ROP GTPase-Dependent Actin Microfilaments Promote

ROP GTPase-Dependent Actin Microfilaments Promote PIN1 Polarization by Localized Inhibition of Clathrin- Dependent Endocytosis Shingo Nagawa1., Tongda Xu1,2., Deshu Lin1,3, Pankaj Dhonukshe4, Xingxing Zhang3, Jiri Friml5, Ben Scheres4, Ying Fu3, Zhenbiao Yang1* 1Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, California, United States of America, 2 Temasek Lifesciences Laboratory Ltd, National University of Singapore, Singapore, 3 State Key Laboratory of Plant Physiology and Biochemistry, Department of Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing, China, 4Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands, 5Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Ghent, Belgium Abstract Cell polarization via asymmetrical distribution of structures or molecules is essential for diverse cellular functions and development of organisms, but how polarity is developmentally controlled has been poorly understood. In plants, the asymmetrical distribution of the PIN-FORMED (PIN) proteins involved in the cellular efflux of the quintessential phytohormone auxin plays a central role in developmental patterning, morphogenesis, and differential growth. Recently we showed that auxin promotes cell interdigitation by activating the Rho family ROP GTPases in leaf epidermal pavement cells. Here we found that auxin activation of the ROP2 signaling pathway regulates the asymmetric distribution of PIN1 by inhibiting its endocytosis. ROP2 inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by the ROP2 effector protein RIC4. Our findings suggest a link between the developmental auxin signal and polar PIN1 distribution via Rho-dependent cytoskeletal reorganization and reveal the conservation of a design principle for cell polarization that is based on Rho GTPase-mediated inhibition of endocytosis. Citation: Nagawa S, Xu T, Lin D, Dhonukshe P, Zhang X, et al. (2012) ROP GTPase-Dependent Actin Microfilaments Promote PIN1 Polarization by Localized Inhibition of Clathrin-Dependent Endocytosis. PLoS Biol 10(4): e1001299. doi:10.1371/journal.pbio.1001299 Academic Editor: Mark Estelle, University of California, San Diego, United States of America Received October 26, 2011; Accepted February 21, 2012; Published April 3, 2012 Copyright: � 2012 Nagawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported by grants from the U.S. National Institute of General Medical Sciences to ZY (GM081451). PD is supported by Dutch Science Organization’s NWO-VENI grant and by Utrecht University starting independent investigator grant. This work was made possible in part, through access to the Optical Biology Core facility of the Developmental Biology Center, a Shared Resource supported in part by the Cancer Center Support Grant (CA-62203) and Center for Complex Biological Systems Support Grant (GM-076516) the at the University of California, Irvine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: ABP1, auxin-binding protein 1; ANTH(C), C-terminal region of AtAP180 protein; BFA, Brefeldin A; CA-ROP2, constitutive active form of ROP2; DN- ROP2, dominant-negative mutant of ROP2; GFP, green fluorescent protein; Ika, Ikarugamycin; NAA, naphthalene acetic acid; PC, pavement cell; PIN, PIN-FORMED; RIC, ROP INTERACTIVE CRIB MOTIF-CONTAINING PROTEIN; ROP, Rho-like GTPases from plants; SD, standard deviation; WT, wild type * E-mail: zhenbiao.yang@ucr.edu . These authors contributed equally to this work Introduction Cell polarity is a conserved cellular property that is necessary for the generation of diverse forms and types of cells in both uni- and multicellular organisms [1,2]. The general design principles that govern the formation of polarity and how they are used to generate diverse forms of polarity is a fundamental issue of developmental mechanisms. In the unicellular yeast, Rho family GTPase-mediated activation of endocytosis is required for cell polarization [3–5]. In contrast, emerging evidence suggests that Rho family GTPase- mediated inhibition of endocytosis is essential for the polarization of cells in somemulticellular tissues as shown in cultured epithelial cells from rat [6] and neuroectodermal epithelial cells from Drosophila [7]. It is unclear whether Rho-mediated inhibition of endocytosis is a common design principle for polarity establishment in multicellular systems and how the inhibition of endocytosis is regulated. In multicellular plants, coordinated polarization of the proposed auxin efflux carriers PIN-FORMED (PIN) proteins within a plant tissue is required for polar auxin transport and formation of auxin gradients, which regulate a wide range of morphogenetic and growth patterns in plants [8–11]. Asymmetric endocytosis and recycling of plasma membrane (PM)-localized PINs have been shown to contribute to the polar PIN localization [12,13], and auxin has been implicated as a self-organizing signal to polarize PIN proteins through its inhibition of clathrin-dependent PIN endocytosis in root cells, which is mediated by the auxin-binding protein 1 (ABP1) putative cell surface auxin receptor [14,15]. We studied auxin regulation of cell polarity formation and PIN1 polarization in Arabidopsis leaf epidermal pavement cells (PCs), which display multipolarity by forming the puzzle-piece appear- ance with interdigitated lobes and indentations [16–20]. Recently we showed that ABP1-dependent auxin signaling promotes the formation of multipolarity in PCs by activating Rho-like GTPases from plants (ROPs) that are associated with the plasma membrane [19,21]. ROPs also regulate other processes mediated by auxin such as root hair development, lateral root formation, and root PLoS Biology | www.plosbiology.org 1 April 2012 | Volume 10 | Issue 4 | e1001299 Administrator Highlight Administrator Highlight Administrator Highlight Administrator Highlight Administrator Highlight Administrator Highlight Administrator Highlight Administrator Highlight Administrator Highlight gravitropic responses [22–24]. In addition, auxin activation of ROPs is associated with auxin regulation of gene expression in the nucleus [25,26]. We found that polar PIN1 localization to the tip of lobes in PCs is dependent upon ROP2, which is activated by auxin in the same PM region where PIN1 is localized [19]. PIN1 is required for ROP2 activation and lobe formation, supporting a role for auxin in self-organizing PIN1 polarization in PCs [19]. How auxin-activated ROP2 regulates PIN1 polarization is unknown. One possible mechanism would be the inhibition of PIN1 endocytosis by activated ROP2, because inactivation of ROP2 leads to PIN1 internalization in PCs [19]. This finding is consistent with the report showing that the expression of constitutively active ROPs inhibited internalization of the endocytosis tracer dye FM-64 in roots and guard cells [27–29]. ROP2 regulates the formation of the multipolarity in PCs by activating RIC4 [17], a member of the ROP INTERACTIVE CRIB MOTIF-CONTAINING proteins (RICs) family of ROP effector proteins [30]. RIC4 promotes the local accumulation of fine cortical actin microfilaments in the tip of PCs and pollen tubes [17,31], and actin dynamics has been implicated in the regulation of auxin transport and PIN endocytosis [32–34]. These observations raise an interesting possibility that the ROP2- RIC4 pathway could regulate PIN1 polarization through endocytic trafficking and the actin cytoskeleton. In this report we show that PIN1 endocytosis is preferentially inhibited in the PM region of lobes and that auxin activation of ROP2 in this region inhibits clathrin-dependent PIN1 endocytosis, allowing PIN1 to be polarized to the ROP2-active region. We further demonstrate that ROP2 promotion of F-actin accumula- tion via its effector protein RIC4 is responsible for its inhibition of PIN1 endocytosis. Our results reveal the conservation of a new design principle for cell polarization, which is based on localized inhibition of endocytosis by Rho GTPase signaling in multicellular plants and animals, and provide new insights into the mechanisms by which Rho GTPases inhibit clathrin-dependent endocytosis of polarity proteins. Our results establish an auxin signaling pathway leading to the polarization of PIN proteins that is essential for pattern formation and morphogenesis in multicellular plants. Results ROP2 Inhibits PIN1 Endocytosis in the Lobe Region of PCs To test the auxin-mediated self-organizing PIN1 polarization, we investigated how auxin-activated ROP2 signaling regulates PIN1 localization to the lobe tip. We first utilized PIN1-green fluorescent protein (GFP) transient expression in leaves of Nicotiana benthamiana (tobacco) plants by the agrobacterium infiltration method [35]. This system allows determining the effect of mutant ROP2 on PIN1-GFP localization independent of PC shape changes, which occur in Arabidopsis rop2 mutants [16,17]. Within 3 d after infiltration, PIN1-GFP was detected in PCs of tobacco leaves and localized to the PM with stronger accumulation at the tips of lobes as in Arabidopsis PCs (Figure 1A, arrow). PIN1-GFP signal was also observed in the cytoplasm as endosome-like vesicles (Figure 1A, arrow). Time-lapse imaging showed that PIN1-GFP appeared to be internalized preferentially in the indentation region but not in the lobe region where stronger PM accumulation of PIN1-GFP was observed (Figures 1A, S1A, and S1B). Both PIN1-GFP and FM4-64 were internalized simultaneously and became colocalized in the same vesicles, confirming that GFP-PIN1 was internalized through endocytosis (Figure S1C). Because PIN1 internalization appears to occur preferentially in the indentation region but not in the lobe region where ROP2 is activated [17,19], we hypothesized that ROP2 activation may inhibit endocytosis of PIN1, allowing PIN1 to be polarized in that region. To visualize PIN1 internalization, we utilized PIN1 fused with the dendra2 photo-convertible fluorescent protein (Figures S2 and S3) [36]. Photo-conversion of PIN1-dendra2 transiently expressed in tobacco or Arabidopsis leaves was conducted using transient high dosage of irradiation with 405-nm laser (Figures S2A and S3A). To confirm whether PIN1-dendra2 expressed in leaves was internalized from the PM, PIN1-dendra2 cells were treated with Brefeldin A (BFA), which inhibits ADP ribosylation factor (ARF) GEF and arrests endosomal recycling, causing internalized PIN1 to accumulate in an aggregate known as BFA bodies in plant cells [14,32]. PIN1-dendra2 at the PM was photo- converted from green to red emission. 30 min after photo- conversion, converted PIN1-dendra2 was observed in BFA bodies, which demonstrated the occurrence of PIN1-dendra2 endocytosis (Figure S2A). To test the effect of ROP2 on PIN1- dendra2 endocytosis, we coexpressed a dominant-negative mutant of ROP2 (DN-ROP2) with PIN1-dendra2 and observed the internalization of the photo-converted signal at the PM. In the lobe regions of PCs expressing PIN1-dendra2 only, PIN1- dendra2 vesicles were rarely formed from the PM (Figure 1B). In contrast, in cells expressing both PIN1-dendra2 and DN-ROP2, numerous PIN1-dendra2 vesicles were formed and pinched off from the PM (Figure 1B, arrowheads). Furthermore, time-lapse imaging showed that DN-ROP2 expression greatly accelerated the decrease in the photo-converted PM signal, which was Author Summary Formation of cell polarity is a process of distributing cellular structures or molecules in an asymmetric manner. This process plays an important role in the generation of diverse cell forms and types. In plants, the quintessential hormone auxin is important for diverse physiological functions, including growth and development of cells and organs. To perform these functions, auxin must be transported and localized to specific regions within the plant. This is partially mediated by polar distribution of the PIN-FORMED (PIN) auxin efflux transporters, which transport auxin outside of the cell and allow for the directional short- and long- distance transport of auxin throughout plant tissues and organs. Although auxin itself has been implicated as a signal to regulate PIN polar distribution, how auxin does so remains to be elucidated. We previously showed that auxin promotes the generation of ‘‘puzzle-piece’’ polarity in leaf epidermal pavement cells, which contain interdigitated lobes and indentations, by activating the ROP (Rho-like GTPases from plants) members of the conserved Rho family of small GTPases. Here, we find that auxin-dependent local activation of ROP2 in the lobe region inhibits PIN1 internalization into the endosomal compartments (or endocytosis), leaving higher levels of PIN1 polar distribution in the lobe region. PIN1 internalization is inhibited by altering the actin cytoskeleton through the ROP2 effector protein RIC4, a protein involved in cytoskeletal remodeling. On the basis of our findings, we propose that the Rho GTPase-mediated inhibition of endocytosis of PIN1 provides a self-organizing mechanism for the polar PIN1 distribution. Rho GTPase-based inhibition of endocytosis is also impor- tant for the formation of cell polarity in animal cells. Thus, we conclude that Rho GTPase signaling to inhibit endocy- tosis is a common mechanism for cell polarization in multicellular organisms. Rho GTPase Inhibits PIN1 Endocytosis PLoS Biology | www.plosbiology.org 2 April 2012 | Volume 10 | Issue 4 | e1001299 quantified by changes in the relative intensity (Figure 1C) or in the absolute intensity (Figure S2B) of the converted signal. In cells coexpressing DN-ROP2 and PIN1-dendra2, the PM PIN1-dendra2 signal was generally weaker compared to cells expressing PIN1- dendra2 alone (Figure 1B). This finding was likely due to the DN- ROP2–mediated induction of endocytosis, but not its general toxic effect, because DN-ROP2 expression did not affect the expression and localization patterns of several endosomal markers (Figure S4). Thus, these results show that DN-ROP2 expression promoted PIN1- dendra2 internalization. To confirm that the effect of DN-ROP2 on PIN1 endocytosis in tobacco cells reflected the function of ROP2 in Arabidopsis, we transiently expressed PIN1-dendra2 in the PCs of wild type (WT) or the rop4-1 rop2 RNAi line, in which ROP2 is down-regulated by RNAi and the functionally redundant ROP4 is knocked out (rop4 R2i) [17]. As expected, photo-converted signal was found in Figure 1. PIN1 endocytosis is inhibited in the lobe region by the ROP2 pathway in PCs. (A) Time-lapse imaging of PIN1-GFP transiently expressed in tobacco PCs. PIN1-GFP signal was imaged using laser scanning confocal microscopy 3 d after the infiltration of tobacco leaves with agrobacterium containing pPIN1-PIN1-GFP. Internalization of PIN1-GFP from the PM (arrowheads) occurred preferentially in the indention region but not in the lobe region (arrow) where stronger PM accumulation of PIN1-GFP was observed. Note that only a cell at the left side of the image expressed PIN1-GFP, and internalization events were only visualized in that cell but not in the cell at the right side of the images. (B) A time-course analysis of PIN1-dendra2 internalization after photo-conversion in tobacco PCs. Signal in the red color represents photo-converted PIN1-dendra2. PIN1-dendra2 was transiently expressed in tobacco PCs for 3 d before photo-conversion was achieved. Coexpression of DN-ROP2 promoted vesicle formation (arrowheads) from the PM and accelerated decrease of PM signal. (C) Quantitative analysis of the PM signal representing photo-converted PIN1-dendra2 shown in (B). Relative intensity was measured as absolute value of intensity divided by value of intensity in the first time point. Error bars represent standard deviation (SD) (n=5). (D) A time-course analysis of PIN1-dendra2 internalization after photo-conversion in Arabidopsis PCs. PIN1-dendra2 (and CA-ROP2) was transiently expressed in WT or rop4 R2i Arabidopsis PCs for 24 h. Formation of vesicles (arrowheads) and decrease of PM signal were accelerated in rop4 R2i cells. Coexpressing CA-ROP2 suppressed both vesicle formation and decrease of PM signal in rop4 R2i cells (n=5). (E) Quantitative analysis of PM signal shown in (D). Measurements of signal were done as in (C). doi:10.1371/journal.pbio.1001299.g001 Rho GTPase Inhibits PIN1 Endocytosis PLoS Biology | www.plosbiology.org 3 April 2012 | Volume 10 | Issue 4 | e1001299 vesicles budding from the PM and decreased rapidly from PM in rop4 R2i cells but not in WT control cells (Figures 1D, 1E, and S3B). Moreover, expressing the constitutive active form of ROP2 (CA-ROP2) in rop4 R2i cells suppressed PIN1-dendra2 internal- ization (Figures 1E and S3B). These results indicate that ROP2/ ROP4 suppresses PIN1 internalization, which supports our hypothesis that active ROP2 inhibits PIN1 endocytosis in the lobe region. We next tested the identity of the PIN1 vesicles induced by DN- ROP2 expression by examining the colocalization with known endocytic markers in plants. Coexpression of DN-ROP2 with PIN1-GFP in tobacco leaves greatly increased the number of PIN1-GFP vesicles in the cytoplasm (Figure 2A and 2B), similar to the PIN1-dendra2 vesicles. Previous studies showed that endocytic trafficking mediated by the Rab5 family of GTPases plays an essential role in various developmental processes including PIN polarization [13,37,38]. Ara7, a Rab5 homolog, resides in an endosomal compartment from which various internalized proteins, such as PIN1, are sorted for targeting to vacuoles or recycling to the PM [39]. In cells coexpressing Venus-Ara7, PIN1-GFP, and DN-ROP2, most PIN1-GFP vesicles overlapped with Venus-Ara7 (Figure S5). Thus, most PIN1 vesicles induced by DN-ROP2 were localized to the endosomal compartment containing Ara7. Taken together our results suggest that activated ROP2 in the lobe region inhibits PIN1 endocytosis in that region. ROP2 Inhibits Endocytosis through the Clathrin- Dependent Pathway Several types of endocytosis have been characterized in yeast or animals [40]. We speculated that the clathrin-dependent endocytic pathway contributed to the PIN1 internalization in PCs because this pathway has been reported to modulate the internalization of PIN proteins in other tissues [15,41,42]. To test this notion, we inhibited clathrin-dependent endocytosis by coexpressing the C-terminal region of AtAP180 protein (ANTH[C]) with PIN1-GFP. The conserved AP180 protein contains both the PIP2-binding domain and the clathrin-binding domain and is essential for the early stage of clathrin-dependent endocytosis [43,44]. ANTH(C), which contains the clathrin-binding domain (ANTH domain), has a dominant- negative effect on the function of AP180 protein and inhibits the clathrin-mediated endocytosis [43]. Overexpression of ANTH(C) greatly reduced the number of PIN1-GFP–associated vesicles and suppressed DN-ROP2 induction of the PIN1-GFP vesicles (Figure 2A and 2B). ANTH(C) did not have a general toxic effect, because its expression did not affect the expression and localization of several other endosomal markers (Figure S4). Treatment with Ikarugamycin (Ika), a specific inhibitor of clathrin-dependent endocytos