Chemical screen of Arabidopsis zygote and proteomics in tobacco BY-2 cells identify general plant cell division inhibitors

  1. Yusuke Kimata
  2. Moé Yamada
  3. Takashi Murata
  4. Keiko Kuwata
  5. Ayato Sato
  6. Takamasa Suzuki
  7. Daisuke Kurihara
  8. Mitsuyasu Hasebe
  9. Tetsuya Higashiyama
  10. Minako Ueda

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Abstract

Cell division is essential for growth and development and involves events such as spindle assembly, chromosome separation, and cell plate formation. In plants, the tools used to control these events at the desired time are still poor because the genetic approach is ineffective owing to a high redundancy and lethality, as well as harmful side effects. Accordingly, we screened cell division-affecting compounds, with a focus on Arabidopsis thaliana zygotes, which individually develop in maternal ovules; the cell division was reliably traceable without time-lapse observations. We then identified the target events of the identified compounds using tobacco BY-2 cells for live-cell imaging and proteomics. As a result, we isolated two compounds, PD-180970 and PP2. PD-180970 disrupts microtubule (MT) organization and, thus, nuclear separation, presumably by inhibiting MT-associated proteins (MAP70). PP2 affected class II Kinesin-12 localization at the phragmoplast emerging site and impaired cytokinesis. Moreover, neither chemical caused irreversible damage to viability but they were effective in multiple plant species such as cucumber ( Cucumis sativus ) and moss ( Physcomitrium patens ). We propose that the combination of chemical screening based on Arabidopsis zygotes and target event specification focusing on tobacco BY-2 cells can be used to effectively identify novel tools and transiently control specific cell division events that are conserved in diverse plant species.

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    Reply to the reviewers

    Revision Plan

    1. General Statements

    We really appreciate the positive comments and suggestions of the reviewers on our submitted manuscript. We think we will be able to solve the issues inquired by reviewers by adding new data and revising the phrases as detailed below.

    2. Description of the planned revisions

    Reviewer #1:

    Major comments

    Localization analysis of a transiently expressed MAP70 transgene with inactivating phosphosite mutations would be important to see whether the identified conserved phosphosites are relevant for MAP70 interaction with MTs. This experiment could be performed rapidly using transient expression in BY-2 cells.

    We agree on the importance of this analysis. Therefore we are currently preparing fluorescent markers of Nt-MAP70-2-like and its phospho-blocked (Ala) version to coexpress with MT and nuclear markers in BY-2 cells. We estimate that we need three more months to complete this experimsnt.

    The authors propose that PP2 blocks phragmoplast formation by preventing phosphorylation of class II Kinesin-12 proteins. In support, authors show that PP2 treatment correlates with a decrease in KIN12A phosphopeptide count (not fully abolished) and its failure to localize to emerging phragmoplasts in BY-2 cells and Physcomitrium. As class II Kinesis-12 proteins have been previously implicated in phragmoplast assembly this is a fairly reasonable hypothesis, but would benefit from the analysis of transgenic KIN12A variants carrying inactivating (A) or potentially activating (D/E) phosphosite mutations. Is loss of phosphorylation sufficient to prevent phragmoplast localization? Can an activated variant rescue PP2-induced KIN12A localization and cell division defects? As above, using transient expression in BY-2 cells would be a fast approach to tackle these questions.

    We are currently preparing fluorescent markers of phospho-blocked (Ala) and phospho-mimic (Asp) versions of KIN12A (PAKRP1) to coexpress with MT and nuclear markers in BY-2 cells. We will check whether they localize to phragmoplast and also test PP2 effects. We would need three more months to complete these analyses.

    Reviewer #2:

    Major comments

    • The manuscript would strongly benefit from being revised by a native english speaker. There are many unusual or awkward formulation, in particular in the abstract.

    We apologize for unnatural sentences. After adding new data and correcting the manuscript, we will ask a native english speaker to revise it.

    Reviewer #3:

    Major comments

    The major concern is lack of evidence to connect MAP70 and MT disruption upon treatment with PD-180970, in contrast to PP2, which was shown to affect localization of Kinesin-12. I wonder if authors could use taxol to stabilize MTs, then observe the localization of MAP70 with application of PD-180970?

    As we responded to reviewer 1, we are preparing the fluorescent marker of Nt-MAP70-2-like to coexpress with MT and nuclear markers in BY-2 cells. By using this multi-color marker, we will test whether PD-180970 affects the localization of MAP70 on MTs, also using taxol. However, in our experiene, taxol is not a very effective inhibitor and may not work in our transient expression system in BY-2 cells. In that case, we will analyze whether phospho-mimic (Asp) version can prevent MT disruption in the presence of PD-180970 to assess the relation of PD-180970, MAP70 and MT disruption.

    I have another concern on the action of PD-180970. PD-180970 appears to affect ubiquitously indispensable proteins for MTs. If PD-180970 disrupt MT by inhibiting phosphorylation of some MAPs, it must need time for turnover of proteins phosphorylated before PD-180970 was applied. In the proteomics experiment, author treated the cells with the compounds for 8-9 hr. On the other hand, in BY-2 cells, PD-18970 disrupted MTs only 30 min after application of PD-180970. I wonder if proteins were replaced during the 30 min. Could authors examine how long it takes to affect interphase MTs? If PD-180970 disrupts MTs in a 5-10 min like oryzalin, it is unlikely that inhibition of phosphorylation of proteins like MAP70 caused MT disruption. Rather, it may inhibit some proteins that have activity to disrupt microtubules but are usually inactivated by phosphorylation or inhibit something directly without phosphorylation.

    We agree that there is no evidence that PD-180970 disrupts MTs by inhibiting phosphorylation of MAP70. In our live-imaging system, in which reagents are added to liquid cultivation medium, the time from the reagent application to the arrival to each cell varies. Therefore, in order to accurately measure the time required for the inhibitor to take effect, it is necessary to design a new assay system, such as using fluorescent dyes to monitor the reagent's diffusion. In addition, since some reactions mediated by protein phosphorylation occur rapidly, minute-order observations might not be sufficient. Therefore, as an alternative strategy to assess the direct involvement of MAP70 phosphorylation on MT stabilization, we will examine whether PD-180970 induces MT disruption using strains expressing the phospho-blocked (Ala) and phospho-mimic (Asp) versions of MAP70 described above.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer #1:

    Minor comments

    The authors identified the analogs PD-166326 and PP1 as potent inhibitors of cell division. For completeness, it would be interesting to include a description of these arrest phenotypes and how they compare with that of PD180870 or PP2.

    We have added the effects of all tested compounds on Arabidopsis embryos in Fig. S3C and Table S1. Based on this data and the results of tobacco BY-2 cells, we have compared the effects of PD-166326 and PD180870, and PP1 and PP2 in Results.

    Although there are two more obvious candidates in the phosphoproteome datasets on which the authors focus on, there is very little discussion on whether the other top hits and whether they might be involved in cell division. On a related note, there is no discussion on the specificity of these compounds and the likelihood of phenotypes unrelated to cell division.

    We have added the information of “Similar proteins in Arabidopsis” and “Description and putative functions” for all identified candidates for PD-180970 and PP2 in Table S2 and S3, respectively. With referring this information, we have added the sections to describe the possible contributions of these candidates on MT organization and phragmoplast formation in Results. In addition, we have described the specificity of these compounds and the phenotypes unrelated to cell division in the section for the results of Arabidopsis roots (Fig. S2A).

    1st results section:

    "...developed into the globular stage without causing morphological defects..."

    Should omit the word "causing" or replace with "any/detectable"

    We have omitted the word "causing".

    Reviewer #2:

    Even if the identification of the kinase(s) targeted by these two compounds is missing, the characterisation of at least two downstream effectors of these elusive kinase(s) inhibited by PD-180970 and PP2 is an important step forward. I would recommend to this point make very clear in the writing (e.g. already in the abstract). Upon a superficial reading, the reader could assume that MAP70s and PAKRP1s are the direct molecular targets of these compounds.

    We appreciate the very positive comments. To clarify this point, in addition to the following responses to each suggestion, we have changed the last sentense of the abstract to “These properties make PD-180970 and PP2 useful tools for transiently controlling plant cell division at key manipulation nodes that are conserved in diverse plant species”.

    Major comments

    • I would modify the title to shift the emphasis from the methodology to the biological targets identified.

    We have changed the title to “Identification of novel compounds inhibiting microtubule organization and phragmoplast formation in diverse plant species”.

    • Concerning MAP70s the authors claim that there is little functional data about this family. Yet, a recent paper (https://www.science.org/doi/10.1126/sciadv.abm4974) identifies MAP70-5 as necessary for the proper organisation of CMTs in the endodermis and its ability to actively remodel to accommodate emergence of the lateral root primordium in Arabidopsis thaliana. This could provide a functional context to test several of the predictions that the authors list in the discussion.

    We have referred this paper in Results and Discussion, as “MAP70-5 was reported to increase MT length* in vitro *and to reorganize cortical MTs to alter the endodermal cell shape for lateral root initiation, suggesting that MAP70-5 mediates dynamic change of MT arrays”.

    Minor comments

    • The narrative would be improved by moving the section "PD-180970 and PP2 do not irreversibly damage viability" before the phosphoproteomic section.

    We have moved the “irreversibly” section to before the “phosphoproteomics” section.

    Reviewer #3:

    Minor comments

    In supplemental data, authors show only 12 or 14 candidates of the target. It is interesting how other MAPs including homologues of MAP70 and Kiesnin-12 in BY-2 cells were scored in the phospho-proteomics assay. I suggest authors show longer lists of proteomics including other MAPs. It would be valuable information for the research community.

    We apologize for not providing the complete dataset. We have added Dataset S1 of total protein sequences that we predicted from published RNA-sea data of BY-2 cells, and all identified proteins of phosphoproteomics assay for PD-180970 and PP2 in Datasets S2 and S3, respectively. We have moved the lists of top candidates to Tables S2 and S3.

    In Abstract, authors should mention that the two compounds reduced phosphorylation level of diverse proteins including MAP70 and Kinesin-12. This is very important results and, otherwise, it may cause misunderstanding of the activity of the compounds. In addition to this, it is better to rephrase the following sentence. "presumably by inhibiting MT-associated proteins (MAP70)" with "presumably by inhibiting phosphorylation of MT-associated proteins (MAP70)."

    To avoid such a misunderstanding, we have changed the descriptions in Abstract to “Phosphoproteomic analysis showed that these compounds reduced phosphorylation level of diverse proteins. In particular, PD-180970 inhibited phosphorylation of the conserved serine residues in MT-associated proteins (MAP70). PP2 significantly reduced the phosphorylation of class II Kinesin-12, and impaired its localization at the phragmoplast emerging site”. Due to this change, the suggested sentence was eliminated. Also in Discussion, we have mentioned the reduction of phosphorylation of various proteins by stating, "we found that PD-180970 and PP2 reduced the phosphorylation levels of diverse proteins. These parts may be further modified depending on the results of the phospho-blocked (Ala) and phospho-mimic (Asp) analyses.

    Page7 line 1st. it would be better to insert "of MAP70 family" after "in the conserved MT-binding domain" because the MT binding domains are unique to the MAP70 family. I could not understand why this is " (2nd line) consistent with PD-18970 severely disrupting all the tested MT structure". At current stage, there is no evidence that dephosphorylation of MAP70 caused the microtubule disruption. I suggest authors remove the sentence (", which was~MT structures").

    We agreed on both points and have corrected them as the reviewer suggested.

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript by Kimata and colleagues describes identification of compounds that inhibit microtubule organization and cell division in plants. In this manuscript, authors screened two chemical libraries and successfully found two compounds, PD-180970 and PP2", as potent inhibitors of cell division. Because the two compounds act as kinase inhibitor in animal cells, authors analyzed their effects on phosphorylation by using BY-2 suspension cells. Among the affected proteins, authors focused on two microtubule-related proteins, MAP70 and Kinesin-12. Authors showed that PD-180970 disrupt all structures of microtubules in BY-2 cells. All members of Arabidopsis MAP70 shared the target residues, although Arabidopsis map70 mutants grew normally. PP2 abolished the localization of Kinesin-12 to phragmoplasts, and inhibit the cell plate formation. Both compounds worked in other plant species including moss. I think highly of the unique screening system using Arabidopsis zygote and BY-2 cells developed by the authors. Although the direct targets and specificity of the compounds are still to be determined, I think the two compounds should become powerful tools in plant cell biology in future. Generally, the manuscript is well written, and the data are of high quality. I have, however, several suggestions as below.

    Major

    The major concern is lack of evidence to connect MAP70 and MT disruption upon treatment with PD-180970, in contrast to PP2, which was shown to affect localization of Kinesin-12. I wonder if authors could use taxol to stabilize MTs, then observe the localization of MAP70 with application of PD-180970?

    I have another concern on the action of PD-180970. PD-180970 appears to affect ubiquitously indispensable proteins for MTs. If PD-180970 disrupt MT by inhibiting phosphorylation of some MAPs, it must need time for turnover of proteins phosphorylated before PD-180970 was applied. In the proteomics experiment, author treated the cells with the compounds for 8-9 hr. On the other hand, in BY-2 cells, PD-18970 disrupted MTs only 30 min after application of PD-180970. I wonder if proteins were replaced during the 30 min. Could authors examine how long it takes to affect interphase MTs? If PD-180970 disrupts MTs in a 5-10 min like oryzalin, it is unlikely that inhibition of phosphorylation of proteins like MAP70 caused MT disruption. Rather, it may inhibit some proteins that have activity to disrupt microtubules but are usually inactivated by phosphorylation or inhibit something directly without phosphorylation.

    Minor

    In supplemental data, authors show only 12 or 14 candidates of the target. It is interesting how other MAPs including homologues of MAP70 and Kiesnin-12 in BY-2 cells were scored in the phospho-proteomics assay. I suggest authors show longer lists of proteomics including other MAPs. It would be valuable information for the research community.

    In Abstract, authors should mention that the two compounds reduced phosphorylation level of diverse proteins including MAP70 and Kinesin-12. This is very important results and, otherwise, it may cause misunderstanding of the activity of the compounds. In addition to this, it is better to rephrase the following sentence. "presumably by inhibiting MT-associated proteins (MAP70)" with "presumably by inhibiting phosphorylation of MT-associated proteins (MAP70)."

    Page7 line 1st. it would be better to insert "of MAP70 family" after "in the conserved MT-binding domain" because the MT binding domains are unique to the MAP70 family. I could not understand why this is " (2nd line) consistent with PD-18970 severely disrupting all the tested MT structure". At current stage, there is no evidence that dephosphorylation of MAP70 caused the microtubule disruption. I suggest authors remove the sentence (", which was~MT structures").

    Significance

    Redundancy of genes prevent researchers from exploring the genetic mechanisms of cell division. Time-specific manipulation of plant cell division by optogenetics or pharmacology has not been established. Identification of compounds that can specifically affect cell division is desired for further investigation of plat cell division. Although the direct targets and specificity of the compounds are still to be determined, I think the screening system and the two compounds identified by the authors should become powerful tools in plant cell biology in future. This work will influence not only plant biologists but also broad readership including cell/developmental biologists and chemical biologists.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Kimata et al. report on the identification and characterisation of two compounds inhibiting cell division in plants. Motivated by the need to circumvent genes function redundancy to study cell division in plants, the authors screened 170 biologically active compounds for inhibitors of the first highly stereotypical division of the Arabidopsis zygote. They identify two compounds PD-180970 and PP2 that very potently block this division. Monitoring the effect of these compounds on microtubules dynamics and using high resolution imaging on BY-2 cell cultures, they conclude that PD-180970 inhibits MT organization while PP2 inhibits phragmoplast formation. These two compounds have reversible effects and are active in several plants species ( Arabidopsis, Tobacco, Cucumber and Physcomitrella). Both compounds targets kinases in animal cells. To shed light on the molecular mechanism perturbed by these compounds, the authors performed a phospho-proteomic profiling of synchronised BY-2 cells upon treatment by either of this compounds, controlled by inactive analogs. They identify two proteins which phosphorylation is severely reduced by the compounds. PD-180970 reduces the phosphorylation of members of the MAP70 at three conserved S residues, two mapping in the microtubule binding domains. PP2 reduces phosphorylation of PAKRP1/KIN12A and PAKRP1L//KIN12B a pair of phragmoplasts-associated kinesins. The authors show that PP2 disrupts the phragmoplast-localisation of the both kinesins, phenocopying the effects of the double mutant pakrp1/pakrp1l and thus providing a likely molecular mechanisms for the effects of PP2.

    Overall the manuscript is solid, the experiments well executed and controlled and the results precisely. The conclusions are supported by the data and the manuscript is clearly structured.

    Even if the identification of the kinase(s) targeted by these two compounds is missing, the characterisation of at least two downstream effectors of these elusive kinase(s) inhibited by PD-180970 and PP2 is an important step forward. I would recommend to this point make very clear in the writing (e.g. already in the abstract). Upon a superficial reading, the reader could assume that MAP70s and PAKRP1s are the direct molecular targets of these compounds.

    Major comments:

    • I would modify the title to shift the emphasis from the methodology to the biological targets identified.
    • Concerning MAP70s the authors claim that there is little functional data about this family. Yet, a recent paper (https://www.science.org/doi/10.1126/sciadv.abm4974) identifies MAP70-5 as necessary for the proper organisation of CMTs in the endodermis and its ability to actively remodel to accommodate emergence of the lateral root primordium in Arabidopsis thaliana. This could provide a functional context to test several of the predictions that the authors list in the discussion.
    • The manuscript would strongly benefit from being revised by a native english speaker. There are many unusual or awkward formulation, in particular in the abstract.

    Minor comments:

    • The narrative would be improved by moving the section "PD-180970 and PP2 do not irreversibly damage viability" before the phosphoproteomic section.

    Significance

    Plant cell biologists interested in cell division and microtubules will find this pre-print enticing. The compounds identified will reveal useful tools to analyse cell division in plants and the manuscript provides a significant technical advance. Although my expertise does not lay in the field of chemical inhibitors of cell division, there are to my knowledge, no compounds that selectively inhibit phragmoplast growth like PP2. The manuscript paves the way for further studies such as genetic suppressor screens to identify the plant kinase(s) targeted by these compounds.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Kimata and colleagues describe the identification of two reversibly acting compounds that affect specific stages of cell division through a chemical screen in in vitro cultured Arabidopsis zygotes. They further characterize the effects of these compounds on cell division using advanced imaging techniques, and demonstrate that they perturb cell cycle progression in multiple plant species and systems, thus acting on conserved pathways. Finally, using phosphoproteomics and transgenic approaches in cultured plant cells the authors identified potential indirect targets of these compounds. The work described is very thorough and well presented, with conclusions supported by the data. In addition it provides information on two compounds that can be broadly applied in plant molecular research and demonstrate the feasibility of an in vitro ovule culture system for chemical screening in plants.

    Major comments

    Localization analysis of a transiently expressed MAP70 transgene with inactivating phosphosite mutations would be important to see whether the identified conserved phosphosites are relevant for MAP70 interaction with MTs. This experiment could be performed rapidly using transient expression in BY-2 cells.

    The authors propose that PP2 blocks phragmoplast formation by preventing phosphorylation of class II Kinesin-12 proteins. In support, authors show that PP2 treatment correlates with a decrease in KIN12A phosphopeptide count (not fully abolished) and its failure to localize to emerging phragmoplasts in BY-2 cells and Physcomitrium. As class II Kinesis-12 proteins have been previously implicated in phragmoplast assembly this is a fairly reasonable hypothesis, but would benefit from the analysis of transgenic KIN12A variants carrying inactivating (A) or potentially activating (D/E) phosphosite mutations. Is loss of phosphorylation sufficient to prevent phragmoplast localization? Can an activated variant rescue PP2-induced KIN12A localization and cell division defects? As above, using transient expression in BY-2 cells would be a fast approach to tackle these questions.

    Minor comments

    The authors identified the analogs PD-166326 and PP1 as potent inhibitors of cell division. For completeness, it would be interesting to include a description of these arrest phenotypes and how they compare with that of PD180870 or PP2.

    Although there are two more obvious candidates in the phosphoproteome datasets on which the authors focus on, there is very little discussion on whether the other top hits and whether they might be involved in cell division. On a related note, there is no discussion on the specificity of these compounds and the likelihood of phenotypes unrelated to cell division.

    1st results section: "...developed into the globular stage without causing morphological defects..." Should omit the word "causing" or replace with "any/detectable"

    Significance

    The work described in this manuscript identified and characterized two compounds that affect specific processes important for cell division in plant cells. Furthermore, the authors demonstrate the feasibility of an in vitro ovule culture system for chemical screening of specific processes in early plant embryos. The identified compounds are effective in multiple tissues in phylogenetically distant land plants and their effects are reversible. These compounds can be useful to, for example, manipulate the microtubule cytoskeleton, cell cycle, or ploidy in different plant models and different contexts, and more specifically to cell biologists studying the molecular mechanisms of cell division.

  5. Version published to 10.1101/2022.04.28.489799v1 on bioRxiv