Author response:
The following is the authors’ response to the original reviews.
eLife Assessment
Plasmodesmata are channels that allow cell-cell communication in plants; based on the functional similarities between facilitated transport within plasmodesmata and into the nucleus, the authors speculate that nuclear pore complex proteins might be involved in plasmodesmata function. If supported, this would transform our understanding of cell-to-cell communication in plants. The authors localize nuclear pore complex proteins to plasmodesmata using proteomics and heterologous overexpression; however, the data are incomplete since key controls for localization, functionality, and expression level of fluorescent protein fusions are absent.
Thank you for the constructive reviews. We have tried to address the comments as outlined below. Specifically, we added new data to the manuscript with respect to the assessment of the protein levels of three independent stable Arabidopsis lines expressing NUP62-GFP from its own promoter using mass spectrometry quantification. These experiments were carried out to evaluate whether the observed PD localization of NUP62-GFP to peripheral puncta might be an artifact caused by inadvertent overexpression and resulting mistargeting. Quantitative analysis shows no indication for significant overexpression of NUP62-GFP.
To assess whether the localization of NUPs is distinct from localization of an ER marker, we have now included a comparison of the NUP43-mVenus localization with that of the mCherry-HDEL luminal ER marker, revealing distinct localization patterns. The peripheral puncta thus do not appear to be due to simple ER accumulation.
To evaluate whether the CPR5-mCitrine fusion is functional, we tested whether the fusion construct was able to complement the loss-of-function cpr5-1 mutant. In two independent complementation lines (cpr5-1/CPR5:CPR5-mCitrine), the roots of 14-d old seedlings were significantly longer compared to the cpr5-1 mutant, and four-week-old plants showed a more WT-like growth phenotype. Although we did not detect CPR5-mCitrine fluorescence, the construct appears to be able to restore the wild type phenotype, indicating that the lines express a functional CPR5 protein.
We have restructured the figures and provided additional information in the figure legends.
Public Reviews:
Reviewer #1 (Public review):
Summary:
Plasmodesmata are channels that allow cell-cell communication in plants; based on the functional similarities between facilitated transport within plasmodesmata and into the nucleus, the authors speculate that nuclear pore complex proteins might be involved in plasmodesmata function. In this manuscript, they localize nuclear pore complex proteins to plasmodesmata using proteomics and heterologous overexpression. They also document a possible plasmodesmata transport defect in a mutant affecting one nuclear pore complex protein.
Strengths:
The main strength of this manuscript is the interesting and novel hypothesis. This work could open exciting new directions in our understanding of plasmodesmata function and cell-cell communication in plants. They also localized many NUPs (12/35 Arabidopsis NUPs).
Weaknesses:
The main weakness of this manuscript is that the data are incomplete. While the authors appropriately and frequently acknowledge caveats to their data, two controls are essential to interpret the results that fluorescently-tagged NUPs localize to the plasmodesmata: (1) assessment of the expression level of these fluorescently-tagged NUPs to determine whether the plasmodesmata localization might be an overexpression artefact;
As we outlined in the manuscript, we also considered the possibility that the peripheral localization could be a consequence of overexpression, in particular in the transient expression system. To be able to control the levels, NUP genes were expressed under the control of the b-estradiol-inducible XVE promoter which allows for b-estradiol dose dependent gene expression (Bashandy et al., 2015; Schlücking et al., 2013). We assessed the dependence of localization on expression levels by studying NUP localization under conditions of reduced estradiol concentrations for induction and shortened incubation time. We validated that the fluorescence was substantially reduced relative to the standard estradiol concentration experiments, however we still detected both nuclear and peripheral localization of the NUPs (Figure 4C-F).
We also considered that in stable transformants the expression of one extra copy of a NUP62-GFP fusion under the control of the native promoter could cause a moderate overexpression and as a consequence lead to artifactual accumulation in the periphery (Figure 3C-E).
To evaluate the level of NUP62-GFP fusion protein relative to untransformed controls, we quantified the levels of NUP62 in three independent transgenic fluorescent WT/NUP62p:NUP62-GFP Arabidopsis lines and in Arabidopsis WT using mass spectrometry (new Figure 3F). The new data indicate that there is no significant increase in NUP protein amounts in the lines expressing the fusion construct relative to WT.
We now write in the revised manuscript (line 200-205):
“NUP62 protein abundance in two-week-old cotyledons of the stable NUP62p:NUP62-GFP transformants was not statistically different to NUP62 protein levels in WT (Figure 3F). Notably, the punctate fluorescence at the cell periphery, encompassing both PD-associated and non-PD-associated localization, were not detectable or absent in roots and young leaves of four-day-old seedlings (Figure 3D). However, it cannot be excluded that the GFP fusion impacts NUP62 localization.” We provide a new Method section for the mass spec analysis of the cotyledons in lines 582-590.
The use of antibodies in wild type tissue would be a potential way to avoid overexpression when trying to detect the localization of NUPs in planta. To investigate the localization of NUPs at physiological expression levels, we attempted to immunolocalize NUPs using antibodies. However, the anti-NUP antibodies available to us were not optimized for immunolocalization and we were unable to detect any fluorescence in the cells at the NPC nor the periphery.
(2) assessment of the function of the fluorescently-tagged NUPs, either by molecular complementation of a knockout mutant phenotype or by biochemical methods to test whether the fluorescently-tagged NUP incorporates into nuclear pore complexes. Conducting these experiments for even one fluorescently-tagged NUP would substantially strengthen this manuscript.
We agree with the reviewer that validation of the functionality of NUP fusion proteins would be valuable. Previously, C-terminally fused Arabidopsis NUPs, such as NUP93a-GFP, GP210-GFP, NUP58-GFP were reported to localize to the nuclear envelope when stably expressed in transgenic Arabidopsis lines (Tamura et al., 2010). As reported for transmembrane NUP GP210 and CPR5 fusion proteins (Gu et al., 2016; Tamura et al., 2010), C-terminally fused GP210 and CPR5 localized to the nuclear envelope but not to the nucleoplasm when expressed heterologously in N.benthamiana (see Figure 3-figure supplement 1). We found several soluble NUPs to also localize to the nucleoplasm (PpNUP98.1, PpNUP62, AtNUP62, AtHOS1) (Figure 1-figure supplement 1, Figure 3, Figure 3-figure supplement 1). Previous studies have reported that several FG NUPs (i.e. NUP98a/b or NUP62) and Y-complex NUPs (i.e. HOS1, NUP96, and NUP107) have been found to also localize in the nucleoplasm rather than specifically to the nuclear envelope when expressed as fusion proteins (Chen et al., 2023; Gallemí et al., 2016; Huang et al., 2024; Lazaro et al., 2012). Of note, for NUP98a, Gallemi and colleagues (2016) discussed the localization to the nucleoplasm as confirmation that, like vertebrate NUP98, Arabidopsis NUP98a is a dynamic NUP rather than just a key structural element of the NPC. HOS1 was reported to interact with ICE1, CO, FVE, and HDA6 in the nucleoplasm (Dong et al., 2006; Jung et al., 2012; Lazaro et al., 2012), indicating that HOS1 might dynamically shuttle between the nuclear pore and nucleoplasm, which could also explain the observed nucleoplasmic localization. In Drosophila, the FG-NUPs NUP98, NUP62, and NUP50 localized in the NPC, and also in the nucleoplasm and interacted with genes (Kalverda et al., 2010). The nucleoplasmic localization could thus have a functional relevance. Yet we cannot rule out, whether soluble NUPs mislocalize in overexpression conditions as we state multiple times in the manuscript.
For this revision, we generated two new independent transgenic Arabidopsis lines stably expressing CPR5-mCitrine under control of its own promoter in the cpr5-1 mutant background (cpr5-1/CPR5p:CPR5-mCitrine). The roots were significantly longer in the two independent transgenic cpr5-1/CPR5p:CPR5-mCitrine Arabidopsis lines compared to the cpr5-1 mutant, and four-week-old plants showed a more WT-like growth phenotype (new Figure 7-figure supplement 1, G–I). However, we could not detect fluorescence in the 10-14 day old seedlings, which could be due to a variety of reasons, such as cleavage of the FP and degradation of the FP without accumulating elsewhere in the cells.
In the new manuscript we write in lines 275-283:
“To assess whether the CPR5-mCitrine fusion protein is functional in Arabidopsis, we tested whether CPR5p:CPR5-mCitrine (including all introns) expression in the cpr5-1 mutant background results in a rescue of the severe growth phenotype of the cpr5-1 loss-of-function mutant (Bowling et al., 1997). Indeed, roots were significantly longer in the two independent transgenic cpr5-1/CPR5p:CPR5-mCitrine Arabidopsis lines compared to the cpr5-1 mutant, and four-week-old plants showed a more WT-like growth phenotype (Figure 7-figure supplement 1, G–I). However, we could not detect fluorescence in 10-14 day old seedlings, which could be due to a variety of reasons, such as cleavage of the FP and degradation of the FP without accumulating elsewhere in the cells. The lack of fluorescence in the transgenic lines requires further investigation.“
Reviewer #2 (Public review):
Summary:
The authors aim to address whether nuclear pore complex components localize and function at PD in plant cells to mediate cell-to-cell communication.
Strengths:
(1) Novelty and Significance:
The core hypothesis, drawing parallels between PD and NPC transport, is highly original and addresses a critical gap in understanding plant intercellular communication. The idea that phase-separated domains formed by FG-NUPs could act as diffusion barriers at PD offers a plausible and sophisticated explanation for their complex transport properties, including size exclusion and facilitated translocation. This could fundamentally change how we view PD function.
(2) Comprehensive Evidence:
The study employs a rigorous and diverse set of experimental approaches, including a comprehensive bioinformatic analysis of both moss and Arabidopsis NUPs in available PD proteomic datasets, extensive imaging analysis of Nup localization in vivo, and functional transport assays using a loss-of-function nup mutant (cpr5). The transport assay is particularly important to provide functional evidence linking CPR5 to PD-mediated transport. The finding that callose levels were not significantly different in cpr5 mutants under these conditions is helpful and supports a distinct, callose-independent mechanism of transport regulation.
(3) Objectivity:
The authors are forthright in discussing the limitations and potential artifacts of their own data, clearly distinguishing between observations and definitive conclusions.
Weaknesses:
While the claims are generally justified as hypotheses or consistent observations, the authors themselves extensively detail the caveats, which are worth reiterating for clarity:
(1) Potential Overexpression Artifacts in Localization:
Although efforts were made to control expression levels, the authors acknowledge that transient overexpression could still lead to NUP accumulation at PD, either as a physiologically relevant accumulation under excess conditions or due to mis-targeting, or even as storage depots. The resolution of confocal microscopy also does not allow for a definitive conclusion on the nature of the location.
We would like to add that in addition to the experiments using estradiol-controlled transient overexpression for localizing NUP fusions, we also provided localization data obtained from Arabidopsis transformants that stably express one extra copy of a NUP62-GFP fusion under the control of the native promoter. In cotyledons, NUP62-GFP localized to the nucleus and in the periphery, and in many cases to PD (Figure 3C-E). In the course of the revision we tested whether the extra copy of NUP62 could cause overexpression that might lead to artifactual accumulation in the periphery.
To evaluate the level of NUP62-GFP fusion protein relative to untransformed controls, we quantified the levels of NUP62 in three independent transgenic fluorescent WT/NUP62p:NUP62-GFP Arabidopsis lines and in Arabidopsis WT using mass spectrometry (new Figure 3F). The new data indicate that there is no significant increase in NUP protein amounts in the lines expressing the fusion construct relative to WT.
We now write in the revised manuscript (lines 200-205):
“NUP62 protein abundance in two-week-old cotyledons of the stable NUP62p:NUP62-GFP transformants was not statistically different to NUP62 protein levels in WT (Figure 3F). Notably, the punctate fluorescence at the cell periphery, encompassing both PD-associated and non-PD-associated localization, were not detectable or absent in roots and young leaves of four-day-old seedlings (Figure 3D). However, it cannot be excluded that the GFP fusion impacts NUP62 localization.“ We provide a new Method section for the mass spec analysis of the cotyledons in lines 582-590.
(2) Proteomics Purity:
The authors note that the presence of NUPs in PD fractions/proteomics cannot definitively rule out contamination, as PD cannot currently be purified to absolute homogeneity and is often contaminated with other organelles, including the nucleus.
We would like to add that despite their low abundance in plant cells, NUPs were found to be enriched in cell wall, and PD fractions relative to total cell extracts (revised Figure 2-supplement 2). To evaluate whether NUP enrichment might be a consequence of contamination by nuclear fractions, for the revision, we evaluated the enrichment of nucleolar proteins and histones. As shown in the revised Figure 2–figure supplement 2, other nuclear proteins did not show a significant enrichment, supporting the notion that NUPs were specifically enriched in PD fractions, consistent with the localization of NUP-FP fusions. We note however, that these data do not demonstrate unambiguously that NUPs are bona fide PD components.
(3) CPR5 Mutant Interpretation:
While cpr5 mutants exhibited reduced macromolecular transport, the authors state that they cannot exclude that the reduced transport is due to secondary effects in the cpr5 mutants, which show rather severe phenotypic defects. This is an important distinction, as CPR5 has known roles in defense responses and hormone signaling that could indirectly influence PD integrity, independent of callose deposition. The lack of effect on small molecule transport is a good control, but the broader pleiotropic effects of cpr5 mutants remain a consideration.
We agree with the assessment of the reviewer. The mutant is compromised in many ways and thus the effects we observe could be indirect. This is stated also in the manuscript (lines 314-317).
(4) Conceptual Distinction between NPC and PD:
The authors correctly point out that while similarities exist, the physical assembly of NUPs at PD must differ from that at the NPC due to the presence of the desmotubule and smaller cytoplasmic sleeve width at PD. Moreover, nucleocytoplasmic transport depends on karyopherin proteins that interact with the NPC central channel to complete the transport. Yet the role of karyopherins in this case is not clear. Therefore, the proposed "PD pore complex" may bear some NPC features, but not be identical.
Reviewer 2 summarized the key concerns that we highlighted and discussed in the manuscript, which addressed differences in PD and NPC architecture. In particular, we noted that one of the major differences in PD is the presence of the desmotubule (in lines 370-372). We also highlighted that we did not detect all NUPs at PD (in lines 375-376). While a negative result, this observation may also be consistent with differences regarding the assembly of NUPs in or near PD vs the NPC. We fully agree with the reviewer that the proposed “PD pore complex” may be not identical to the NPC, and we also discussed that the NUPs seen at PD could represent sites of accumulation in the ER near PD.
Reviewer #3 (Public review):
Summary:
This manuscript presents a step towards testing the hypothesis that plasmodesmata have homology to nuclear pores. The similarities between the two structures have long been noted as both structures allow the transport of proteins and nucleic acids, and both structures are composed of curved membranes. The manuscript has identified nuclear pore proteins (NUPs) in plasmodesmal protein fractions and uses live imaging in a non-endogenous system and functional assays of a mutant to propose that this might be a bona fide association.
The conclusions the authors seek to draw are that: NUPs are present in plasmodesmal protein fractions; NUPs localise at plasmodesmata; NUPs might form a pore-gating complex at plasmodesmata, regulating non-specific (2xGFP) and specific (SHR) transport through plasmodesmata
The authors then use these conclusions to propose the possibility that phase separation mediates transport through plasmodesmata. If there is phase separation at plasmodesmata or a nuclear pore-like complex, it would revolutionise the community. However, this data is insufficient to act as a cornerstone for such a discovery.
Strengths:
The strength of the manuscript lies in the boldness and novelty of the idea.
Weaknesses:
The weaknesses lie in the lack of informative controls. The authors' own assessments of their data suggest they agree with this - in their abstract alone, they point out that the transport defects they observe might be off-target effects, and suggest there is a requirement in the future to determine whether the NUPs are bona fide PD components.
Across the proteomic and live imaging experiments, the conclusions could be stronger if they compared the NUP localisation and accumulation with ER proteins - the question of whether NUPs behave like other ER proteins is not addressed. As NUPs reside in the nuclear envelope, continuous with the ER, and the ER traverses plasmodesmata, a comparison between the NUPs and ER proteins would be extremely informative.
We agree with the comments of the reviewer. To assess whether NUPs show localization patterns that are similar to ER proteins, we transiently co-expressed NUP43-mVenus fusions with the mCherry-HDEL luminal ER marker in N.benthamiana. Comparison of the localization patterns reveals distinct patterns of NUP43-mVenus and mCherry-HDEL (see the new Figure5, new Figure 5-figure supplement 1). NUP43-mVenus appears to be associated with the ER, however restricted to subregions that partially overlay with aniline blue-labeled pit fields (new Figure 5, new Figure 5-figure supplement 1).
In the new version of the manuscript, we write (lines 209-214):
“We assessed whether NUP localization is distinct from ER localization in N. benthamiana leaves that heterologously co-expressed NUP43-mVenus and the ER luminal marker mCherry-HDEL. The localization patterns of NUP43-mVenus and of the mCherry-HDEL luminal ER marker were clearly distinct (Figure 5, Figure 5-figure supplement 1). NUP43-mVenus may be associated to the ER, however restricted to subregions of the ER, which partially overlay with aniline blue-labeled pit fields (Figure 5, Figure 5-figure supplement 1).”
Regarding the proteomic identification of NUPs in plasmodesmal fractions, the authors place significant weight on their own metric for PD enrichment, the PD score. As I understand it, this a metric derived from addition of two factors: a two component enrichment score that is the difference between intensity of peptides of a given protein in the PD fraction and cell wall fraction, added to the difference between intensity of peptides of a given protein in the PD fraction and total cell fraction, and a feature score that is a factor that describes representation of protein domains contained in said given protein in the plasmodesmal fraction relative to the representation of that domain in proteins in the whole proteome. The features chosen for analysis are not indicated, and the feature factor, as I understand it, is a score common to all proteins with a given feature. While each of the factors carries a measure of meaning and information, I do not understand how adding them is mathematically or biologically meaningful.
The feature score was defined based on PD proteome analysis previously described (Gombos et al., 2023). Features of known PD proteins were extracted and weighted against the entire Arabidopsis proteome. Structural features included Pfam domains PF00722 (GHL), PF06955 (XET_C), PF08372 (PRT_C), PF00335 (Tetraspanin), and PF00168 (C2 domain). Subcellular localization features included plasma membrane (PM), endoplasmic reticulum (ER), extracellular space (EX), and cell wall (CW). Functional features were assigned according to MapMan categories bin 10, 15, 26, and 30. To clarify the approach, we added a more detailed explanation to the feature score in the Methods of the revised manuscript.
We agree with the reviewer that experimental values and feature factors represent two distinct, independent parameters. The PD score aims to identify proteins that are not only experimentally enriched in the plasmodesmal fraction but also share structural features characteristic of bona fide plasmodesmata-associated proteins, reducing the number of false positive candidates driven by either parameter alone in PD proteome lists. From a mathematical standpoint, we combined the two normalized factors in the PD score by summation, treating them as contributing equally to a protein’s PD association tendency.
Conclusion:
The conclusions of the study are not fully supported in the absence of ER controls. Of note, the imaging is ambiguous because the proteins do not show a discrete plasmodesmal association. This is a localisation reminiscent of cortical ER association and needs to be further investigated to determine whether it is a true and specific plasmodesmal association.
We agree with the reviewer’s comments. In the revised version of the manuscript, we have now included a comparison of the NUP43-mVenus localization with that of the mCherry-HDEL luminal ER marker, which reveals distinct localization patterns (see new Figure5, new Figure 5-figure supplement 1). NUP43-mVenus may be associated with the ER; however, NUP43 is restricted to subregions of the ER, which partially overlay with aniline blue-labeled pit fields (new Figure 5, new Figure 5-figure supplement 1). Whether NUP localization is distinct from cortical ER requires further investigation.
The conclusions drawn from Figure 1, Figure Supplement 4 are confusing. The text describing this data says that "NUPs were enriched in cell wall and PD fractions compared to total cell extract, while the abundance of other nuclear envelope proteins was unaffected by the PD purification and showed no enrichment in PD fractions". However, the data show that there is no difference in the normalised protein intensity for the NUPs across TC, CW, and PD fractions. The only sample that shows enrichment in PDs is the PDLP/MCTPs.
To address this point, we rephrased the text (line 146-152). Among all NUPs identified in our PD proteome, 75% were more abundant in PD fractions (Figure 2-figure supplement 2), exceeding the proportions observed in TC (60%) and CW (~50%) fractions. In contrast, other nuclear proteins such as nuclear envelope proteins, nucleolar proteins, or histones showed PD intensities that fell within or overlapped the ranges observed in TC or CW. The native abundance of NUPs was lower compared to that of proteins from other compartments, which may explain why the enrichment significance was not statistically significant (p = 0.24 for PD vs. TC). By comparison, the corresponding p-values for other nuclear compartment proteins were higher, ranging from 0.5 to 0.9.
Regarding the possibility that there is a pore-gating complex at plasmodesmata. If NUPs are specifically located at plasmodesmata, this is a strong hypothesis. The authors approach this functionally by assaying for protein and dye movement through plasmodesmata in the cpr5 mutants. These experiments suggest that cpr5 mutants have reduced transport through plasmodesmata for both proteins, but not for a smaller dye. They infer that the latter finding suggests that the cpr5 mutant has no alterations in plasmodesmal number, but this is completely unsupported - in their introduction, the authors identify how PD structure can modify transport capacity, so there are many technical and biological phenomena that could explain these data.
We wrote in the manuscript: “The cpr5 mutants showed no detectable defect in small molecule transport indicative of WT-like PD density and preservation of the capability to mediate small molecule transport as shown by ‘Drop-ANd-See’ trans-leaf diffusion assays.”
Indeed, we did not study PD density by e.g. quantification of a PD-marker fluorescence. Theoretically, PD density might be changed and permeabilities adjusted by unknown mechanisms to allow for WT-like small molecule transport. Strikingly, we observed transport differences for larger cargo. As we cannot exclude potential changes in PD density, we have rewritten and deleted the conclusion on PD density and now write: “The cpr5 mutants showed no detectable defect in small molecule transport indicative of preservation of the capability to mediate small molecule transport as shown by ‘Drop-ANd-See’ trans-leaf diffusion assays”. (Lines 310-312)
I note for their DANS assays that the diffusion of dye from ad- to abaxial surface varies in the path followed (indicated by the asymmetry of the surfaces) and is not consistent within a leaf, let alone between leaves. This presents challenges in quantification and data interpretation that have not been addressed, and so the data cannot be confidently concluded to be an indicator of a different phenomenon rather than a less sensitive measure of the same.
Indeed, in our hands, the spread of the small molecule dye did not proceed radially and was very often asymmetrical. Therefore, we quantified the fluorescent area by identifying pixels with fluorescence above a threshold, instead of determining a diameter of the fluorescent area. We describe the analysis in the figure legend and briefly mention it in the method section.
“Fluorescent areas on the abaxial side were identified using auto threshold and Fiji YEN-algorithm with user modifications. The same threshold setting was used for the adaxial side. The extent of dye diffusion was quantified by the ratio between the areal spread of fluorescence on the abaxial side and the areal spread of fluorescence on the adaxial side.” (Figure 7)
Furthermore, to avoid any positional artifacts in the comparison between different plants and genotypes, we only assessed the 4th leaf and 24 hours later the 5th leaf with the same labelling position on the leaf.
Further, as the authors themselves acknowledge, altered protein movement might also arise from an off-target developmental phenotype. Many proteins have been shown to have no association with plasmodesmata but an indirect effect on their function. This hasn't been investigated and so cannot be ruled out.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
This is a really interesting hypothesis, but the support is incomplete.
(1) P. 5 "Although the single insertion Arabidopsis lines tested here should have FG-NUP62-GFP levels closer to native conditions than the heterologous overexpression of FG-NUP62-mVenus in N. benthamiana, it cannot be excluded that the levels in tested lines are still higher than the native levels, or that the fluorescence protein fused to the NUP affects localization." I appreciate the authors' cautious interpretation of their results, but they could exclude both of these possibilities. The first is relatively easy: test the expression level of the transgene compared to endogenous NUP expression; although transcript and protein levels are not tightly correlated, this can give some estimate of whether the transgene is overexpressed. The second would be to conduct complementation assays of a knockout mutant. I understand that this would be difficult if nup mutants are lethal, but it is pretty common practice to transform heterozygotes and isolate homozygotes expressing fluorescent protein to conduct complementation assays. Anyhow, there is a defect in the cpr5 mutants that the authors could assess in complementation assays. Alternatively, the authors could use biochemical approaches to determine whether FP-tagged NUPs are incorporated into nuclear pore complexes. These three experiments, even for only one NUP, would provide compelling evidence that the authors are localizing a functional NUP fusion protein at near-native expression levels. This is essential to support their speculation that NUPs play a biological role in PD.
Thank you for these three important recommendations: the quantification of NUP FP expression, the complementation of a mutant phenotype with NUP FP expression, and the assessment whether NUP FPs are incorporated into the NPC.
First, to evaluate the abundance of NUP62-GFP fusion protein relative to untransformed controls, we quantified the abundance levels of NUP62 in three independent transgenic fluorescent WT/NUP62p:NUP62-GFP Arabidopsis lines and in Arabidopsis WT using mass spectrometry (new Figure 3F). The new data indicate that there is no significant increase in NUP protein amounts in the lines expressing the fusion construct relative to wild type.
We now write in the revised manuscript (line 200-205):
“NUP62 protein abundance in two-week-old cotyledons of the stable NUP62p:NUP62-GFP transformants was not statistically different to NUP62 protein levels in WT (Figure 3F). Notably, the punctate fluorescence at the cell periphery, encompassing both PD-associated and non-PD-associated localization, were not detectable or absent in roots and young leaves of four-day-old seedlings (Figure 3D). However, it cannot be excluded that the GFP fusion impacts NUP62 localization.” We provide a new Method section for the mass spec analysis of the cotyledons in lines 582-590.
Second, to tested whether a NUP fusion is functional we assessed whether CPR5-mCitrine can complement the cpr5-1 mutant phenotype in complementation lines. We generated two new independent transgenic Arabidopsis lines stably expressing CPR5-mCitrine under control of its own promoter in the cpr5-1 mutant background (cpr5-1/CPR5p:CPR5-mCitrine). The roots were significantly longer in the two independent transgenic cpr5-1/CPR5p:CPR5-mCitrine Arabidopsis lines compared to the cpr5-1 mutant, and four-week-old plants showed a more WT-like growth phenotype (new Figure 7-figure supplement 1, G–I). However, we could not detect fluorescence in the 10-14 day old seedlings, which could be due to a variety of reasons, such as cleavage of the FP and degradation of the FP without accumulating elsewhere in the cells.
In the new manuscript we write in lines 275-283:
“To assess whether the CPR5-mCitrine fusion protein is functional in Arabidopsis, we tested whether CPR5p:CPR5-mCitrine (including all introns) expression in the cpr5-1 mutant background results in a rescue of the severe growth phenotype of the cpr5-1 loss-of-function mutant (Bowling et al., 1997). Indeed, roots were significantly longer in the two independent transgenic cpr5-1/CPR5p:CPR5-mCitrine Arabidopsis lines compared to the cpr5-1 mutant, and four-week-old plants showed a more WT-like growth phenotype (Figure 7-figure supplement 1, G–I). However, we could not detect fluorescence in 10-14 day old seedlings, which could be due to a variety of reasons, such as cleavage of the FP and degradation of the FP without accumulating elsewhere in the cells. The lack of fluorescence in the transgenic lines requires further investigation.”
Third, to assess whether NUP FP fusions are also detectable specifically in nuclei, we have provided example images for potential nuclear localization of NUP62-GFP in the stable Arabidopsis line (Figure 3C), and for AtGP210-mVenus, AtNUP98b-mVenus, AtCPR5-mCitrine, and At NUP43-mCitrine in transient expression experiments in N. benthamiana (Figure 3-figure supplement 1).
(2) The rationale for experiments was sometimes unclear. For example, why study Physcomitrium NUPs, then switch to Arabidopsis? Why use heterologous overexpression lines for SIM, rather than the stable Arabidopsis line for NUP62-GFP?
Our initial work focused on the PD proteome in Physcomitrium patens. We had identified NUPs in PD-enriched fractions of the moss (Gombos et al., 2023). To evaluate whether this was a specific feature of the moss, or a technical artifact of PD enrichment in moss extracts, we extended the study to Arabidopsis thaliana and subsequently focused on the higher plant. The text in the manuscript reflects this flow.
The NUP62-GFP stable transgenic Arabidopsis line was generated after the SIM experiments with CPR5-mCitrine. We plan to follow the suggestion of the reviewer to perform SIM experiments with the stable Arabidopsis NUP62p:NUP62-GFP lines.
(3) The organization of the figures was confusing. Why present transient Physco NUP localization, and also Arabidopsis proteomics in Figure 1? Why split the results on transient localization of Arabidopsis NUPs in benth across Figures 2 & 3?
We reorganized the Figures and created a separate proteome main figure (now Figure 2 with 2 figure supplements). We classified Arabidopsis NUPs in FG-NUPs and structural NUPs. Thus, we present the data also in two separate Figures: Figure 3 and supplements, dedicated to FG-NUPs, and Figure 4, dedicated to structural NUPs. According to the NPC, FG-NUPs play a direct role in transport facilitation, setting them apart from the structural NUPs.
(4) Why are several NUPs localized to the interior of the nucleus and not restricted to the nuclear membrane (e.g., Figure 1 Sup 1 top two rows, Figure 2)? How does this unusual nuclear localization alter the authors' interpretation of their results?
We observed that the transmembrane NUPs tested localized to the nuclear envelope and not to the nucleoplasm (see Figure 3-figure supplement 1 for example AtGP210 and AtCPR5). We found several soluble NUPs to also localize to the nucleoplasm (PpNUP98.1, PpNUP62, AtNUP62, AtHOS1). Previous studies had reported that several FG NUPs (i.e. NUP98a/b or NUP62) and Y-complex NUPs (i.e. HOS1, NUP96, and NUP107) also localized in the nucleoplasm rather than specifically to the nuclear envelope when expressed as fusion proteins (Chen et al., 2023; Gallemí et al., 2016; Huang et al., 2024; Lazaro et al., 2012). Of note, for NUP98a, Gallemi and colleagues (2016) discussed the localization to the nucleoplasm as confirmation that, like vertebrate NUP98, Arabidopsis NUP98a is a dynamic NUP rather than just a key structural element of the NPC. HOS1 was reported to interact with ICE1, CO, FVE, and HDA6 in the nucleoplasm (Dong et al., 2006; Jung et al., 2012; Lazaro et al., 2012), indicating that HOS1 might dynamically shuttle between the nuclear pore and nucleoplasm, which could also explain the observed nucleoplasmic localization. In Drosophila, the FG-NUPs NUP98, NUP62, and NUP50 localized in the NPC, and also in the nucleoplasm and interacted with genes (Kalverda et al., 2010). The nucleoplasmic localization could thus have a functional relevance. Yet we cannot rule out, whether soluble NUPs mislocalize in overexpression conditions as we state multiple times in the manuscript.
(5) Figure legends are insufficiently detailed. Figure legends should be sufficiently detailed to explain the figure without consulting the main text. For example,
(a) Figure 1A, 3C don't describe the cell type or even the organism that is being imaged. Are Physco proteins expressed in Physco? Arabidopsis? Benth? Leaves?
We added the missing information including cell types and organism.
(b) In Figure 1 Supplement 3, many abbreviations are not defined (HC, MC, etc).
We now define the abbreviations in the figure legend.
(c) In Figure 2B, the legend says "At least 15 images from 3 biological replicates were analyzed for each NUP", but there are MANY more than 15 datapoints in Figure 2B. What do the points represent?
We obtained at least three independent replicates for each data set we show here. We analyzed 15 ROIs derived from three biological replicates of AtNUP50b. In the other cases, a larger number of experiments was performed resulting in more ROIs being analyzed.
(d) For all microscopy images, are they single images or reconstructions (e.g., maximum projections)?
We now specify single confocal optical section or maximum projections.
Reviewer #2 (Recommendations for the authors):
(1) PD index shall be measured for data in Figures 3D and 3E.
To address this question, we have performed PD index quantification for the data in Figures 4D and 4E and added the information to the main text (lines 178-184):
“In leaves transiently expressing NUP43-mCitrine or CPR5-FP fusions, the fluorescence intensity correlated with the estradiol concentration used, with decreased fluorescence intensity for samples where 2µM estradiol was applied versus the intensity in samples exposed to 20µM estradiol (Figure 4 D,E). Notably, the fluorescence ratio between periphery and nucleus did not differ significantly after expression induction by 2 µM compared to 20 µM β-estradiol (Figure 4F) and PD localization was not eliminated (example for localization of NUP43-mCitrine in Figure 4C; PD index(NUP43, 2µM) = 1.42, PD index(CPR5, 2µM) = 1.40).”
(2) The expression level of the native promoter-driven Nup62-GFP shall be measured and compared with the native level using RT-qPCR. Even if this turns out to be an overexpression line, it would still be useful to support the hypothesis.
To evaluate the level of NUP62-GFP fusion protein relative to untransformed controls, we quantified the levels of NUP62 in three independent transgenic fluorescent WT/NUP62p:NUP62-GFP Arabidopsis lines and in Arabidopsis WT using mass spectrometry (new Figure 3F). The new data indicate that there is no significant increase in NUP protein amounts in the lines expressing the fusion construct relative to WT.
We now write in the revised manuscript (line 200-205):
“NUP62 protein abundance in two-week-old cotyledons of the stable NUP62p:NUP62-GFP transformants was not statistically different to NUP62 protein levels in WT (Figure 3F). Notably, the punctate fluorescence at the cell periphery, encompassing both PD-associated and non-PD-associated localization, were not detectable or absent in roots and young leaves of four-day-old seedlings (Figure 3D). However, it cannot be excluded that the GFP fusion impacts NUP62 localization.“ We provide a new Method section for the mass spec analysis of the cotyledons in lines 582-590.
(3) Last sentence in the introduction: Nup136 has been considered as the plant homolog of Nup153.
In the manuscript we wrote:
“The majority of the FG-NUPs were conserved, with only three FG-NUPs lost in the green lineage (NUP153, POM121, NUP358).“
As the FG-NUP136 is the plant homolog to NUP153, we now write (lines 90-92):
“The majority of the FG-NUPs were conserved, with two FG-NUPs apparently lost in the green lineage (POM121, NUP358).“
Reviewer #3 (Recommendations for the authors):
(1) Generally, my interpretation of the images in this manuscript is that many of the localisations are not clean and discrete plasmodesmal associations and are rather more consistent with cortical ER association. As the ER is a component of plasmodesmata, the ER is continuous with the nuclear envelope, and the authors also predict and show ER localisation of one of their key NUPs, CPR5 in Figure 4B. This is not necessarily surprising. However, what becomes essential is that the authors need to determine whether NUPs behave any differently from other ER proteins. To that end, I think co-localisations with ER-located proteins would be helpful in interpreting these ambiguous localisations.
To address this point, we performed additional colocalization experiments using an ER marker. In the new version of the manuscript, we now include a comparison of the NUP43-mVenus localization with that of the mCherry-HDEL luminal ER marker, which reveals distinct localization patterns (see new Figure5, new Figure 5-figure supplement 5-1). NUP43-mVenus may be associated with the ER; however, NUP43 is restricted to subregions of the ER, which partially overlay with aniline blue-labeled pit fields (new Figure 5, new Figure 5-figure supplement 1).
(2) The super-resolution images of CPR5 show some clear structures peripheral to plasmodesmata. However, again, I would like to see what an ER protein looks like at this location, as the ER feeds into the plasmodesmata. Is this a specific structure or a general feature of the localisation of an ER protein?
Since mCherry-HDEL (see above) did not show a similar localization or enrichment at PD, we did not perfrom SIM analyses with the marker.
(3) The authors support their use of the PD score using validated PD proteins as the positive control and contaminants from mitochondria and other organelles as the negative control. No mention is made of where ER proteins are classified. The ER passes through plasmodesmata but might also represent a contaminating pool. As NUPs reside in the nuclear envelope, continuous with the ER, a comparison between the NUPs and ER proteins would be extremely informative.
To evaluate a potential enrichment of ER proteins in the plasmodesmata fraction, we analyzed ER protein enrichment and added the new data as a graph in Figure 2-figure supplement 2. ER-resident proteins did not show significant enrichment in the cell wall fraction relative to total cell extract, while displaying a slight but consistent enrichment in the plasmodesmata fraction. Notably, NUPs enrichment was higher in both cell wall fraction and plasmodesmata fraction compared to transmembrane ER-resident proteins. While ER membrane co-purification cannot be entirely excluded, the enrichment of NUPs in the plasmodesmata fraction may not be due to desmotubule membrane carryover alone. The analysis was incorporated into the revised manuscript (lines 152-155).
(4) Regarding the data analysis and use of the Kruskal-Wallis test, the Kruskal-Wallis test tests differences in the distribution of the data, not differences in the mean or median values. In many cases, it can be inferred that the median changes when the data distribution does, but this is not as confident an inference for means. There are other methods available to compare the means of such datasets.
We used the Kruskal–Wallis test for statistical comparison of more than two nonparametric data sets. However, we did not state in the manuscript that we performed a Dunns´ test for the post hoc pairwise comparison after the Kruskal-Wallis test. In the revised manuscript, we added this information in the Methods, Results and Figure legends. For the bombardment experiment data, we now added mean bootstrapping, as used previously in this context (Johnston and Faulkner, 2021). Mean bootstrap analysis for the bombardment data set was performed with n=5000 resamples and we provide the p values and confidence intervals in the figure legend (Figure 7 B):
“Mean fluorescent cell counts: n(WT) = 2.67, n(cpr5-T3) = 1.59, n(cpr5-1) = 0.68; median fluorescence cell counts: n(WT) = 2, n(cpr5-1) = 0, n(cpr5-T3) = 1. Based on Bonferroni-corrected Dunn´s test for pairwise comparison after Kruskal-Wallis test: a indicates significant difference to WT with p(cpr5-1) < 10-15; b indicates significant difference to WT with p(cpr5-T3) = 0.0004; c indicates p(cpr5-1 vs. cpr5-T3) = 0.0002. Mean bootstrap analysis according to (Johnston and Faulkner, 2021) with 95% confidence interval (CI) and bootstrap resampling of B = 5000: CIWT vs. cpr5-1 [1 x 10-5 , 0.001], p(cpr5-1) = 0002 ; CIWT vs. cpr5-T3 [1 x 10-5 , 0.001], p(cpr5-T3) = 0.0002; CIcpr5-1 vs. cpr5-T3, p(cpr5-1 vs. cpr5-T3) = 0.0002 [1 x 10-5 , 0.001].“
(5) The comments that estradiol induction prevents over-expression, or allows for controlled expression, are not experimentally supported or widely established outside this manuscript. I suggest they tone this claim down.
As outlined above the reduction in estradiol concentration lead to reduced fluorescence intensity for the NUP-FP fusions as one would expect; here notably with a reduction at both nuclei and periphery (Figure 4C-F). The system has been used previously in the Simon lab, from whom we obtained the constructs. There is substantial literature regarding the use of the b-estradiol-inducible XVE promoter system, specifically for b-estradiol dose-dependent gene expression in N. benthamiana leaves (Bashandy et al., 2015; Bleckmann et al., 2010; Borghi, 2010; Schlücking et al., 2013). We assessed the dependence of localization on expression levels by studying NUP localization with a lower estradiol concentration for induction and shortened incubation time. Interestingly, despite the apparent lower expression, we still find NUPs at PD.
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