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Freeze-fracture-etching Electron Microscopy for Facile Analysis of Yeast Ultrastructure
电镜冷冻断裂蚀刻技术用于酵母超微结构的简易分析   

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Abstract

We describe a streamlined method that enables the quick observation of yeast ultrastructure by electron microscopy (EM). Yeast cells are high-pressure frozen, freeze-fractured to cut across the cytoplasm, and freeze-etched to sublimate ice in the cytosol and the organelle lumen. The cellular structures delineated by these procedures are coated by a thin layer of platinum and carbon deposited by vacuum evaporation, and this platinum–carbon layer, or replica, is observed by transmission EM. The method differs from the deep-etching of pre-extracted samples in that intact live cells are processed without any chemical treatment. Lipid droplets made of unetchable lipid esters are observed most prominently, but other organelles–the nucleus, endoplasmic reticulum, Golgi, vacuoles, mitochondria–and their mutual relationships can be analyzed readily. It is of note that the entire procedure, from quick-freezing to EM observation, can be performed within a day.

Keywords: Freeze-fracture(冷冻-断裂), Etching(蚀刻), Electron microscopy(电子显微镜检查), Quick freezing(快速冷冻), Lipid droplet(脂滴), Organelle(细胞器), Yeast(酵母)

Background

Budding yeast (Saccharomyces cerevisiae) is probably the most frequently used model organism, and application of a wide assortment of experimental techniques and the presence of sophisticated genome-wide databases have significantly facilitated research advancement (Botstein and Fink, 2011). The microscopic imaging of yeast, however, is not always carried out in a satisfactory manner due to its relatively small size and round shape. The presence of the cell wall is a problem for conventional electron microscopy (EM) in particular, because it hampers the penetration of reagents used in sample preparation. Freeze-substitution EM of quick-frozen yeast is currently considered the best method for ultrastructural observation (Giddings et al., 2001), but the method has drawbacks, namely, the membrane structures are not clearly visible, some cellular components may not be retained during the substitution process in organic solvents, and the procedure takes at least several days before observation is possible.

In a recent study to examine lipophagy in stationary-phase yeast, we used freeze-fracture-etching to analyze the yeast ultrastructure (Tsuji et al., 2017). Canonical freeze-fracture EM has been used to observe wide areas of membranes in two dimensions. By adding the etching process after freeze-fracturing, the cellular ultrastructure–the lipid droplets and interorganellar relationships in particular–was readily observed. Deep-etching combined with quick-freezing has been used successfully to analyze the cytoskeleton in pre-extracted samples (Heuser and Salpeter, 1979). The method described here is different in that intact live cells are quick-frozen and processed, thereby preserving the membrane organelles. Furthermore, with slight modification, the distribution of proteins and lipids on the nanoscale can also be analyzed (Cheng et al., 2014).

Materials and Reagents

  1. 200 mesh EM grid (Electron Microscopy Sciences, catalog number: M200-CR )
  2. 50 mesh EM grid (Electron Microscopy Sciences, catalog number: G50-Cu )
  3. Polyvinyl formvar (Nisshin EM, catalog number: 602 )
  4. Copper foil: 20 μm in thickness (Nilaco, catalog number: CU-113213 )
  5. 10 μl pipette tip (Thermo Fisher Scientific, catalog number: 3510 )
  6. Aluminum disc: 3 mm in diameter and 0.3 mm in thickness (Engineering Office M. Wohlwend, catalog number: 242 )
  7. Platinum-carbon (Pt/C) (Leica Microsystems, catalog number: 16771798 )
  8. Carbon (C) (Leica Microsystems, catalog number: 16771797 )
  9. Saccharomyces cerevisiae
  10. Acetone (CH3COCH3) (KANTO KAGAKU, catalog number: 01026-70 )
  11. Liquid nitrogen
  12. Household bleach (6% sodium hypochlorite)

Equipment

  1. 50-100 ml flask (IWAKI, catalog numbers: 4980FK50 , 4980FK100 )
  2. Razor blade (Feather, catalog number: FAS-10 )
  3. Forceps (EM Japan, catalog number: T7330 )
  4. Hole puncher (Carla Craft, catalog number: SD-15-3 )
  5. Incubator shaker (TAITEC, catalog number: BR-22FP )
  6. High-pressure freezing machine (Bal-Tec, model: HPM 010 )
  7. Freeze-fracture apparatus (Balzers, model: BAF400 )
  8. Electron beam gun (Balzers, model: EK552 )
  9. Crystal thickness monitor (Balzers, model: QSG 301 )
  10. Stereoscopic microscope (Leica Microsystems, model: Leica MZ6 )
  11. Transmission electron microscope (JEOL, model: JEM-1011 )

Procedure

The outline of the method is depicted in Figure 1.


Figure 1. The outline of the method. After ‘quick-freezing’ of yeasts, the cytoplasm is exposed by ‘fracturing’ of frozen cells with cooled knife. The ‘etching’ procedure induces sublimation of water in the cytosol and the organelle lumen. This makes lipid droplets stand out because lipid esters in the lipid droplet core do not sublimate. Vacuum evaporation of platinum and carbon onto the surface makes a ‘replica’ of the cellular ultrastructure.

  1. Quick freezing
    1. Prepare round-shaped copper discs of 3 mm in diameter using a hole puncher. Clean the discs by soaking them in acetone (Figures 2A and 2B).
    2. Concentrate yeast cells by centrifugation at 1,500 x g for 1 min.
    3. Place yeast cells on an EM grid (200 mesh) by dipping the grid into a pellet or spreading ~0.6 μl of pellet on the grid using a pipette tip (Figure 2B).


      Figure 2. Tools used for quick-freezing and freeze-fracture-etching. A. Copper foil (20 μm thick). The left portion (arrow) has already been used to prepare round-shaped discs. B. The sample sandwich subjected to quick-freezing: a 200 mesh EM grid impregnated with yeast cells is placed between a 20-μm-thick copper disc and an aluminum disc. C. A specimen table of Balzers BAF400. The sample sandwiches are placed in the two round indentations (arrows).

    4. Sandwich the EM grid impregnated with yeast cells between a 20-μm-thick copper foil and a flat aluminum disc (Figure 2B) (see Note 1).
    5. Quick-freeze yeast cells using an HPM 010 high-pressure freezing machine (or a similar device) according to the manufacturer’s instructions.
    6. Keep the frozen samples in liquid nitrogen until they are transferred to the cold specimen stage of a freeze-fracture apparatus.

  2. Freeze-fracturing and etching
    1. Mount the frozen sample onto a specimen table of the freeze-fracture apparatus (Figures 2C and 3). Here, the procedure for the Balzers BAF400 will be described, but it can easily be adapted to other devices.


      Figure 3. Freeze-fracture apparatus (BAF400) for freeze-fracture-etching. Electron beam guns for carbon and platinum deposition are located at 80° and 20° to the specimen surface, respectively. Specimen table is set on the temperature-controlled specimen stage.

    2. Transfer the specimen table to the pre-cooled specimen stage of the freeze-fracture apparatus (Figure 3). The specimen stage needs to be cooled below -120 °C before this transfer.
    3. Keep the specimen temperature at -120 °C for 10 min, and then at -102 °C for 3 to 5 min by using the temperature controller of the specimen stage.
    4. Cool the knife to the lowest-possible temperature (e.g., near the temperature of liquid nitrogen).
    5. After the vacuum reaches below ~5 x 10-7 mbar, fracture the specimens at -102 °C by separating the copper disc from the aluminum disc using the pre-cooled knife (see Note 2).
    6. Keep the fractured specimens at -102 °C for another 2 min to induce sublimation of water from the fractured surface.
    7. Evaporate platinum-carbon (Pt/C) and carbon (C) onto the specimen using the electron beam gun. First, evaporate 2 to 4 nm of Pt/C at an angle of 20° to the specimen surface, then 10 nm of C at an angle of 80°. Rotate the specimen stage at maximum speed during evaporation. Control the thickness of the Pt/C and C deposition using a crystal thickness monitor (Figure 3).
    8. Break the vacuum, bring the specimen into the atmosphere, and transfer the freeze-fracture replicas to household bleach to digest biological materials for more than 2 h.
    9. Rinse the replicas with water.
    10. Mount the replicas on the formvar-coated EM grids under a stereoscopic microscope. The formvar-coated EM grids are prepared by a standard protocol (Slot and Geuze, 2007).
    11. Observe the replicas by transmission EM (see Note 3).

Data analysis

  1. Yeast cells are fractured in a random manner. Roughly speaking, approximately 70 to 80% of the yeast cells are freeze-fractured along the plasma membrane. For analysis of cytoplasmic organelles, the rest of the cells showing the cross-fractured cytoplasm are selected. Within the cytoplasm, lipid droplets are invariably cross-fractured and observed as round protruding structures whereas other organelles may be either freeze-fractured along the limiting membrane to show the convex or concave surface, or cross-fractured to reveal the internal structure (Figure 4).


    Figure 4. Freeze-fracture-etching EM of yeast in the stationary phase. A. The plasma membrane (PM) and the vacuolar membrane show the two-dimensional freeze-fractured plane whereas the lipid droplet (LD) is cross-fractured. A number of small vesicles (arrows) are also observed. CW: cell wall. B. Two LDs adhere to the vacuolar membrane (arrowheads). A cross-fractured mitochondrion (MT) is observed. Scale bars = 0.2 μm.

  2. Damage to the cellular ultrastructure may occur when ice crystals form due to improper operation of the high-pressure freezing machine or the freeze-fracture apparatus, or by poor handling of frozen samples, for example, failure to keep samples in liquid nitrogen. Suboptimal replicas may form as a result of unsatisfactory vacuum evaporation. Samples exhibiting these problems can be readily identified by EM and should be excluded from further analyses.
  3. In most experiments, three replicates, prepared by independent quick-freezing and freeze-fracture-etching sessions, are analyzed for each yeast specimen.

Notes

  1. Liquid (or culture medium) must be kept to a minimum volume so that it will not spread to the outer side of the copper foil, which would result in improper freezing.
  2. The knife must be cooled to the coldest possible temperature before fracturing samples.
  3. EM grids must be completely dried before EM observation.

Acknowledgments

The condition of the etching procedure was modified from Heuser and Salpeter (1979). The authors thank Dr. John E. Heuser for his kind advice on technical details. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of the Government of Japan to TF (25111510, 15H02500, 15H05902) and TT (15K18954, 17K15544).

References

  1. Botstein, D. and Fink, G. R. (2011). Yeast: an experimental organism for 21st Century biology. Genetics 189(3): 695-704.
  2. Cheng, J., Fujita, A., Yamamoto, H., Tatematsu, T., Kakuta, S., Obara, K., Ohsumi, Y. and Fujimoto, T. (2014). Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries. Nat Commun 5: 3207.
  3. Giddings, T. H., Jr., O’Toole, E. T., Morphew, M., Mastronarde, D. N., McIntosh, J. R. and Winey, M. (2001). Using rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis. Methods Cell Biol 67: 27-42.
  4. Heuser, J. E. and Salpeter, S. R. (1979). Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane. J Cell Biol 82(1): 150-173.
  5. Tsuji, T., Fujimoto, M., Tatematsu, T., Cheng, J., Orii, M., Takatori, S. and Fujimoto, T. (2017). Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole. Elife 6: e25960.
  6. Slot, J. W. and Geuze, H. J. (2007). Cryosectioning and immunolabeling. Nat Prot 2: 2480-2491.

简介

我们描述了一种流线型的方法,可以通过电子显微镜(EM)快速观察酵母超微结构。 酵母细胞是高压冷冻的,冷冻破碎以跨细胞质切割,并冷冻蚀刻以升华胞质溶胶和细胞器腔内的冰。 通过这些方法描绘的细胞结构涂覆有通过真空蒸发沉积的铂和碳薄层,并且通过传输EM观察该铂 - 碳层或复制品。 该方法不同于预提取样品的深刻蚀,因为完整的活细胞在没有任何化学处理的情况下进行处理。 最突出的是观察到由不可获得的脂质酯制成的脂滴,但是可以容易地分析其他细胞器 - 细胞核,内质网,高尔基体,空泡,线粒体及其相互关系。 值得注意的是,从快速冷冻到EM观察的整个过程都可以在一天之内完成。
【背景】萌发酵母(Saccharomyces cerevisiae)可能是最常用的模型生物,应用各种各样的实验技术和复杂的全基因组数据库的存在大大促进了研究进展(Botstein和Fink,2011)。然而,由于其相对较小的尺寸和圆形,酵母的显微成像并不总是以令人满意的方式进行。细胞壁的存在对于常规电子显微镜(EM)尤其是一个问题,因为它妨碍样品制备中使用的试剂的渗透。快速冷冻酵母的冷冻替代EM目前被认为是超微结构观察的最佳方法(Giddings et al。,2001),但该方法存在缺点,即膜结构不清晰可见,有些细胞成分可能不被保留在有机溶剂中的取代过程中,该程序至少需要几天才能进行观察。
  最近在固定相酵母中检查脂肪的研究中,我们使用冷冻断裂蚀刻来分析酵母超微结构(Tsuji et al。,2017)。通常的冷冻断裂EM已被用于观察两个方面的广泛的膜。通过在冷冻压裂后添加蚀刻工艺,特别是细胞超微结构 - 脂滴和组织间关系 - 很容易观察到。深层蚀刻结合快速冷冻已被成功地用于分析提取前样品中的细胞骨架(Heuser and Salpeter,1979)。这里描述的方法不同之处在于完整的活细胞被快速冷冻和加工,从而保留了膜细胞器。此外,通过轻微的修改,蛋白质和脂质在纳米尺度上的分布也可以被分析(Cheng et al。,2014)。

关键字:冷冻-断裂, 蚀刻, 电子显微镜检查, 快速冷冻, 脂滴, 细胞器, 酵母

材料和试剂

  1. 200目EM网格(电子显微镜科学,目录号:M200-CR)
  2. 50目EM网格(电子显微镜科学,目录号:G50-Cu)
  3. 聚乙烯塑料(Nisshin EM,目录号:602)
  4. 铜箔:厚度20μm(Nilaco,目录号:CU-113213)
  5. 10μl移液头(Thermo Fisher Scientific,目录号:3510)
  6. 铝盘:直径3毫米,厚度0.3毫米(工程办公室M.Whwwend,目录号:242)
  7. 铂 - 铂(Pt / C)(Leica Microsystems,目录号:16771798)
  8. 碳(C)(Leica Microsystems,目录号:16771797)
  9. 酿酒酵母
  10. 丙酮(CH 3 COCH 3)(KANTO KAGAKU,目录号:01026-70)
  11. 液氮
  12. 家用漂白剂(6%次氯酸钠)

设备

  1. 50-100ml烧瓶(IWAKI,目录号:4980FK50,4980FK100)
  2. 剃刀刀片(羽毛,目录号:FAS-10)
  3. 镊子(EM日本,目录号:T7330)
  4. 打孔机(Carla Craft,目录号:SD-15-3)
  5. 孵化器(TAITEC,目录号:BR-22FP)
  6. 高压冷冻机(Bal-Tec,型号:HPM 010)
  7. 冷冻断裂装置(Balzers,型号:BAF400)
  8. 电子束枪(Balzers,型号:EK552)
  9. 水晶厚度监视器(Balzers,型号:QSG 301)
  10. 立体显微镜(Leica Microsystems,型号:Leica MZ6)
  11. 透射电子显微镜(JEOL,型号:JEM-1011)

程序

该方法的大纲如图1所示

图1.方法的概要在酵母“快速冻结”之后,细胞质通过冷冻刀“冻结”冷冻细胞而暴露。 “蚀刻”程序引起细胞质和细胞器腔内水的升华。这使得脂质液滴脱颖而出,因为脂滴核心中的脂质酯不升华。铂和碳在表面上的真空蒸发是细胞超微结构的“复制”。

  1. 快速冷冻
    1. 使用穿孔机准备直径为3毫米的圆形铜圆盘。通过将其浸入丙酮来清洁光盘(图2A和2B)。
    2. 通过以1,500×g离心1分钟浓缩酵母细胞。
    3. 将酵母细胞置于EM网格(200目)上,将网格浸入沉淀物中,或使用移液管吸头在网格上铺展〜0.6μl颗粒(图2B)。


      图2.用于快速冷冻和冷冻断裂蚀刻的工具。 :一种。铜箔(20μm厚)。左部分(箭头)已经用于制备圆形盘。 B.将经过快速冷冻的样品夹心物:将浸渍有酵母细胞的200目EM网格置于20μm厚的铜盘和铝盘之间。 C. Balzers BAF400的样品台。样品三明治放在两个圆形凹痕(箭头)中。

    4. 将20微米厚的铜箔和扁平铝盘之间用酵母细胞浸渍的EM网格进行三明治(图2B)(见注1)。
    5. 根据制造商的说明,使用HPM 010高压冷冻机(或类似装置)快速冷冻酵母细胞。
    6. 将冷冻样品保存在液氮中,直至转移到冷冻断裂装置的冷试样阶段
  2. 冷冻压裂和蚀刻
    1. 将冷冻样品装入冷冻断裂装置的样品台(图2C和3)。在这里,将介绍Balzers BAF400的程序,但可以轻松适应其他设备

      图3.用于冷冻断裂蚀刻的冷冻断裂装置(BAF400)。碳和铂沉积的电子束枪分别位于样品表面的80°和20°。样品台设置在受温度控制的样品台上。

    2. 将样品台转移到冷冻断裂装置的预冷样品台(图3)。在转移前,样品台需要冷却至-120°C以下。
    3. 将样品温度保持在-120℃10分钟,然后使用样品台的温度控制器在-102℃保持3〜5分钟。
    4. 将刀子冷却到液氮温度附近的最低温度(例如)。
    5. 在真空度达到5×10 -7以上的条件下,使用预冷刀将铜盘从铝盘上分离(见注2),使试样在-102℃断裂。 br />
    6. 将断裂的样品保持在-102℃另外2分钟,以从裂缝表面引起水的升华。
    7. 使用电子束枪将铂 - 碳(Pt / C)和碳(C)蒸发到样品上。首先,以与试样表面20°的角度蒸发2〜4nm的Pt / C,然后以80°的角度蒸发10nm的C。在蒸发过程中以最大速度旋转样品台。使用晶体厚度监测器控制Pt / C和C沉积的厚度(图3)。
    8. 打破真空,将样品放入大气中,将冻干复制品转移到家用漂白剂中,消化生物材料2小时以上。
    9. 用水冲洗副本。
    10. 在立体显微镜下将复制品安装在形式涂覆的EM网格上。通过标准方案(Slot和Geuze,2007)制备表面涂覆的EM网格。
    11. 通过传输EM观察复制品(见注3)

数据分析

  1. 酵母细胞以随机方式断裂。粗略地说,约70-80%的酵母细胞沿着质膜冻结断裂。为了分析细胞质细胞器,选择显示交叉裂隙细胞质的其余细胞。在细胞质内,脂质液滴总是交叉断裂并被观察为圆形突出结构,而其他细胞器可以沿着极限膜冷冻断裂,以显示凸表面或凹面,或交叉断裂以显示内部结构(图4 )

    图4.固定相中酵母的冻结 - 腐蚀EM。A.质膜(PM)和液泡膜显示二维冷冻断裂面,而脂滴(LD )是交叉断裂的。还观察到许多小囊泡(箭头)。 CW:细胞壁。 B.两个LD粘附于液泡膜(箭头)。观察到交叉断裂的线粒体(MT)。刻度棒=0.2μm。

  2. 由于高压冷冻机或冷冻断裂装置的不正确的操作,或由于冷冻样品的处理差,例如不能将样品保持在液氮中而形成冰晶,可能会导致细胞超微结构的损害。由于不充分的真空蒸发,可能形成次佳的副本。展示这些问题的样品可以通过EM轻松识别,并应排除在进一步的分析之外
  3. 在大多数实验中,对每个酵母样品分析了通过独立快速冷冻和冷冻 - 断裂 - 蚀刻阶段制备的三个重复。

笔记

  1. 液体(或培养基)必须保持最小体积,使其不会扩散到铜箔的外侧,这将导致不正确的冻结。
  2. 压裂样品前,必须将刀片冷却至最冷的温度。
  3. EM电网在EM观察前必须完全干燥

致谢

蚀刻过程的条件由Heuser和Salpeter(1979)进行了修改。作者感谢John E. Heuser博士关于技术细节的善意建议。这项研究得到了日本政府教育,文化,体育,科学和技术部科学研究资助计划(25111510,15H02500,15H05902)和TT(15K18954,17K15544)的支持。

参考

  1. Botstein,D。和Fink,GR(2011)。  酵母菌:21世纪生物学的实验生物。遗传学 189(3):695-704。
  2. Cheng,J.,Fujita,A.,Yamamoto,H.,Tatematsu,T.,Kakuta,S.,Obara,K.,Ohsumi,Y.and Fujimoto,T。(2014)。酵母和哺乳动物自噬体表现出不同的磷脂酰肌醇3-磷酸不对称。 Nat Commun 5:3207.
  3. Giddings,TH,Jr.,O'Toole,ET,Morphew,M.,Mastronarde,DN,McIntosh,JR and Winey,M。(2001)。  使用快速冷冻和冷冻置换来制备酵母细胞进行电子显微镜和三维分析。 > Methods Cell Biol 67:27-42。
  4. Heuser,JE和Salpeter,SR(1979)。组织的快速冷冻,深刻和旋转复制的鱼雷突触后膜中的乙酰胆碱受体。 82(1):150-173。
  5. Tsuji,T.,Fujimoto,M.,Tatematsu,T.,Cheng,J.,Orii,M.,Takatori,S.and Fujimoto,T。(2017)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/28590904”target =“_ blank”> Niemann-Pick C型蛋白通过在酵母液泡中扩大筏状膜结构域来促进微小肠蛋白。 Elife 6:e25960。
  6. Slot,JW和Geuze,HJ(2007)。  冷冻切片和免疫标记。 Nat Prot 2:2480-2491。
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Copyright Tsuji and Fujimoto. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tsuji, T. and Fujimoto, T. (2017). Freeze-fracture-etching Electron Microscopy for Facile Analysis of Yeast Ultrastructure. Bio-protocol 7(18): e2556. DOI: 10.21769/BioProtoc.2556.
  2. Tsuji, T., Fujimoto, M., Tatematsu, T., Cheng, J., Orii, M., Takatori, S. and Fujimoto, T. (2017). Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole. Elife 6: e25960.
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