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Isolation of Growth Cones from Mouse Brain
从小鼠脑部分离神经元生长锥   

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Abstract

Growth cones are motile structures at the tips of growing neurites, which play an essential role in regulation of growth and navigation of growing axons and dendrites of neurons in the developing nervous system. This protocol describes isolation of growth cones from the brain tissue from young mice. Growth cones isolated using this protocol have been extensively characterized using electron microscopy (Pfenninger et al., 1983) and may be used for any kind of subsequent biochemical and/or functional analyses, including Western blot analysis of protein expression (Westphal et al., 2010), analysis of the activity of growth cone-accumulated enzymes (Leshchyns’ka et al., 2003; Li et al., 2013), and analysis of the endocytosis and exocytosis rates (Chernyshova et al., 2011).

Keywords: Neurons(神经元), Growth cone(生长锥), Neurite outgrowth(轴突生长), Differential centrifugation(差速离心), Biochemical analysis(生化分析)

Materials and Reagents

  1. Mouse brains extracted from 1-3 days old mice, frozen in liquid nitrogen and kept at -80 °C (for up to 1 year)
  2. Sucrose
  3. Purified (e.g. using Milli-Q system from Millipore) water kept at 4 °C
  4. Mini EDTA-free Protease Inhibitor Cocktail Tablets (Roche Applied Science, catalog number: 05892791001 )
  5. PMSF (Sigma-Aldrich, catalog number: P7626 )
  6. Ethanol
  7. 80% sucrose (see Recipes)
  8. Homogenization buffer (see Recipes)
  9. 0.75 M sucrose buffer (see Recipes)
  10. 1 M sucrose buffer (see Recipes)
  11. 2.33 M sucrose buffer (see Recipes)

Equipment

  1. Potter homogenizer (Thermo Fisher Scientific, catalog number: 08-414-14A )
  2. 1 ml plastic pipette (Sarstedt, model: 86.1180 )
  3. Bench top centrifuge with an angle rotor, for example Allegra X-15R (Beckman Coulter)
  4. Ultracentrifuge with a swing rotor, for example L-60 ultracentrifuge with SW40Ti rotor (Beckman) or HIMAC CP100WX ultracentrifuge with P40ST rotor (Hitachi)
  5. Centrifuge tube 13PA (Hitachi, catalog number: 332901A )

Procedure

  1. Prepare 80% sucrose in advance and keep it at 4 °C.
  2. Prepare buffers for homogenization and centrifugation immediately before preparation of growth cones and place them on ice.
  3. Take 10 brains from the -80 °C freezer and place them on ice. Proceed immediately to the next step.
  4. Transfer brains to the Potter homogenizer and add homogenization buffer. Use 1 ml of buffer for homogenization per 1 brain.
  5. Homogenize brains.
    Note: If you isolate growth cones from several experimental groups, use the same numbers of strokes to homogenize brains in each group.
  6. Centrifuge homogenate at 1,660 x g for 15 min at 4 °C using the bench top centrifuge.
  7. Collect the supernatant.
  8. Prepare a discontinuous 0.75/1.0/2.33 M sucrose density gradient. To prepare the gradient, carefully pipette into a centrifuge tube 1 ml of ice cold 2.33 M sucrose buffer (bottom), then 3 ml of ice cold 1.0 M sucrose buffer, and then 4 ml of ice cold 0.75 M (top) sucrose buffer. Volumes are given for the centrifuge tube 13PA. To prevent intermixing of the sucrose layers during the gradient preparation, tilt the tube and place the tip of the pipette against the wall of the tube at the top of the tube (Figure 1). Release the solutions to the tube slowly.   

     
    Figure 1. Position of the centrifuge tube and pipette during formation of a sucrose gradient

  9. Load the supernatant on top of the gradient and centrifuge at 242,000 x g for 60 min at 4 °C.
  10. Collect the growth cone enriched fraction at the interface between the load and 0.75 M sucrose (Figure 2A). The growth cone depleted fraction can be collected between 0.75 M and 1.0 M sucrose (Figure 2A) and may be used as non-growth cone membranes. To collect the growth cone-enriched fraction, squeeze the bulb of a 1 ml plastic pipette, then carefully place the tip of the pipette into the layer of the sucrose gradient containing growth cones (Figure 2B), and then slowly release the bulb of the pipette to allow the growth cone-containing solution to flow into the pipette. Then carefully remove the pipette containing growth cones from the centrifuge tube and release the growth cone fraction into a clean centrifuge tube. Repeat if required.

     
    Figure 2. Collection of the growth cone enriched fraction from a sucrose gradient. A: Distribution of the sucrose layers and the interfaces containing fractions enriched in growth cones and non-growth cone membranes in the centrifuge tube after the centrifugation. B: Position of the tip of a plastic pipette during collection of the growth cone-enriched fraction.
     
  11. Re-suspend the growth cone fraction in the buffer for homogenization by adding this buffer to the tube to fill it to capacity. Centrifuge at 100,000 x g for 40 min at 4 °C.
  12. Collect the pellet containing growth cones, re-suspend it in 50 μl of the homogenization buffer and freeze at -80 °C. Important, the homogenization buffer has to contain protease inhibitors as described in the section Recipes. Growth cones thus obtained can be stored at -80 °C for up to 1 week for Western blot analysis. Growth cones for functional analyses (e.g. analysis of exo- and endocytosis) have to be used immediately and cannot be frozen.
    Note: To check the growth cone isolation efficiency, the growth cone enriched fraction can be analyzed by Western blot. The growth cone fraction have to be enriched in growth-associated protein (GAP-43) and the neural cell adhesion molecule (NCAM) when compared to brain homogenates and non-growth cone membranes. Non-growth cone membranes, which also contain Golgi membranes, have to be enriched in Golgi matrix protein GM130, while the growth cone fraction should have only low levels of this protein due to the presence of Golgi-derived vesicles in growth cones.

Recipes

  1. 80% sucrose
    For 500 ml of the solution
    Weigh 400 g of sucrose in a clean 500 ml Erlenmeyer flask.
    Fill the flask with water up to a 500 ml mark.
    Put the flask on a magnetic stirrer hotplate and mix with a magnetic stirrer until the sucrose is dissolved. You may heat the solution up to 50 °C to increase the sucrose solubility.
    The solution can be stored at 4 °C for up to 1 month.  
  2. Homogenization buffer
    5 mM Tris-HCl
    0.32 M sucrose
    1 mM MgCl2
    For 100 ml of buffer
    13.6 ml of 80% sucrose
    0.1 ml of 1 M MgCl2
    0.5 ml of 1 M Tris-HCl buffer (pH 7.4)
    85.8 ml of water
    Mix well and keep on ice. Add protease inhibitors just before usage. Use one tablet of the EDTA-free protease inhibitor cocktail and 10 μl of 1 mM PMSF for 10 ml of buffer. Tablets require some time to be dissolved.  
  3. 0.75 M sucrose
    For 100 ml of buffer
    32 ml of 80% sucrose
    0.1 ml of 1 M MgCl2
    0.5 ml of 1 M Tris-HCl buffer (pH 7.4)
    67.4 ml of water
    Mix well and keep on ice.
  4. 1 M sucrose
    For 100 ml of buffer
    42.7 ml of 80% sucrose
    0.1 ml of 1 M MgCl2
    0.5 ml of 1 M Tris-HCl buffer (pH 7.4)
    56.7 ml of water
    Mix well and keep on ice.
  5. 2.33 M sucrose
    For 100 ml of buffer
    99.4 ml of 80% sucrose
    0.1 ml of 1 M MgCl2
    0.5 ml of 1 M Tris-HCl buffer (pH 7.4)
    Mix well and keep on ice.

References

  1. Chernyshova, Y., Leshchyns'ka, I., Hsu, S. C., Schachner, M. and Sytnyk, V. (2011). The neural cell adhesion molecule promotes FGFR-dependent phosphorylation and membrane targeting of the exocyst complex to induce exocytosis in growth cones. J Neurosci 31(10): 3522-3535.
  2. Leshchyns'ka, I., Sytnyk, V., Morrow, J. S. and Schachner, M. (2003). Neural cell adhesion molecule (NCAM) association with PKCbeta2 via betaI spectrin is implicated in NCAM-mediated neurite outgrowth. J Cell Biol 161(3): 625-639.
  3. Li, S., Leshchyns'ka, I., Chernyshova, Y., Schachner, M. and Sytnyk, V. (2013). The neural cell adhesion molecule (NCAM) associates with and signals through p21-activated kinase 1 (Pak1). J Neurosci 33(2): 790-803.
  4. Pfenninger, K. H., Ellis, L., Johnson, M. P., Friedman, L. B. and Somlo, S. (1983). Nerve growth cones isolated from fetal rat brain: subcellular fractionation and characterization. Cell 35(2 Pt 1): 573-584.
  5. Westphal, D., Sytnyk, V., Schachner, M. and Leshchyns'ka, I. (2010). Clustering of the neural cell adhesion molecule (NCAM) at the neuronal cell surface induces caspase-8- and -3-dependent changes of the spectrin meshwork required for NCAM-mediated neurite outgrowth. J Biol Chem 285(53): 42046-42057.

简介

生长锥是生长神经突的尖端的运动结构,其在调节发育中的神经系统中生长的轴突和神经元的树突的生长和导航中发挥重要作用。 该方案描述了来自幼鼠的脑组织的生长锥的分离。 使用该方案分离的生长锥已经使用电子显微镜广泛地表征(Pfenninger等人,1983),并且可以用于任何种类的随后的生物化学和/或功能分析,包括蛋白质表达的蛋白质印迹分析(Westphal et al。 2010),分析生长锥积累酶的活性(Leshchyns'ka et al。,2003; Li et al。,2013),以及内吞作用和胞吐作用率的分析(Chernyshova等,2011)。

关键字:神经元, 生长锥, 轴突生长, 差速离心, 生化分析

材料和试剂

  1. 从1-3天龄小鼠中提取的小鼠脑,在液氮中冷冻并保持在-80℃(至多1年)
  2. 蔗糖
  3. 纯化(例如使用Millipore的Milli-Q系统)保持在4℃的水
  4. 微型无EDTA蛋白酶抑制剂混合片(Roche Applied Science,目录号:05892791001)
  5. PMSF(Sigma-Aldrich,目录号:P7626)
  6. 乙醇
  7. 80%蔗糖(见配方)
  8. 均质缓冲液(参见配方)
  9. 0.75M蔗糖缓冲液(见配方)
  10. 1M蔗糖缓冲液(见配方)
  11. 2.33 M蔗糖缓冲液(见配方)

设备

  1. Potter匀浆器(Thermo Fisher Scientific,目录号:08-414-14A)
  2. 1ml塑料移液管(Sarstedt,型号:86.1180)
  3. 具有角转子的台式离心机,例如Allegra X-15R(Beckman Coulter)
  4. 具有摆动转子的超速离心机,例如具有SW40Ti转子(Beckman)的L-60超速离心机或具有P40ST转子(Hitachi)的HIMAC CP100WX超速离心机
  5. 离心管13PA(日立,目录号:332901A)

程序

  1. 提前准备80%蔗糖,并保持在4℃
  2. 在制备生长锥之前立即制备用于匀浆和离心的缓冲液,并将其置于冰上
  3. 从-80°C冰箱取10个大脑,并将它们放在冰上。 立即进行下一步。
  4. 将大脑转移到Potter匀浆器中并加入匀浆缓冲液。 每1只大脑使用1ml缓冲液匀浆
  5. 均匀化大脑。
    注意:如果你隔离几个实验组的生长锥,使用相同数量的笔划来均化每个组中的大脑。
  6. 使用台式离心机在4℃下在1660×g离心匀浆15分钟。
  7. 收集上清液。
  8. 制备不连续的0.75/1.0/2.33 M蔗糖密度梯度。为了制备梯度,小心地移液管离心管1ml冰冷的2.33M蔗糖缓冲液(底部),然后3ml冰冷的1.0M蔗糖缓冲液,然后4ml冰冷的0.75M(顶部)蔗糖缓冲液。为离心管13PA给出体积。为了防止在梯度制备期间蔗糖层的混合,倾斜管并且将移液管的尖端抵靠在管顶部的管壁上(图1)。缓慢释放溶液到管中。  

     
    F igure 1. rifuge管和移液器在蔗糖梯度形成期间的位置

  9. 将上清液加载到梯度的顶部,并在4℃下以242,000×g离心60分钟。
  10. 收集生长锥富集部分在负载和0.75 M蔗糖之间的界面(图2A)。可以在0.75M和1.0M蔗糖之间收集生长锥消耗的部分(图2A),并且可以用作非生长锥膜。为了收集生长锥富集部分,挤压1ml塑料吸管的灯泡,然后小心地将吸管的尖端放入含有生长锥的蔗糖梯度层(图2B),然后缓慢释放移液管以允许含生长锥的溶液流入移液管。然后小心地从离心管中取出含有生长锥的移液管,并将生长锥组分释放到干净的离心管中。如果需要,重复。

     
    图2.来自蔗糖梯度的生长锥富集级分的收集 A:在离心管中蔗糖层和富含生长锥和非生长锥膜的含有级分的界面的分布,离心。 B:在收集生长锥富集级分期间塑料移液管的尖端的位置。
     
  11. 通过将该缓冲液加入到管中以将其填充至容量,将缓冲液中的生长锥部分再悬浮用于匀浆。在4℃下以100,000×g离心40分钟。
  12. 收集含有生长锥的沉淀,将其重悬在50μl的匀浆缓冲液中并在-80℃下冷冻。重要的是,匀浆缓冲液必须含有蛋白酶抑制剂,如"食谱"一节中所述。由此获得的生长锥可以在-80℃储存长达1周用于Western印迹分析。必须立即使用用于功能分析(例如外泌和胞吞作用的分析)的生长锥,并且不能冷冻。
    注意:为了检查生长锥分离效率,可以通过Western印迹分析生长锥富集的部分。当与脑匀浆和非生长锥膜相比时,生长锥部分必须富集生长相关蛋白(GAP-43)和神经细胞粘附分子(NCAM)。还包含高尔基体膜的非生长锥膜必须富含高尔基体基质蛋白GM130,而生长锥部分应当仅具有低水平的该蛋白,这是由于在生长锥中存在高尔基体衍生的囊泡。 em>

食谱

  1. 80%蔗糖 对于500ml溶液
    在干净的500ml锥形瓶中称取400g蔗糖 向烧瓶中加水至500毫升。
    将烧瓶置于磁力搅拌器加热板上,并用磁力搅拌器混合,直到蔗糖溶解。您可以将溶液加热至50°C以提高蔗糖溶解度。
    溶液可以在4℃下储存长达1个月。  
  2. 均匀化缓冲液
    5mM Tris-HCl
    0.32 M蔗糖 1mM MgCl 2
    对于100毫升的缓冲区
    13.6ml 80%蔗糖 0.1ml的1M MgCl 2溶液 0.5ml的1M Tris-HCl缓冲液(pH7.4) 85.8ml水
    混合好,并保持在冰上。 在使用前添加蛋白酶抑制剂。 使用一片无EDTA的蛋白酶抑制剂混合物和10μl的1mM PMSF用于10ml缓冲液。 片剂需要一些时间才能溶解。  
  3. 0.75 M蔗糖 对于100毫升的缓冲区
    32ml 80%蔗糖 0.1ml的1M MgCl 2溶液 0.5ml的1M Tris-HCl缓冲液(pH7.4) 67.4ml水
    充分混合并保持冰。
  4. 1 M蔗糖
    对于100毫升的缓冲区
    42.7ml 80%蔗糖 0.1ml的1M MgCl 2溶液 0.5ml的1M Tris-HCl缓冲液(pH7.4) 56.7ml水
    充分混合并保持冰。
  5. 2.33 M蔗糖
    对于100毫升的缓冲区
    99.4ml 80%蔗糖 0.1ml的1M MgCl 2溶液 0.5ml的1M Tris-HCl缓冲液(pH7.4) 充分混合并保持冰。

参考文献

  1. Chernyshova,Y.,Leshchyns'ka,I.,Hsu,S.C.,Schachner,M.and Sytnyk,V。(2011)。 神经细胞粘附分子促进FGFR依赖性磷酸化和膜靶向的exocyst复合物诱导胞吐生长锥。 J Neurosci 31(10):3522-3535。
  2. Leshchyns'ka,I.,Sytnyk,V.,Morrow,J.S。和Schachner,M。(2003)。 神经细胞粘附分子(NCAM)与PKCbeta2通过betaI血影的关联牵连NCAM介导的神经突生长。 J Cell Biol 161(3):625-639
  3. Li,S.,Leshchyns'ka,I.,Chernyshova,Y.,Schachner,M。和Sytnyk,V。(2013)。 神经细胞粘附分子(NCAM)通过p21激活的激酶1(Pak1)结合并发出信号, 。 J Neurosci 33(2):790-803。
  4. Pfenninger,K.H.,Ellis,L.,Johnson,M.P.,Friedman,L.B.and Somlo,S。(1983)。 从胎鼠大脑分离的神经生长锥:亚细胞分离和表征。 35(2 Pt 1):573-584。
  5. Westphal,D.,Sytnyk,V.,Schachner,M。和Leshchyns'ka,I.(2010)。 神经细胞粘附分子(NCAM)在神经元细胞表面的聚类诱导胱天蛋白酶-8和 -3依赖性的变化所需的NCAM介导的神经突生长。 J Biol Chem 285(53):42046-42057
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Leshchyns’ka, I. and Sytnyk, V. (2013). Isolation of Growth Cones from Mouse Brain. Bio-protocol 3(15): e853. DOI: 10.21769/BioProtoc.853.
  2. Li, S., Leshchyns'ka, I., Chernyshova, Y., Schachner, M. and Sytnyk, V. (2013). The neural cell adhesion molecule (NCAM) associates with and signals through p21-activated kinase 1 (Pak1). J Neurosci 33(2): 790-803.
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