Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis

Abstract

Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects of plant growth, development, intra- and inter-cellular signaling, nutrient uptake and pathogen defense. Despite these significant roles, little is known about the precise molecular details of how it functions in planta. In order to facilitate the direct quantitative study of plant CME, here we review current routinely used methods and present refined, standardized quantitative imaging protocols which allow the detailed characterization of CME at multiple scales in plant tissues. These include: (i) an efficient electron microscopy protocol for the imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed characterization of the ultra-structure of clathrin-coated vesicles; (ii) a detailed protocol and analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal interplay of endocytosis components during single CME events; (iii) a semi-automated analysis to allow the quantitative characterization of global internalization of cargos in whole plant tissues; and (iv) an overview and validation of useful genetic and pharmacological tools to interrogate the molecular mechanisms and function of CME in intact plant samples.

The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis

“…A Zeiss LSM-800 confocal microscope was to examine the effect of FM4-64 uptake in 5-day old Col-0 seedlings. Seedlings were incubated for 6 hours at either room temperature or 35°C for six hours and then incubated with 2 µM FM4-64 in AM+ media for 5 minutes, washed twice in 1/2 MS and 1% sucrose media and imaged and analyzed as specified previously ( 30 ). A 40x water immersion objected was used.…”

“…7-day old seedlings were incubated at either 25°C or 37°C for 6 hours prior to imaging. Root epidermal cells were imaged and analyzed as described previously ( 30 ); this provided unbiased lifetimes, densities and fluorescence profiles of endocytosis proteins in samples subjected to the experimental temperature conditions.…”

“…Structured illumination microscopy (SIM) was conducted on 7-day old seedlings expressing TPLATE-GFP or AP2A1-GFP and CLC2-TagRFP. Root samples were prepared as described previously ( 30 ), but high-precision 1.5 coverslips were used (Thorlabs, #CG15CH), and epidermal cells in the elongation zone were selected for imaging. For 3D-SIM, an OMX BLAZE v4 SIM (Applied Precision) was used.…”

Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana

“…Three days after germination, the Arabidopsis seedlings were mounted in liquid ½MS under a cover glass or under a small piece of growth medium agar in a chamber with a cover glass bottom. In most cases, the meristematic root zone was imaged, but other regions were imaged in some experiments as indicated (Johnson et al ., 2020). Confocal images were obtained with a Zeiss LSM 700 using a ×100/NA 1.46 oil objective lens.…”

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

“…This is an important distinction since the dynamic of single molecules can sometimes show different behavior than their associated structure within the cell. For example, we found that the clathrin light chain 2 (CLC2) shows static punctuated structures at the membrane with very little lateral diffusion when observed with classical FP and tracking analyses [40][41][42] . In contrast, at the molecular level assessed by sptPALM, we observed that a fraction of CLC2 molecules are diffusing within the membrane 15 .…”