基于显微镜的测定，用于使用SNARE运输研究和分析回收内体
Summary
回收内体是内体管网络的一部分。在这里，我们提出了一种使用GFP-STX13作为细胞器标记来量化回收内体动力学的方法。
Abstract
回收内体（REs）是由所有细胞类型中的早期/分选内体产生的管状囊泡细胞器。这些细胞器在黑素体的生物发生中起着关键作用，黑素体是由黑素细胞产生的溶酶体相关细胞器。RE在形成期间将黑素细胞特异性货物输送到过早的黑素体。在Hermansky-Pudlak综合征的几个突变体中观察到的REs的产生阻塞，导致皮肤，头发和眼睛色素减退。因此，研究REs的动力学（参见数量和长度）对于了解这些细胞器在正常和疾病条件下的功能是有用的。在这项研究中，我们的目标是使用常驻SNARE STX13测量RE动力学。
Introduction
黑色素色素的生物合成发生在黑素体中，黑素体是一种与常规溶酶体共存的黑素细胞特异性溶酶体相关细胞器（LRO）。内吞系统在黑色素体的生物发生中起着关键作用，黑素体是肤色和防止电离辐射的光保护所必需的1，2，3。在此过程中，黑色素合成酶在早期/分选内体上被分选，然后通过称为循环内体（REs）的管状或囊泡内体运输到过早的黑素体4，5，6，7，8，9，10。这些细胞器的靶向和融合调节全功能色素沉着黑素体的成熟7，11，12，13，14。在Hermansky-Pudlak综合征中观察到，这些细胞器形成或对这些细胞器的货物分拣中的缺陷导致眼皮白化病和其他临床表型15，16。
在这里，我们描述了一种基于显微镜的简单技术来研究和分析RE。在这种方法中，我们利用了一种跨膜蛋白Qa-SNARE Syntaxin（STX）13，它存在于循环内体17上，并且在黑素细胞中分选内体和黑素体之间的循环12，18。此外，删除N端非结构化调节域（即SynN或STX13Δ129）允许SNARE卡在黑素体中，黑素体测量向黑素体的前向运输途径12。我们在研究中使用了已知的回收内体标志物Rab GTPase（Rab）11111111。对野生型黑素细胞中的蛋白质GFP-STX13WT，GFP-STX13Δ129，mCherry-Rab11和TYRP1进行荧光成像，然后定量其相对定位，除了它们靶向黑素体外，还将提供REs的性质和动力学。因此，这是一种简单的技术，可用于可视化和测量黑素细胞中RE的动力学。
Protocol
该协议涉及黑素细胞的接种，然后转染质粒。进一步的步骤包括固定，免疫染色，成像和分析细胞，以测量RE的长度和数量。该协议的详细说明如下。 1. 在预处理的盖玻片上接种小鼠黑素细胞 用基底膜基质培养基（在完全RPMI培养基中为1：20：20：1：20）涂覆在培养皿（即35毫米培养皿中4- 5个培养皿）中的玻璃盖玻片，并在组织培养罩中干燥15分钟。使用前…
Representative Results
STX13Δ129突变体定位到黑素体的定量分析STX13在小鼠野生型黑素细胞中的免疫荧光显微镜显示，GFP-STX13WT定位为囊泡和管状结构，GFP-STX13Δ129定位为细胞表面外的环状结构（图1A）。此外，细胞内环状GFP-STX13Δ129显示出与黑素体蛋白TYRP1（图1A）和明场成像黑素体（数据未显示）的共定位<sup c…
Discussion
回收内体是一组内吞细胞器，它们介导所有细胞类型中货物到细胞表面的回收21，22，23，24，25。在黑色素细胞等特殊细胞类型中，这些细胞器部分地将其运输路线转移到黑素体以进行生物发生3，16，26。?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了生物技术部的支持（BT / PR32489 / BRB / 10 / 1786 / 2019到SRGS）;科学与工程研究委员会（CRG/2019/000281给SRGS）;DBT-NBACD（BT/HRD-NBA-NWB/38/2019-20 至 SRGS）和 IISc-DBT 合作计划（至 SRGS）。该部门的基础设施得到了DST-FIST，DBT和UGC的支持。AMB 由 DBT-JRF （DBT/2015/IISc/NJ-02） 提供支持。
Materials
| anti-TYRP1 antibody (TA99) | ATCC | HB-8704 | |
| Fluoromount-G | Southern Biotech | 0100-01 | |
| Lipofectamine 2000 | ThermoFisher Scientific | 11668-500 | |
| Matrigel matrix | BD Biosciences | 356231 | |
| OPTI-MEM | ThermoFisher Scientific | 022600-050 | |
| Phorbol-12-myristate-13-acetate | Sigma-Aldrich | P8139 | |
| RPMI Medium 1640 | ThermoFisher Scientific | 31800-022 |
References
- Dell’Angelica, E. C. The building BLOC(k)s of lysosomes and related organelles. Current Opinion in Cell Biology. 16 (4), 458-464 (2004).
- Raposo, G., Marks, M. S. Melanosomes–dark organelles enlighten endosomal membrane transport. Nature Reviews in Molecular Cell Biology. 8 (10), 786-797 (2007).
- Ohbayashi, N., Fukuda, M. Recent advances in understanding the molecular basis of melanogenesis in melanocytes. F1000Research. 9, (2020).
- Theos, A. C., et al. Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes. Molecular Biology of the Cell. 16 (11), 5356-5372 (2005).
- Di Pietro, S. M., et al. BLOC-1 interacts with BLOC-2 and the AP-3 complex to facilitate protein trafficking on endosomes. Molecular Biology of the Cell. 17 (9), 4027-4038 (2006).
- Setty, S. R., et al. BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles. Molecular Biology of the Cell. 18 (3), 768-780 (2007).
- Delevoye, C., et al. AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis. Journal of Cell Biology. 187 (2), 247-264 (2009).
- Bultema, J. J., Ambrosio, A. L., Burek, C. L., Di Pietro, S. M. BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles. Journal of Biological Chemistry. 287 (23), 19550-19563 (2012).
- Sitaram, A., et al. Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes. Molecular Biology of the Cell. 23 (16), 3178-3192 (2012).
- Nag, S., et al. Rab4A organizes endosomal domains for sorting cargo to lysosome-related organelles. Journal of Cell Science. 131 (18), (2018).
- Dennis, M. K., et al. BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery. Journal of Cell Biology. 209 (4), 563-577 (2015).
- Jani, R. A., Purushothaman, L. K., Rani, S., Bergam, P., Setty, S. R. STX13 regulates cargo delivery from recycling endosomes during melanosome biogenesis. Journal Cell Science. 128 (17), 3263-3276 (2015).
- Shakya, S., et al. Rab22A recruits BLOC-1 and BLOC-2 to promote the biogenesis of recycling endosomes. EMBO Reports. 19 (12), 45918 (2018).
- Bowman, S. L., et al. A BLOC-1-AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers. Journal of Cell Biology. 220 (7), 202005173 (2021).
- Wei, M. L. Hermansky-Pudlak syndrome: a disease of protein trafficking and organelle function. Pigment Cell Research. 19 (1), 19-42 (2006).
- Bowman, S. L., Bi-Karchin, J., Le, L., Marks, M. S. The road to lysosome-related organelles: Insights from Hermansky-Pudlak syndrome and other rare diseases. Traffic. 20 (6), 404-435 (2019).
- Prekeris, R., Klumperman, J., Chen, Y. A., Scheller, R. H. Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes. Journal of Cell Biology. 143 (4), 957-971 (1998).
- Mahanty, S., et al. Rab9A is required for delivery of cargo from recycling endosomes to melanosomes. Pigment Cell Melanoma Research. 29 (1), 43-59 (2016).
- Delevoye, C., et al. Recycling endosome tubule morphogenesis from sorting endosomes requires the kinesin motor KIF13A. Cell Reports. 6 (3), 445-454 (2014).
- Ha, L., et al. ARF functions as a melanoma tumor suppressor by inducing p53-independent senescence. Proceedings of the National Academy of Science U. S. A. 104 (26), 10968-10973 (2007).
- Soldati, T., Schliwa, M. Powering membrane traffic in endocytosis and recycling. Nature Reviews Molecular Cell Biology. 7 (12), 897-908 (2006).
- Grant, B. D., Donaldson, J. G. Pathways and mechanisms of endocytic recycling. Nature Reviews Molecular Cell Biology. 10 (9), 597-608 (2009).
- Hsu, V. W., Prekeris, R. Transport at the recycling endosome. Current Opinion in Cell Biology. 22 (4), 528-534 (2010).
- Taguchi, T. Emerging roles of recycling endosomes. Journal of Biochemistry. 153 (6), 505-510 (2013).
- Goldenring, J. R. Recycling endosomes. Current Opinion in Cell Biology. 35, 117-122 (2015).
- Delevoye, C., Marks, M. S., Raposo, G. Lysosome-related organelles as functional adaptations of the endolysosomal system. Current Opinion in Cell Biology. 59, 147-158 (2019).
- Hsu, V. W., Bai, M., Li, J. Getting active: protein sorting in endocytic recycling. Nature Reviews in Molecular Cell Biology. 13 (5), 323-328 (2012).
- Desfougeres, Y., D’Agostino, M., Mayer, A. A modular tethering complex for endosomal recycling. Nature Cell Biology. 17 (5), 540-541 (2015).
- Le, L., Sires-Campos, J., Raposo, G., Delevoye, C., Marks, M. S. Melanosome biogenesis in the pigmentation of mammalian skin. Integrated Computational Biology. 61 (4), 1517-1545 (2021).
Cite This Article
Bhatt, A. M., Setty, S. R. G. The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking. J. Vis. Exp. (180), e63087, doi:10.3791/63087 (2022).