Changes in seam number and location induce holes within microtubules assembled from porcine brain tubulin and in Xenopus egg cytoplasmic extracts
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eLife assessment
The study, using cryo-electron tomography represents a valuable study to the research community, to raise awareness that in vitro-assembled microtubules have more lattice defects than microtubules assembled in cell extracts. However the evidence supporting the claims was incomplete in places and there was not enough data. It is not clear how generalizable these findings are regarding tubulin assembly into microtubules.
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Abstract
Microtubules are tubes of about 25 nm in diameter that are critically involved in a variety of cellular functions, including motility, compartmentalization, and division. They are considered as pseudo-helical polymers whose constituent αβ-tubulin heterodimers share lateral homotypic interactions, except at one unique region called the seam. Here, we used a segmented sub-tomogram averaging strategy to reassess this paradigm and analyze the organization of the αβ-tubulin heterodimers in microtubules assembled from purified porcine brain tubulin in the presence of GTP and GMPCPP, and in Xenopus egg cytoplasmic extracts. We find that in almost all conditions, microtubules incorporate variable protofilament and/or tubulin subunit helical-start numbers, as well as variable numbers of seams. Strikingly, the seam number and location vary along individual microtubules, generating holes of one to a few subunits in size within their lattices. Together, our results reveal that the formation of mixed and discontinuous microtubule lattices is an intrinsic property of tubulin that requires the formation of unique lateral interactions without longitudinal ones. They further suggest that microtubule assembly is tightly regulated in a cytoplasmic environment.
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Author Response
Reviewer #1 (Public Review):
In this manuscript, the author characterizes the lattice of kinesin-decorated microtubule reconstituted from porcine tubulins in vitro and Xenopus egg extract using cryo-electron tomography and subtomogram averaging. Using the SSTA, they looked at the transition in the lattice of individual microtubules. The authors found that the lattice is not always uniform but contains transitions of different types of lattices. The finding is quite interesting and probably will lead to more investigation of the microtubule lattice inside the cells later on for this kind of lattice transition.
The manuscript is easy to read and well-organized. The supporting data is very well prepared.
Overall, it seems the conclusion of the author is justified. However, the manuscript appears to show a lack of …
Author Response
Reviewer #1 (Public Review):
In this manuscript, the author characterizes the lattice of kinesin-decorated microtubule reconstituted from porcine tubulins in vitro and Xenopus egg extract using cryo-electron tomography and subtomogram averaging. Using the SSTA, they looked at the transition in the lattice of individual microtubules. The authors found that the lattice is not always uniform but contains transitions of different types of lattices. The finding is quite interesting and probably will lead to more investigation of the microtubule lattice inside the cells later on for this kind of lattice transition.
The manuscript is easy to read and well-organized. The supporting data is very well prepared.
Overall, it seems the conclusion of the author is justified. However, the manuscript appears to show a lack of data. Only 4 tomograms are done for the porcine microtubules. Increasing the data number would make the manuscript statistically convincing.
One tomogram can contain one to several tens of microtubules. For example, 64 microtubules were analyzed in the Xenopus-DMSO dataset obtained on 5 tomograms, versus 24 microtubules for the GTP-dataset obtained on 4 tomograms (see Table 1). Hence, taking the number of tomograms to assess the statistical relevance of our work cannot be considered as a valid criterion. Tomograms are taken randomly on the EM-grid sample, solely based on ice quality and the covering of microtubules in the holes as determined at low magnification before tomographic acquisition. No prior knowledge of the structure and lattice-type organization of the microtubules can be obtained before acquisition. It appears to us that a more pertinent criterion is the number of events that we characterized, specifically lattice-type transitions along individual microtubules. In the dataset mentioned by the referee (see Figure 2-figure supplement 3-4 and Table I), 24 microtubules were analyzed and further divided into 195 segments, providing an equivalent number of individual 3D reconstructions. For each 3D reconstruction, almost all lateral interactions could be characterized in terms of lattice-type, i.e., 2091 of the B-type, 460 of the A-type, and 112 not determined (essentially at transition regions). Most importantly, we document in this specific dataset 119 transitions in lattice-type, which we think is sufficient to characterize such molecular events and provide solid statistics for this dataset. Adding the GMPCPP and Xenopus data, we end-up with 938 individual 3D reconstructions (not including the full-length microtubule volumes), 12 463 lateral interactions analyzed (A-, B-, or ND-type), and the observation of 172 lattice-type transitions. Therefore, we respectfully disagree with the referee stating that our work lacks data.
To highlight the quantity of data used in our work, we have modified the following sentences: L124-131: ' Analysis of 24 microtubules taken on 4 tomograms, representing 195 segments of ~160 nm length (i.e., 2664 lateral interactions), allowed us to characterize 119 lattice type transitions with an average frequency of 3.69 µm-1 (Table 1), but with a high heterogeneity' L160-164: ' Analysis of 31 GMPCPP-microtubules taken on 6 tomograms, representing 338 segments of ~150 nm in length (i.e., 3236 lateral interactions), and using the same strategy as in the presence of GTP (Figure 5—figure supplement 1-2) revealed a transition frequency of 1.25 µm-1 (Table 1), i.e., ~3 fold lower than microtubules assembled in the presence of GTP.' L200-203: ' A total of 64 microtubules taken on 5 tomograms were analyzed in the Xenopus-DMSO dataset (i.e., 419 segments from which we characterized 5446 lateral interactions), and 15 microtubules taken on one tomogram for the Xenopus Ran-dataset (i.e., 86 segments from which we characterized 1118 lateral interactions), (Table 1).'
In addition, having the same transition with the missing wedge orientation randomly from different subtomograms will allow a better average of transition without the missing wedge artifact.
In this work, we did not aim at averaging transitions. Transitions in lattice-types are highly heterogeneous in nature, and we wonder what additional information an averaging strategy would have provided. Conversely, each transition is a unique event that we characterized to obtain useful statistics, and the missing data at high angle inherent to electron tomography were not an obstacle to fulfill this task.
Another thing that I found lacking is the mapping of the transition region/alignment in the raw data.
In Figure 4, we clearly show the correspondence between the segmented sub-tomogram averages (SSTA) and the raw filtered images at the transition region. This is also the case in Figure 5 where the SSTA (Figure 5A) are compared with the raw tomogram (Figure 5B), and where we clearly visualize the holes that result from the transitions in lattice types.
However, it is not easy for me or the reader to understand how each segment is oriented relative to each other apart from the simplified seam diagrams in the figures, and also the orientation of the seam corresponding to the missing wedge in the average. With these improvements, I think the conclusion of the manuscript will be better justified.
The segmentation process is explained in Figure 2-figure supplement 2 and in the Materials and Methods section, which shows that each segment is linearly related to the next. Small rotations can happen between individual segments, and it is important to check that the same protofilaments are followed during the initial modeling (see the online tutorial referenced in the manuscript for full-length microtubules). The segment models are derived from that of the full-length microtubule, as explained in the Materials and Methods section, using a new routine (splitIntoNsegments) implemented into the PEET program. In addition, a detailed protocol describing our SSTA strategy will be submitted following publication of our manuscript.
Reviewer #2 (Public Review):
Differences in protofilament and subunit helical-start numbers for in vitro polymerized and cellular microtubules have previously been well characterized. In this work, Guyomar et al. analyze the fine organization of tubulin dimers within the microtubule lattice using cryo-electron tomography and subtomogram averaging. Microtubules were assembled in vitro or within Xenopus egg cytoplasmic extracts and plunge frozen after addition of a kinesin motor domain to mark the position of tubulin dimers. By generating subtomogram averages of consecutive sections of each microtubule and manually annotating their lattice geometry, the authors quantified changes in lattice arrangement in individual microtubules. They found in vitro polymerized microtubules often contained multiple seams and lattice-type changes. In contrast, microtubules polymerized in the cytoplasmic extract more frequently contained a single seam and fewer lattice-type transitions.
Overall, their segmented subtomogram averaging approach is appropriately used to identify regions of lattice-type transition and quantify their abundance. This study provides new data on how often small holes in the lattice occur and suggests that regulators of microtubule growth in cells also control lateral tubulin interactions. However, not all of the claims are well supported by their data and the presentation of their main conclusions could be improved.
1 - We have corrected approximative claims and conclusions where necessary. In particular, we now discuss separately the Xenopus-DMSO and the Xenopus-Ran egg extract samples, and have modified our conclusions accordingly. We also deposited onto the EMPIAR all tomograms and PEET models to reproduce the 938 segmented sub-tomogram averages analyzed in this study (see new Supplementary file 2).
Reviewer #3 (Public Review):
Protofilament number changes have been observed in in vitro assembled microtubules. This study by Guyomar and colleagues uses cryo-ET and subtomogram averaging to investigate the structural plasticity of microtubules assembled in vitro from purified porcine brain tubulin at high concentrations and from Xenopus egg extracts in which polymerization was initiated either by addition of DMSO or by adding a constitutively active Ran. They show that the microtubule lattice is plastic with frequent protofilament changes and contains multiple seams. A model is proposed for microtubule polymerization whereby these lattice discontinuities/defects are introduced due to the addition of tubulin dimers through lateral contacts between alpha and beta tubulin, thus creating gaps in the lattice and shifting the seam. The study clearly shows quantitatively the lattice changes in two separate conditions of assembling microtubules. The high frequency of defects they observe under their microtubule assembly conditions is much higher than what has been observed in vivo in intact cells. Their observations are clear and supported by the data, but it is not at all clear how generalizable they are and whether the defect frequencies they see are not a result of the assembly conditions, dilutions used and presence of kinesin with which the lattice is decorated. The study definitely has implications for mechanistic studies of microtubules in vitro and raises the question of how these defects vary for protocols from different labs and between different tubulin preparations.
1 - High tubulin concentration: It has been documented by many laboratories since the discovery of tubulin and the characterization of its assembly properties that a sufficient concentration of free tubulin is necessary to self-assemble microtubules. This is called the critical concentration for self-assembly (the CC, i.e., the critical concentration to overcome the nucleation barrier), and has been reported to be in the range 14~25 µM in the presence of GTP depending on laboratories. For example, in the seminal work of Mitchison and Kirschner the CC was estimated at 14 µM (Fig. 5 of ref. (Mitchison & Kirschner, 1984b)) and self-assembly was induced at concentrations in the range 32-59 µM (Mitchison & Kirschner, 1984a). Our own estimate of the CC for porcine brain tubulin was 21 µM (Fig 2C of (Weis et al., 2010)), and we routinely use a tubulin concentration slightly above the CC when we aim at robust microtubule self-assembly. Hence, we argue that 40 µM, which is ~twice the CC, cannot be considered as a "very high" tubulin concentration to induce microtubule self-assembly.
2 - Protofilament number and lattice-type transitions in cells: While microtubules with protofilament numbers different than 13 have been observed in different cell types and species (reviewed in (Chaaban & Brouhard, 2017)), we are aware of only one recent study where changes in protofilament numbers along individual microtubules have been reported in cells (Foster et al., 2021), but with no statistics concerning their frequencies. Hence, we cannot compare changes in protofilament number frequencies in Xenopus egg extracts with those that occur in intact cells. Concerning lattice-type transitions, we are not aware of any previous study that documented such features, whether in vitro or in cells.
3 - Generalization of our results, source of tubulin and protocols: Multi-seams in microtubules assembled in vitro have been reported by several groups in the past (see our Introduction, L49-62), starting from (Kikkawa et al., 1994), the Milligan group (Dias & Milligan, 1999; Sosa et al., 1997), and more recently by the Sindelar group (Debs et al., 2020). In Kikkawa et al. (1994), the authors purified tubulin from porcine brain by three cycles of assembly/disassembly followed by phosphocellulose chromatography. Assembly was carried out at 24 µM in the presence of Taxol. In Sosa and Milligan (1996-1997), the authors used a commercial source (Cytoskeleton) and assembled the microtubules at 30 µM in the presence of Taxol. In Debs et al. (2020), the authors used tubulin purified from porcine brain according to (Castoldi & Popov, 2003), as we did, to assemble GMPCPP microtubules, and bovine brain tubulin (Cytoskeleton) to assemble Taxol-stabilized microtubules. Noticeably, they used an initial tubulin concentration of 100 µM to initiate microtubule polymerization and then added Taxol to continue the reaction.
We add to these previous studies that microtubules with different numbers of seams are not unique ones, but that both the number and location of seams can vary within individual microtubules. The reason why this was not observed before is that the analytical tools used in those previous studies were not suited to reveal this structural heterogeneity within individual microtubules. By contrast, the SSTA approach that we designed was specifically developed towards this aim. Even in the recent work by Debs et al. (2020) that provides the most comprehensive characterization of multi-seams in microtubules assembled in vitro and that obtained a seam distribution very similar to ours (compare their Figure 3C with our new Figure 10C for GDP microtubules, dark blue bars), their protofilament-based approach could not reveal changes in the number and location of seams within individual microtubules. Yet, they probably could have done it if they had asked whether segments with different seam numbers had been extracted from the same microtubules.
Here, we designed a specific approach to tackle the structural heterogeneity of individual lattices that permitted this discovery. Not only do we confirm results obtained by others, but we also propose a molecular mechanism that explains how multi-seams form in microtubules assembled in vitro and how they change in location in a cytoplasmic environment. By doing so, we propose a novel molecular event - formation of unique lateral interactions without longitudinal ones - that was not envisioned before, and which to our opinion, must be incorporated in further modelling studies concerning microtubule nucleation and assembly, including the mechanism of dynamic instability (see the Ideas and speculation section).
4 - Dilution: A 50X dilution was used only for Xenopus egg cytoplasmic extracts to decrease their density on the EM grid just before freezing. These conditions were settled by cryo-fluorescence microscopy to ensure that we had the adequate density of microtubules onto the EM-grid (Figure 7 and Figure 2—figure supplement 1D). Of note, the microtubules analyzed by SSTA were assembled in extracts that were not supplemented with fluorescent tubulin. While we could imagine that dilution may induce the removal of dimers from the microtubule lattice, we cannot foresee how this could change the register between tubulin subunits within the microtubule lattice.
5 - Kinesin decoration: Like many other laboratories (see the Table in Figure 3 of (Manka & Moores, 2018)), we use the non-processive motor domain of kinesin 1 to decorate microtubules, with the aim to differentiate the - and -tubulin monomers within the microtubule lattice. In particular, it has been shown that lattice parameters such as the protofilament skew and lattice spacing are unmodified when kinesin motor domains are added to GMPCPP- or GDP-microtubules (Zhang et al., 2015, 2018). In addition, we cannot envisage how this non processive motor added to preformed microtubules could change the registry of the -tubulin heterodimers within the microtubule lattice.
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eLife assessment
The study, using cryo-electron tomography represents a valuable study to the research community, to raise awareness that in vitro-assembled microtubules have more lattice defects than microtubules assembled in cell extracts. However the evidence supporting the claims was incomplete in places and there was not enough data. It is not clear how generalizable these findings are regarding tubulin assembly into microtubules.
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Reviewer #1 (Public Review):
In this manuscript, the author characterizes the lattice of kinesin-decorated microtubule reconstituted from porcine tubulins in vitro and Xenopus egg extract using cryo-electron tomography and subtomogram averaging. Using the SSTA, they looked at the transition in the lattice of individual microtubules. The authors found that the lattice is not always uniform but contains transitions of different types of lattices. The finding is quite interesting and probably will lead to more investigation of the microtubule lattice inside the cells later on for this kind of lattice transition.
The manuscript is easy to read and well-organized. The supporting data is very well prepared.
Overall, it seems the conclusion of the author is justified. However, the manuscript appears to show a lack of data. Only 4 tomograms are …
Reviewer #1 (Public Review):
In this manuscript, the author characterizes the lattice of kinesin-decorated microtubule reconstituted from porcine tubulins in vitro and Xenopus egg extract using cryo-electron tomography and subtomogram averaging. Using the SSTA, they looked at the transition in the lattice of individual microtubules. The authors found that the lattice is not always uniform but contains transitions of different types of lattices. The finding is quite interesting and probably will lead to more investigation of the microtubule lattice inside the cells later on for this kind of lattice transition.
The manuscript is easy to read and well-organized. The supporting data is very well prepared.
Overall, it seems the conclusion of the author is justified. However, the manuscript appears to show a lack of data. Only 4 tomograms are done for the porcine microtubules. Increasing the data number would make the manuscript statistically convincing. In addition, having the same transition with the missing wedge orientation randomly from different subtomograms will allow a better average of transition without the missing wedge artifact.
Another thing that I found lacking is the mapping of the transition region/alignment in the raw data. However, it is not easy for me or the reader to understand how each segment is oriented relative to each other apart from the simplified seam diagrams in the figures, and also the orientation of the seam corresponding to the missing wedge in the average. With these improvements, I think the conclusion of the manuscript will be better justified.
-
Reviewer #2 (Public Review):
Differences in protofilament and subunit helical-start numbers for in vitro polymerized and cellular microtubules have previously been well characterized. In this work, Guyomar et al. analyze the fine organization of tubulin dimers within the microtubule lattice using cryo-electron tomography and subtomogram averaging. Microtubules were assembled in vitro or within Xenopus egg cytoplasmic extracts and plunge frozen after addition of a kinesin motor domain to mark the position of tubulin dimers. By generating subtomogram averages of consecutive sections of each microtubule and manually annotating their lattice geometry, the authors quantified changes in lattice arrangement in individual microtubules. They found in vitro polymerized microtubules often contained multiple seams and lattice-type changes. In …
Reviewer #2 (Public Review):
Differences in protofilament and subunit helical-start numbers for in vitro polymerized and cellular microtubules have previously been well characterized. In this work, Guyomar et al. analyze the fine organization of tubulin dimers within the microtubule lattice using cryo-electron tomography and subtomogram averaging. Microtubules were assembled in vitro or within Xenopus egg cytoplasmic extracts and plunge frozen after addition of a kinesin motor domain to mark the position of tubulin dimers. By generating subtomogram averages of consecutive sections of each microtubule and manually annotating their lattice geometry, the authors quantified changes in lattice arrangement in individual microtubules. They found in vitro polymerized microtubules often contained multiple seams and lattice-type changes. In contrast, microtubules polymerized in the cytoplasmic extract more frequently contained a single seam and fewer lattice-type transitions.
Overall, their segmented subtomogram averaging approach is appropriately used to identify regions of lattice-type transition and quantify their abundance. This study provides new data on how often small holes in the lattice occur and suggests that regulators of microtubule growth in cells also control lateral tubulin interactions. However, not all of the claims are well supported by their data and the presentation of their main conclusions could be improved.
-
Reviewer #3 (Public Review):
Protofilament number changes have been observed in in vitro assembled microtubules. This study by Guyomar and colleagues uses cryo-ET and subtomogram averaging to investigate the structural plasticity of microtubules assembled in vitro from purified porcine brain tubulin at high concentrations and from Xenopus egg extracts in which polymerization was initiated either by addition of DMSO or by adding a constitutively active Ran. They show that the microtubule lattice is plastic with frequent protofilament changes and contains multiple seams. A model is proposed for microtubule polymerization whereby these lattice discontinuities/defects are introduced due to the addition of tubulin dimers through lateral contacts between alpha and beta tubulin, thus creating gaps in the lattice and shifting the seam. The …
Reviewer #3 (Public Review):
Protofilament number changes have been observed in in vitro assembled microtubules. This study by Guyomar and colleagues uses cryo-ET and subtomogram averaging to investigate the structural plasticity of microtubules assembled in vitro from purified porcine brain tubulin at high concentrations and from Xenopus egg extracts in which polymerization was initiated either by addition of DMSO or by adding a constitutively active Ran. They show that the microtubule lattice is plastic with frequent protofilament changes and contains multiple seams. A model is proposed for microtubule polymerization whereby these lattice discontinuities/defects are introduced due to the addition of tubulin dimers through lateral contacts between alpha and beta tubulin, thus creating gaps in the lattice and shifting the seam. The study clearly shows quantitatively the lattice changes in two separate conditions of assembling microtubules. The high frequency of defects they observe under their microtubule assembly conditions is much higher than what has been observed in vivo in intact cells. Their observations are clear and supported by the data, but it is not at all clear how generalizable they are and whether the defect frequencies they see are not a result of the assembly conditions, dilutions used and presence of kinesin with which the lattice is decorated. The study definitely has implications for mechanistic studies of microtubules in vitro and raises the question of how these defects vary for protocols from different labs and between different tubulin preparations.
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