bims-mecosi 2023-08-13 papers

Issue of 2023–08–13
two papers selected by
Verena Kohler, University of Graz

Contact (Thousand Oaks). 2023 Jan-Dec;6:6 25152564231189064

Interorganellar Communication Through Membrane Contact Sites in Toxoplasma Gondii.

Apicomplexan parasites are a group of protists that cause disease in humans and include pathogens like Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, the etiological agent of toxoplasmosis and one of the most ubiquitous human parasites in the world. Membrane contact sites (MCSs) are widespread structures within eukaryotic cells but their characterization in apicomplexan parasites is only in its very beginnings. Basic biological features of the T. gondii parasitic cycle support numerous organellar interactions, including the transfer of Ca2+ and metabolites between different compartments. In T. gondii, Ca2+ signals precede a series of interrelated molecular processes occurring in a coordinated manner that culminate in the stimulation of key steps of the parasite life cycle. Calcium transfer from the endoplasmic reticulum to other organelles via MCSs would explain the precision, speed, and efficiency that is needed during the lytic cycle of T. gondii. In this short review, we discuss the implications of these structures in cellular signaling, with an emphasis on their potential role in Ca2+ signaling.

Keywords: Toxoplasma gondii; apicomplexan; calcium; membrane contact sites

DOI: https://doi.org/10.1177/25152564231189064

Curr Protoc. 2023 Aug;3(8): e854

Applying Optical Tweezers with TIRF Microscopy to Quantify Physical Interactions Between Organelles in the Plant Endomembrane System.

Eleanor M Fletcher, Benji C Bateman, Stanley W Botchway, Andrew D Ward, Imogen A Sparkes.

Plant organelles are associated with each other through tethering proteins at membrane contact sites (MCS). Methods such as total internal reflection fluorescence (TIRF) optical tweezers allow us to probe organelle interactions in live plant cells. Optical tweezers (focused infrared laser beams) can trap organelles that have a different refractive index to their surrounding medium (cytosol), whilst TIRF allows us to simultaneously image behaviors of organelles in the thin region of cortical cytoplasm. However, few MCS tethering proteins have so far been identified and tested in a quantitative manner. Automated routines (such as setting trapping laser power and controlling the stage speed and distance) mean we can quantify organelle interactions in a repeatable and reproducible manner. Here we outline a series of protocols which describe laser calibrations required to collect robust data sets, generation of fluorescent plant material (Nicotiana tabacum, tobacco), how to set up an automated organelle trapping routine, and how to quantify organelle interactions (particularly organelle interactions with the endoplasmic reticulum). TIRF-optical tweezers enable quantitative testing of putative tethering proteins to reveal their role in plant organelle associations at MCS. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Microscope system set-up and stability Basic Protocol 2: Generation of transiently expressed fluorescent tobacco tissue by Agrobacterium-mediated infiltration Basic Protocol 3: Setting up an automated organelle trapping routine Basic Protocol 4: Quantifying organelle interactions.

Keywords: TIRF; endoplasmic reticulum; optical tweezers; plant organelles

DOI: https://doi.org/10.1002/cpz1.854