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Labeling of the Intestinal Lumen of Caenorhabditis elegans by Texas Red-dextran Feeding
德克萨斯宏右旋糖酐喂食法标记秀丽隐杆线虫肠腔   

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Molecular Biology of the Cell
Oct 2014

Abstract

In this method, the intestinal lumen of Caenorhabditis elegans (C. elegans) is labeled with a fluorescent fluid-phase marker, Texas Red-dextran. Since dextran conjugates are membrane impermeable, animals fed with it show a red fluorescent signal in the lumen of the intestine. Texas Red-dextran in the lumen is not efficiently endocytosed by intestinal cells and is not effectively transported to the body cavity paracellularly. It is useful to determine whether round-shaped membrane structures are invaginations from the apical membrane or cytoplasmic vesicles. If the barrier function of the intestinal epithelium is impaired, Texas Red-dextran can leak from the intestinal lumen to the body cavity. Therefore, this method can be used to visualize apical membrane morphology in intestinal cells and to investigate the barrier properties of the intestinal epithelium.

Keywords: C. elegans (线虫), Intestine (肠), Apical membrane (顶膜)

Materials and Reagents

  1. C. elegans strain
    GK70 unc-119 (ed3) III; dkIs37[Pact-5-GFP-pgp-1;unc-119(+)]
  2. Escherichia coli OP50 strain (obtained from the Caenorhabditis Genetics Center)
  3. Texas Red-dextran (40,000 MW) (Life Technologies, Molecular Probes®, catalog number: D-1829 )
  4. Levamisole hydrochloride (Sigma-Aldrich, catalog number: L9756 )
  5. Agarose (Nacalai Tesque, catalog number: 01158-85 )
  6. Egg buffer (see Recipes)
  7. M9 buffer (see Recipes)
  8. Agarose pads (see Recipes)

Equipment

  1. Siliconized tubes (1.7 ml) (National Scientific Supply, catalog number: 20172-945 )
  2. Tabletop centrifuge (Hitachi Koki, model: CT15RE )
  3. Confocal laser scanning microscopy system (Olympus, model: FV1000 )
  4. Four clean glass slides and a coverslip

Procedure

  1. Place synchronized L1 larvae on an Escherichia coli (E. coli) OP50-seeded plate and incubate them for 3 days at 20 °C.
  2. Pick approximately 30-40 young adult animals from an OP50-seeded plate and transfer them into 500 μl of egg buffer in 1.7 ml siliconized tubes.
  3. Allow the animals to settle with gravity for a few minutes. Remove the supernatant and wash with 500 μl of egg buffer 3 times.
  4. Remove most of the supernatant, leaving approximately 10-20 μl of worm suspension and add 100 μl of egg buffer containing 1 mg/ml Texas Red-dextran.
  5. Incubate the animals for 90 min at room temperature with occasional gentle agitation every 30 min.
  6. Collect the animals by centrifuging at 400 x g for 2 min and discard the supernatant. Wash with 1 ml of egg buffer at least 3 times.
    Note: Remove as much Texas Red-dextran in the worm suspension as possible.
  7. Add 1 M levamisole hydrochloride to the worm suspension to a final concentration 10~20 mM.
  8. Transfer the worm suspension to a 1.5% agarose pad on a glass slide and cover it with a coverslip.
  9. Observe the animals with confocal laser scanning microscopy (see Figure 1).


    Figure 1. Animals expressing GFP-PGP-1 were fed Texas Red-dextran. GFP-PGP-1 and Texas Red-dextran labeled the intestinal apical membrane (large arrowheads) and the intestinal lumen (large arrows), respectively. Texas Red-dextran that is taken up by feeding is restricted to the intestinal lumen when the junctional seals of intestinal epithelium cells are intact (Carberry et al., 2012). A. RNAi-mediated knockdown of C. elegans chaperonin subunit (CCT-5) disorganized the microvillus actin filament and microtubules, leading to aberrant morphology of the intestinal apical membrane (Saegusa et al., 2014). B. In such animals, the apical membrane, labeled by GFP-PGP-1 (C. elegans P-glycoprotein homolog), formed bubble-like membrane structures invaginated from the apical membrane (B, small arrows). Such structures contain Texas Red-dextran (B, small arrows). By contrast, GFP-PGP-1-containing vesicles underneath the apical membrane (B, small arrowheads) did not contain Texas Red-dextran, indicating that these are exocytic vesicles. Images were obtained using an FV1000 confocal microscopy system. GFP-PGP-1; excitation: 473 nm, detection: 490-540 nm. Texas-Red dextran; excitation: 559 nm, detection: 575 to 675 nm. The scale bars indicate 10 μm.

Notes

  1. Although Texas Red-dextran is not efficiently taken up by the intestinal cells in this feeding method, some other apical endocytic markers such as rhodamine-dextran (Mr 40K) and Texas Red BSA are reported to be available to visualize endocytic vesicles (Grant et al., 2001).
  2. Depletion of tight junction components such as claudins (CLC-1-4 in C. elegans) causes the infiltration of dextran conjugates from the intestinal lumen into the body cavity (Asano et al., 2003).

Recipes

  1. Egg buffer
    118 mM NaCl
    48 mM KCl
    2 mM CaCl2
    2 mM MgCl2
    25 mM HEPES
    pH 7.3
    Note: This solution is more physiologically isotonic than M9 for C. elegans tissues (Grant et al., 2001).
  2. M9 buffer
    42 mM Na2HPO4
    22 mM KH2PO4
    86 mM NaCl
    1 mM MgSO4
  3. Agarose pads (prepare beforehand)
    1. Melt 1.5% agarose in M9 buffer in a microwave oven and transfer it to a glass tube.
    2. Keep the agarose in a molten state by placing the tube in a heat block at 65 °C.
    3. Place a clean glass slide between two glass slides sealed with pieces of labeling tape over both ends.
    4. Drop the 1.5% agarose onto the center glass slide and place another clean slide on top, perpendicular to the other three slides.
    5. Gently remove the top perpendicular slide after the agarose solidifies.
    6. Use the agarose before it dries up.

Acknowledgments

This work has been supported by the JSPS KAKENHI Grant Number 26291036, the Sumitomo Foundation, the Naito Foundation, and the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Ken Sato). The protocol has been adapted from Saegusa et al. (2014).

References

  1. Asano, A., Asano, K., Sasaki, H., Furuse, M. and Tsukita, S. (2003). Claudins in Caenorhabditis elegans: their distribution and barrier function in the epithelium. Curr Biol 13(12): 1042-1046.
  2. Carberry, K., Wiesenfahrt, T., Geisler, F., Stöcker, S., Gerhardus, H., Überbach, D., Davis, W., Jorgensen, E., Leube, R. E. and Bossinger, O. (2012). The novel intestinal filament organizer IFO-1 contributes to epithelial integrity in concert with ERM-1 and DLG-1. Development 139(10): 1851-1862.
  3. Grant, B., Zhang, Y., Paupard, M. C., Lin, S. X., Hall, D. H. and Hirsh, D. (2001). Evidence that RME-1, a conserved C. elegans EH-domain protein, functions in endocytic recycling. Nat. Cell Biol. 3(6): 573-579.
  4. Saegusa, K., Sato, M., Sato, K., Nakajima-Shimada, J. and Harada, A. and Sato, K. (2014). Caenorhabditis elegans chaperonin CCT/TRiC is required for actin and tubulin biogenesis and microvillus formation in intestinal epithelial cells. Mol. Biol. Cell 25(20): 3095-3104.

简介

在这种方法中,秀丽隐杆线虫(秀丽隐杆线虫)的肠腔内用荧光液相标记德克萨斯红葡聚糖标记。 由于葡聚糖结合物是不透膜的,所以喂食的动物在肠腔中显示出红色荧光信号。 德克萨斯州的红葡萄糖在管腔中不能有效地被肠细胞内吞,并且不能被有效地输送到细胞外的体腔。 确定圆形膜结构是否是从顶端膜或细胞质囊泡的入内是有用的。 如果肠上皮的屏障功能受损,德克萨斯红葡聚糖可以从肠腔泄漏到体腔。 因此,该方法可用于显示肠细胞中的顶端膜形态,并研究肠上皮的阻隔性。

关键字:线虫, 肠, 顶膜

材料和试剂

  1. C。 elegans 应变
    GK70 unc-119 (ed3)III; dkIs37 [Pact-5-GFP-pgp-1; unc-119(+)]
  2. 大肠杆菌 OP50菌株(获自Caenorhabditis Genetics Center)
  3. 德克萨斯红葡聚糖(40,000MW)(Life Technologies,Molecular Probes ,目录号:D-1829)
  4. 盐酸左旋咪唑(Sigma-Aldrich,目录号:L9756)
  5. 琼脂糖(Nacalai Tesque,目录号:01158-85)
  6. 蛋缓冲区(参见配方)
  7. M9缓冲区(请参阅配方)
  8. 琼脂糖垫(见配方)

设备

  1. 硅化管(1.7ml)(National Scientific Supply,目录号:20172-945)
  2. 台式离心机(Hitachi Koki,型号:CT15RE)
  3. 共聚焦激光扫描显微镜系统(Olympus,型号:FV1000)
  4. 四个干净的玻璃载玻片和盖玻片

程序

  1. 将同步的L1幼虫放置在大肠杆菌(大肠杆菌)OP50接种板上,并在20℃下孵育3天。
  2. 从OP50接种的板中挑取约30-40只成年动物,并将其转移到在1.7ml硅化试管中的500μl蛋缓冲液中。
  3. 让动物在重力下沉降几分钟。 取出上清液,用500μl卵缓冲液洗涤3次
  4. 除去大部分上清液,留下约10-20μl的蠕虫悬浮液,并加入100μl含有1mg/ml德克萨斯红葡聚糖的蛋缓冲液。
  5. 在室温孵育动物90分钟,偶尔轻轻搅拌每30分钟。
  6. 通过在400×g离心2分钟收集动物,并弃去上清液。 用1ml鸡蛋缓冲液洗涤至少3次。
    注意:尽可能减少蠕虫悬浮液中的德克萨斯红葡聚糖。
  7. 向蜗杆悬浮液中加入1M盐酸左旋咪唑,最终浓度为10〜20mM
  8. 将蜗杆悬浮液转移到载玻片上的1.5%琼脂糖垫上,盖上盖玻片
  9. 用共焦激光扫描显微镜观察动物(见图1)

    图1.表达GFP-PGP-1的动物饲喂德克萨斯红葡聚糖。 GFP-PGP-1和德克萨斯红葡聚糖标记的肠顶端膜(大箭头)和肠腔), 分别。当肠上皮细胞的连接密封完整时,通过饲喂摄取的德克萨斯红葡聚糖被限制在肠腔中(Carberry等人,2012)。 A. RNAi介导的C的敲低。秀丽隐杆线虫亚单位(CCT-5)使微小杆菌肌动蛋白丝和微管解体,导致肠顶端膜的异常形态(Saegusa等人,2014)。在这样的动物中,由GFP-PGP-1(秀丽隐杆线虫P-糖蛋白同源物)标记的顶膜形成从顶膜卷入的气泡样膜结构(B,小箭头)。这种结构含有德克萨斯红葡聚糖(B,小箭头)。相比之下,含有GFP-PGP-1的囊泡下面的根尖膜(B,小箭头)不包含德克萨斯红葡聚糖,表明这些是胞吐囊泡。使用FV1000共焦显微镜系统获得图像。 GFP-PGP-1;激发:473nm,检测:490-540nm。德克萨斯 - 红葡聚糖;激发:559nm,检测:575〜675nm。比例尺表示10μm。

笔记

  1. 虽然德克萨斯红葡聚糖在该喂养方法中没有被肠细胞有效摄取,但是一些其它的顶端内吞标记物如罗丹明葡聚糖(emamine)和德克萨斯据报道,红色BSA可用于可视化内吞小泡(Grant等人,2001)。
  2. 紧密连接组分例如密蛋白(CLC-1-4,秀丽隐杆线虫)的消耗引起葡聚糖缀合物从肠腔渗入体腔(Asano等人, ,2003)。

食谱

  1. 蛋缓冲区
    118 mM NaCl
    48 mM KCl
    2mM CaCl 2 2 / 2mM MgCl 2/
    25 mM HEPES
    pH 7.3
    注意:这种溶液比线虫组织的M9更具生理等渗性(Grant et al。,2001)。
  2. M9缓冲区
    42mM Na 2 HPO 4
    22mM KH 2 PO 4 sub/
    86 mM NaCl
    1mM MgSO 4
  3. 琼脂糖垫(预先准备)
    1. 在微波炉中在M9缓冲液中融化1.5%琼脂糖,并将其转移到玻璃管
    2. 通过将管放置在65℃的加热块中使琼脂糖保持在熔融状态。
    3. 将干净的载玻片放在两个玻璃片之间,两端贴上标签带
    4. 将1.5%琼脂糖放在中心载玻片上,放置另一个 清洁滑块在顶部,垂直于其他三个幻灯片
    5. 琼脂糖凝固后轻轻取下顶部垂直的载玻片。
    6. 在干燥之前使用琼脂糖。

致谢

这项工作得到了JSPS KAKENHI Grant号26291036,住友基金会,内藤基金会和医学和药物研究所的莫希达纪念基金会(到Ken Sato)的支持。 该协议已经改编自Saegusa等人(2014)。

参考文献

  1. Asano,A.,Asano,K.,Sasaki,H.,Furuse,M。和Tsukita,S。(2003)。 Caenorhabditis elegans中的紧密连接蛋白:它们在上皮中的分布和屏障功能。 Curr Biol 13(12):1042-1046
  2. Carberry,K.,Wiesenfahrt,T.,Geisler,F.,Stöcker,S.,Gerhardus,H.,Uberbach,D.,Davis,W.,Jorgensen,E.,Leube,REand Bossinger, )。 新型肠丝组织器IFO-1有助于上皮完整性与ERM-1和DLG- 发展 139(10):1851-1862
  3. Grant,B.,Zhang,Y.,Paupard,M.C.,Lin,S.X.,Hall,D.H.and Hirsh,D。(2001)。 证据RME-1,一个守恒的。 elegans EH结构域蛋白,在胞吞循环中的功能。 Cell Biol。 3(6):573-579。
  4. Saegusa,K.,Sato,M.,Sato,K.,Nakajima-Shimada,J.and Harada,A.and Sato,K。(2014)。 Caenorhabditis elegans chaperonin CCT/TRiC is 需要肌动蛋白和微管蛋白生物发生和微肠在肠上皮细胞中的形成。 Biol。 Cell 25(20):3095-3104。
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Copyright: © 2015 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. Saegusa, K. and Sato, K. (2015). Labeling of the Intestinal Lumen of Caenorhabditis elegans by Texas Red-dextran Feeding . Bio-protocol 5(16): e1564. DOI: 10.21769/BioProtoc.1564.
  2. Saegusa, K., Sato, M., Sato, K., Nakajima-Shimada, J. and Harada, A. and Sato, K. (2014). Caenorhabditis elegans chaperonin CCT/TRiC is required for actin and tubulin biogenesis and microvillus formation in intestinal epithelial cells. Mol. Biol. Cell 25(20): 3095-3104.
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