Author response:
The following is the authors’ response to the original reviews.
eLife Assessment
This important study provides new insight into the regulation of cell organization and division in Trypanosoma brucei through the control of a kinesin motor protein by a polo-like kinase. The authors present solid evidence from rigorous biochemical and imaging analyses showing that phosphorylation modulates kinesin function and cellular organization. However, direct in vivo evidence that PLK phosphorylates kinesin-G is lacking.
We performed experiments to investigate the effect of PLK inhibition on the phosphorylation of KIN-G in vivo in trypanosome cells by immunoprecipitation and mass spectrometry. The new results showed that treatment of trypanosome cells with GW843682X, a potent TbPLK inhibitor validated previously in procyclic trypanosomes, reduced the phosphorylation levels on Thr301 and Ser569 of KIN-G by ~27% and 100%, respectively. The partial reduction in Thr301 phosphorylation after GW843682X treatment could be attributed to slower dephosphorylation of phosphorylated Thr301 after GW843682X was added to the cell culture. Nonetheless, these new results demonstrated that KIN-G is an in vivo substrate of PLK.
Public Reviews:
Reviewer #1 (Public review):
Summary:
This manuscript identifies the orphan kinesin KIN-G as a substrate of Polo-like kinase (TbPLK) in Trypanosoma brucei and demonstrates that phosphorylation of Thr301 inhibits KIN-G microtubule binding and disrupts its cellular function. Using a combination of in vitro kinase assays, phosphosite mapping, microtubule binding and gliding assays, and in vivo complementation with phosphomimetic and phosphodeficient mutants, the authors link TbPLK-mediated regulation of KIN-G to defects in centrin arm integrity, FAZ elongation, Golgi organization, flagellum positioning, and division plane placement. The study provides a mechanistic advance in understanding how TbPLK regulates centrin arm biogenesis and integrates KIN-G into the growing regulatory network controlling hook complex and FAZ assembly. Overall, the work is technically strong, internally consistent, and builds logically on previous studies from this group and others.
Strengths:
A major strength of the manuscript is the clear mechanistic link between phosphoryltion of Thr301 and loss of microtubule binding activity. The use of phosphomimetic (T301D) and phosphodeficient (T301A) mutants in an RNAi-rescue framework provides a clean and convincing demonstration of functional relevance in vivo. The integration of biochemical assays with detailed cell biological phenotyping (centrin arm length, FAZ elongation, basal body segregation, and cytokinesis markers) is particularly effective and makes the central conclusion robust. The observed phenotypic cascade from centrin arm defects to FAZ and division plane abnormalities is also well aligned with existing models of trypanosome morphogenesis.
Weaknesses:
My (more or less main) concern relates to the interpretation of the Golgi phenotype. The conclusion that phosphorylation of KIN-G "impairs Golgi biogenesis" is currently based on fluorescence microscopy using TbGRASP and Sec13 markers and on quantification of the number and distribution of Golgi/ERES puncta in binucleated cells. While these data convincingly demonstrate altered Golgi/ERES number and spatial organization, they do not distinguish between true defects in Golgi biogenesis or duplication and alternative possibilities such as fragmentation, vesiculation, or mislocalization of Golgi membranes. Given the central role of Golgi-centrin arm organization in the proposed model, ultrastructural analysis (for example, by EM or electron tomography) would greatly strengthen this aspect of the study by providing direct evidence for structural alterations of the Golgi and its association with the centrin arm and ERES. Such data would elevate this part of the manuscript from a descriptive fluorescence phenotype to a true structural cell biological insight. I appreciate that this experiment goes beyond the current dataset, but it would substantially enhance the mechanistic depth of the Golgi-related conclusions and strengthen the causal chain linking centrin arm defects to Golgi abnormalities. However, I have to confess, the inclusion of such data would make this reviewer particularly enthusiastic about the work. If this is not feasible, I would recommend tempering the wording of "Golgi biogenesis" to a more conservative description, such as altered Golgi organization or duplication, and explicitly acknowledging the limitations of fluorescence-based analysis for this conclusion.
Thanks for these very constructive comments, which are very well taken. We totally agree with this reviewer on these points. Since it is not feasible for us to perform EM or electron tomography, we have revised the manuscript to describe the effect of KIN-G phosphorylation on the Golgi as “altered Golgi duplication” rather than “Golgi biogenesis”. We also explicitly acknowledge the limitations of fluorescence-based analysis of the Golgi for this conclusion and suggest that further characterization with EM or electron tomography would allow one to reveal the potential structural alterations of the Golgi and its association with the centrin arm.
An additional conceptual point concerns the dual role of TbPLK in centrin arm regulation. TbPLK is known to promote centrin arm biogenesis through phosphorylation of TbCentrin2, yet in this study, TbPLK phosphorylation of KIN-G negatively regulates centrin arm assembly. This dual positive and negative regulatory role is intriguing but could be discussed more explicitly. The manuscript would benefit from a clearer conceptual framework addressing how phosphorylation of KIN-G might serve as a temporal or spatial switch to restrain KIN-G activity at specific stages of centrin arm assembly.
This is a great point. However, we are not sure whether the previous work on TbPLK phosphorylation of TbCentrin2 could lead to the conclusion that TbPLK promotes centrin arm biogenesis through phosphorylation of TbCentrin2. In the published work (de Graffenried et al., MBoC, 2013), trypanosome cells expressing the phospho-deficient mutant TbCentrin2-S54A only showed minor growth defects, exhibiting growth defects after 5 days (de Graffenried et al., MBoC, 2013). Cells expressing the phosphomimic mutant TbCentrin2S54D, however, showed very strong growth defects (de Graffenried et al., MBoC, 2013). The effects of TbPLK phosphorylation on TbCentrin2 appear to be quite similar to that of TbPLK phosphorylation on KIN-G, although the KIN-G-T301A mutant does not have growth defects (up to 5 days in our experiments). It appears that the primary role of TbPLK in regulating TbCentrin2 and KIN-G is negative regulation. Nonetheless, we have added more discussion on these regulatory roles of TbPLK in the revised manuscript.
Finally, a schematic model summarizing the proposed regulatory pathway from TbPLK phosphorylation of KIN-G to centrin arm assembly, FAZ elongation, division plane placement, and Golgi organization would aid the reader.
Thanks for this suggestion. We made a schematic model to summarize the roles of KIN-G and its regulation by TbPLK. This is included in Figure 8.
Reviewer #2 (Public review):
Summary:
The authors identify KIN-G as an in vitro substrate for phosphorylation by TbPLK and show that several of the in vitro P-ated sites, including T310, overlap with P-ation sites seen in live cells. The authors further show that PLK-mediated P-ation inhibits KIN-G binding to microtubules in vitro, as does a KIN-G-T301D mutant, and that expression of a KIN-G-T301D Phospho-mimic in T. brucei phenocopies KIN-G RNAi knockdowns, producing defects in cell division, morphogenesis of the centrin arm, FAZ and other cellular structures, as well as a misplaced cytokinesis furrow.
Understanding cytoskeletal rearrangements that drive cell division in T. brucei is an important and unresolved problem, so the work addresses important questions that are of great interest. PLK and KIN-G have previously been shown to be important for cell division and morphogenesis of cytoskeletal structures that drive cell division in T. brucei. The current work advances our understanding by suggesting a potential mechanism by which PLK and KIN-G might participate, namely through PLK-dependent P-ation to control KIN-G MT binding activity.
Strengths:
The authors use a rigorous combination of biochemistry, phosphoproteomics, cell biology, and mutant analysis to support their conclusion that PLK-mediated P-ation of KIN-G negatively regulates KIN-G microtubule binding, and this may explain the observation that a KIN-G T301 phosphomimic mutant blocks cell division and perturbs biogenesis of cytoskeletal structures that drive cell division and morphogenesis. Combining rigorous and informative in vitro studies with mutant analysis in live cells is a great strength. The work is solid and important, though a few pieces are needed to fully connect the in vitro findings with the in vivo observations, as detailed below.
Weaknesses:
Overall, I find this work to be solid and to provide an important addition to our understanding of mechanisms controlling cell division in T. brucei. The biochemistry, in particular, is rigorous and convincingly demonstrates PLK can P-ate KIN-G, altering its MT-binding ability. Analysis of phospho-mutants of KIN-G in live T. brucei supports the conclusion that P-ation of KIN-G at T301 negatively affects KIN-G function in vivo. I think, however, that the results fall short of supporting the title, because, although the data convincingly show that PLK can phosphorylate KIN-G at T301 in vitro, and that T301 is P-ated in vivo, they do not formally demonstrate (nor even test) whether PLK is the kinase responsible for this phosphorylation in vivo (experiments to address this seem quite feasible). I also do not see where the authors try to reconcile the absence of phenotype for KIN-G-T301A with the implied importance of KIN-G phosphorylation by PLK in cell division, which calls into question the need for P-ation of KIN-G-T301 in cell division. Suggestions for addressing these concerns are provided below.
My two main questions are:
(1) What is the biological relevance of KIN-G P-ation at T301?
(a) The authors report no defect for the KIN-G-T301A mutant, so what then is the need for T301 P-ation, if the cell gets along fine without it? One step toward addressing this would be to ask what fraction of KIN-G shows P-ation at T301. Although published studies indicate P-ation at T301, it isn't known what percentage of KIN-G in the cell is P-ated. One might anticipate, for example, that T301-P is a small minority of the population in asynchronous cultures and that T301 P-ation increases at specific cell cycle stages.
This is a great point that is very well taken. We also had been puzzled by the observation of no growth defects of T301A mutant. This comment enlightened us. From the new experiments we performed to compare the phosphorylated peptides of KIN-G in cells treated and non-treated with the PLK inhibitor GW843682X, we calculated the percentage of phosphorylated Thr301 in non-GW843682X-treated cells. We found that the percentage of peptides containing the phosphorylated Thr301 is ~14% of the total Thr301-containing peptides (Fig. 1H). This result indicates that T301-P is indeed a small minority of the population in the asynchronous trypanosome cells. It is possible that T301 phosphorylation may occur at a specific cell cycle stage such as early S-phase, during which PLK and KIN-G co-localize at the centrin arm.
(b) Published work links PLK to cell division, FAZ elongation, etc.. The current work suggests that one role of PLK is to P-ate KIN-G at T301. In contrast, however, the current work also indicates that P-ation of KIN-G at T301 is unnecessary for normal cell division, FAZ elongation, etc..
Yes, previous work discovered essential roles of TbPLK in basal body segregation, centrin arm biogenesis, FAZ elongation, and cytokinesis. These functions of TbPLK correlate with TbPLK’s localization to multiple subcellular structures, the basal body, the centrin arm, and the new FAZ tip, and are attributed to the regulation of its substrates at these structures. At the basal body, TbPLK phosphorylates SPBB1, which is required for basal body segregation. Defects in basal body segregation can lead to defective flagellum positioning and FAZ elongation. At the new FAZ tip, TbPLK regulates the cytokinesis regulator CIF1, which is required for cytokinesis. At the centrin arm, TbPLK phosphorylates TbCentrin2 at S54 and KIN-G at T301 (and S569, which was newly identified as an in vivo TbPLK site and has not yet been characterized). However, cells expressing TbCentrin2-S54A have very weak growth defects, and cells expressing KIN-G-T301A have no detectable growth defects. In contrast, cells expressing TbCentrin2-S54D and cells expressing KIN-G-T301D have strong growth defects. Therefore, the essential role of TbPLK in centrin arm biogenesis apparently is not attributed to the phosphorylation of TbCentrin2 and KIN-G. It is possible that phosphorylation of other centrin arm-localized protein(s) by TbPLK may be essential for centrin arm biogenesis, but this possibility remains to be explored.
(c) Some experiments or at least commentary on points a and b above would strengthen the paper.
We performed experiments and presented the data in Fig. 1H. We also included commentary in the revised manuscript on the points about the potential role of T301 phosphorylation. Thanks for these great comments that significantly improved the manuscript.
(2) Is PLK the kinase that P-ates Kin-G T301 in vivo?
(a) The authors show PLK P-ates T301 (and other residues) in vitro, and that T-301 is P-ated in vivo. To bring the analysis full circle, it would be informative to examine KIN-G P-ation in a PLK mutant or upon inhibition of PLK with published inhibitors. This seems to be a very doable experiment with the tools available.
We treated trypanosome cells with a potent PLK inhibitor GW843682X, which was previously demonstrated to inhibit TbPLK activity in vitro and mimic TbPLK knockdown in vivo in trypanosomes, and immunoprecipitated KIN-G for mass spectrometry. We compared the KIN-G peptides identified by mass spectrometry from trypanosome cells treated with or without GW843682X, and found that two phosphosites (T301 and S569) were reduced by ~27% and 100%, respectively, after GW843682X treatment (Fig. 1G). These results provided evidence to support that TbPLK phosphorylates KIN-G in vivo.
Reviewer #3 (Public review):
Summary:
Here, the authors investigate the role of the Trypanosoma brucei polo-like kinase TbPLK in the function of flagellum-associated cellular structures in trypanosomes. They set out to test the hypothesis that a key substrate of TbPLK is the kinesin protein KIN-G, and that TbPLK phosphorylation of KIN-G regulates its functions in cells.
Strengths:
Using in vitro biochemistry with purified proteins, the authors convincingly demonstrate that TbPLK phosphorylates KIN-G at 29 sites. Moreover, they convincingly show that phosphorylation at one site, T301, impairs the binding of purified KIN-G to purified microtubules. Using immunofluorescence-based imaging approaches, they also show that TbPLK colocalizes with KIN-G at centrin arms during the early S-phase of the cell cycle. Centrin arms are structures that are located near the basal body and flagellum and are important for new flagellum biogenesis, Golgi positioning, and cell division. To evaluate the function of KIN-G phosphorylation in cells, they depleted KIN-G by RNAi, simultaneously expressed phospho-mimetic (T301D) and phospho-ablative mutant proteins, and used immunofluorescence to examine the impact on flagellum-associated cellular structures. They show that expression of the phospho-mimetic mutant KIN-G-T301D causes the following defects: reduced cell proliferation, disruption of centrin arm and Golgi biogenesis, impairment of FAZ elongation and flagellum positioning, and misplacement of the cell division plane. The data convincingly support the conclusion that KIN-G phosphorylation on T301 plays an important role in regulating the cellular functions of this kinesin motor protein.
Weaknesses:
Some of the broader conclusions are not directly supported by the data. For example, the title states "Polo-like kinase phosphorylation of the orphan kinesin KIN-G negatively regulates centrin arm biogenesis in Trypanosoma brucei," but the data do not directly address the specific role of TbPLK in phosphorylating KIN-G in cells. Moreover, some of the more specific conclusions in the paper, for example, that "phosphorylation of KIN-G" causes various cellular defects, are a bit of an overstatement. The supporting data rely on the expression of a phospho-mimetic mutant of KIN-G. Presumably, phosphorylation in cells is a normal part of KIN-G regulation, and it is not just phosphorylation, but rather hyperphosphorylation that is being mimicked by the mutant. Some rewording of the specific conclusions is warranted, and the broader conclusion would be better supported with additional experimental evidence.
This is a great point that is very well taken. We performed new experiments to address the in vivo phosphorylation of KIN-G by TbPLK. We treated cells with a potent PLK inhibitor, GW843682X, and then immunoprecipitated KIN-G for mass spectrometry to identify changes in phosphorylation. We found that the phosphorylation levels of T301 and S569 were reduced by ~27% and 100%, respectively, confirming that these two sites are in vivo TbPLK phosphosites.
We also calculated the ratio of phospho-T301 versus non-phospho-T301 in non-treated cells and found that phospho-T301 accounts for ~14% of the total KIN-G protein. This new result suggests that it is the hyperphosphorylation that causes growth defects. We have revised the manuscript accordingly to reflect this point.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
Several statements use rather strong causal language (for example, "thereby impairing Golgi biogenesis, FAZ elongation, and division plane placement"). While the phenotypic correlations are convincing, direct causality is largely inferred from prior literature. Slightly tempering this wording could improve precision. It would also be helpful to state explicitly in figure legends the number of cells analyzed per condition and the number of independent experiments for each quantified phenotype.
Thanks for these constructive comments, which we agree and appreciate greatly. We have revised the manuscript accordingly to improve precision. The total number of cells analyzed, and the number of independent experiments were included in the figure legends.
Reviewer #2 (Recommendations for the authors):
Minor comments for improving the text are:
(1) The paper overall is clearly written. However, the Discussion starts with a solid sentence, then becomes a bit diffuse in discussing a wide range of PLK activities that were not addressed in the current work. That detracts attention a bit from the central contributions of this paper.
Thanks for this comment. We have deleted the discussion about TbPLK activities that were published previously.
(2) At least two places in the text state apparent contradictions.
(a) p.5 and Figure 2C. The authors say microtubule gliding speed was "...insignificantly reduced..." by the TbPLK-K70R mutant, yet they then state that motility was "interfered with". If the effect is "insignificant", why do they claim there is an effect?
(b) p6 and Figure 3C. The authors report KIN-G-T301A impact on microtubule gliding activity is insignificant, but then say this mutation reduces the motility of KIN-G. These statements are contradictory.
We meant to say that there was a slight but insignificant effect. We agree that such statements are somewhat contradictory and, hence, have been deleted. Thanks.
(3) p. 8, and Figure 7. "ventral side" and "leading edge" are not defined but are used to describe the KIN-G RNAi phenotype.
We have deleted the wording “ventral side”, as it is not necessary. Thanks.
(4) Figure 7B. Please explain the labeling - the new flagellum daughter is indicated as having the old posterior, while the old flagellum daughter cell is indicated as having the new cell posterior. This is counterintuitive to a reader not intimately familiar with the T. brucei cell division process.
Thanks very much. We included two sentences in the revised manuscript to explain this point.
The sentences read as follows: “The nascent posterior is formed near the mid-portion of the NFD cell through microtubule bundling and cytoskeleton remodeling during late stages of the cell cycle (Wheeler et al., 2013). Consequently, the NFD cell inherits the old, existing cell posterior, whereas the OFD cell inherits the newly formed or nascent cell posterior.”
(5) Figure 4, 5, and 7: "% Cells" is reported. Please indicate the total number of cells that were examined.
The total number of cells were included in the figure legends.
Reviewer #3 (Recommendations for the authors):
(1) The manuscript should be carefully edited for minor grammatical errors.
Thanks. We have carefully proofread the manuscript and corrected the grammatical errors.
(2) A general conclusion is that TbPLK phosphorylation of KIN-G in cells is critical for regulating its motor activity. However, this relies on the expression of phospho-mimetic mutants, which bypass TbPLK. Thus, there really is no direct evidence provided to support the specific role of TbPLK other than the in vitro phosphorylation data. Some additional experiments to assess the specific role of TbPLK in phosphorylating KIN-G in cells would lend support for the general conclusion. Is it possible to deplete or inhibit TbPLK and show that this impacts the phosphorylation of KIN-G in cells?
This is a great point that is very well taken. We performed a new experiment by inhibiting TbPLK with a potent PLK inhibitor, GW843682X, and then immunoprecipitating KIN-G for mass spectrometry to identify the phosphorylation levels before and after GW843682X treatment. We were able to confirm that T301 and S569 phosphorylation was reduced after treatment. This confirms that TbPLK phosphorylates T301 and S569 of KIN-G in vivo in trypanosome cells.
Specific:
(1) Figure 2: For microtubule gliding assays, representative videos should be included as supplementary data. Also, when the data do not show a significant difference between KIN-G and KIN-G + TbPLD-K70R, then the authors should not state there is a slight difference, as this is not supported by the data.
We have deleted the statement. We have included the representative videos for all the experiments presented in Figures 2 and 3. Thanks.
(2) Figure 3: As stated above, for microtubule gliding assays, representative videos should be included. Moreover, for KIN-G-T301A, the authors say that the gliding activity was "moderately, but insignificantly, reduced." Again, if the difference is not significant, it cannot be concluded that there is a difference compared with the wild-type protein.
We have deleted the statement. Thanks.
(3) Some of the headings in the Results section are not accurate. Regarding the in vivo results, the heading "Phosphorylation of Thr301 in KIN-G by TbPLK causes defective cell proliferation" seems to be an overstatement. It may be more accurate to state that "Expression of a phospho-mimetic mutant of KIN-G causes defective cell proliferation." The same comment applies to the other headings that follow this one. Presumably, there is a population of phosphorylated KIN-G in cells, and phosphorylation/dephosphorylation is a normal part of its regulation. In the text, the authors might more accurately conclude that hyperphosphorylation causes the defects they are seeing.
This is a great point. We agree and as we responded above, we have revised the manuscript, from the title to the main text, to reflect the point that hyperphosphorylation of T301 by TbPLK causes the defects. Thanks very much for these great comments!
