使用扩张显微镜对波多细胞蛋白尼腓林、阿克汀和波多辛的成像
Summary
所介绍的方法使荧光标记的细胞蛋白具有扩展显微镜的可视化，从而在传统显微镜上实现 70 nm 的分辨率。
Abstract
由球状内皮、球状地下膜和卵石细胞组成的球状过滤器的破坏导致白蛋白尿。多细胞足部过程包含与细胞细胞适配器蛋白（如波多辛）结合的活性素束。这些适配蛋白，如波多辛，将球状狭缝隔膜的骨干，如肾素，与细胞链球菌素连接起来。研究这些蛋白质和其他多囊蛋白的本地化和功能对于理解球状过滤器在健康和疾病中的作用至关重要。所呈现的协议使用户能够在传统显微镜上实现超分辨率成像的细胞中的行为素、波多辛和肾上腺素的可视化。首先，细胞被传统的免疫荧光技术所染色。然后，样品中的所有蛋白质都与膨胀的水凝胶共价锚定。通过用蛋白酶K消化，结构蛋白在最后一步被切开，从而允许凝胶的同位素肿胀。在水中透析样品可使样品膨胀4-4.5倍，样品可以通过传统的荧光显微镜进行成像，潜在分辨率为70纳米。
Introduction
白蛋白尿是心血管风险的代孕参数，是球状过滤器1中断的结果。球状过滤器由受精内皮、球状地下室膜和由政治细胞形成的狭缝隔膜组成。主要和次要的足部过程的负细胞包裹在毛细细壁的球状2。足部过程的微妙结构由皮质作用素束保持，皮质作用束也作为多个狭缝隔膜蛋白和其他适配蛋白2的锚。狭缝隔膜的骨干蛋白称为肾素，以同性亲性的方式与对立的 podocyt 的肾素分子相互作用。通过不同的适配器蛋白，肾上腺素与作用细胞球菌2，3有关。肾素编码基因NPHS1的突变导致芬兰4型肾综合征。
肾素的相互作用蛋白之一是波多辛，一种类似发夹的蛋白质的口腔素家族3。波多辛招募肾上腺素脂质筏，并将其链接到行动细胞球菌5。波多辛由NPHS2基因编码。NPHS2中的突变导致类固醇耐肾病综合征6。
为了可视化和共同本地化行为适应蛋白，可以使用免疫荧光技术。不幸的是，光的衍射屏障将传统荧光显微镜的分辨率限制在200-350纳米7。新型显微镜技术，如刺激发射耗竭（STED）8，光激活定位显微镜（PALM）9，随机光学重建显微镜（STORM或dSTORM）或地面状态删除显微镜，然后单个分子返回（GSDIM）9，10，11，使分辨率高达约10纳米。然而，这些超级分辨率技术需要非常昂贵的显微镜，训练有素的人员，因此在许多实验室中是不可用的。
扩展显微镜 （ExM） 是一种新颖而简单的技术，能够使用传统显微镜实现超分辨率成像，并有可能提供给大型研究团体12。在蛋白质保留扩张显微镜（proExM）中，兴趣样本（细胞或组织）是固定的，并沾染了氟磷13。然后，样品中的蛋白质由小分子（6-（丙烯酰）氨基酸、苏奇尼米酰酯、AcX）共同锚定成可膨胀的水凝胶13。通过酶酶K（ProK）的酶消化，蛋白质和氟磷在膨胀13后在凝胶中保持其相对位置。凝胶膨胀后，样品膨胀至4.5倍（体积膨胀90倍），有效横向分辨率约为60-70纳米（300纳米/4.5）。这项技术的修改甚至可以允许10倍的扩展（1000倍体积扩展），使分辨率20-30纳米的传统显微镜14，15，16。
老鼠和人类肾脏的球状结构已经通过ExM17可视化。在本文中，我们提出了一个详细的 ProExM 协议，使用传统的荧光显微镜可视化细胞内 F-actin 和 actin 适配器蛋白 podocin 的超分辨率图像。
Protocol
Representative Results
Discussion
提出的方法使研究者能够可视化细胞蛋白，如波多辛、肾上腺素和细胞骨质成分，例如F-actin。在此协议中，变质的 cos7 细胞用作研究狭缝隔膜蛋白与 F-actin 相互作用的模型。不幸的是，不朽的足细胞系不能表达足够的内源性狭缝隔膜蛋白19。
使用这种方法，细胞蛋白可以使用传统的荧光显微镜以纳米尺度分辨率进行可视化。协议中最关键的步骤是：1） 用 Ac…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢布兰卡·杜夫尼亚克和尼古拉·库尔的出色技术援助。
Materials
| Acrylamide >99% | Sigma-Aldrich | A3553-100G | |
| 6-((Acryloyl)amino)hexanoic acid, succinimidyl ester, Acryloyl-X, SE | invitrogen | A-20770 | store up to 4 months |
| APS | Sigma-Aldrich | A3678-25G | |
| Deckgläser (cover glasses) | Engelbrecht | K12432 | 24x32mm #1.0 |
| Diamont cutter | VWR | 201-0392 | for cutting the cover slips |
| Guanidine HCl | Sigma-Aldrich | G3272-100G | 8M Stock can be kept at RT |
| Marten hair paintbrush | Leon Hardy | 3 (770) | |
| "Menzel" Deckgläser (cover glasses) | Thermo Fischer | 15654786 | 24x24mm #1.5 |
| N,N`-Methylenbisacrylamide | Sigma-Aldrich | M7256-25G | |
| Objektträger UniMark | Marienfeld | 703010 | |
| Proteinase K | New England Biolabs | P8107S | |
| Sodium Acrylate | Sigma-Aldrich | 408220 | check purity |
| Sodium Bicarbonate | Sigma-Aldrich | S5761 | |
| Staining chamber | produced at the university's workshop | ||
| TEMED | ROTH | 2367.1 | |
| 6-Well glass bottom plates | Cellvis | P06-1.5H-N | |
| Antibodies | |||
| Actin-ExM 546 | chrometra | non-available | 1:40 |
| Anti Podocin produced in rabbit | Sigma | P-0372-200UL | 1:200 |
| Donkey anti guinea-pig CF633 | Sigma | SAB4600129-50UL | 1:200 |
| Goat anti rabbit 488 | Life Technologies | A11034 | 1:1000 |
| Guinea pig anti nephrin | Origene | BP5030 | 1:100 |
| Software | |||
| FIJI | |||
| Visiview | |||
| microscope | |||
| AXIO Observer Z1 | Zeiss | non-available |
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