Single-molecule tracking reveals the dynamics of Ipl1 recruitment to the kinetochores and spindles in S. cerevisiae

  1. Nitesh Kumar Podh
  2. Ayan Das
  3. Akriti Kumari
  4. Kirti Garg
  5. Rashmi Yadav
  6. Kirti Kashyap
  7. Sahil Islam
  8. Anupam Gupta
  9. Gunjan Mehta

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Abstract

Aurora kinase B, Ipl1 in Saccharomyces cerevisiae , is the master regulator of cell division required for checkpoint regulation, spindle assembly and disassembly, chromosome segregation, and cytokinesis. Decades of research employed ensemble averaging methods to understand its dynamics and function; however, the dynamic information was lost due to population-based averaging. Here, we use single-molecule imaging and tracking (SMIT) to quantify the recruitment dynamics of Ipl1 at the kinetochores and spindles in live cells. Our data suggest that Ipl1 is recruited to these locations with different dynamics. We have demonstrated how the recruitment dynamics of Ipl1 at the kinetochores during metaphase changes in the presence and absence of tension across the kinetochore, in the absence of protein phosphatase 1 (Glc7), and the absence of its known recruiters (Ctf19 and Bub1). The SMIT of other chromosome passenger complex members suggests its hierarchical assembly at the kinetochore. Hence, SMIT provides a dynamic view of the Ipl1 trafficking at the kinetochores and spindles.

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    Referee #3

    Evidence, reproducibility and clarity

    In their manuscript titled "Single-molecule tracking reveals the dynamics of Ipl1 recruitment to the kinetochores and spindles in S. cerevisiae", authors Podh, Mehta, and colleagues track single molecules of members of the chromosomal passenger complex (CPC) to determine the dynamics of the complex during chromosome biorientation. The authors tagged members of the CPC with a HaloTag to titrate the number of fluorophores to image single molecules and performed microscopy with high temporal resolution and measure residence lifetimes and diffusion rates of the complex. Furthermore, they used mutations that disrupt different localization pathways for the complex to determine which lifetimes are associated with these pathways. Next, they arrested the cells in metaphase and treated the cells with a microtubule inhibitor to determine the effect of disrupting kinetochore-microtubule connections on CPC dynamics. In this way, they determined that the more long-lived CPC molecules are at unstable connections. Finally, they compared the dynamics of the different complex members and saw that two of the members (Nbl1 and Bir1) have more long-lived associations near kinetochores than the other two members (Sli15 and Ipl1). Overall, the study is quite interesting, as the dynamics of the CPC near the kinetochore are highly relevant for the function of the complex. However, I have some concerns about the methodology and conclusions.

    Major comments:

    1. My largest concerns relate to the categorization of the fluorescent molecules. As far as I can tell from the methods, foci are only counted if they are "bound", meaning that they remain within a certain radius for a minimal number of frames. This requirement appears to assume diffusive movement. However, frequent directed movement would be expected for CPC molecules localized to the inner centromere, kinetochore or spindle. Firstly, the spindle itself would be subject to dynein-directed movements to position it near the bud neck during metaphase. Second, the kinetochores themselves would be undergoing directed movements from microtubule dynamics. These movements are quite rapid and would certainly take place over the timescale of the microscopy experiments performed in this study (See PMC1366782 for example). Kinetochores undergoing these directed movements would likely be the most relevant to CPC function, as these would still be undergoing biorientation.

    To establish the parameters used for assessing bound molecules, the authors used histone H3. However, this would not be an appropriate measure for dynamics at the spindle/kinetochore for the reasons stated above, especially if the measurements were taken from cells that were not in metaphase. As an additional control, the authors could use cell lines with GFP-labeled centromeres in metaphase cells and subject them to the same analyses. Since these are not single molecules, all of the foci would be expected to have lifetimes for the full 40s duration of image collection. Any foci that fall short of this duration would indicate that the lifetimes of some single molecules are not measured for their full duration.

    1. The authors repeatedly state that the double exponential decay equations fit the survival probability distributions "well". However, there is no statistical measurements of the fits presented. How much better does double exponential decay fit as compared to single exponential decay? In figure 2A (metaphase), both single and double decay curves are shown in the figure, but they seem to overlap to the point of being nearly indistinguishable. The methods section mentions that F-tests were performed for the fits, but I cannot find the results of the tests.

    On a related note, some of the curves don't seem to fit the data well at all. Figures 2C, 3B, and S3 have especially bad fits. Is there an explanation for this? Would a different method fit these data better? For figure 3B, the data show that over 10 percent of the tracks last longer that 10 seconds. This is much higher than for any other condition, yet the authors conclude that there is no specifically bound fraction since the curve doesn't match the data. This is a substantial issue for the interpretation of these results.

    1. For the interpretation of the differences in lifetimes for Bir1/Nbl1 vs. Sli15/Ipl1, the authors conclude that the longer lifetime of the former indicates earlier recruitment to the "kinetochore". A simpler explanation may be that there are different subcomplexes that have different recruitment dynamics. For example, a complex with all four subunits may have a longer lifetime than one that is just Sli15/Ipl1 due to different recruitment methods (Sgo1-dependent vs Ctf19-dependent). The lifetimes of Sli15 or Ipl1 molecules would therefore be a combination of both recruitment methods.

    Minor comments:

    1. In figure 3B (without tension), the ROI and tracks numbers are likely switched based off of the numbers for the other graphs.
    2. In the introduction (Page 4 line 15), the authors conclude about their result from depleting Glc7 that "fast exchange of Ipl1 is essential to keep the Glc7 away from its kinetochore substrates." I'm not sure what this statement means, as it is unlcear what "its" is referring to Ipl1 or Glc7. Either way, I don't think the authors can conclude anything about keeping Glc7 away without looking at the localization of Glc7 itself.
    3. On page 7 line 2 the authors claim to track Ipl1 on "kinetochores" in metaphase. Later on the same page they clarify that they cannot differentiate between inner centromere, inner kinetochore, and outer kinetochore. I would think that they also can't distinguish these with microtubule binding. The authors again claim to be observing kinetochore localization on page 9 line 2. This is confusing, and a more accurate term for the localization should be adopted.
    4. The authors claim that Glc7 is "required for" fast turnover (page 11 line 5), yet they still see many instances of fast turnover following its depletion.
    5. The authors assume that the molecules have a longer lifetime are the ones "involved in phosphorylation" (page 12 line 21 and 24). This claim would need to be justified, as short periods of localization could be sufficient for phosphorylation of substrates.
    6. On page 12 line 23, the authors state numbers for how long it takes Ipl1 to "find its target sites". I cannot find where these numbers come from
    7. On page 13 line 15 the authors claim that tension evicts Ipl1 from kinetochores. However, tension was not specifically tested, as benomyl treatment will create unattached kinetochores.

    Significance

    General assessment: The strengths of the study are in quantifying the dynamics of the CPC at localizations near the kinetochore in a new and informative way. The limitations lye in not knowing where the CPC molecules are localized in relation to the kinetochore, which would have provided mechanistic insights into the recruitment pathways. Although this limits the ability to derive strong mechanistic insights from the results, the numbers themselves are valuable and interesting.

    Advance: Previous studies on CPC localization come from conventional fluorescence microscopy that observes the average of many molecules. This manuscript uses a single molecule technique to observe the residence lifetime and diffusion rate of CPC complex members.

    Audience: This study would be of general interest to researchers that study chromosome biorientation and segregation.

    Reviewer's expertise: Chromosomal passenger complex, budding yeast, microscopy

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    Referee #2

    Evidence, reproducibility and clarity

    This report investigates, using single-molecule approaches, the dynamics of Ipl1(Aurora B)and other Chromosomal Passenger Complex (CPC) components in budding yeast mitosis. The work is largely descriptive, and identifies a number of (confirmatory) results regarding the connections between Ipl1/CPC, its centromere/kinetochore receptors and GLC7/PP1. In addition, experimental rigor is lacking in certain instances, casting doubt on the conclusions made.

    Significance

    Generally, this paper provides some interesting suggestions but falls short in expanding our knowledge significantly. As such the impact of this study will be minor. The used methodology is hard to critically gauge due to strong statements which seem unsubstantiated. In addition, the manuscript is written in a way that appears to generate 'controversy/contrast' with earlier work, which seems unwarranted and unnecessary. Some technical questions remain, which will need to be addressed before publication.

    Specific points:

    • page 1, line 16, change 'the master regulator' to ' a master regulator'.
    • line 18-20, this statement is unnecessary
    • page 2, line 15-17, why does their overlapping ..... make studying Aur-B challenging?
    • page 4, line 11, remove 'the' before Ipl1
    • line 13-16, this is an unclear statement

    Results:

    • in general, why are experiments done in heterozygously tagged diploids? Is there a risk that the untagged, wt allele obscures behavior? Also, viability assays in this situation are not informative about functionality of the used alleles. These experiments will need to be done in 1)haploids or 2) homozygous diploids page 6, line 14-25: this paragraph makes strong statements about the behavior and function of certain Ipl1 pools. These statements seems unsubstantiated by solid data. The whole paper relies on these extrapolations so care should be taken. What evidence are the conclusions based on? Also, how can the authors actually distinguish KI, centromere and spindle pools with confidence here? By looking at tubulin-CloverGFP this seems unrealistic: in fact the imaging that is shown fails to recapitulate classical spindle MT patterns (rather, the whole cell lights up - what's up here?). What about co-localization with other factors? Additional/alternative experiments are needed here. If not, it remains unclear what the analysis downstream of these images signifies. page 12, line 1-6: how can the authors arrive on these conclusions (i.e. Bir1/Nbl1 arrives first -whatever that may even imply vis-a-vis molecular behavior?). These conclusions are not substantiated.
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    Referee #1

    Evidence, reproducibility and clarity

    The CPC is involved in important functions during mitosis, including kinetochore assembly, chromosome biorientation, spindle assembly checkpoint and cytokinesis. In this report, the authors studied the dynamics of the CPC at the kinetochore and mitotic spindle in yeast using single-molecular tracking. They conclude that Ipl1 (the catalytic subunit of the CPC) shows different residence times A) between the kinetochore and the spindle, B) between Ctf19- and Bub1-mediated recruitment to the kinetochore, C) with and without tension during the metaphase arrest, D) in the presence and absence of Glc7 phosphatase, and E) between Bir1-Nbl1 and Sli15-Ipl1.

    These are interesting findings, which provide people working in this research field with useful information. However, some clarifications and additional evidence are required regarding the following points:

    1. The statement about the pink and blue fractions (Page 6, Line 18-20): What is the evidence supporting this statement? More explanations and/or reference citations are required.
    2. Different dynamics between the kinetochore and the spindle (Page 7. Line 5-6): Is this difference due to the different locations in cells or the difference in the cell cycle (metaphase vs anaphase)? Is the dephosphorylation of Sli15, which promotes the relocalization of CPC from the kinetochore to the spindle at the anaphase onset (e.g. Pereira et al Science 2003), involved in this difference? What happens to the CPC dynamics if non-phosphorylatable Sli15 localizes to the spindle during metaphase?
    3. The statement about the Ipl1 dynamics at different kinetochore sites (Page 7, Line 24-25): It is not clear how solid this conclusion is. If they use other kinetics models, do they reach different conclusions? I do not think the functional redundancy of CPCs at three locations would necessarily support the conclusion because they may still redundantly support biorientation and cell viability even if they show different dynamics at the three locations.
    4. The conclusion about Ipl1 dynamics under tension (Page 10, Line 7-8): The kinetochores should also be under tension in metaphase in cycling cells. However, Fig 3C (left) shows the pink fraction (specific bound fraction) is still present in this condition. How do they explain the discrepancy between metaphase arrest (Fig 3B, left) and metaphase in cycling cells (Fig 3C, left)?
    5. Figure 4 should be cited in the last section of Results (Page 11-12).

    Significance

    Given the importance of CPC in the regulation of mitosis, it is useful knowledge for experts that the CPC shows different dynamics depending on different localization sites and under different conditions. However, this work does not tell us much about whether or how the different dynamics of the CPC are important to its function. It is also not clear what molecular mechanisms (e.g. phosphorylation or other post-translational modifications, different affinity between proteins) directly cause the different dynamics of the CPC. Therefore, in my view, the scientific significance is rather limited.

  5. Version published to 10.1101/2024.08.01.606162v1 on bioRxiv