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, phosphorylatesribosomal S6 protein (RPS6) increasing this anabolic process. Therefore, S6K and RPS6 phosphorylation aregenerally 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-specificlevel, there is a huge gap in plants due to the lack of alternative techniques for the evaluation of TOR activity as thereare for other organisms such as mammals. Here, we describe an in vivo protocol to detect S6K phosphorylation intissues/cells of model photosynthetic organisms such as Arabidopsis thaliana and Chlamydomonas reinhardtii. Ourproposed method consists of the immunolocalization of a phosphorylated target of TOR kinase using a fluorescentsecondary 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 handlingdifferent samples at the same time. In addition, different ultrastructural cell markers can also be used, such as fornucleus and cell wall detection, allowing a detailed analysis of cell morphology. To our knowledge, this is the firstprotocol to detect TOR activity in situ in photosynthetic organisms; we consider that it will pave the research on theTOR kinase, opening new possibilities to better understand its complex signaling.