–rare earth element complexes activate endocytosis

Lihong Wanga,b,1, Mengzhu Chenga,1, Qing Yanga,1, Jigang Lic, Xiang Wanga, Qing Zhoub, Shingo Nagawad,e, Binxin Xiab, Tongda Xud,e, Rongfeng Huangd,e, Jingfang Hea, Changjiang Lic, Ying Fuc, Ying Liua, Jianchun Baoa, Haiyan Weia, Hui Lie,f, Li Tane, Zhenhong Gue, Ao Xiaa, Xiaohua Huanga,2, Zhenbiao Yangg,h, and Xing Wang Dengi,2

aNational and Local Joint Engineering Research Center of Biomedical Functional Materials, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, School of Chemistry and Materials Science, Nanjing Normal University, 210023 Nanjing, China; bState Key Laboratory of Food Science and Technology, Jiangnan University, 214122 Wuxi, China; cChina State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, 100193 Beijing, China; dFujian Agriculture and Forestry University–University of California, Riverside Joint Center for Horticultural Biology and Metabolomics, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, 350002 Fuzhou, China; eShanghai Center for Plant Stress Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, 201602 Shanghai, China; fSchool of Life Sciences, East China Normal University, 200241 Shanghai, China; gCenter for Plant Cell Biology, Institute of Integrative Genome Biology, University of California, Riverside, CA 92521; hDepartment of Botany and Plant Sciences, University of California, Riverside, CA 92521; and iState Key Laboratory of Protein and Plant Gene Research, The Peking-Tsinghua Center for Life Sciences, School of Advanced Agricultural Sciences and School of Life Sciences, Peking University, 100871 Beijing, China

Contributed by Xing Wang Deng, May 29, 2019 (sent for review February 12, 2019; reviewed by Liwen Jiang and Enrique Rojo) Endocytosis is essential to all , but how cargoes are challenges, such as in the identification and visualization of cargo selected for internalization remains poorly characterized. Extra- receptor and their interaction with cargoes in vivo (1–4). cellular cargoes are thought to be selected by transmembrane By visualizing the dynamic of cargo or its receptor protein using receptors that bind intracellular adaptors proteins to initiate stimulated emission depletion (STED) microscopy, and in- endocytosis. Here, we report a mechanism for clathrin-mediated vestigating the mechanism for the endocytosis of rare earth el- endocytosis (CME) of extracellular lanthanum [La(III)] cargoes, ements (REEs), e.g., lanthanum [La(III)], we have identified a which requires extracellular arabinogalactan proteins (AGPs) that mechanism for the activation of CME and cargo recognition. are anchored on the outer face of the plasma membrane. AGPs

The REEs in the periodic table of elements comprise 15 lan- PLANT BIOLOGY were colocalized with La(III) on the cell surface and in La(III)- thanide elements plus scandium and yttrium, which have similar Arabidopsis induced endocytic vesicles in cells. Superresolu- properties in atomic radius and charge (5) and are considered to tion imaging showed that La(III) triggered AGP movement across be nonessential elements of living organisms. REEs are exten- the plasma membrane. AGPs were then colocalized and physically sively used in agriculture, industry, national defense, environ- associated with the μ subunit of the intracellular adaptor protein 2 mental protection, medicine, etc. (6, 7). For decades, REEs have (AP2) complexes. The AGP-AP2 interaction was independent of been ingredients of fertilizers for the improvement of plant growth CME, whereas AGP’s internalization required CME and AP2. More- and crop yields mainly via foliage spraying, but the mechanisms for over, we show that AGP-dependent endocytosis in the presence of La(III) also occurred in human cells. These findings indicate that extracellular AGPs act as conserved CME cargo receptors, thus chal- Significance lenging the current paradigm about endocytosis of extracellular cargoes. Due to the vital role for eukaryotes, clathrin-mediated endo- cytosis (CME) has attracted increasing attention, but great arabinogalactan proteins | endocytosis | extracellular cargo | lanthanum | challenges still stand. Here, by overcoming the biggest chal- superresolution imaging lenge in CME research, we visualized the dynamic of extracel- lular cargo receptor protein by using stimulated emission ndocytosis, including clathrin-mediated endocytosis (CME), depletion microscopy. We identified an unconventional mech- Eis a fundamental cellular process in , animals, and anism for extracellular cargoes, here specifically rare earth el- microorganisms (1, 2). By internalizing extracellular cargoes and ements (REEs). We showed that arabinogalactan proteins membrane-integral or -associated proteins, CME plays an es- (AGPs) act as extracellular cargo receptors and move across the sential role in various cellular processes, such as , plasma membrane to initiate endocytosis. REEs promote the cell-polarity formation, cell-fate determination, , and cross-membrane translocation of its extracellular cargo re- cell movement (1, 2). Consequently, the mechanisms for cargo ceptor AGPs to activate their endocytosis. Our data thus pro- recognitions and CME machinery and processes have been ex- vide insights into the mechanism for the activation of CME, the tensively studied (1–4). A suite of adaptor proteins have been biological role of AGPs, and the cellular mechanisms of REE shown to be involved in the recognition of cargoes or cargo re- actions in plants. ceptors at the cytoplasmic side of the plasma membrane (PM). Author contributions: X.H. and X.W.D. designed research; L.W., M.C., Q.Y., X.W., B.X., One of the best-characterized and conserved adaptor proteins R.H., J.H., C.L., and Y.L. performed research; L.W., M.C., Q.Y., J.L., Q.Z., S.N., T.X., Y.F., J.B., belongs to adaptor protein 2 (AP2) complexes consisting of H.W., H.L., L.T., Z.G., A.X., X.H., and X.W.D. analyzed data; and L.W., M.C., Q.Y., J.L., X.H., 4 subunits, α, β, σ, and μ subunits (2–4). Upon activation by Z.Y., and X.W.D. wrote the paper. protein–protein interaction or modification such as phosphory- Reviewers: L.J., The Chinese University of Hong Kong; and E.R., Consejo Superior de lation, membrane cargo proteins are recognized by AP2, which Investigaciones Científicas. then recruits clathrin subunit proteins for clathrin coat assembly The authors declare no conflict of interest. (2–4). It is well established that extracellular cargoes are recog- Published under the PNAS license. nized by transmembrane receptors, whose cytoplasmic domains 1L.W., M.C., and Q.Y. contributed equally to this work. interact with CME adaptor proteins to initiate cargo endocytosis 2To whom correspondence may be addressed. Email: [email protected] or (2–4). Despite these advances, major gaps remain in our un- [email protected]. derstanding of CME, particularly regarding the mechanisms This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. underpinning the recognition of cargoes and the regulation of 1073/pnas.1902532116/-/DCSupplemental. CME. Furthermore, the current research on CME faces major Published online June 25, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1902532116 PNAS | July 9, 2019 | vol. 116 | no. 28 | 14349–14357 Downloaded by guest on September 26, 2021 REEs to enter plant cells and their action mechanisms to promote with La(III), in which the outlines of the endocytic compartments plant growth remain poorly characterized. On the other hand, the were drawn by AGPs labeled by immunogold particles (Fig. 1B widespread applications of REEs have also resulted in the massive and SI Appendix,TableS1). The observed sizes of the endocytic accumulation of REEs in the global environment (including soil, compartments were in the range of 100 to 200 nm (Fig. 1B), which water, and atmosphere) and living organisms at an unprecedented were consistent with those of the EMARG images (Fig. 1A). The speed (8–10). Therefore, the pollution of REEs is rapidly emerg- results were also consistent with the previously published size ing as a universal threat to ecological integrity and function, as well ranges of CME determined by TEM that has a resolution down to as human health (11), highlighting the urgent need for establishing the nanometer level and thus is commonly used to determine the guidelines to limit the concentration of REEs in the ecosystem. To size of CME vesicles in living organisms (1, 2, 19). Meanwhile, accomplish this goal, it is imperative that we have a clear quali- compared with untreated cells, La(III) treatment greatly increased tative and quantitative examination about how REEs are absorbed the labeling of AGPs on the PM (especially the invaginated PM by and act on plants, especially in , which are directly sprayed domains) apparently undergoing endocytosis (Fig. 1 B and D and with REEs in agricultural application. Recently, by using inter- SI Appendix,TableS1), suggesting that the colocalization of disciplinary techniques, including electron microscopic autoradi- La(III) and AGPs on the PM may induce AGP internalization. ography (EMARG) of radioactive La, cerium (Ce), and terbium AGPs belong to a large family of ubiquitously (Tb) [140La(III), 141Ce(III), and 160Tb(III)], we directly observed found in plants (20, 21). Normally, AGPs are tethered to the the life cycle of REEs in plants and surprisingly found that REEs, outer leaflet of the PM by a glycosylphosphatidylinositol (GPI) firstly anchored on the PM in the form of the nanoscale particles, anchor and may cross-link with some components of the activated endocytosis in leaf cells and then entered leaf cells with (GPI-anchored AGPs) in plant cells (20, 21). AGPs play crucial the help of some biomolecules (12, 13). Our findings have roles in cell growth, patterning, reproductive develop- attracted much attention from a wide range of disciplines, e.g., ment, postembryonic patterning, signal transduction, and medicine (14), agriculture (15), ecology (16), and cell biology (17, tube growth in plants (20, 21). To date, no evidence is available 18). At the same time, new questions have been raised about the for a role of AGPs in the regulation of endocytosis in plants, mechanisms for the REE regulation of endocytosis. How do REEs animals, and microorganisms. We thus asked whether AGPs cause the activation of endocytosis, what are the receptors for could initiate the endocytosis in the presence of La(III) in plant REEs (the primary target of REEs) to initiate the endocytosis, and leaves. The high functional redundancy is anticipated for AGPs how do these receptors interact with adaptors and then activate due to the large AGP gene family (i.e., 85 AGP genes in the endocytosis in leaf cells? Arabidopsis genome) encoding AGPs with the incredible het- In this study, by employing a double-imaging method involving erogeneity of backbone and (20, 21). To circum- isotopically labeled 140La(III) and immunogold-labeled potential vent the difficulties in genetic approaches to analyze the redundant La(III) receptors, we show that arabinogalactan proteins (AGPs) functions, we suppressed the functions of AGPs by using β-glucosyl were colocalized with La(III) on the cell surface and in La(III)- , a widely used inhibitor of AGPs (22, 23). Endocy- induced endocytic vesicles in Arabidopsis leaf cells. Super- tosis was visualized by staining the cell membrane with N-(3- resolution imaging with STED microscopy, combined with im- triethylammoniumpropyl)-4-(4-diethylaminophenylhexatrienyl) munocytochemical, biochemical, and genetic analyses, show that (FM4-64), a membrane-impermeable fluorescent styryl dye (24, La(III) triggered the movement of AGPs from the outer face of 25), and GFP-tagged components of the CME machinery, e.g., the PM across the PM into the cytoplasmic face to interact with GFP-tagged clathrin light chain (CLC1-GFP) (26). CLC1-GFP the μ subunit of the intracellular AP2, a CME adaptor complex. and FM4-64 dye colabeled both of the PM and the La(III)- AGP-mediated La(III) endocytosis was also observed in human induced endomembrane compartment visualized by confocal cells. These findings thus establish AGPs as the primary con- laser-scanning microscopy (CLSM), by which the dynamic pro- served targets of La(III) to initiate CME of La(III) and chal- cess of endocytosis could be observed, although CLSM cannot lenge the current paradigm about the CME of extracellular determine the sizes of endocytic vesicles due to its technical lim- cargoes. Furthermore, our findings provide insight into the itation (e.g., diffraction limit) (27). The endomembrane com- mechanisms by which REEs impact biological processes, a sub- partment was only observed when leaf cells were treated with ject that has puzzled scientists for over a century. La(III) (Fig. 2A). Time-lapse imaging using spinning-disk confocal microscopy showed that La(III) treatment induced the budding of Results and Discussion CLC1-GFP labeled large vesicles from the PM (Movie S1). These AGPs Play a Critical Role in Mediating La(III)-Induced CME in Leaf observations suggest that La(III) treatment may induce CME, Cells. In our previous studies, we found that REEs (La, Ce, resulting in the formation of the endomembrane compartments. and Tb) could activate endocytosis in horseradish leaf cells (12, These compartments were found in ∼20% of leaf cells in the 13). We found that La(III) treatment can activate endocytosis in presence of 30 μM La(III), with an average of 5 to 7 compart- Arabidopsis (wild type, Col-0) leaves as well; therefore, in this ments (red circles) per cell (Fig. 2B, Top), and in ∼80% of leaf study we choose La(III) as a representative REE to study the cells, with 3 to 17 compartments per cell in the presence of 80 μM cellular mechanism underlying REEs-activated endocytosis in La(III) (Fig. 2B, Top). Interestingly, treatments with the β-glucosyl the model plant Arabidopsis. To identify the primary target of Yariv reagent drastically blocked La(III)-activated endocytosis REEs, we use a double-imaging method that involves iso- (Fig. 2B, below). Only 1 to 2 endocytic compartments in each cell topically labeled 140La(III) and immunogold-labeled potential were observed in cells treated with β-glucosyl Yariv reagent (Fig. targets. Based on the EMARG observations that La(III) treat- 2B, below). These results suggest that AGPs play a critical role in ment can activate endocytosis (Fig. 1A), the expression of several initiating CME in leaf cells induced by certain concentrations of genes encoding AGPs (SI Appendix, Fig. S1) and the postulation La(III). Moreover, larger vesicles were observed in the CLSM that AGPs and La(III) might form complexes (5), we asked images than those in the EMARG and TEM images (Fig. 1 A and whether AGPs could be the potential REE targets. We colabeled B) due to the fusion of the endocytic vesicles in CLSM images and La(III) with 140La(III) and AGPs with immunogold in Col-0 leaf lower spatial resolution of CLSM (19). cells and visualized them using a transmission electron micro- scope equipped with an energy-dispersive spectrometer (TEM- AGPs and La(III) Act Together to Trigger CME in Leaf Cells. The EDS). By using this double-imaging method, we observed that colocalization of La(III) with AGPs on the PM and the re- AGPs were distributed on the cell surface and in endocytic quirement of AGPs for La(III)-triggered endocytosis suggest compartments in the La(III)-treated Col-0 leaf cells together that AGPs act on the early events of the endocytosis, possibly as

14350 | www.pnas.org/cgi/doi/10.1073/pnas.1902532116 Wang et al. Downloaded by guest on September 26, 2021 PLANT BIOLOGY

Fig. 1. AGPs are the targets of La(III) that activates endocytosis in Arabidopsis leaf cells. (A) The representative EMARG images of Col-0 leaf cells treated with 0, 30, or 80 μM La(III) containing radioactive 140La(III) for 12 h from 3 biological replicates. White points are La(III). Bar = 100 nm. (B, top row) The repre- sentative TEM images of Col-0 leaf cells treated with 0, 30, and 80 μM La(III) and successively immunostained with anti-AGP antibody (JIM13) and gold-labeled anti-JIM13 antibody from 3 biological replicates. Irregular small dots indicated with white arrows are La(III). Large dark dots display AGPs labeled with immunogold. (Scale bar, 100 nm.) TEM allowed unambiguous observation of the distribution sites for both La(III) and AGPs. (B, middle row) Enlarged view showing more clear morphologies from the area marked with the black square in the top row. (Scale bar, 20 nm.) The TEM images show colocalization of AGPs and La(III) on the PM. (B, bottom row) The contents of La and gold (Au) labeling AGPs were determined by EDS. Among the possible sites where La(III) and AGPs were colocated on the PM, such as regions marked with red squares in the middle row, we randomly selected 10 sites to detect the contents of La and Au. The contents of La and Au shown in this row were the average values of the detected La and Au contents in the 10 sites. EDS detected La and Au from their same distribution sites, further showing that La(III) and AGPs were colocalized on the PM and might form their complexes on the PM. (C) The repre- sentative TEM images of β-glucosyl Yariv reagent-treated Col-0 leaf cells treated with 0, 30, or 80 μM La(III) for 12 h and successively immunostained with JIM13 and gold-labeled anti-JIM13 antibody from 3 biological replicates. Large dark dots display AGPs labeled with immunogold. (Scale bar, 100 nm.) (D) Quantitative analysis of gold particles on the PM in Col-0 leaf cells treated without or with β-glucosyl Yariv reagent. Data are represented as means ± SEM. *P < 0.05 (2-tailed unpaired t test). Mean values were obtained from 15 images taken from 3 independent experiments. Chl, chloroplast; CW, cell wall.

Wang et al. PNAS | July 9, 2019 | vol. 116 | no. 28 | 14351 Downloaded by guest on September 26, 2021 Fig. 2. AGPs initiate the CME of Arabidopsis leaf cells after treatment La(III). (A) The representative CLSM images of Col-0 leaf cells expressing CLC1-GFP treated with 0, 30, or 80 μM La(III) for 12 h and stained with FM4-64 from 3 biological replicates. Induced endocytic vesicles are indicated with white ar- rowheads. (Scale bar, 10 μm.) The further dynamic results from time-lapse imaging are shown in Movie S1.(B) The representative CLSM images of Col-0 leaf cells treated with 0, 30, or 80 μM La(III) for 12 h and stained with FM4-64. Induced endocytic vesicles are indicated with white arrowheads. The leaf cells were treated without or with β-glucosyl Yariv reagent before La(III) treatment. (Scale bar, 10 μm.) (C and D) The representative CLSM images of the leaf cells in Col-0 and DEX-inducible Venus–CANTH lines treated with 0, 30, or 80 μM La(III) for 12 h from 3 biological replicates. (Scale bar, 6 μm.) The leaves of Col-0 and DEX- inducible Venus–CANTH lines treated with 0, 30, or 80 μM La(III) for 12 h were stained with FM4-64 (red) and JIM13 coupled with secondary antibody (rabbit anti-rat IgG heavy chain & light chain (H&L) [FITC]) (green) and observed under CLSM for evaluation of AGP’s CME. (E) The frequency of endocytosis and the amount of endocytic vesicles in Col-0 and DEX-inducible Venus–CANTH lines treated with 0, 30, or 80 μM La(III) for 12 h. A representative result of 3 in- dependent experiments is presented (n ≥ 10). Data are represented as means ± SEM, *P < 0.05 (2-tailed unpaired t test).

direct targets or cocargoes of La(III). If so, AGPs would be in- labeling of AGPs showed that La(III) treatment dramatically ternalized by endocytosis together with La(III). To visualize induced the accumulation of AGPs along the PM as punctates AGP endocytosis, AGPs were successively immunostained with and in endocytic vesicles (Fig. 2 C and E); these effects were anti-AGP antibody (JIM13) and fluorescence-labeled anti- stronger with 80 μM La(III) treatment compared with 30 μM JIM13 antibody in cells costained with FM4-64 (Fig. 2 C and D). La(III) (Fig. 2 C and E). These results are consistent with the This method allowed the simultaneous visualization of endocytic results from immunogold labeling of AGPs (Fig. 1B). Furthermore, events and AGP dynamics using CLSM. Immunofluorescence treatment with β-glucosyl Yariv reagent blocked the accumulation

14352 | www.pnas.org/cgi/doi/10.1073/pnas.1902532116 Wang et al. Downloaded by guest on September 26, 2021 (CANTH) was fused with the Venus protein (31–33). The con- served AP180 is essential for the early stage of CME (31, 33); thus, overexpression of CANTH is known to specifically block CME (31, 32). DEX-induced overexpression of Venus–CANTH blocked the internalization of AGPs but had no effect on their accumulation on the PM induced by La(III) treatment (Fig. 2D). Furthermore, no endocytic compartments were observed in La(III)-treated Venus–CANTH cells (Fig. 2D), indicating that Venus–CANTH effectively blocked La(III)-induced CME in these cells. The induction of Venus–CANTH also abolished La(III)-triggered CME in leaf cells without AGP antibody (JIM13) staining, excluding the possibility that this antibody could interference with La(III)-triggered CME (SI Appendix, Fig. S2). The results further demonstrated that AGPs are in- volved in the activation of CME, which is in agreement with the inhibition of La(III)-triggered endocytosis by β-glucosyl Yariv reagent (Fig. 2B).

La(III)-Triggered AGP Movement across the PM during the Initiation of CME. Because AGPs are secretory proteins localized to the outer leaflet of the PM via a GPI anchor (20, 21), they may act as cocargoes or cargo receptors to initiate CME. Based on the existing models of CME in plants and other systems (28–30), the extracellular cargoes must be recognized by a transmembrane cargo receptor, whose intracellular domain interacts with the intracellular adaptor proteins. To understand how AGPs may work as cocargoes or cargo receptors for CME, we used the

superresolution STED microscopy to visualize the dynamic be- PLANT BIOLOGY haviors of AGPs during La(III)-induced CME (27, 34, 35). To overcome the technical challenges presented by plant cells such as the cell wall (>250 nm thickness) for superresolution imaging (36), we performed the STED imaging of AGPs by using the Fig. 3. AGPs migrate across the PM after La(III) treatment. (A and B) The Arabidopsis protoplasts. La(III)-treated protoplasts were in- leaves of Col-0 and DEX-inducible Venus–CANTH lines were treated with 0 or cubated with (secondary antibody)-labeled anti-AGP antibody, 30 μM La(III). Twelve hours later, the protoplasts were isolated from the and the location of AGPs in protoplast was shown by observing respective leaves, stained with JIM13 and secondary antibody (goat anti-rat the fluorescence signals of the secondary antibody. Then, time- IgG [Alexa Fluor 555]), and observed under STED microscopy (∼108 s) to lapse imaging of stained AGPs was conducted. As shown in Fig. visualize the dynamic behavior of AGPs during La(III)-induced CME. The 3 and Movie S2, AGPs were localized on the surface of the images presented in A and B are a representative time series of images taken protoplasts and rarely crossed the PM in the control cells that from Movie S2 (n = 10 frames; t = 108 s), which showed the time-lapse im- aging of AGPs moving across the PM in the leaf cells of Col-0 and DEX- were not treated with La(III). In La(III)-treated Col-0 proto- inducible Venus–CANTH lines at the indicated times after 12-h treatment plasts, PM-associated AGPs were dramatically increased, and with 0 or 30 μM La(III). (Scale bar, 2.5 μm.) the majority of them were found to move across the membrane and enter the cytoplasm (Fig. 3A, Movie S2, and SI Appendix, Fig. S3). It appears that AGPs were spanning the PM on their of AGPs on the PM (Fig. 1 C and D). The green fluorescence that way into the cytoplasm (Fig. 3A, Movie S2, and SI Appendix, Fig. labeled punctate AGPs was superimposed on the FM4-64 red S3). Intriguingly, in the DEX-induced protoplasts from the Venus– fluorescence that labeled both of the PM and the endosomal CANTH lines, La(III) treatment also induced AGPs to cross the compartment (Fig. 2C, Right columns in middle and bottom rows), PM (or inserted into the membrane), but most of them did not indicating that AGPs can serve as a marker for endocytosis in these completely enter the cytoplasm (Fig. 3B, Movie S2,andSI Ap- cells. This result was consistent with that from the TEM for pendix, Fig. S3) and were apparently stuck in the PM (Fig. 3B, immunogold-labeled AGPs (Fig. 1B). In addition, after 30 μM Movie S2,andSI Appendix,Fig.S3). Quantitative analysis (SI La(III) treatment, all of the endosomal compartments showed Appendix, Fig. S3 and Table S2) showed that ∼20% of leaf cells in AGP signals (Fig. 2C, Right columns in middle row). After 80 μM La(III)-treated Col-0 displayed AGP movement across the PM, La(III) treatment, most of endosomal compartments showed and in these cells, ∼90% AGPs on the cell surface crossed the PM. AGP signals, while some endosomal compartments showed no In leaf cells of Venus–CANTH lines treated with La(III), almost AGP signals (Fig. 2C, Right columns in bottom row). These all AGPs on the cell surface spanned the PM but failed to enter vesicles without AGP signal might result from other La(III) re- the cytoplasm. ceptors, which awaits further investigation. Taken together, our Because the specific antibody of AGPs (JIM13) may not in- results suggest that La(III) and AGPs act together to trigger terfere with La(III)-triggered CME (SI Appendix, Fig. S2), these endocytosis in leaf cells. observations suggest that La(III) induced AGPs to move across Because La(III)-induced endosomal compartments costained the PM, but these AGPs in the PM were not internalized if the with CLC1-GFP, we hypothesize that the AGP-dependent in- assembly of clathrin coats was blocked by Venus–CANTH. The ternalization of La(III) is mediated by CME, which is known to behavior of AGPs translocating across the PM drives the trans- be the main endocytic pathway in plants (28–30). We further membrane transport and internalization of primary and sec- tested whether CME is involved in La(III)-activated AGP in- ondary antibodies bound to AGPs. These observations raised a ternalization. To inhibit CME, we generated transgenic lines very exciting question: how can AGPs move across the PM without harboring a dexamethasone (DEX)-inducible Venus–CANTH the assembly of clathrin coats, i.e., without the membrane in- construct, in which the C-terminal region of AtAP180 proteins vagination after the clathrin proteins are recruited to the inner

Wang et al. PNAS | July 9, 2019 | vol. 116 | no. 28 | 14353 Downloaded by guest on September 26, 2021 face of the membrane? This unexpected discovery apparently accumulate in human organs and cells in the form of nanoparticles contradicts with our conventional knowledge about AGPs (20, to a harmful level (11, 37). The residual REEs (such as gadolinium) 21). Given GPI anchoring of AGPs to the outer leaflet of the PM, in human body results in nephrogenic systemic fibrosis (37). AGPs the most likely interpretation of these findings is that La(III) in- in human are obtained through diet, mostly plant-based diet (38, duced the conformational changes in AGPs, causing AGPs to flip- 39), bind or adhere to the cell surface (40, 41), and perform im- flop to the inner side of the PM. portant functions such as reducing the cholesterol in the blood (42), regulating immunity (43), and serving as targeted-drug car- La(III) Induced the Physical Association of AGPs with the μ Subunit of riers to deliver drug to target organs rapidly (39). Therefore, we the Intracellular AP2 to Initiate CME. If AGPs move across the PM, asked whether La(III) induced the activation of AGP-dependent they could recruit adaptor proteins to initiate the assembly of endocytosis in human cells. To test this, we used human embryonic clathrin coat proteins (2, 3). To date, the primary adaptor pro- kidney 293 cells (HEK293) and observed that the endocytosis was teins in plant CME are the AP2 family proteins; other adaptor indeed activated in HEK293 in the presence of La(III), and the proteins, such as ENTH/ANTH family proteins, and accessory activation was more evident when the concentrations of La(III) proteins, such as TPLATE complex (TPC), have also been were increased (SI Appendix,Fig.S6A). Treatments with β-glucosyl reported (2–4). We first costained AGPs (green) and the μ Yariv reagent drastically blocked La(III)-activated endocytosis (SI subunit of intracellular AP2 (blue) in both Col-0 and DEX- Appendix,Fig.S6B). In mammalian cells, except for AGPs, no inducible Venus–CANTH lines using anti-AGP and anti-μ an- glycoproteins have been reported that can be inhibited by tibodies conjugated to fluorescein isothiocyanate (FITC) and β-glucosyl Yariv (39, 42, 43), while fetal bovine serum containing Alexa Fluor405, respectively. In the absence of La(III), only AGPs (44, 45) was added in the culture medium (46). So, it small amounts of AGPs and AP2 were localized on the cell surface, seems likely that AGPs also activate and regulate REEs’ endo- while they exhibit distinct localization sites in Col-0 leaf cells (Fig. cytosis in human cells. The co-IP assay data showed that AGPs 4A, Right image in first row). La(III) treatment induced the ex- and AP2 physically interacted in HEK293 cells after La(III) pression of AGPs and AP2, respectively (Fig. 1B and SI Appendix, treatment (SI Appendix, Fig. S6C), further demonstrating that Fig. S4). Consequently, La(III) treatment induced the colocaliza- AGP-mediated endocytosis also occurred in human cells. Thus, tion of AGPs and AP2 on both the membrane and the endocytic AGP-dependent endocytosis in the presence of La(III) appears compartments (Fig. 4A, second row). In the DEX-treated Venus– to be a universal mechanism. Moreover, we did not observe the CANTH lines, AGPs and AP2 were colocalized only on the PM of big ring structures formed in HEK293 cells upon La(III) treat- leaf cells in the presence of La(III) (Fig. 4B). These results dem- ments. We reason that the structures of endocytic vesicles may onstrate that La(III) induced its codistribution with AGPs and differ across species or even in different organs of the same or- AP2 in a physical proximity on the PM and that their subsequent ganism (1, 47, 48). internalization is dependent upon clathrin assembly. To summarize, we found that extracellular AGPs, upon in- Among the AP2 subunits, cargoes commonly bind directly to duction by La(III), move across the PM and physically interact the μ subunit of AP2, thus initiating the CME (2). To determine with the AP2 complexes to initiate the endocytosis of AGPs/ whether the μ subunit of AP2 is involved in AGP-mediated ini- La(III) (Figs. 1–5 and SI Appendix, Figs. S1–S8 and Tables S1– tiation of CME in leaf cells, we examined endocytosis in the S4). Our findings demonstrate an unprecedented mechanism for mutant of AP2 μ subunit (ap2μ-1) with or without β-glucosyl the activation of CME by proposing that AGPs serve as un- Yariv reagent treatments in the absence or presence of La(III). conventional receptors for extracellular La(III) cargo to initiate We found that La(III)/AGP-initiated endocytosis was greatly CME (Fig. 5). Based on our previous studies that REEs have diminished in the knockout (transfer DNA insertional mutant) similar life cycles and cellular behaviors in plant cells (12, 13), of the μ subunit (Fig. 4C). Moreover, the complementation of and the chemical principle that the REEs in the periodic table of the μ subunit of AP2 recovered La(III)-activated endocytosis elements have similar physical and chemical properties (5), we (Fig. 4C). Thus, AGPs may initiate the endocytosis via their di- suggest that our conclusions in this study may represent the rect interaction with the μ subunit of AP2. Indeed, coimmuno- general action mechanisms for all REEs in plant cells (Fig. 5). precipitation (co-IP) assay data showed that AGPs and AP2 This mechanism for CME activation seem to occur across the physically interacted in both Col-0 and DEX-induced Venus– eukaryotic kingdoms. Thus, our findings support a paradigm for CANTH lines but only after La(III) treatment (Fig. 4D). The the activation of CME by extracellular cargoes, in which extra- distinct band for AGPs was not observed in coimmunoprecipitated cellular cargo receptors migrate across the PM to interact with proteins from ap2μ-1 mutant after La(III) treatment (Fig. 4D), AP2. These findings also raise some exciting questions: how can which strongly supports the conclusion that AGPs and AP2 AGPs, which are heavily modified by bulky and physically interacted in Col-0 after La(III) treatment. Moreover, are typically tethered to the extracellular cell surface via a GPI by bioinformatics prediction and molecular dynamic simulation, anchor, move across the PM, and how does REE binding initiate we found that some AGPs expressed in leaves contain the rec- this transmembrane movement? The transmembrane migration ognition site of AP2 (SI Appendix, Table S3) and might directly occurs before clathrin assembly, as shown using the STED interact with AP2 to form the stable complexes (SI Appendix, Fig. superresolution microscopy in cells where this process was S5), which awaits further investigation. We next used anti-AP2 blocked by CANTH expression (Fig. 3). Flip-flopping of REE- antibody to immunoprecipitate the active AGPs that formed activated AGPs is a possible explanation for the transmembrane complexes with AP2 after La(III) treatment and determined the movement. From a structural perspective, it is possible that after content of La(III) in the active AGP molecules by using induc- AGPs containing transmembrane domain act as receptors to tively coupled plasma mass spectrometry. We found that La(III) coordinate with the hydrous ions of REE(III), the introduction was present in the active AGP molecules, further demonstrating of hydrous ions of REE(III) increases the hydrophilicity of AGPs the formation of AGP-La(III) complexes (Fig. 4E). Taken to- (SI Appendix, Fig. S5). This may speed up the flip-flopping of gether, our results demonstrate that La(III) treatment induced membrane lipids, resembling the rapid flip-flopping of endo- the formation of the La(III)-AGP-AP2 complexes and their plasmic reticulum membrane lipids driven by transmembrane subsequent internalization by CME. proteins after their hydrophilicity is increased (49–51), and thus drive the transmembrane movement of AGPs (Fig. 3). Alterna- AGP-Dependent Endocytosis in the Presence of La(III) Also Occurred in tively, La(III)-activated AGPs could interact with a transmembrane Human Cells. REEs are ubiquitous in atmospheric particulate protein, which then may bind the AP2 complexes. Moreover, it was matters, especially in developing countries (9), and are found to reported that some of the AGPs are expressed in specific tissues

14354 | www.pnas.org/cgi/doi/10.1073/pnas.1902532116 Wang et al. Downloaded by guest on September 26, 2021 PLANT BIOLOGY

Fig. 4. La(III) triggers AGPs endocytosis by interacting with AP2 in leaf cells. (A) The representative CLSM images of coimmunostained AGPs (green) and AP2 (blue) in the cells of Col-0 leaves treated with 0 or 30 μM La(III) for 12 h from 3 biological replicates. The leaves after 12-h treatment with 0 or 30 μM La(III) were stained with JIM13 coupled with secondary antibody (rabbit anti-rat IgG H&L [FITC]) and monoclonal rabbit anti-AP2M1 antibody (EP2695Y) coupled with donkey anti-rabbit IgG H&L (Alexa Fluor 405), and observed under CLSM for evaluation of AGP’s CME. (Scale bar, 6 μm.) (B) The representative CLSM images of coimmunostained AGPs (green) and AP2 (blue) in the leaf cells of DEX-inducible Venus–CANTH lines treated with 30 μM La(III) for 12 h. (Scale bar, 6 μm.) (C) CLSM images of the leaf cells in the mutant of AP2 μ subunit (ap2μ-1, SALK-083693C) and its complementated line treated with 0 or 30 μM La(III) for 12 h and stained with FM4-64. Induced endocytic vesicles are shown with white arrowheads. The leaf cells of ap2μ-1 were treated without or with β-glucosyl Yariv reagent before La(III) treatment. (Scale bar, 10 μm.) (D) AGPs directly interacted with AP2 as tested by co-IP from Col-0, DEX-inducible Venus–CANTH lines and ap2μ-1, and Western blot analysis. In this assay, the anti-AP2M1 antibody (EP2695Y) that specifically binds to the μ subunit of AP2 was used to precipitate AP2. The + and − symbols mean that protein extracts were incubated with and without anti-AP2M1 antibody, respectively, before being in- cubated with protein G Sepharose beads. Input factions were the extracted total proteins, and co-IP fractions were the immunoprecipitated AGPs and AP2 in the extracted proteins. (E) The content of La(III) in the immunoprecipitates (by anti-AP2M1 antibody) obtained from the co-IP assay of Col-0 leaves treated with 0 or 30 μM La(III) for 12 h. *P < 0.05 (2-tailed unpaired t test).

Wang et al. PNAS | July 9, 2019 | vol. 116 | no. 28 | 14355 Downloaded by guest on September 26, 2021 Fig. 5. An unprecedented mechanism for the initiation of CME. (A) Mechanism for CME initiated by traditional cargo. Traditional cargo is a transmembrane receptor protein. When cargo receives the endocytic signal, it exposes the recognition domain to recruit and interact with AP2. Subsequently, AP2 recruits clathrin to assemble clathrin-coated vesicle. (B) Unprecedented mechanism for CME initiated by AGPs in presence of REEs. Originally, AGPs are secreted proteins localized to the outer leaflet of the PM via a GPI anchor, but when they encounter La(III), they change their structure to transmembrane form. This form of AGPs exposes the recognition domain to recruit and interact with AP2 and then recruit clathrin to assemble clathrin-coated vesicle. These further showed that secretory AGPs an- chored in the outer leaflet of the PM via a GPI anchor act as the transmembrane receptor proteins and perform the function of transmembrane receptor proteins.

(such as AGP6 and AGP11 in pollen) (52). Whether AGPs in (9). This action greatly increases the risk of heavy metal stress pollen tubes have the same role as AGPs in leaves should be in- in plants and enhances the accumulation of pollutants in the vestigated in future studies by using the related mutants. human body via the food chain. Obviously, once pollutants Explosive usage of REEs in the modern industry and agricul- accumulate in plant cells via REEs-initiated endocytosis in leaf ture urgently demands our understanding of how REEs enter the cells, the health of animals and human beings will be seriously biological systems and their impacts on ecosystems and human threatened. Our current study also solves the mystery of how health. We found that AGPs and REEs also activate and regulate REEs sprayed on the foliage of plants in agriculture may enter the endocytosis in human cells (SI Appendix,Fig.S6). Meanwhile, plant cells via endocytosis; that is, AGPs are the primary targets REEs are heavy metal elements in the periodic table of elements of REEs to initiate the CME in the leaf cells. This knowledge (5). Thus, REEs-initiated endocytosis opens floodgates for heavy may help to design new strategies to manipulate plants for metal elements to enter plant cells (11) because REEs and heavy optimal REE accumulation and for limiting heavy metal ac- metal elements are ubiquitous in atmospheric particulate matters cumulation in living organisms.

14356 | www.pnas.org/cgi/doi/10.1073/pnas.1902532116 Wang et al. Downloaded by guest on September 26, 2021 Materials and Methods ACKNOWLEDGMENTS. We are grateful to J. M. Zhou (The University of British Columbia) for help with immunofluorescent assay, X. F. Han and Z. D. Xiao Plant materials, growth conditions and La(III) treatment, plasmid construc- (Southeast University) for help with STED, and X. Ma (Jiangnan University) for tion, chemical solutions and treatments, real-time PCR analysis, EMARG offering the HEK293 cell line. We also thank C. H. Huang (Peking University), measurement of La(III) subcellular distribution, AGP immunolocalization and Z. J. Guo (Nanjing University), and G. B. Jiang (Research Center for Eco- EDS measurement, FM4-64 internalization assay, STED microscopy determi- Environmental Sciences, Chinese Academy of Sciences) for their valuable nation, AGPs and AP2 immunolocalization assay, co-IP assay for the AGP-AP2μ suggestions on the project. This work was supported by National Natural Science Foundation of China Grants 21371100, 21501068, and 31170477; Ph.D. Programs interaction, the measurement of the La level in AGP-La(III) complexes, chemical Foundation of Ministry of Education of China Grant 20130093120006; the calculation, endocytosis in HEK293 cells, and statistical analysis are described Priority Academic Program Development of Jiangsu Higher Education Institutions; in SI Appendix, SI Materials and Methods. and National Institute of General Medical Sciences Grant GM100130.

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