Immunofluorescence for Detection of TOR Kinase Activity In Situ in Photosynthetic Organisms.

Abstract

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 Arabidopsis thaliana and Chlamydomonas reinhardtii . 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.

Plant phenotyping relevance

植物・藻類組織内のTOR活性をリン酸化S6Kの免疫蛍光と共焦点顕微鏡で細胞レベルに測定する新規プロトコルであり、植物の生理状態取得法が中心である。

Code and data availability

This is a methods/protocol paper describing an immunofluorescence procedure for detecting TOR kinase activity in Arabidopsis and Chlamydomonas. No public phenotype/trait datasets, plant images, author analysis code, trained models, or paper-specific data deposits are mentioned. The only URL besides the license is NIH's

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