).


    Figure 2. Lysis of Dictyostelium cells using a 5 micron pre-assembled syringe filter. Cell suspension was spread on a clean coverslip before (A) and after (B) lysis and viewed under 20x objective of a negative phase contrast microscope. Intact cells appear dark (red arrowheads). Around 80-90% lysis is obtained using the preassembled filter.

  12. High speed centrifugation
    The cell lysate is then layered over a 1 ml cushion of LB/25% sucrose in an MLS-50 rotor tube and centrifuged in a Beckman MLS-50 rotor at 180,000 x g for 20 min at 4 °C (See Note 13). The LBPs, being more buoyant than other cell organelles and/or debris, do not pellet and are collected from the interface of the cell lysate and LB/25% sucrose as shown in Figures 3A and 3B. The top layer of the gradient is the cytosolic fraction. It is important to collect LBPs along with the cytosolic fraction otherwise their motility is compromised.
  13. Collection of LBPs
    A 1 ml syringe with needle is used to puncture the tube and phagosomes along with the cytosol are aspirated into the syringe (Figure 3B).


    Figure 3. Extraction of LBPs from cell lysate. A. Image showing separation of LBPs on sucrose gradient post centrifugation. Membrane components are generally pelleted at the bottom whereas the cytosol is present at the top layer. LBPs float at the 15-25% sucrose interphase; B. Procedure for collection of LBPs from the gradient interphase using a syringe needle. It is important to remove the needle while transferring LBPs from the syringe into the collection tube as shown in C.

  14. While transferring the aspirated solution from the syringe to a microfuge tube, it is important to remove the syringe needle (Figure 3C) otherwise the membrane might get disrupted.
  15. The phagosome/cytosol mixture is snap frozen in 36 µl aliquots in liquid nitrogen immediately after collection and stored in liquid nitrogen for up to a week. Each aliquot is rapidly thawed and used for one in vitro motility assay. Any unused thawed aliquot should be discarded.

Notes

  1. While ordering Dictyostelium cells, it is recommended to order them as spores plated on SM agar plate. Spores are more resistant to temperature fluctuations that might occur during transportation. The cells can easily be revived from the spores by picking and adding them to fresh HL-5 media kept at 22 °C.
  2. HL5 media is autoclaved at 120 °C, 20 psi pressure for 20 min. It is essential to remove the medium from the autoclave at the end of the 20 min. If left in the autoclave for prolonged duration, the media caramelizes (dark orange as opposed to a normal straw color) and is sub-optimal for cell culture usage.
  3. Following preparatory steps should be followed to perform the experiment in minimum amount of time:
    1. Keep centrifuges required at steps 7, 9, 10 and 12 at 4 °C.
    2. Dispense 100 ml HL5 media in a 500 ml conical flask (1 flask in case of early phagosome and 2 for late phagosomes) and pre-warm at 22 °C. 
    3. Dispense 330 ml 1x Sorensen’s buffer in a JA-10 jar and keep on ice (1 in case of early phagosome and 2 for late phagosomes).
  4. Bead stock solution should not be more than a year old. Phagosomes prepared from older bead stocks show reduced or no motility. Also, bead solution to be used for phagocytosis is prepared fresh before extraction.
  5. Stock solution of pepstatin A (1 mg/ml) is prepared in methanol. It is highly recommended not to add glacial acetic acid to the solution as it compromises the motility of LBPs.
  6. It is important that the cell culture temperature is always maintained at 22 °C. Motility inside Dictyostelium cells is highly sensitive to temperature and a change of even 2-3 °C from the optimum can disrupt motility.
  7. It is important to maintain the range of optimal cell density for the extraction protocol. Over-confluent cells show reduced motility whereas cell count below the lower limit often leads to difficulty during syringe lysis of cells. In case of lower cell density (3-4 x 106 cells/ml), two 100 ml suspension cultures can be used provided cells from both the cultures show robust intracellular motility. Using cells from an over-confluent suspension (density < 8 x 106 cells/ml) is not recommended since the cells will begin the developmental phase of their life cycle. In vitro motion has only been characterized for LBPs isolated from vegetatively growing cells.
  8. Bead washing with HL-5 medium serves a dual purpose: (a) It removes azide and other preservatives which are present in the bead stock solution. (b) It coats the beads with a layer of HL-5 medium facilitating the process of bead uptake by cells during the phagocytosis step. Skipping this washing step therefore would lead to suboptimal phagocytosis and compromise the yield. Also, azide in the stock solution could potentially kill phagosome motility.
  9. Synchronization step at 4 °C causes attachment of beads onto the cell surface but does not allow the initiation of phagocytosis. Thus, when phagocytosis is allowed to begin at 22 °C, it ensures a synchronized uptake of beads by cells. This is critical to isolate phagosomes in a stage-specific manner.
  10. It is crucial to strictly adhere to the pulse time duration, especially while isolating early phagosomes. In Dictyostelium, the early stage of phagosome maturation lasts for 5 min and any delay beyond this time point will cause a switch to late phagosome stage, which is also reflected in the phagosome motility.
  11. Improper washing of cells after pulse/chase leads to the presence of non-phagocytosed beads in the final phagosome extract. These beads cannot be distinguished from phagosomes under DIC microscope and are immotile during the motility assay. Hence, their presence can lead to a false conclusion of compromised phagosome motility.
  12. Cell lysis is the most crucial step in the extraction protocol. The success of this step is directly correlated with phagosome yield and the quality of phagosome motility. Therefore, it is imperative that this step be performed carefully.
  13. The total volume of the cushion and the cell lysate should at least be half of the maximum capacity of the ultra-centrifugation tube (for MLS-50, the capacity is 5 ml). Under circumstances when the above condition is not met, the cushion volume can be increased to 2 ml.

Recipes

  1. Sorensen’s buffer
    15 mM KH2PO4
    2 mM Na2HPO4
    Adjust pH to 6.0
    Note: This buffer is used for washing of cells to remove media and non-phagocytosed beads. We prepare a 10x stock of this buffer, which is autoclaved and stored at 4 °C. Before each extraction, 1 L of 1x buffer is prepared and chilled for an hour before usage.
  2. Cell lysis buffer (LB/30% sucrose)

    Note: All stock solutions are prepared in ddH2O water if not stated otherwise. Stock solutions of protease inhibitor cocktail, pepstatin A and DTT stored at -20 °C; PMSF and Benzamidine stocks are prepared fresh. All of these are added to the buffer prior to the cell lysis step. It is important to carefully monitor the pH of this buffer. Deviations from pH 8.0 are detrimental for phagosome motility.
  3. Centrifugation cushion buffer (LB/25% sucrose)
    Same composition as the cell lysis buffer except that it contains 25% (w/v) sucrose instead of 30% (w/v) sucrose 

Acknowledgments

We acknowledge funding through an International Senior Research Fellowship from the Wellcome Trust UK (grant WT079214MA). We also acknowledge funding from the Wellcome Trust – Department of Biotechnology Alliance, India (Senior Fellowship grant IA/S/11/2500255 to RM and Early career Fellowship grant IA/E/15/1/502298 to PS).

References

  1. Barak, P., Rai, A., Dubey, A. K., Rai, P. and Mallik, R. (2014). Reconstitution of microtubule-dependent organelle transport. Methods Enzymol 540: 231-248.
  2. Blocker, A., Severin, F. F., Burkhardt, J. K., Bingham, J. B., Yu, H., Olivo, J. C., Schroer, T. A., Hyman, A. A. and Griffiths, G. (1997). Molecular requirements for bi-directional movement of phagosomes along microtubules. J Cell Biol 137(1): 113-129.
  3. Gotthardt, D., Dieckmann, R., Blancheteau, V., Kistler, C., Reichardt, F. and Soldati, T. (2006). Preparation of intact, highly purified phagosomes from Dictyostelium. Methods Mol Biol 346: 439-448.
  4. Harrison, R. E., Brumell, J. H., Khandani, A., Bucci, C., Scott, C. C., Jiang, X., Finlay, B. B. and Grinstein, S. (2004). Salmonella impairs RILP recruitment to Rab7 during maturation of invasion vacuoles. Mol Biol Cell 15(7): 3146-3154.
  5. Harrison, R. E. and Grinstein, S. (2002). Phagocytosis and the microtubule cytoskeleton. Biochem Cell Biol 80(5): 509-515.
  6. Rai, A., Pathak, D., Thakur, S., Singh, S., Dubey, A. K. and Mallik, R. (2016). Dynein clusters into lipid microdomains on phagosomes to drive rapid transport toward lysosomes. Cell 164: 722-734.
  7. Sun, J., Deghmane, A. E., Soualhine, H., Hong, T., Bucci, C., Solodkin, A. and Hmama, Z. (2007). Mycobacterium bovis BCG disrupts the interaction of Rab7 with RILP contributing to inhibition of phagosome maturation. J Leukoc Biol 82(6): 1437-1445.
  8. Vieira, O. V., Botelho, R. J. and Grinstein, S. (2002). Phagosome maturation: aging gracefully. Biochem J 366(Pt 3): 689-704.

简介

芽顶端分生组织(SAM)是通过细胞分裂不断更新自身的细胞的集合,并且还向新发育的器官提供细胞。已知CLAVATA(CLV)3肽调节转录因子WUSCHEL(WUS)以保持未分化细胞的数量恒定并维持SAM的大小。在非细胞自主信号级联中的CLV3和WUS的交互反馈控制确定SAM中的干细胞命运(多能性的维持,或者,分化成子细胞)。 Ca 2 + 是在许多信号传导途径中起重要作用的第二信使。连接CLV3结合其受体和WUS表达的信号系统没有很好地描绘。我们显示Ca 2 + 参与SAM大小的CLV3调节。我们使用的方法之一是测量SAM的大小。在这里,我们提供了一个详细的协议,如何用Nomarski显微镜测量拟南芥SAM大小。将代表SAM的最大"面"的二维圆顶的面积用作SAM大小的代表。在存在和不存在Ca 2+通道阻断剂Gd 3+和sLV 3肽的情况下对野生型(WT)拟南芥进行研究。 ,以及缺乏功能性CLV3( clv3 )或编码Ca 2+ 2+导电离子通道的基因('dnd1 ')的基因型。
关键字: 拟南芥,射击顶端分生组织,苗发育,细胞信号,幼苗

> Nomarski显微镜广泛用于研究拟南芥SAM大小。用于SAM观察的其他显微技术是耗时的,并且需要将树脂嵌入树脂中,然后切片或甚至更复杂的显微镜。 Nomarski显微镜,连同组织清除技术是快速和方便的整个组织成像。通常使用Nomarski显微镜对SAM尺寸测量的发布方法进行简要描述。在这里,我们提供了一个修改的协议,详细的一步一步指南,包括解剖拟南芥SAM组织,通过样品制备的Nomarski显微镜和SAM大小测量。
...

关键字:吞噬体成熟, 动力蛋白, 驱动蛋白, 体外运动, 盘基网柄菌

材料和试剂

  1. 玻璃盖玻片
  2. 1.5 ml微量离心管
  3. 用于溶解细胞(5μm孔径,Supor膜直径32mm)(Pall,目录号:4650)的预装配Acrodisc 注射器过滤器
  4. 1-2毫升溶解容量的注射器
  5. 1 ml注射器用针(26 G)收集LBPs
  6. AX-2菌株细胞(dictyBase,目录号:DBS0238585)(参见注释1)
  7. 用于细胞培养的HL-5培养基:根据制造商的说明书(参见注释2)制备的具有葡萄糖的HL-5培养基(ForMedium TM ,目录号:HLG0102)
  8. 聚苯乙烯珠:750nm直径的羧化聚苯乙烯珠(Polysciences,目录号:07759-15)(参见注释4)
  9. 青霉素 - 链霉素(Penstrep)(10,000μg/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140-122)
  10. 蛋白酶抑制剂混合物(不含EDTA的完全)(Roche Diagnostics,目录号:11836145001)
  11. 用于快速冷冻的液氮
  12. 胃酶抑素A(MP Biomedical,目录号:2195368)
  13. 甲醇
  14. KH 2 PO 4
  15. Na HPO 4
  16. Tris
  17. EGTA
  18. 蔗糖
  19. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43819)
  20. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:78830)
  21. 苄脒盐酸盐(Sigma-Aldrich,目录号:434760)
  22. Sorensen的缓冲区(参见配方)
  23. 细胞裂解缓冲液(见配方)
  24. 离心缓冲垫(见配方)

设备

  1. 旋转振动器
  2. 差示干涉对比(DIC)显微镜(Nikon Instruments,型号:TE2000U或类似)
  3. 具有用于观察和计数细胞的10x和20x物镜的细胞培养显微镜
  4. 水浴超声器(Branson 1510MT超声波清洗器,频率40kHz,10分钟)
    注意:此产品已停产。
  5. 造粒细胞临床离心机
  6. 在22℃下振荡培养箱
  7. 高压灭菌的500ml锥形瓶用于摇动悬浮培养
  8. 带有JA-10转子的Beckman Coulter离心机
    1. JA-10转子(Beckman Coulter,目录号:369687)
    2. Jars(Beckman Coulter,目录号:355605)
  9. 带MLS-50转子的Beckman Coulter台式超速离心机
    1. MLS-50转子(Beckman Coulter,目录号:367280)
    2. Ultra-Clear离心管(Beckman Coulter,目录号:344057)

程序

注意:本协议中提到的步骤应在4℃的步入式冷室中在冰上进行(除非另有说明)。如果没有这样的房间,所有步骤都应严格执行。

  1. 培养盘基网柄菌细胞
    在含有Penstrep(100μg/ml工作浓度)的HL-5悬浮培养基中,在22℃和150rpm下,在摇动培养箱中培养AX-2细胞(参见注释6)。吞噬体提取物制备的最佳细胞密度为4-8×10 6个细胞/ml。 100ml悬浮培养物(或4-8×10 8个细胞)通常足以进行一次制备(参见注释7)。
  2. 在每个准备之前,从微量干涉对比(DIC)显微镜的100倍目标下,将来自培养物(50μl)的细胞的小等分试样放在玻璃盖玻片上以观察细胞内细胞器的运动性。显示了细胞内运动性的视频(视频1)。具有差的细胞内运动性和/或过多液泡的细胞处于应激下,不应该使用。在细胞内的大膜结构容易观察到空腔(图1B) 如果细胞看起来健康,在进行提取程序之前,需要执行注3中所述的准备任务。

    <! - flashid2056v137开始 - >
    视频1.细胞器在细胞器中的细胞内运动。 将细胞悬浮液置于干净的玻璃盖玻片上, 5分钟,然后在DIC显微镜的100倍物镜下成像。使用在LabVIEW上编写的定制程序以30帧/秒采集电影,之后减少到10帧/秒。电影实时运行。细胞器似乎在细胞内以定向的方式移动。体内细胞器的运动(如在视频中观察到的)是分离高度活动的LBP的必要条件。
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    图1.健康与不健康的盘基网柄细胞的比较。将来自最佳密度(A)或高度密集(B)培养物的细胞悬浮液置于干净的玻璃盖玻片上,允许粘附到表面3-5分钟,然后在DIC显微镜的100x物镜下成像。强调细胞很少粘附于盖玻片,呈现球形并显示大的空泡而不是健康的细胞。这样的细胞还显示高度扩散的细胞内运动性,因此不能用于体外重建实验。

  3. 珠粒准备
    材料部分中提到的750nm珠储备溶液具有1.08×10 11个颗粒/ml的珠密度。将200μl珠子储备溶液置于1.5ml微量离心管中,通过在4℃下以10,000×g离心5分钟使珠粒化。弃去上清液,将珠粒再悬浮于1ml HL-5培养基中(见注8)。再次重复该洗涤步骤,将最终的珠粒再悬浮于500μlSorensen缓冲液中。为了避免珠的结块,将它们在超声处理水浴中超声处理10分钟,并保持在冰上直到进一步使用。
  4. 通过将悬浮培养物在50ml Falcon管中在900×g下在室温下离心3分钟来收集细胞。将细胞沉淀立即储存在冰上并重悬于5ml冰冷的Sorensen缓冲液中
  5. 同步
    将洗过的珠子溶液(500μl)加入到细胞中,并将珠子细胞悬浮液在旋转振荡器上轻轻摇动(见注9),在4℃温育20分钟。
  6. 脉冲
    同步后,将珠细胞悬浮液加入到保持在500ml锥形瓶中的100ml HL-5培养基中,在22℃下启动珠吸收。在22℃和150rpm下在振荡培养箱中进行孵育。为了分离早期吞噬体,使用5分钟的脉冲持续时间(参见注释10)。为了分离晚期吞噬体,用珠子将细胞脉冲15分钟
  7. 为了停止脉冲,将100ml细胞悬浮液直接加入保持在JA-10离心瓶中的330ml冰冷的Sorensen缓冲液中。然后通过在Beckman JA-10转子中在4℃下以900×g离心5分钟使细胞沉淀。
  8. Chase
    将细胞沉淀重悬于5ml冰冷的HL-5培养基中,然后加入到保持在500ml锥形瓶中的100mlHL-5培养基中,在22℃下启动追踪。对于追踪,将细胞在22℃和150rpm下在振荡培养箱中孵育。为了分离早期吞噬体,不需要追踪,并且可以直接进行步骤10,而获得晚期吞噬体,追踪细胞45分钟以完成吞噬体成熟的过程。在追逐期结束之前,我们通常取盖玻片上的50微升细胞,以检查细胞是否具有吞噬珠,并确保LBPs在体内运动(视频2)。

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    视频2.在盘基网柄中的LBPs的运动性。 将追加步骤后的50μl细胞悬浮液置于干净的玻璃盖玻片上,使其粘附于表面3-5分钟,然后在DIC显微镜的100倍物镜下成像。使用在LabVIEW上编写的定制程序以30帧/秒采集电影,之后减少到10帧/秒。电影实时运行。高度折射的乳胶珠(现在的LBP)可以容易地与其它内源性细胞器区分开,并在体内显示出强的运动。
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  9. 为了停止追踪,将细胞悬浮液直接加入保存在JA-10离心瓶中的330ml冰冷的Sorensen缓冲液中。然后通过在Beckman JA-10转子中在4℃下以900×g离心5分钟使细胞沉淀。
  10. 去除非吞噬珠子
    追踪后(或对于脉冲后的早期吞噬体),将细胞沉淀用冰冷的Sorensen缓冲液洗涤三次以除去任何未吞噬的珠。在每个洗涤步骤期间,将细胞沉淀重悬于50ml Sorensen缓冲液中,并在4℃下以900×g离心5分钟(参见注释11)。
  11. 细胞裂解
    在最后的洗涤步骤后,称重细胞沉淀,然后以1:1(w/v)比例的裂解缓冲液(LB/30%蔗糖,加入抑制剂并保存在冰上)重悬。然后通过穿过含有5μm孔径膜的预装配的注射器过滤器的一次通道裂解细胞悬浮液。将细胞裂解物收集在微量离心管中并保存在冰上。使用细胞培养显微镜的20x物镜在盖玻片上检查裂解效率(图2)。为了获得最佳产量,需要70-80%的细胞裂解(参见注释12)

    图2.使用5微米预组装的注射器过滤器裂解盘基网柄细胞。在(A)和(B)裂解之前将细胞悬浮液涂布在干净的盖玻片上,在负相位差显微镜的20x物镜下观察。完整细胞呈现黑色(红色箭头)。使用预装过滤器可获得约80-90%的裂解。

  12. 高速离心
    然后将细胞裂解物在MLS-50转子管中在1ml LB/25%蔗糖垫上分层,并在Beckman MLS-50转子中以180,000×g在4℃下离心20分钟(见附注13)。如图3A和3B所示,比其它细胞器和/或碎片更具浮力的LBP不沉淀并从细胞裂解物和LB/25%蔗糖的界面收集。梯度的顶层是胞质部分。重要的是收集LBP以及胞浆部分,否则它们的运动性受损
  13. LBP的收集
    使用具有针的1ml注射器刺穿管,并且将吞噬体与细胞质一起吸入注射器中(图3B)。


    图3.从细胞裂解物中提取LBP A.图像显示离心后蔗糖梯度上LBP的分离。膜组分通常在底部沉淀,而细胞溶胶存在于顶层。 LBPs漂浮在15-25%蔗糖相间; B.使用注射器针从梯度界面收集LBP的程序。重要的是,在将LBP从注射器转移到收集管中时取出针头,如C所示
  14. 当将吸出的溶液从注射器转移到微量离心管时,重要的是取出注射器针头(图3C),否则膜可能被破坏。
  15. 吞噬体/细胞质混合物在收集后立即在液氮中快速冷冻在36μl等分试样中,并在液氮中储存长达一周。将每个等分试样快速解冻并用于一次体外移动试验。任何未使用的解冻的等分试样应该被丢弃。

笔记

  1. 在订购盘基网柄细胞时,建议订购它们作为在SM琼脂平板上接种的孢子。孢子对运输过程中可能发生的温度波动更具抵抗力。通过挑选并添加到保持在22℃的新鲜HL-5培养基中,可以容易地从孢子中恢复细胞。
  2. 将HL5培养基在120℃,20psi压力下高压灭菌20分钟。必须在20分钟结束时从高压釜中除去介质。如果长时间放在高压灭菌器中,介质焦糖化(深橙色,而不是正常的稻草色),并且不适合细胞培养。
  3. 遵循以下准备步骤,在最短的时间内进行实验:
    1. 步骤7,9,10和12需要在4°C下保持离心机
    2. 在500ml锥形瓶中分配100ml HL5培养基(1个早期吞噬体的培养瓶,2个晚期吞噬体),并在22℃预热。
    3. 在JA-10瓶中分配330ml 1x Sorensen缓冲液,并保持在冰上(1是早期吞噬体,1是晚期吞噬体)。
  4. 珠母液不应超过一年。从更大的珠股制备的吞噬体显示降低的或没有运动性。此外,用于吞噬的珠溶液在提取之前是新鲜制备的
  5. 胃酶抑素A(1mg/ml)的储备溶液在甲醇中制备。强烈建议不要向溶液中加入冰醋酸,因为它会损害LBP的运动性
  6. 重要的是细胞培养温度始终保持在22℃。盘基网柄内的运动对温度高度敏感,甚至2-3°C的最佳变化会破坏运动性。
  7. 重要的是保持提取方案的最佳细胞密度的范围。过度融合的细胞显示降低的运动性,而细胞计数低于下限通常导致细胞注射器溶解期间的困难。在较低的细胞密度(3-4×10 6个细胞/ml)的情况下,可以使用两种100ml悬浮培养物,只要来自两种培养物的细胞显示强的细胞内运动性。不推荐使用来自过度铺满悬浮液的细胞(密度<8×10 6细胞/ml),因为细胞将开始其生命周期的发育阶段。 体外运动仅针对从营养生长细胞分离的LBP进行表征。
  8. 用HL-5培养基洗涤珠子具有双重目的:(a)除去存在于珠子储备溶液中的叠氮化物和其它防腐剂。 (b)它用一层HL-5培养基涂覆珠,促进吞噬作用步骤期间细胞吸收珠的过程。因此跳过这个洗涤步骤将导致次优的吞噬作用并损害产量。此外,储备溶液中的叠氮化物可能潜在地杀死吞噬体运动性
  9. 在4℃的同步步骤导致珠附着到细胞表面上,但是不允许吞噬开始。因此,当允许吞噬作用在22℃开始时,其确保细胞同时吸收珠子。这对于以阶段特异性方式分离吞噬体是至关重要的
  10. 严格遵守脉搏持续时间是至关重要的,特别是在分离早期吞噬体时。在盘基网柄中,吞噬体成熟的早期阶段持续5分钟,并且超过该时间点的任何延迟将导致切换到晚期吞噬体阶段,这也反映在吞噬体运动性中。
  11. 在脉冲/追踪后不适当地洗涤细胞导致最终吞噬体提取物中存在非吞噬珠。这些珠不能在DIC显微镜下与吞噬体区分开,并且在运动性测定期间是immotile的。因此,它们的存在可导致吞噬体运动性受损的假结论
  12. 细胞裂解是提取方案中最关键的步骤。这一步的成功与吞噬体产量和吞噬体活动的质量直接相关。因此,必须小心执行此步骤。
  13. 衬垫和细胞裂解物的总体积应该至少是超离心管的最大容量的一半(对于MLS-50,容量为5ml)。在不满足上述条件的情况下,衬垫体积可以增加到2ml

食谱

  1. Sorensen的缓冲区
    15mM KH 2 PO 4 sub/
    2mM Na 2 HPO 4
    将pH调节至6.0
    注意:该缓冲液用于洗涤细胞以除去培养基和非吞噬珠。我们准备10x这种缓冲液的原液,将其高压灭菌并储存在4℃。在每次提取之前,制备1L 1x缓冲液,并在使用前冷冻1小时。
  2. 细胞裂解缓冲液(LB/30%蔗糖)

    注意:如果没有另外说明,所有储备溶液在ddH 2 O水中制备。蛋白酶抑制剂混合物,胃酶抑素A和DTT储存在-20℃的储备溶液;新鲜制备PMSF和苄脒储备液。所有这些在细胞裂解步骤之前加入到缓冲液中。仔细监测该缓冲液的pH是重要的。与pH 8.0的偏差对吞噬体运动性是有害的。
  3. 离心缓冲缓冲液(LB/25%蔗糖)
    除了含有25%(w/v)蔗糖而不是30%(w/v)蔗糖之外,与细胞裂解缓冲液具有相同的组成。

致谢

我们通过来自英国Wellcome Trust的国际高级研究奖学金(拨款WT0792​​14MA)认可资助。我们还确认来自惠康信托 - 印度生物技术联盟部(高级研究金资助IA/S/11/2500255到RM和早期职业奖学金资助IA/E/15/1/502298到PS)的资金。

参考文献

  1. Barak,P.,Rai,A.,Dubey,AK,Rai,P.and Mallik,R。(2014)。  Reconstitution of microtubule-dependent organelle transport。 Methods Enzymol 540:231-248。
  2. Blocker,A.,Severin,FF,Burkhardt,JK,Bingham,JB,Yu,H.,Olivo,JC,Schroer,TA,Hyman,AA和Griffiths,G。(1997)。< a class ="ke -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/9105041"target ="_ blank">吞噬体沿微管的双向运动的分子需求。 Cell Biol 137(1):113-129
  3. Gotthardt,D.,Dieckmann,R.,Blancheteau,V.,Kistler,C.,Reichardt,F.and Soldati,T。(2006)。  从盘基网柄中制备完整的,高度纯化的吞噬体。 346:439-448。
  4. Harrison,RE,Brumell,JH,Khandani,A.,Bucci,C.,Scott,CC,Jiang,X.,Finlay,BB和Grinstein,S。(2004)。  沙门氏菌损害RILP在入侵液泡成熟期间向Rab7募集。 Mol Biol Cell 15(7):3146-3154。
  5. Harrison,RE和Grinstein,S.(2002)。  吞噬作用和微管细胞骨架。生化细胞生物学 80(5):509-515。
  6. Rai,A.,Pathak,D.,Thakur,S.,Singh,S.,Dubey,AK和Mallik,R。(2016)。  Dynein簇到吞噬体上的脂质微结构域中以驱动快速转运到溶酶体。 164: 734.
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引用:D’Souza, A., Sanghavi, P., Rai, A., Pathak, D. and Mallik, R. (2016). Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays. Bio-protocol 6(23): e2056. DOI: 10.21769/BioProtoc.2056.
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