Effects of fluorescent tags and activity status on the membrane localization of ROP GTPases

Abstract

Plant-specific Rho-type GTPases (ROPs) are master regulators of cell polarity and development. Over the past 30 years, their localization and dynamics have been largely examined with fluorescent proteins fused at the amino terminus without investigating their impact on protein function. The moss Physcomitrium patens genome encodes four rop genes. In this study, we introduce a fluorescent tag at the endogenous amino terminus of ROP4 in wild-type and rop1,2,3 triple mutant via homologous recombination and demonstrate that the fluorescent tag severely impairs ROP4 function and inhibits its localization on the plasma membrane. This phenotype is exacerbated in mutants lacking ROP-related GTPase-activating proteins. By comparing the localization of nonfunctional and functional ROP4 fusion reporters, we provide insight into the mechanism that governs the membrane association of ROPs.

Keywords: Cell polarity; ROP GTPase; fluorescent protein tagging; membrane trafficking; moss.

Figure 1.

N-terminal tagging with a fluorescent protein tag strongly impairs ROP4 function in the rop1,2,3 triple mutant background. a Representative moss colonies of rop1,2,3 triple mutant and ROP4 knock-in (KI) lines. The ROP4 knock-in lines were generated by inserting the coding sequence of green fluorescent protein mNeongreen (mNG) immediately downstream of the ROP4 start codon via homologous recombination. Four independent lines were obtained and exhibited similar growth defects. Scale bar: 5 mm. b verification of KI lines via PCR amplification and sequencing. DNA sequencing confirmed the correct integration of mNG without introducing additional mutations. c morphology of protonema cells of rop1,2,3 triple mutant (top) and ROP4 KI lines (bottom) under a 10× lens (left) or 40× lens (right) labeled by Lifeact-mCherry reporters. Cells in the KI lines were imaged on a glass slide, forming dispersed clusters under the pressure of the coverslip. Scale bars: left, 100 µm; right, 20 µm. d quantification of cell length, cell width, and length/width ratio. Data are presented as violin plots, showing the quartiles (thin lines) and the median (central line). Statistical analyses were performed using two-tailed student’s t-tests. ns, not significant. ****, p < 0.0001.

Figure 2.

The localization of ROP4 at the plasma membrane is altered by an N-terminal mNG tag (N-mNG). a overlay of predicted structures of moss PpROP4 and yeast ScCDC42 and the experimentally obtained structures of Arabidopsis AtROP4 and human HsRhoA. All proteins exhibit a similar conformation. The N-terminal (N-ter) and C-terminal (C-ter) regions are indicated. The loop region of PpROP4 containing glycine 134 (G134) and alanine 135 (A135) is highlighted. The fusion of an mNG tag between G134 and A135 (swmNG) does not impair ROP4 function. b the localization N-mNG-PpROP4 and PpROP4-swmNG. The average membrane/cytosol intensity ratio for each genome type is shown. N-mNG-PpROP4, n = 18 cells; PpROP4-swmNG, n = 13 cells, mean ± SD. c intensity profile of N-mNG-PpROP4 from protonema cell tip to the subapical region along the plasma membrane. Intensity values were measured from 10 cells and shown as mean ± SD. d intensity plot of PpROP4-swmNG from protonema cell tip to the subapical region. Intensity values were measured from 11 cells and shown as mean ± SD. Note that there is a sharp decrease of signals around 15 µm distant from the tip. e the predicted structures of N-mNG-PpROP4 and PpROP4-swmNG. f-h the predicted dimeric structures of N-mNG-PpROP4 and PpROP4-swmNG with PpRopGEF4, PpRopGAP1, and PpRopGDI1. i-k the predicted tetrameric structures of PpRopGAP1 with PpROP4, N-mNG-PpROP4, and PpROP4-swmNG. The CRIB domain of PpRopGAP1 is shown in magenta. The dimeric interaction interfaces are highlighted in dashed boxes. l-n the predicted tetrameric structures of PpRopGEF4 with PpROP4, N-mNG-PpROP4, and PpROP4-swmNG. The two PpRopGEF4 subunits (A/B) are differently colored to facilitate visualization.

Figure 3.

Loss-of-function of ROP-related GAPs impairs membrane localization of N-mNG-PpROP4 and PpROP4-swmNG and a model for the trafficking of ROPs to the plasma membrane. a the localization of PpROP4-swmNG in wild-type (WT) and ropgap, ren septuple mutant. b quantification of membrane/cytosol intensity ratio of PpROP4-swmNG. c the localization N-mNG-PpROP4 in WT, ropgap, ren mutant and REN overexpressor lines. The average number of observed cytosolic puncta (arrows, mean ± SD) is shown. d quantification of membrane/cytosol intensity ratio of N-mNG-PpROP4. Note that the intensity ratio of N-mNG-PpROP4 is much lower than that of PpROP4-swmNG in WT cells in fig. 3b. Data in b and d are presented as box-and-whisker plots, showing the interquartile range (box), the median (horizontal line), minimum and maximum values (whiskers), and individual data points. The numbers of cells used for quantification are shown at the bottom. Scale bar in a and c: 10 µm. Statistical analyses were performed using two-tailed student’s t-tests. ns, not significant. **, p < 0.01. ****, p < 0.0001. e FM4–64-labeled vesicles do not colocalize with N-mNG-PpROP4 puncta in the ropgap, ren mutant. f percentage of N-mNG-PpROP4 puncta that show colocalization with FM4–64-labeled vesicles. Data are shown as mean ± SD (153 particles from six cells). g a proposed model for ROP GTPase trafficking to the plasma membrane. ROPs are anchored to the ER membrane after translation, prenylation, and proteolysis of the AAX tail in the cytosol. Guanine nucleotide dissociation inhibitors (GDIs) can extract ROPs (likely in both GDP-bound and GTP-bound forms) from the ER to the cytosol and drop off them at the plasma membrane. Both processes are reversible. The membrane-anchored ROPs also travel through the exocytosis pathway to the plasma membrane and can be retrieved through endocytosis. The increase of active ROPs enhances their dissociation from the plasma membrane likely via GDI extraction and has a minor effect on exocytosis (red arrows), leading to a net decrease of membrane-associated ROPs. N-terminal fluorescent tags may impair the clustering of ROPs and their stable association with the plasma membrane, thus increasing the probability of dissociation. This phenotype is exacerbated with the increase of ROP activity and causes protein aggregation in the cytosol (red arrows).