Lando Ana P, De Marco María A, Cumino Andrea C, Martínez-Noël Giselle M A
Instituto de Investigaciones en Biodiversidad y Biotecnología (INBIOTEC) and FIBA, Vieytes 3103, Mar del Plata, Argentina.
Departamento de Química, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata (UNMdP), Funes 3350, Nivel 2, Mar del Plata, Argentina.
Bio Protoc. 2024 Dec 20;14(24):e5140. doi: 10.21769/BioProtoc.5140.
The target of rapamycin (TOR) is a central hub kinase that promotes growth and development in all eukaryote cells. TOR induces protein synthesis through the phosphorylation of the S6 kinase (S6K), which, in turn, phosphorylates ribosomal S6 protein (RPS6) increasing this anabolic process. Therefore, S6K and RPS6 phosphorylation are generally used as readouts of TOR activity. Protein phosphorylation levels are measured by a western blot (WB) technique using an antibody against one specific phosphosite in cell extracts. However, at the tissue/cell-specific level, there is a huge gap in plants due to the lack of alternative techniques for the evaluation of TOR activity as there are for other organisms such as mammals. Here, we describe an in vivo protocol to detect S6K phosphorylation in tissues/cells of model photosynthetic organisms such as and . Our proposed method consists of the immunolocalization of a phosphorylated target of TOR kinase using a fluorescent secondary antibody by confocal microscopy. The protocol involves four main steps: tissue/cell fixation, permeabilization, and incubation with primary and secondary antibodies. It is an easy technique that allows handling different samples at the same time. In addition, different ultrastructural cell markers can also be used, such as for nucleus and cell wall detection, allowing a detailed analysis of cell morphology. To our knowledge, this is the first protocol to detect TOR activity in situ in photosynthetic organisms; we consider that it will pave the research on the TOR kinase, opening new possibilities to better understand its complex signaling. Key features • The protocol is an easy and non-destructive method to detect S6K phosphorylation at the cellular level for plants and algae. • First method for in situ immunolocalization of target proteins of TOR kinase in photosynthetic organisms.
雷帕霉素靶蛋白（TOR）是一种核心枢纽激酶，可促进所有真核细胞的生长和发育。TOR通过磷酸化S6激酶（S6K）来诱导蛋白质合成，而S6K又会磷酸化核糖体S6蛋白（RPS6），从而增强这一合成代谢过程。因此，S6K和RPS6的磷酸化通常被用作TOR活性的检测指标。蛋白质磷酸化水平通过蛋白质免疫印迹（WB）技术进行测定，该技术使用针对细胞提取物中一个特定磷酸位点的抗体。然而，在组织/细胞特异性水平上，由于缺乏像在哺乳动物等其他生物中那样用于评估TOR活性的替代技术，植物领域存在巨大差距。在此，我们描述了一种用于检测模式光合生物（如 和 ）组织/细胞中S6K磷酸化的体内实验方案。我们提出的方法包括通过共聚焦显微镜使用荧光二抗对TOR激酶的磷酸化靶标进行免疫定位。该方案包括四个主要步骤：组织/细胞固定、通透处理以及与一抗和二抗孵育。这是一种简单的技术，能够同时处理不同样本。此外，还可以使用不同的超微结构细胞标记物，如用于细胞核和细胞壁检测的标记物，从而对细胞形态进行详细分析。据我们所知，这是首个在光合生物中原位检测TOR活性的实验方案；我们认为它将为TOR激酶的研究铺平道路，为更好地理解其复杂信号传导开辟新的可能性。关键特性 • 该方案是一种在细胞水平上检测植物和藻类中S6K磷酸化的简单且无损的方法。 • 光合生物中TOR激酶靶蛋白原位免疫定位的首个方法。
