Single-particle tracking photoactivated localization microscopy of membrane proteins in living plant tissues

Abstract

This protocol describes how to perform single-particle tracking Photo-Activated Localization Microscopy (sptPALM) of membrane proteins in living plant tissues. The procedure covers all stages from sample preparation to data analysis. TWEET A new Protocol for single particle tracking Photo-Activated Localization Microscopy (sptPALM) of membrane proteins in live plant tissues. #PALM #super-resolution @YvonJaillais @RDPlab COVER TEASER sptPALM of membrane proteins in live plant tissues 2

Nanodomain-mediated lateral sorting drives polarization of the small GTPase ROP2 in the plasma membrane of root hair cells

“…In methods using photoactivatable fluorescent proteins (FPs), e.g. sptPALM (Manley et al, 2008;Bayle et al, 2021), the density of fluorophores can be tuned by adjusting the power of the activating laser and it is possible to obtain relatively long tracks. In contrast, FP density in ToBF cannot be tuned, is generally higher and varies over the course of a time-lapse measurement, as well as between different samples.…”

Imaging the living plant cell: From probes to quantification

“…Nanodomains are by definition small, and often their size is below the diffraction limit of optical microscopy. Several techniques have been used to visualize nanodomains in the living plant plasma membrane and to probe their dynamics, notably Total Internal Resonance Fluorescence Microscopy (TIRFM), PhotoActivated Localization Microscopy (PALM), and Single Particle Tracking (SPT) techniques (Table 1) (Martiniere et al, 2012;Hosy et al, 2015;Gronnier et al, 2017;Wang et al, 2018;Martiniere et al, 2019;Platre et al, 2019;Zhang et al, 2019;Smokvarska et al, 2020;Bayle et al, 2021;Noack et al, 2021). These methods have revealed a number of nanodomain-resident proteins, such a Remorins, Flotilins, HYPERSENSITIVE INDUCED REACTION proteins (HIRs), and receptor-like kinases (Li et al, 2012;Bucherl et al, 2017;Daněk et al, 2020;Gronnier et al, 2020;Jaillais and Ott, 2020;Gouguet et al, 2021;Martinière and Zelazny, 2021), as well as some proteins with a dynamic association with nanodomains, such as small GTPases from the RHO-OF-PLANTs (ROP) family (Platre et al, 2019;Smokvarska et al, 2020;Bayle et al, 2021;Fuchs et al, 2021;Smokvarska et al, 2021).…”

“…Second, membrane trafficking is fast, with certain compartments moving tens of micrometers per minute, notably due to cytoplasmic streaming (Luo et al, 2015). In term of resolution, there are more and more examples of super-resolution microscopy methods used in plants (Komis et al, 2015b;Komis et al, 2015a;Schubert, 2017;Shaw et al, 2019;Bayle et al, 2021). These methods can provide large gains in resolution, such as PALM (Hosy et al, 2015;Gronnier et al, 2017;Martiniere et al, 2019;Platre et al, 2019;Bayle et al, 2021) and Stimulated Emission Depletion Microscopy (STED) (Kleine-Vehn et al, 2011;Demir et al, 2013) or provide ultrafast high resolution imaging, such as super-resolution confocal live imaging microscopy (SCLIM) (Table 1) (Naramoto et al, 2014;Uemura et al, 2014;Uemura et al, 2019;Shimizu et al, 2021).…”

“…To image events that occur at or close to the plasma membrane, the technique of choice is TIRF microscopy (or derivatives of the TIRF technique such as variable angle epifluorescence microscopy (VAEM -VA-TIRFM), which is a very sensitive technique because it does not collect any out-of-focus light (Table 1) Gronnier et al, 2017;Johnson and Vert, 2017;Platre et al, 2019;Johnson et al, 2020;Narasimhan et al, 2020;Smokvarska et al, 2020;Bayle et al, 2021). TIRF microscopy has mainly been used to study endocytosis, but also cellulose synthesis and cytoskeleton dynamics, and it can be combined with structured illumination microscopy (SIM) to achieve fast superresolved acquisition (Table 1) (Johnson et al, 2021).…”

Single-Molecule Imaging in Living Plant Cells: A Methodological Review

“…Since the imaging speed of PALM and STORM is slow, they are not suitable for high-speed single-molecule tracking (>1 μm 2 /s). Recently, PALM was successfully used to track slow-moving proteins in living roots [ 90 ]. It can be expected that the combined applications of TIRF-SIM [ 91 ] and STED-FCS [ 92 ] will be used in plant research in the near future.…”