Density Gradient Centrifugation for Enrichment and Identification of GFP-tagged Chitosomal Microvesicles of Filamentous Fungi

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Molecular Microbiology
Feb 2015



Density gradient centrifugation has been utilized to characterize the subcellular distribution of physiologically relevant enzymes in yeasts and filamentous fungi (Leal-Morales et al., 1988; Martínez et al., 1989; Kamada et al., 1991). This approach is now potentiated by protein tagging and live imaging techniques, which make possible to relate a single protein with, for example, a discrete population of intracellular vesicles and their in vivo dynamics (Verdín et al., 2009; Fajardo-Somera et al., 2013; Sánchez-León et al., 2015). Here, we describe the density gradient centrifugation and fractionation analysis of cell-free homogenates of a Neurospora crassa (N. crassa) strain that expresses CHS-6 chitin synthase fused to the green fluorescent protein (Riquelme et al., 2007).

Keywords: Neurospora crassa (粗糙脉孢菌), Chitosomes (chitosomes), Vesicles (囊泡), Fractionation (分馏)

Materials and Reagents

  1. Tygon R-3603 tubing, I.D. 1/16 inch (Saint-Gobain, catalog number: AAC00002 )
  2. Grade 1 Qualitative Filter Papers (GE Healthcare, catalog number: 1001-090 )
  3. Neurospora crassa conidia
  4. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  5. Sodium phosphate monobasic (NaH2PO4.H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S369-500 )
  6. Sodium phosphate dibasic anhydrous (Na2HPO4) (Sigma-Aldrich, catalog number: S-7907 )
  7. Protease Inhibitor Cocktail, Complete ULTRA tablets EDTA-free (Roche Diagnostics, catalog number: 04693132001 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 04693132001 ”.
  8. Glass beads (500 μm) (Bio Spec Product, catalog number: 11079105 )
  9. 10% (w/v) sucrose in 33 mM (final) phosphate buffer (pH 8.2) (steam sterilize and store at 4 °C until use)
  10. 65% (w/v) sucrose in 33 mM (final) phosphate buffer (pH 8.2) (steam sterilize and store at 4 °C until use)
  11. D-Biotin (Faga Labs, catalog number: CAS-58-85-5 )
  12. Vogel’s complete medium (see Recipes)
  13. 50x salt solution (see Recipes)
  14. Trace element solution (see Recipes)
  15. 100 mM phosphate buffer (pH 8.2) (see Recipes)
  16. Laemmli buffer (see Recipes)


  1. Braun MSK 50 ml shaking bottles (LABEQUIP LTD, catalog number: 8541302 )
  2. VWR® Standard Hot Plate Stirrers (VWR International, catalog number: 12365-382 )
  3. Quick-seal centrifuge tube (Beckman Coulter, catalog number: 344326 )
  4. Shaker incubator (Lab-LineX, model: Orbit Environ Shaker )
  5. Vacuum filtration system
  6. Braun MSK cell homogenizer (LABEQUIP LTD, catalog number: 953030 )
  7. Bright field microscope (Olympus, model: Vanox-S )
  8. Beckman L8-70M Ultracentrifuge (pre-cooled at 4 °C) (Beckman Coulter)
  9. Type 70Ti rotor (pre-cooled at 4 °C) (Beckman Coulter)
  10. GM-40 Linear Gradient maker (C. B. S. Scientific)
  11. Peristaltic (Multi-staltic) pump (Buchler Lab, catalog number: N/A )
  12. Tube sealer (Beckman Coulter, catalog number: 342420 )
  13. Density gradient fractionator (ISCO, 185)
    Note: This model is not available anymore. Brandel BR-186 Gradient Fractionator with Syringe Pump can be used instead.
  14. CIGNETTM Fraction collector (ISCO)
    Note: This item is also discontinued, but Bio-Rad 2110 Fraction Collector can be used instead.
  15. 6505 W UV-Vis Spectrophotometer (Bibby-scientific, Jenway)
  16. Refractometer (ZEISS, catalog number: 12230 )


  1. Homogenate (lysate) preparation
    1. Inoculate 400 ml Vogel’s Complete Medium (Vogel, 1956) with Neurospora crassa conidia (1 x 106 conidia/ml) in a 1 L flask. Incubate for 14-20 h at 30 °C, 200 rpm.
    2. Harvest mycelium by vacuum filtration through Whatman filter paper number 1. Wash twice with 200 ml cold sterile distilled H2O to eliminate remnant culture media and then twice with 50 ml cold 33 mM phosphate buffer (pH 8.2) to equilibrate mycelium for the next step.
    3. Mix 10 g wet mycelium and 10 ml 5% (w/v) sucrose in 33 mM phosphate buffer (pH 8.2), supplemented with a protease inhibitor cocktail (1 tablet/50 ml) into a Braun bottle and add 20 g of glass beads (500 μm; 2 g/g mycelium). Keep all solutions, tubes and bottles at 4 °C to prevent protein degradation.
    4. Homogenize in a Braun homogenizer 4 x 30 sec in presence of a CO2 stream directed to the Braun bottle so that a temperature increase during homogenization is prevented. The CO2 line must be firmly attached to the metallic arm of the Braun homogenizer so that the CO2 stream can be directed to the bottle. After homogenization, verify the broken cells by observing the sample in a bright field microscope.
    5. Transfer homogenate into a 50 ml tube (on ice) and centrifuge at 1,000 x g (Rav), 10 min, 4 °C.
    6. Save the supernatant at 4 °C using a glass pipette to avoid the lipid layer formed on top. This will be the homogenate or lysate, which would contain mitochondria, microvesicles, ribosomes, and soluble proteins. Discard pellet containing whole cells, cell walls, and nuclei.

  2. Density gradient centrifugation
    1. Construct a 10-65% sucrose linear gradient (10% and 65% sucrose stock solutions must be prepared in final 33 mM phosphate buffer, pH 8.2) in a quick-seal centrifuge tube using a linear gradient maker and a peristaltic pump set up to obtain a flow rate of 3.5 ml/min (Figure 1).
    2. Gently layer the N. crassa homogenate (supernatant saved in step A6) on top of the 10-65% sucrose gradient by using a Pasteur pipette via single drops close to the surface of the sucrose gradient. Grease the centrifuge inlet to seal and cap the centrifuge tube. Centrifuge at 184,000 x g (Rav), for 4 h and 4 °C in a pre-cooled Beckman rotor 70Ti.
    3. Fractionate the gradient from the top with an ISCO fractionation system using 70% (w/v) sucrose as chase at a flow rate of 6 ml/min. Make sure no air bubbles form in the tube system that conducts the chase sucrose to the gradient or else they will break it. Collect 2 ml fractions (3 fractions/min) into ice-cold tubes and keep at 4 °C.
    4. Characterize each fraction by measuring absorbance at 280 nm, refraction index (to infer the density) and, ideally, an enzyme activity associated with intracellular particles or vesicles (Figure 2A). Refractometers output the refractive index, which can be converted to density (g/ml) using this table:
    5. Mix 50 to 100 μl of each fraction with Laemmli buffer (2x), boil for 5 min and store at -20 °C until analysis by Western blot (Figure 2B).

      Figure 1. Preparation and fractionation of sucrose density gradients. A. To prepare the density gradients, sucrose stock solutions are independently poured into each column of the gradient maker (keep locked the Teflon valve that connects one column to the other). A magnetic stir bar is placed in the 10% sucrose solution column, from which a silicon tube (purged of any bubble) exits towards the peristaltic pump and, via a capillary tube attached to the tip of the silicon one, is introduced into the quick-seal centrifuge tube down to the bottom. After turning the peristaltic pump on, the Teflon valve is opened to allow the sucrose solutions mixing. Then, increasing concentration sucrose flows to the quick-seal tube. Lower concentration sucrose arrives first to the tube, which is pushed up by higher concentration sucrose (the capillary tip is always touching the quick-seal tube bottom). Immediately before the transfer of total volume of the sucrose gradient to the quick-seal tube, turn off the peristaltic pump and take out the capillary tube very gently to avoid breaking the gradient. The remaining volume of the quick-seal tube is filled up with the mycelial lysate. B. After centrifugation and isopycnic separation of mycelial homogenates, the gradient is carefully fractionated using a 70% sucrose solution as chase. This solution is injected into the quick-seal tube from the bottom so that the gradient is integrally pushed up toward the exit tubing and the fraction collector. Before fractionation, it is critical to purge any bubble from the syringe that contains the chase solution and the tubing that conducts it to the bottom of the quick-seal tube; otherwise, the gradient could be broken. Collected fractions are subsequently characterized (enzyme activity, refractive index, absorbance, etc.) and stored at 4 °C.

      Figure 2. Density gradient centrifugation of homogenates of N. crassa expressing CHS-6 chitin synthase co-translationally fused to GFP (Riquelme et al., 2007). A. Density (inferred from the refractive index), total protein (Abs 280 nm) and chitin synthase activity were estimated for each fraction of the density gradient collected. Chitin synthase activity sedimented at 1.13 g/ml, the buoyant density of chitosomes. B. Western blot analysis demonstrated the presence of CHS-6-GFP around fraction 13 (1.13 g/ml). C. Live imaging of a N. crassa hyphae by confocal microscopy showed that CHS-6-GFP localizes at the tip mainly at the core of the Spitzenkörper (an apical body that directs the growth of the hypha), where microvesicles (chitosomes) accumulate, as confirmed by transmission electron microscopy (Riquelme et al., 2002). Scale bar, 5 μm


  1. Vogel’s complete medium
    Dilute 50x salt solution 50 fold with distilled water
    Sucrose 15 g/L
    0.5% yeast extract
    0.5% N-Z case (casamino acids)
    Steam sterilize and store at room temperature
  2. 50x salt solution
    Na3C6H5O7.5.5 H2O
    Sodium citrate
    420 mM
    Potassium phosphate monobasic anhydrous
    1.8 M
    Ammonium nitrate anhydrous
    1.2 M
    Magnesium sulfate heptahydrate
    40.5 mM
    Calcium chloride dihydrate
    34 mM
    Trace element solution*

    500 μl
    Biotin solution (0.1 mg/ml, in ethanol)

    250 μl
    Bring up to 100 ml with ddH2O
    Add 2 ml of chloroform as preservative
    Alternatively, steam sterilize and store at 4 °C
  3. Trace element solution
    Note: Concentrations in brackets are final concentrations.
    Citric Acid.H2O
    5.0 g (238 mM)
    5.0 g (174 mM)
    1.0 g (25.5 mM)
    0.25 g (14 mM)
    0.05 g (2.3 mM)
    H3BO3 anhydrous
    0.05 g (8 mM)
    0.05 g (2.3 mM)
    Bring up to 100 ml with ddH2O
    Add 1 ml of chloroform for storage at room temperature
  4. 100 mM phosphate buffer (pH 8.2)
    1.23 g
    12.92 g
    up to 1 L
  5. Laemmli buffer
    0.5 M Tris (pH 6.8)
    1.25 ml
    10% SDS
    2.0 ml
    2.5 ml
    9.5 ml
    Bromophenol blue
    1 mg
    Stored at -20 °C
    Add 25 μl β-mercaptoethanol to 475 μl Laemmli buffer before use
    Dilute samples 1:2 in Laemmli buffer and boil for 4 min


This work was supported by Mexican National Council for Science and Technology (CONACYT) grants CB-222375, CB2008-105600-Q, and U45818-Q.


  1. Fajardo-Somera, R. A., Bowman, B. and Riquelme, M. (2013). The plasma membrane proton pump PMA-1 is incorporated into distal parts of the hyphae independently of the Spitzenkörper in Neurospora crassa. Eukaryot Cell 12(8): 1097-1105.
  2. Kamada, T., Bracker, C. E., Lippman, E. and Bartnicki-García, S. (1991). Unexpected destruction of chitosomal chitin synthetase by an endogenous protease during sucrose density gradient purification. J Cell Sci 99 (Pt 3): 565-570.
  3. Leal-Morales, C. A., Bracker, C. E. and Bartnicki-García, S. (1988). Localization of chitin synthetase in cell-free homogenates of Saccharomyces cerevisiae: chitosomes and plasma membrane. Proc Natl Acad Sci U S A 85(22): 8516-8520.
  4. Martínez, J. P., Gimenez, G., Bracker, C. E. and Bartnicki-García, S. (1989). Sedimentation properties of chitosomal chitin synthetase from the wild-type strain and the 'slime' variant of Neurospora crassa. Biochim Biophys Acta 990(1): 45-52.
  5. Riquelme, M., Roberson, R. W., McDaniel, D. P. and Bartnicki-Garcia, S. (2002). The effects of ropy-1 mutation on cytoplasmic organization and intracellular motility in mature hyphae of Neurospora crassa. Fungal Genet Biol 37(2): 171-179.
  6. Riquelme, M., Bartnicki-García, S., González-Prieto, J. M., Sánchez-León, E., Verdín-Ramos, J. A., Beltrán-Aguilar, A. and Freitag, M. (2007). Spitzenkörper localization and intracellular traffic of green fluorescent protein-labeled CHS-3 and CHS-6 chitin synthases in living hyphae of Neurospora crassa. Eukaryot Cell 6(10): 1853-1864.
  7. Sánchez-León, E., Bowman, B., Seidel, C., Fischer, R., Novick, P. and Riquelme, M. (2015). The Rab GTPase YPT-1 associates with Golgi cisternae and Spitzenkörper microvesicles in Neurospora crassa. Mol Microbiol 95(3): 472-490.
  8. Verdín, J., Bartnicki-García, S. and Riquelme, M. (2009). Functional stratification of the Spitzenkörper of Neurospora crassa. Mol Microbiol 74(5): 1044-1053.
  9. Vogel, H. J. (1956). A convenient growth medium for Neurospora (medium N). Microbiol Genet Bull 13:42-43.


已经使用密度梯度离心来表征生物相关酶在酵母和丝状真菌中的亚细胞分布(Leal-Morales等人,1988;Martínez等人,1989) ; Kamada等人,1991)。 这种方法现在通过蛋白质标记和实时成像技术加强,这使得可能将单个蛋白质与例如胞内囊泡的离散群体及其体内动力学联系起来(Verdín等人 al。,2009; Fajardo-Somera等人,2013;Sánchez-León等人,2015)。 在这里,我们描述了表达CHS-6几丁质合酶的粗糙脉孢菌( N.crassa )菌株的无细胞匀浆的密度梯度离心和分级分析 绿色荧光蛋白(Riquelme等人,2007)。

关键字:粗糙脉孢菌, chitosomes, 囊泡, 分馏


  1. Tygon R-3603管,I.D。 1/16英寸(Saint-Gobain,目录号:AAC00002)
  2. 1级定性过滤纸(GE Healthcare,目录号:1001-090)
  3. 分生孢子菌
  4. 蔗糖(Sigma-Aldrich,目录号:SO389)
  5. 磷酸二氢钠(NaH 2 PO 4 PO 4,H 2 O 2)(Thermo Fisher Scientific,Fisher Scientific,目录号码:S369-500)
  6. 磷酸氢二钠无水(Na 2 HPO 4)(Sigma-Aldrich,目录号:S-7907)
  7. 蛋白酶抑制剂混合物,完全ULTRA片剂,无EDTA(Roche Diagnostics,目录号:04693132001)
  8. 玻璃珠(500μm)(Bio Spec Product,目录号:11079105)
  9. 在33mM(最终)磷酸盐缓冲液(pH8.2)中的10%(w/v)蔗糖(蒸汽灭菌并在4℃储存直到使用)
  10. 在33mM(最终)磷酸盐缓冲液(pH 8.2)中的65%(w/v)蔗糖(蒸汽消毒并在4℃储存直到使用)
  11. D-生物素(Faga Labs,目录号:CAS-58-85-5)
  12. Vogel的完整介质(见配方)
  13. 50x盐溶液(见配方)
  14. 微量元素溶液(参见配方)
  15. 100 mM磷酸盐缓冲液(pH 8.2)(参见配方)
  16. Laemmli缓冲区(参见配方)


  1. Braun MSK 50ml摇瓶(LABEQUIP LTD,目录号:8541302)
  2. VWR 标准热板搅拌器(VWR International,目录号:12365-382)
  3. 快速离心管(Beckman Coulter,目录号:344326)
  4. 摇床培养箱(Lab-LineX,型号:Orbit Environ Shaker)
  5. 真空过滤系统
  6. Braun MSK细胞匀浆器(LABEQUIP LTD,目录号:953030)
  7. 明场显微镜(奥林巴斯,型号:Vanox-S)
  8. Beckman L8-70M超速离心机(在4℃预冷)(Beckman Coulter)
  9. 型号70Ti转子(在4℃预冷)(Beckman Coulter)
  10. GM-40线性梯度仪(C. B. S. Scientific)
  11. 蠕动(多效)泵(Buchler Lab,目录号:N/A)
  12. 管密封器(Beckman Coulter,目录号:342420)
  13. 密度梯度分馏器(ISCO,185)
    注意:此模型不可用。可以使用带有注射泵的Brandel BR-186梯度分级器。
  14. CIGNET TM 馏分收集器(ISCO)
    注意:此项目也已停用,但可以使用Bio-Rad 2110馏分收集器。
  15. 6505W紫外 - 可见分光光度计(Bibby-scientific,Jenway)
  16. 折射计(ZEISS,目录号:12230)


  1. 匀浆(裂解物)制备
    1. 用脉孢菌接种400ml Vogel's完全培养基(Vogel,1956) crassa分生孢子(1×10 6个分生孢子/ml)。孵育14-20小时 ?在30℃,200rpm
    2. 通过真空过滤收获菌丝体 Whatman滤纸编号1.用200ml冷无菌洗涤两次 蒸馏H 2 O以除去残余培养基,然后用50μL蒸馏两次 ml冷33mM磷酸盐缓冲液(pH 8.2)以平衡菌丝体 下一步。
    3. 在33℃下混合10g湿菌丝体和10ml 5%(w/v)蔗糖 ?mM磷酸盐缓冲液(pH 8.2),补充有蛋白酶抑制剂 鸡尾酒(1片/50ml)装入Braun瓶中,并加入20g玻璃 珠(500μm; 2g/g菌丝体)。保持所有的解决方案,管和瓶 ?4℃以防止蛋白质降解。
    4. 在博朗均匀化 均化器在指向Braun的CO 2流的存在下4×30秒 ?使得均化期间的温度升高 防止。 CO <2>线必须牢固地连接到金属臂 Braun均化器,使得CO 2物流可以被引导至 瓶子。均质后,通过观察破碎的细胞来验证 样品在明视场显微镜。
    5. 将匀浆转移到50ml管中(在冰上),并在1,000×g(R av av),10分钟,4℃下离心。
    6. 保存上清液在4°C使用玻璃吸管,以避免 脂质层。这将是匀浆或裂解物,其 将含有线粒体,微泡,核糖体和可溶性 蛋白质。丢弃含有全细胞,细胞壁和细胞核的沉淀。

  2. 密度梯度离心
    1. 构建10-65%蔗糖线性梯度(10%和65%蔗糖储备液 溶液必须在最终的33mM磷酸盐缓冲液,pH 8.2中制备) ?快速离心管使用线性梯度仪和a 蠕动泵设置以获得3.5ml/min的流速(图1)。
    2. 轻轻地分层 N。 crassa 匀浆(步骤A6中保存的上清液) ?通过使用巴斯德移液管通过在10-65%蔗糖梯度的顶部 单滴接近蔗糖梯度的表面。润滑脂 离心机入口密封和盖离心管。离心机 在预冷却的Beckman转子70Ti中在4℃和4℃下进行4小时。
    3. 使用ISCO分馏从顶部分馏梯度 系统,使用70%(w/v)蔗糖作为追逐,流速为6ml/min。使 ?确保在进行追踪的管系统中没有形成气泡 蔗糖到梯度,否则会破坏它。收集2毫升 馏分(3个馏分/分钟)加入到冰冷的管中并保持在4℃
    4. 通过测量280nm处的吸光度表征每个级分, 折射率(推断密度),理想地,酶活性 ?与细胞内颗粒或囊泡相关(图2A)。 折射计输出折射率,可以转换 密度(g/ml): http://主页。。
    5. 混合 用Laemmli缓冲液(2x)洗涤50至100μl每种级分,煮沸5分钟 并储存在-20℃直到通过Western印迹分析(图2B)

      图1.蔗糖密度梯度的制备和分级。 A. ?为了制备密度梯度,蔗糖储备溶液是 独立注入梯度制作器的每一列(保持锁定 ?连接一列到另一列的特氟龙阀)。磁性 搅拌棒置于10%蔗糖溶液柱中, 硅管(清除任何气泡)朝向蠕动泵排出 并且通过附接到硅尖端的毛细管 引入快速离心管中直到底部。后 ?打开蠕动泵,打开特氟龙阀门以允许 蔗糖溶液混合。然后,增加浓度蔗糖 流向快速密封管。较低浓度蔗糖首先到达 到管,其被更高浓度的蔗糖( 毛细管尖总是接触快速密封管底部)。 在即将转移蔗糖梯度的总体积之前 到快速密封管,关闭蠕动泵并取出 非常缓和毛细管,以避免打破梯度。剩下的 ?快速密封管的体积用菌丝体裂解物填充。乙。 ?离心和菌丝体匀浆分离后, 使用70%蔗糖溶液将梯度小心分离 追。将该溶液注入快速密封管中 使得梯度被一体地向上推向出口 管和馏分收集器。在分馏之前,它是至关重要的 以从包含追加溶液的注射器吹扫任何气泡 和将其传导到快速密封管的底部的管道; 否则,梯度可能会断开。收集的馏分是 随后表征(酶活性,折射率, 吸光度,等)),并在4℃下保存

      图2.密度梯度 离心匀浆的N。 crassa 表达CHS-6几丁质 合酶与GFP共翻译融合(Riquelme等人,2007)。 A。 密度(从折射率推测),总蛋白(Abs 280nm) ?和几丁质合酶活性 密度梯度收集。几丁质合酶活性沉淀于1.13 g/ml,壳聚糖的浮力密度。 B.蛋白质印迹分析 证明了在级分13(1.13g/ml)周围存在CHS-6-GFP。 C.实时成像。 crassa 菌丝通过共聚焦显微镜显示 ?CHS-6-GFP位于尖端主要在Spitzenk?rper的核心 (指导菌丝生长的顶端体),其中 微泡(壳寡糖)积累,如通过传播所证实的 电子显微镜(Riquelme等人,2002)。比例尺,5μm


  1. Vogel的完整媒介
    用蒸馏水稀释50倍盐溶液50倍 添加:
  2. 50x盐溶液
    Na H 3 H 6 H 6 H 5 O 7 H 6 H 5 O
    420 mM
    KH 2 PO 4
    1.8 M
    NH 4 3
    1.2 M
    MgSO 4。 。 O
    40.5 mM
    CaCl 2 2H O
    34 mM

    用ddH 2 O 2/b达到100ml 加入2ml氯仿作为防腐剂
  3. 微量元素溶液
    柠檬酸 H sub 2 O
    ZnSO 4 。 7H O
    Fe(NH 4)2子(SO 4)子部分2子部分。 > 2 O
    CuSO 4 H O
    MnSO 4 H sub 2 O
    H BO 3
    Na 2 MoO 4 sub 。 2H 2
    用ddH 2 O 2/b达到100ml 加入1ml氯仿,在室温下贮存
  4. 100mM磷酸盐缓冲液(pH8.2)
    NaH 2 2 PO 4 4 H <2> O
    Na HPO 4
    H sub 2 O
    最多1 L
  5. Laemmli缓冲区
    0.5 M Tris(pH 6.8)
    1.25 ml
    2.0 ml
    2.5 ml
    ddH sub 2 O
    9.5 ml
    1 mg




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引用:Verdín, J., Sánchez-León, E., Fajardo-Somera, R., Morales, C. A., Bartnicki-García, S. and Riquelme, M. (2015). Density Gradient Centrifugation for Enrichment and Identification of GFP-tagged Chitosomal Microvesicles of Filamentous Fungi. Bio-protocol 5(19): e1611. DOI: 10.21769/BioProtoc.1611.