Rolfe, D. | bioRxiv Science

bioRxiv2018

The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis.

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport and cellular signalling. Recent models suggest that for these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signalling 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 sub-diffraction-limited Airyscan confocal microscopy. In addition, single particle tracking reveals that these proteins move at different rates within the membrane. We show that the 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 affecting single particle dynamics of two 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 differentially regulating protein cluster size and dynamics thereby contributing to the formation and functionality of membrane nanodomains.\n\nOne sentence summarySize and mobility of protein nanodomains in the plant plasma-membrane are regulated by interaction with the cell wall extracellular matrix.\n\nSignificance statementThe plant plasma membrane acts as the front line for cellular perception of the environment. As such, a large number of signalling and transport proteins which perceive or transport environmental signals, developmental cues and nutrients are located within it. Recently, a number of studies have revealed that proteins located within the plasma membrane do not simply freely diffuse within its plane. Rather, proteins are localized in nanometer sized structures called nanodomains. In addition to the plasma-membrane, plant cells also have an extracellular matrix, the cell wall. Here we have shown that the cell wall has a role in regulating the dynamics and size of plasma membrane nanodomains for proteins involved in morphogenesis (PIN3) and pathogen perception (FLS2).

cell biology↗

bioRxiv2018

Solid immersion microscopy readily and inexpensively enables 12 nm resolution on plunge-frozen cells

Super-resolution fluorescence microscopy achieves 20-30 nm resolution by using liquid-immersion objectives to optimize light collection and chemical sample fixation to minimize image blurring. It is known that fluorophore brightness increases substantially under cryogenic conditions and that cryo-fixation is far superior in preserving ultrastructure. However, cryogenic conditions have not been exploited to improve resolution or sample quality because liquid immersion media freezes at the objective, losing its optical properties. Here, simply by replacing the immersion fluid with a low-cost super-hemispherical solid immersion lens (superSIL), we effortlessly achieve <8 nm localisation precision and 12 nm resolution under cryogenic conditions in a low-cost, low-tech system. This is to our knowledge the best resolution yet attained in biological samples. Furthermore, we demonstrate multicolour imaging and show that the inexpensive setup outperforms 10-fold more costly super-resolution microscopes. By also removing the barrier to total internal reflection fluorescence imaging of mammalian cells under cryogenic conditions, superSIL microscopy delivers a straightforward route to achieve unmatched nanoscale resolution on both bacterial and mammalian cell samples, which any laboratory can effortlessly and inexpensively implement.

biophysics↗