SO
评审
Shuhei Ota
  • Biotech Engineer, National Institute for Environmental Studies
研究方向
  • Plant Science, Cell Biology
Immunofluorescence for Detection of TOR Kinase Activity In Situ in Photosynthetic Organisms
利用免疫荧光法原位检测光合生物中TOR激酶活性
作者:Ana P. Lando, María A. De Marco, Andrea C. Cumino and Giselle M. A. Martínez-Noël日期:12/20/2024,浏览量:455,Q&A: 0

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.

Detection and Quantification of Programmed Cell Death in Chlamydomonas reinhardtii: The Example of S-Nitrosoglutathione
在莱茵衣藻中检测和定量程序性细胞死亡:以S-硝基谷胱甘肽为例
作者:Lou Lambert and Antoine Danon日期:08/05/2024,浏览量:472,Q&A: 0

Chlamydomonas (Chlamydomonas reinhardtii) is a unicellular model alga that has been shown to undergo programmed cell death (PCD) that can be triggered in response to different stresses. We have recently shown that Chlamydomonas is particularly well suited to the study and quantification of PCD. We have shown for the first time that S-nitrosoglutathione (GSNO), a nitric oxide (NO) donor, is able to induce PCD and can be used as a study system in Chlamydomonas. In this article, we provide a simple and robust protocol for quantifying GSNO-induced PCD, which can be adapted to any other treatment. We explain how to detect NO production in the cell following GSNO treatment. We show how PCD can be identified simply by analyzing the degradation profile of genomic DNA. We also provide an easy and reproducible cell death quantification protocol, which makes it possible to follow the course of PCD over time and highlight very fine differences in the number of affected cells between different samples.

Improved Methods for Acetocarmine and Haematoxylin Staining to Visualize Chromosomes in the Filamentous Green Alga Zygnema (Charophyta)
用醋酸洋红和苏木精染色法观察丝状绿藻Zynema(轮藻门)染色体的改进方法
作者:Nina Rittmeier and Andreas Holzinger日期:08/20/2023,浏览量:474,Q&A: 0

Genome sizes of Zygnema spp. vary greatly, being unknown whether polyploidization occurred. The exact number of chromosomes in this genus is unknown since counting methods established for higher plants cannot be applied to green algae. The massive presence of pectins and arabinogalactan proteins in the cell wall interferes with the uptake of staining solutions; moreover, cell divisions in green algae are not restricted to meristems as in higher plants, which is another limiting factor. Cell divisions occur randomly in the thallus, due to the intercalary growth of algal filaments. Therefore, we increased the number of cell divisions via synchronization by changing the light cycle (10:14 h light/dark). The number of observed mitotic stages peaked at the beginning of the dark cycle. This protocol describes two methods for the visualization of chromosomes in the filamentous green alga Zygnema. Existing protocols were modified, leading to improved acetocarmine and haematoxylin staining methods as investigated by light microscopy. A freeze-shattering approach with liquid nitrogen was applied to increase the accessibility of the haematoxylin dye. These modified protocols allowed reliable chromosome counting in the genus Zygnema.


Key features

• Improved method for chromosome staining in filamentous green algae.

• Optimized for the Zygnema strains SAG 698-1a (Z. cylindricum), SAG 698-1b (Z. circumcarinatum), and SAG 2419 (Zygnema ‘Saalach’).

• This protocol builds upon the methods of chromosomal staining in green algae developed by Wittmann (1965), Staker (1971), and Fujii and Guerra (1998).

• Cultivation and synchronization: 14 days; fixation and permeabilization: 24 h; staining: 1 h; image analysis and chromosome number quantification: up to 20 h.

Rhizoctonia solani Infection Assay of Young Sugar Beet and Arabidopsis plantlets
甜菜和拟南芥幼苗丝核枯菌侵染试验
作者:Fredrik Dölfors, Louise Holmquist, Georgios Tzelepis and Christina Dixelius日期:01/20/2022,浏览量:2724,Q&A: 1

Rhizoctonia solani is a soil-borne fungus, which rarely produces any spores in culture. Hence, all inoculation procedures are based on mycelia, often as a coat on cereal kernels, placed in close vicinity to the plant to be infected. In this protocol, an inoculation method is described where the fungus is first allowed to infest a perlite-maize flour substrate for 10 days, followed by thorough soil mixing to generate uniform fungal distribution. Pre-grown seedlings are then replanted in the infested soil. Plant materials can be harvested, five (sugar beet) and ten days (Arabidopsis) post infection, followed by a rapid cleaning step ahead of any nucleic acid preparation. Commercial DNA or RNA extraction kits can be used or, if higher DNA yield is required, a CTAB extraction method. Our purpose was to develop a reliable and reproducible protocol to determine the infection levels in planta upon infection with R. solani. This protocol is less laborious compared to previous ones, improves the consistency of plant infection, reproducibility between experiments, and suits both a root crop and Arabidopsis.



Graphic abstract:



Overview of the R. solani infection procedure.


An in vitro Coupled Assay for PEPC with Control of Bicarbonate Concentration
一种控制碳酸氢盐浓度的磷酸烯醇式丙酮酸羧化酶的体外偶联分析
作者:Nicholas R. Moody, Chatawal Phansopal and James D. Reid日期:12/20/2021,浏览量:1608,Q&A: 0

Phosphoenolpyruvate carboxylase (PEPC) catalyzes a critical step in carbon metabolism in plants and bacteria, the irreversible reaction between bicarbonate and phosphoenolpyruvate to produce the C4 compound oxaloacetate. This enzyme is particularly important in the context of C4 photosynthesis, where it is the initial carbon-fixing enzyme. Many studies have used kinetic approaches to characterize the properties of PEPCs from different species, different post-translational states, and after mutagenesis. Most of these studies have worked at a fixed saturating concentration of bicarbonate. Controlling the concentration of bicarbonate is difficult at low concentrations because of equilibration with atmospheric CO2. We describe here a simple, repeatable, and gas-tight assay system for PEPC that allows bicarbonate concentrations to be controlled above ca. 50 µM.


Buoyant Density Fractionation of Small Extracellular Vesicle Sub-populations Derived from Mammalian Cells
哺乳动物细胞来源的胞外小囊泡亚群浮力密度分级
作者:Morayma M. Temoche-Diaz, Matthew J. Shurtleff and Randy Schekman日期:08/05/2020,浏览量:4135,Q&A: 0
Small extracellular vesicles (sEVs) encompass a variety of distinct vesicles that are secreted to the extracellular space. Many methodologies currently used for EV isolation (e.g., differential ultracentrifugation concluding in a high-speed pellet, precipitation by macromolecular crowding agents or size excusion chromatography–SEC) do not fractionate distinct sEV sub-populations. Samples obtained by the aforementioned methods are usually used for characterization and physiological studies. However the fraction that contains the molecule of interest or is the carrier of a specific activity is unknown. Therefore isolating distinct sEV sub-populations is critical to understand EV function. The goal of this procedure is to purify distinct sEV sub-populations based on slight differences in their buoyant density. Moreover, this technique also allows sEVs purification from vesicle-free RNA-protein complexes co-isolating in the high-speed pellet or by the use of crowding agents. This protocol describes cultivation of mammalian cells for sEV collection, sEV sedimentation, buoyant density fractionation of sEV sub-populations and immunoblots for sEV markers. This protocol can be used to fractionate distinct sEV sub-populations produced by a variety of mammalian cells.
Analysis of Exosome Transfer in Mammalian Cells by Fluorescence Recovery after Photobleaching
光漂白后荧光恢复技术分析哺乳动物细胞外泌体转移
During the course of evolution, prokaryote and eukaryote cells have developed elegant and to some extent analogous strategies to communicate with each other and to adapt to their surrounding environment. Eukaryotic cells communicate with each other through direct interaction via juxtracrine signaling and/or by secreting soluble factors. These secreted factors can subsequently act on the cell itself (autocrine signaling) or interact with neighboring (paracrine signaling) and distant (endocrine signaling) cells. The transmission of signals between cells and tissues has been traditionally thought to be regulated by a protein-based signaling system. Typically, proteins destined for secretion into the extracellular milieu by exocytosis contain a canonical secretion-targeting sequence (Théry et al., 2002). However, proteins with a non-continuous and stimulus-dependent secretion, proteins that do not contain a canonical secretion-targeting sequence, and species that might be too labile within the extracellular environment (DNA, mRNA, peptides, metabolites, miRNA and other RNA species), can be secreted in small membranous extracellular vesicles (EVs) in a specific manner (Hagiwara et al., 2014). Exosomes represent one broad class of these secreted membrane vesicles with a diameter of 30-130 nm (Cocucci et al., 2009; Théry et al., 2009; Kowal et al., 2014), which are formed inside the secreting cells in endosomal compartments called multivesicular bodies. Molecules loaded into exosomes as well as the intensity of the exosome transfer between cells are important parameters for the subsequent conditioning of recipient cells. Current knowledge on secretion of exosomes and their internalization in recipient cells remains incomplete. It is known that secretion intensity of exosomes varies according to the cellular type and its physiological state (Garcia et al., 2016). Moreover, the different combination of transmembrane proteins on the surface of exosomes that facilitate the adhesion to the cell-extracellular matrix vary the avidity with which a recipient cell captures exosomes (Hoshino et al., 2015). Here, we have developed an in vitro system by which the transfer of exosomes between cells in co-culture can be quantified using FRAP (‘Fluorescence Recovery After Photobleaching’) technology. This protocol has been used to analyze the effects of exosome transfer of hypoxia inducible factor 1-α (HIF-1α) in Mesenchymal Stem Cells (MSC; HIF-MSC) to Human Umbilical Cord Vein Endothelial Cells (HUVEC) (Gonzalez-King et al., 2017).