Preparation of Clathrin-Coated Vesicles From Arabidopsis thaliana Seedlings

Clathrin coated vesicles (CCVs) mediate endocytosis of plasma membrane proteins and deliver their content to the endosomes for either subsequent recycling to the plasma membrane or transport to the vacuole for degradation. CCVs assemble also at the trans-Golgi network (TGN) and is responsible for the transport of proteins to other membranes. Oligomerization of clathrin and recruitment of adaptor protein complexes promote the budding and the release of CCVs. However, many of the details during plant CCV formation are not completely elucidated. The analysis of isolated CCVs is therefore important to better understand the formation of plant CCVs, their cargos and the regulation of clathrin-mediated transport processes. In this article, we describe an optimized method to isolate CCVs from Arabidopsis thaliana seedlings.


INTRODUCTION
Plants regulate their development to the changing environment by sensing and responding to environmental signals. Plasma membrane associated receptors, transporters and lipids play an important role in coordinating extracellular signals with intracellular responses. The regulation of plasma membrane proteins is therefore critical for optimal plant development and growth. Besides transcriptional control, endocytosis and subsequent endosomal trafficking of plasma membrane proteins is also essential for correct environmental responses (Paez Valencia et al., 2016). The endocytosis of a membrane protein can be either dependent on clathrin (clathrinmediated endocytosis [CME]) or independent (McMahon and Boucrot, 2011;Sandvig et al., 2011Sandvig et al., , 2018Baisa et al., 2013;Mayor et al., 2014;Robinson, 2015). Clathrin build a coat around the endocytosed vesicle to form a clathrin coated vesicle (CCV). The clathrin coat is formed by multiple units composed of three clathrin heavy chains (CHCs) and three clathrin light chains (CLCs) that oligomerize together to form three-legged structures called triskelia.
Several accessory proteins, in concert with the heterotetrameric adaptor complex ADAPTIN PROTEIN 2 (AP2), link the clathrin coat to the plasma membrane bilayer and recognize specific cargo proteins for endocytosis. Another important adaptor complex in Arabidopsis is the eight core-component complex TPLATE (TPC) that is important for clathrin recruitment at the plasma membrane and for the initiation of CME (Van Damme et al., 2011;Gadeyne et al., 2014).
Once the clathrin adaptors have selected the cargos and the clathrin coat is assembled to a clathrin coated pit (CCP), the local membrane curvature resolves into a CCV upon scission from the plasma membrane. Once CCVs are released into the cytoplasm, they undergo uncoating before delivering their cargos to the trans-Golgi network (TGN)/early endosomal compartment (EE) (Dettmer et al., 2006). The endocytosed cargos can either be recycled back to the plasma membrane or transported to the vacuole for degradation (Viotti et al., 2010;Paez Valencia et al., 2016;Reynolds et al., 2018). Clathrin is also recruited onto the TGN by adaptor complexes AP1 and AP3. CCVs can be generated from the surface of the TGN and are responsible for post-Golgi trafficking to the vacuole and for transport to the plasma membrane (Robinson and Pimpl, 2014;Rosquete et al., 2018).
Research in the past decade has shown that clathrin-mediated transport is essential for important physiological processes in plants. Although many regulatory factors have been identified, we still lack in-depth understanding on the nature of CCV cargos and the exact mechanisms of clathrin-dependent vesicle trafficking. One limitation, so far, was the difficulty to isolate and analyze CCVs from different Arabidopsis mutants. CCVs were originally isolated and purified from pig brains in which they are highly abundant (Pearse, 1975). In the original protocol, CCVs were enriched by differential centrifugation followed by linear sucrose density gradient centrifugations. This protocol was further improved by the use of isotonic linear D 2 O/Ficoll gradient (Pearse, 1980(Pearse, , 1982. Two protocols for CCV isolation from suspension-cultured carrot cells and from Pisum sativum cotyledons were described (Depta and Robinson, 1986;Harley and Beevers, 1989). Both involve sucrose step-gradient centrifugation and linear D 2 O/Ficoll gradient centrifugation. Later, a method to isolate CCVs from Arabidopsis thaliana suspension-cultured cells was established based on these protocols (Reynolds et al., 2014).
In this article, we describe a modified method for CCV isolation from Arabidopsis seedlings. The protocol enables to circumvent the time-consuming steps required for the preparation of Arabidopsis suspension-cultured cells and allows isolation of CCVs directly from entire Arabidopsis seedlings.

Reagents, Solutions and Buffers
All the solutions were prepared using ddH 2 0 and chemicals of analytical quality based on the previous publication (Reynolds et al., 2014) with some modifications. Solutions indicated with an asterisk ( * ) are supplemented with the following protease inhibitors: 500 µM PMSF and 7.3 µM pepstatin A, 2.1 µM leupeptin-hemisulfate and 1 mM DTT.
• 500 mM PMSF and 7.3 mM pepstatin A solved in DMSO.

Methods
For the isolation of CCVs, membranes are first enriched from plant total extracts by ultracentrifugation. The membrane fraction is then separated on a sucrose step gradient and further separated on a D 2 O/Ficoll linear gradient. Medium density fractions are subsequently subjected to ultracentrifugation to obtain a pellet that contains the isolated intact CCVs.
Keep all the solutions, mortars and pestles at 4 • C overnight and pre-chill centrifuges prior to use. Prepare solutions indicated with an asterisk ( * ) immediately before use. The overview of the whole procedure is schematically presented in Figure 1. The isolation of CCVs will take 2 days including the preparation of solutions and all centrifugation steps. The subsequent SEM analysis should be carried out with freshly isolated CCVs. If this is not possible, the isolated CCVs can be stored overnight at 4 • C and then processed for the SEM analysis. The preparation of the SEM samples take half a day including the imaging. The prepared grids for SEM analysis can be kept overnight in a desiccator with vacuum applied and imaged the following day.

Preparation of D 2 O/Ficoll Gradient for Ultracentrifugation
(

CCV Purification
(1) Prepare 30 g of 7-days-old seedlings. In a cold room, grind 5 g of the seedlings in a clean mortar with 5 mL ice cold CIB * . Collect the lysate and keep in a 50 mL tube on ice.
Repeat until all seedlings are processed. Distribute the lysate equally into two 50 mL conical tubes. Save 500 µL in a 1.5 mL tube and label as "Lysate" for later analysis.
Carefully collect the supernatant with a 25 mL disposable sterile pipette into a fresh 50 mL conical tube and keep on ice.
(3) To increase the yield of CCVs, transfer the pellets from the two tubes to a mortar and grind the pellets with 6 mL CIB * and collect the lysate in a fresh 50 mL tube. Centrifuge the lysate for 10 min at 1,000 × g at 4 • C. Carefully collect the supernatant and add it to the previously obtained supernatant from step 2. Save 500 µL of sample in a 1.5 mL tube and label as "S1" for later analysis. (4) In a cooled high-speed centrifuge, centrifuge the supernatant for 25 min at 30,000 × g at 4 • C. Save 500 µL of sample in a 1.5 mL tube and label as "S30" for later analysis. (5) Transfer the supernatant to two 26.3 mL polycarbonate ultracentrifuge tubes and centrifuge for 1 h at 120,000 × g at 4 • C. (6) While waiting for the centrifugation, prepare the tubes for the sucrose step gradient. Prepare 2.5 mL 50% (w/v) sucrose solution * , 11 mL 25% (w/v) sucrose solution * and 7 mL 12% (w/v) sucrose solution * .
(7) Prepare two Ultra-Clear centrifuge tubes (14 × 95 mm), and first layer gently 1 mL of 50% (w/v) sucrose solution * . Slowly add 5 mL of 25% sucrose solution * and subsequently add 3 mL of 12% sucrose solution * into each tube without disturbing the other layers. Keep the tubes in a tube rack at 4 • C until use. (8) Discard the supernatant. (9) Resuspend the two pellets in 1 mL CIB * each. Transfer the homogenate into two 1.5 mL tubes and centrifuge for 10 min at 16,000 × g at 4 • C. (10) Transfer the supernatant to a 15 mL conical tube.
Resuspend the pellets remaining in the 1.5 mL tubes with 1.5 mL of CIB * each. Centrifuge 10 min at 16,000 × g at 4 • C. Pool the supernatant together. This should yield a final volume of approximately 5 mL. Save 200 µL in a 1.5 mL tube and label as "SGL" for "sucrose gradient load" for later analysis. (11) Carefully layer 2.4 mL of the supernatant on each of the sucrose step gradient prepared in step 7. Centrifuge 50 min at 116,000 × g at 4 • C. Note: During this step, carefully handle the ultracentrifuge tubes to avoid disturbing the gradient. Select slow acceleration and slow deceleration. (12) After the centrifugation, collect the 12% (w/v) layer and the 12%/25% (w/v) interface. Transfer each of the collected layers into a fresh 26.3 mL polycarbonate ultracentrifuge tube. Note: Avoid taking the 25% (w/v) fraction to prevent contamination with denser membrane structures. (13) Dilute the content of both 26.3 mL polycarbonate ultracentrifuge tube with 13 mL CIB * each. Sucrose concentration should be less than 5% (w/v). Centrifuge 50 min at 200,000 × g at 4 • C. Note: Measure the refractive index of the diluted content using a refractometer. Add more CIB * to dilute the sucrose concentration to below 5% (w/v), if necessary. (14) Discard the supernatant and resuspend each of the two pellets in 300 µL CIB * . Transfer the homogenate into two 1.5 mL tubes and centrifuge for 5 min at 16,000 × g at 4 • C. Centrifuge 14 h at 80,000 × g at 4 • C. Note: During this step, carefully handle the ultracentrifuge tubes to avoid disturbing the gradients. Select slow acceleration and slow deceleration.
(2) Immobilize carbon-coated 200-mesh nickel grids by clasping with non-magnetic negative steel forceps. (3) In an appropriate fume hood, on a bit of plastic film mix one part of the CCV fraction and three part of 4% (w/v) OsO 4 solution (e.g., 1 µL CCV fraction and 3 µL OsO 4 solution).
Mix thoroughly by pipetting. (4) Carefully deposit 1 µL of the CCV mixture (step 3, above) on the face of each of the immobilized grids. Let dry under the fume hood. (5) Place a droplet of Nano-W ready-to-use 2% solution onto a plastic film. Transfer 10 µL of Nano-W ready-to-use 2% solution on each of the dried mixture-coated grids. Incubate for 1 min. Using a wedge of Whatman paper, wick away excess fluid until the grids are nearly dry. Let dry under the fume hood. Note: Avoid touching the surface of the grids with paper. Accordingly, touch the edges of the Nano-W ready-touse 2% solution droplet and remove as much solution as possible. (6) Image the grids using a scanning electron microscope in scanning transmission electron microscope (STEM) mode with 30 kV. A 25,000 × magnification (calculated for a Polaroid 545) is sufficient to distinguish CCVs from uncoated vesicles.

RESULTS AND DISCUSSION
To determine the presence of intact CCVs, we examined isolated CCVs from 7-day-old wild-type seedlings under a scanning electron microscope. Negative staining and scanning electron microscopy analysis of the sample showed the presence of intact CCVs (Figure 2). Since uncoated vesicles were also present, we determined the relative amount of CCVs to the total number of intact membrane structures. We observed 60.3% CCVs in two independent experiments (n = 1714 vesicles).
To further evaluate the purity of CCVs we analyzed the obtained samples by immunoblotting using an anti-CHC antibody and antibodies specific to intracellular organelles ( Figure 3A) Together, these results suggest that CCVs were successfully isolated without major contamination of other organelles.
We conducted the same experiment using 7-d-old seedlings expressing the late endosome marker mRFP-ARABIDOPSIS RAB GTPase 7 (ARA7) under the control of its native promoters (Inada et al., 2016). When analyzed with the same antibodies used in Figure 3A, mRFP-ARA7 fractionated differently ( Figure 3B). Although, faint signals of mRFP-ARA7 were still visible in the CCV enriched fraction, in contrast to CHC, mRFP-ARA7 was not enriched when compared to the total lysate. Although there are reports of flat clathrin patches on the surface of endosomes (Smith and Farquhar, 1966;Prekeris et al., 1998;Prekeris et al., 1999;Raiborg et al., 2001Raiborg et al., , 2002Sachse et al., 2002;Williams and Urbe, 2007), to date there is no evidence of CCV formation from ARA7-positive late endosomal compartments.

CONCLUSION
The method presented here is suitable for the isolation of CCVs from Arabidopsis seedlings grown under standard conditions. This enables the quantitative and morphological analyses on CCVs and CCV-associated proteins from different Arabidopsis mutant backgrounds or upon treatment with compounds of interest. We verified the successful enrichment and isolation of intact CCVs applying this method by immunoblotting and scanning electron microscopy.

AUTHOR CONTRIBUTIONS
NM, TB, M-KN, and EI optimized the method. NM, TB, and ML conducted negative staining and analysis by SEM. NM and EI wrote the manuscript with the help of ML.

FUNDING
The work in the authors' laboratory was supported by funds from the German Science Foundation (DFG: IS 221/4-1 and SFB969/C08).