Volume 18, Issue 24, Pages 1907-1916 (December 2008) A Tip-Localized RhoGAP Controls Cell Polarity by Globally Inhibiting Rho GTPase at the Cell Apex  Jae-Ung Hwang, Vanessa Vernoud, Amy Szumlanski, Erik Nielsen, Zhenbiao Yang  Current Biology  Volume 18, Issue 24, Pages 1907-1916 (December 2008) DOI: 10.1016/j.cub.2008.11.057 Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 1 Loss-of-Function Mutation in REN1 Causes Severe Male Gametophyte Defect (A) A schematic of the strategy for ROP1 enhancer and suppressor mutant screen. A homozygote Arabidopsis line of LAT52::GFP-ROP1 was subjected to T-DNA insertion mutagenesis. From basta-resistant T1 plants, we expected to identify ROP1 “enhancer” and “suppressor” mutants as shown. Some putative “suppressor” mutants might be false positive because of cosuppression between the endogenous ROP1 gene and the GFP-ROP1, which could be distinguished by reduction or loss of GFP fluorescence. Grey color indicates GFP-ROP1 expression. (B) Representative in vitro-germinated pollen tubes from ren1-1/+ (ren1-1) plants and two alleles (ren1-2 and ren1-3) compared with those of wild-type (WT), 12d.8, and ren1 (ren1-1 LAT52::GFP-ROP1). A typical pollen tube from each mutant line is highlighted in white. Pollen grain (g) and pollen-tube tip (t) are indicated. Scale bar represents 50 μm. The ren1-3 pollen tubes were cultured in a germination medium with 0.5 mM [CaCl2 + Ca(NO3)2], and others were cultured on standard germination medium for 6–8 hr. (C) Pollen grains from isolated dehisced WT, ren1-1, and 12d.8 anthers. Pollen grains were examined immediately after release from anthers onto the germination medium. A significant portion of pollen grains from ren1-1 anthers were found to have already developed swollen tubes (indicated by arrowheads). Scale bar represents 50 μm. (D) T-DNA insertion loci of three alleles of ren1. Solid and gray blocks indicate exons and introns of REN1 (At4g24580), respectively. In ren1-1, two copies of T-DNA are inserted in the second intron in a tandem repeat. Arrows indicate the gene-specific primers used for RT-PCR analysis. (E) Suppression of ren1-1 by expression of full-length REN1 coding sequence. Comparison of in vitro pollen-tube phenotypes of WT, ren1-1/+, and ren1-1 LAT52::REN1 (ren1-1/ren1-1 LAT52::REN1/LAT52::REN1). Pollen was germinated for 8 hr on standard germination medium, and typical ren1-1 pollen tubes are indicated by arrows. Scale bar represents 50 μm. Current Biology 2008 18, 1907-1916DOI: (10.1016/j.cub.2008.11.057) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 2 REN1 Is a RhoGAP for ROP1 (A) REN1 interacts with GTP-bound ROP1. GST-CA-rop1 (CA) or GST-DN-rop1 (DN) were loaded with GTP or GDP, respectively, and incubated with MBP-REN1 or MBP-REN1 (1–429 aa). CA- or DN-rop1-bound REN1 or REN1 deletion mutants were pulled down with amylose-conjugated agarose beads and quantified by western blotting. Upper panel shows western blotting reacted with ROP antibody (IB). Lower panel shows a Coommassie-blue-stained gel image for loading control. (B) REN1 promotes the GTP hydrolysis of ROP1. In vitro GTPase activity of ROP1 was measured as described in Wu et al. [23]. The intrinsic GTPase activity of GST-ROP1 was minimal. For testing the GAP activity of REN1, His-REN1 or His-REN1(R244L) was added to GST-ROP1 as described in Supplemental Experimental Procedures. His-REN1 or His-REN1(R244L) themselves do not induce GTP hydrolysis when tested in the absence of GST-ROP1. GST-ROP1 tends to show multiple turnover reactions rather than the typical single turnover reaction, as observed on in vitro GAP activity assay for RopGAP1 [23]. In our assay condition, GDP-GTP exchange on ROP1 may be unusually fast and spontaneous. (C) Partial suppression of GFP-ROP1-overexpression-induced depolarized pollen-tube growth by REN1 overexpression. When co-overexpressed with GFP-ROP1 in tobacco pollen tubes, REN1 partially suppressed the ROP1-overexpression-induced pollen-tube phenotype, whereas REN1(R244L) slightly enhanced it. More than 20 tubes with significant GFP fluorescence were measured. The changes in GFP-ROP1-overexpression phenotype were not due to altered GFP-ROP1-expression levels, because similar GFP fluorescence was seen in tubes expressing REN1 and REN1(R244L). Asterisks indicate significant difference from control tubes expressing GFP-ROP1 alone (p < 0.05). Representative result of three independent experiments is presented. (D) The GAP-dead mutant REN1(R244L) failed to complement ren1-1. Introduction of the LAT52::REN1(R244L) construct into ren1-1 plants did not increase basta-resistance segregation ratio of T2 ren1-1 plants (Table S3), nor did it restore ren1 pollen tubes to normality (n = 4 independent lines). Representative pollen-tube images from wild-type (WT), ren1-1 (ren1-1/+), ren1-1 LAT52::REN1 (ren1-1/ren1-1 LAT52::REN1/LAT52::REN), and ren1-1 LAT52::REN1(R244L)[ren1-1/+ LAT52::REN1(R244L)/LAT52::REN1(R244L)]. Pollen tubes were observed 5 hr after pollen grains were placed onto the standard germination medium. Scale bar represents 50 μm. (E) ROP localization in ren1-1 pollen tubes compared with WT tubes. ROP1 was delocalized to a broader region of PM in the ren1-1 pollen tube. Pollen tubes were chemically fixed and immunostained with ROP antibody. Scale bar represents 10 μm. (F) REN1 control of the tip-localized ROP1 activity in Arabidopsis pollen tubes. To visualize the in vivo ROP1 activity, we transformed Arabidopsis plants with LAT52::GFP-RIC4. RIC4 specifically binds with the PM-localized active form of ROP1, which reflects the PM-localized ROP1 activity [11]. In pollen tubes of the WT background, GFP-RIC4 mainly localized in the cytosol, forming some aggregates. In contrast, GFP-RIC4 apparently localized in a broad area of PM in ren1-1 tubes. Scale bar represents 10 μm. The rightmost images are zoomed to the PM area indicated with arrowheads. Inset shows line scans of GFP-RIC4 intensity profiles across the PM area indicated with arrowheads. The position of PM is indicated by an arrow. “a.u.” denotes “arbitrary unit.” (G) Similar to ren1-1, pollen grains expressing CA-rop1 germinated prematurely and developed severely swollen tubes. Shown is pollen directly after its release from the isolated anthers (0 hr) and after 5 hr of incubation on the germination medium (5 hr). Scale bar represents 25 μm (0 hr) and 50 μm (5 hr), respectively. Current Biology 2008 18, 1907-1916DOI: (10.1016/j.cub.2008.11.057) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 3 REN1 Is Localized to Exocytic Vesicles in the Pollen-Tube Apex (A) REN1 is localized to the apex of pollen tubes. The localization of endogenous REN1 was observed by immunolocalization with specific REN1 antibody, which was raised against the C-terminal region (530–920 aa) of REN1 and affinity purified. Representative images of ren1-1 (left) and wild-type (right) pollen tubes are presented. (B) GFP-REN1 shows similar localization in Arabidopsis pollen tubes. REN1 is localized in or close to the growth region in the apical PM, where active ROP1 is localized. Scale bar represents 5 μm. The intensity profiles of GFP-REN1 and immunolabeled endogenous ROP following the longitudinal axis (an arrow in the overlay image) are presented at the right. The position of PM is indicated by an arrow. (C and D) REN1 localization in the clear zone overlapped with that of FM4-64-labeled recycling exocytic vesicles (C) and YFP-RabA4D-labeled exocytic vesicles (D). Medium containing FM4-64 was applied to GFP-REN1 pollen tubes 1 hr before observation. YFP-RabA4D pollen tubes were fixed and immunostained with REN1 antibody and Alexa594-conjugated secondary antibody. Scale bars represent 5 μm. (E) GFP-REN1 targeting to the tube apex was affected in raba4d pollen tubes. GFP-REN1 still localized to the cytosol near the tube apex but did not do so in an inverted-cone shape (arrow), which indicates that targeting of GFP-REN1-associated vesicles is affected by raba4d mutation. However, GFP-REN1 association with the vegetative nuclear envelope was not altered (asterisk). Pollen of raba4d LAT52::GFP-REN1 was cultured in the germination medium with 0.001% borate, in which the raba4d-induced growth-polarity defect was more pronounced as compared with that in the standard germination medium. Current Biology 2008 18, 1907-1916DOI: (10.1016/j.cub.2008.11.057) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 4 REN1 Targeting to the Pollen-Tube Apex Is Mediated by Dynamic F-Actin and Vesicular Trafficking (A) GFP-REN1 accumulation to the clear zone was disrupted with short-term treatments of 0.5 nM LatB or 0.1 μg/ml BFA. GFP-REN1-expressing pollen tubes (ren1-1 LAT52::GFP-REN1) were incubated with LatB- or BFA-containing medium 20–30 min before observation. Scale bar represents 5 μm. (B) Both LatB and BFA induced severe growth defect in ren1-1 LAT52::GFP-REN1 pollen tubes when added to the pollen-germination medium. Representative pollen tubes are highlighted. However, LatB and BFA at the same concentration did not significantly affect wild-type (WT) pollen-tube growth, except that growth rate was reduced. Pollen grain (g) and pollen-tube tip (t) are indicated. Scale bars represent 50 μm. (C) ren1-3 and raba4d synergistically affected pollen-tube growth. Pollen from ren1-3/+ raba4d/+ plants was cultured on the standard germination medium, on which pollen tubes from single ren1-3 or raba4d mutants did not display tip swelling. Six hours after incubation, the widest diameter for WT, ren1-3, and raba4d tubes was 7.9 ± 0.2 μm (mean ± SE, n = 91), 10.6 ± 0.2 μm (n = 162), and 10.4 ± 0.2 μm (n = 39), respectively. However, approximately 20% of pollen tubes from ren1-3/+ raba4d/+ flowers developed balloon-like tips (highlighted), with diameters ≥ 20 um (n = 84/439). Scale bar represents 50 μm. Current Biology 2008 18, 1907-1916DOI: (10.1016/j.cub.2008.11.057) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 5 REN1-Vesicle Targeting to the Tip Oscillates ahead of Tip Growth (A–C) The tip localization of GFP-REN1 oscillates with growth rate in growing Arabidopsis pollen tubes. GFP-REN1 localization was observed by time-lapse imaging of Arabidopsis pollen tubes (ren1-1 LAT52::GFP-REN1). The GFP intensity of a 2-μm-wide region was line scanned along the longitudinal axis of an Arabidopsis pollen tube (A). From the resulting intensity profiles, the mean GFP intensity of the area within 2 μm from the cell margin was calculated (shaded area in the intensity profile). The net tip growth was calculated by net advance of the cell margin between two consecutive images. Cell margin was determined where GFP intensity turns above the background signal. Representative GFP-REN1 signal oscillation in the tip is presented with an oscillation curve of tip growth (B) and in false-colored sequential images (C; red indicates high fluorescence and blue indicates low fluorescence). Numbers at the bottom (C) are the elapsed time (s). Scale bar represents 5 μm. (D) A model for REN1 regulation of ROP1 activity in the pollen-tube tip. Left, localized ROP1 activation (red circles) in the tube apical PM laterally amplifies, probably via a positive-feedback mechanism (1 and 2). Active ROP1 induces the assembly of F-actin (indicated by lines) and promotes accumulation of REN1-associated exocytotic vesicles in the tube apex (2). Once vesicles fuse to the PM, REN1 is targeted to the PM and inactivates PM-localized active ROP1 (3). This activity prevents excess ROP1 activation and depolarization of tip growth. After ROP1 inactivation, REN1 returns to the cytosol, thus allowing ROP1 activity to increase again (4). The REN1-mediated negative-feedback mechanism is activated behind the apical ROP activation by ∼60° and prevents excess ROP activation in the apical PM (Right). Current Biology 2008 18, 1907-1916DOI: (10.1016/j.cub.2008.11.057) Copyright © 2008 Elsevier Ltd Terms and Conditions