Abstract
Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.
Highlights
Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling
We chose to use Airyscan imaging and total internal reflection fluorescence-single particle (TIRF-SP) imaging as they do not involve the use of special fluorophores required for photo-activated localization microscopy (PALM) or a high power depletion laser used in stimulated emission depletion (STED) which causes damage of aerial tissue in plants due to the presence of light absorbing chloroplasts
We have shown that several plasma-membrane proteins form nanodomains which can be resolved with subdiffraction-limited imaging
Summary
The cell wall regulates dynamics and size of plasmamembrane nanodomains in Arabidopsis. We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains. We have shown that the cell wall has a role in regulating the dynamics and size of plasmamembrane nanodomains for proteins involved in morphogenesis (PIN3) and pathogen perception (FLS2). We chose to use Airyscan imaging and total internal reflection fluorescence-single particle (TIRF-SP) imaging as they do not involve the use of special fluorophores required for photo-activated localization microscopy (PALM) or a high power depletion laser used in STED which causes damage of aerial tissue in plants due to the presence of light absorbing chloroplasts. We hypothesize that the constraint of the cell wall on PM proteins and differential regulation by the actin and microtubule cytoskeletons can contribute to PM organization by altering protein dynamics and nanodomain size
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