Microtubules are important cellular filaments that are comprised of αβ tubulin heterodimers. The tubulin heterodimers are stacked end-to-end to form structures known as protofilaments, which associate laterally to form the hollow-tube structure of the microtubule (Mitchison and Kirschner, 1984). The α/β polarity of the tubulin dimer induces microtubule polarity, such that the microtubule end with β-tubulin exposed forms the fast-growing, dynamic ‘plus-end’ of the microtubule (Desai and Mitchison, 1997; Mitchison and Kirschner, 1984). In solution, the β-tubulin subunit binds to a GTP nucleotide, which then hydrolyzes to GDP after incorporation into the microtubule lattice. This delayed hydrolysis leads to a high concentration of GTP tubulin at the growing microtubule plus-end, commonly referred to as the ‘GTP-cap’ (Desai and Mitchison, 1997). The presence of the GTP-cap creates a distinct region that is present exclusively at growing microtubule ends (Maurer et al., 2011; Maurer et al., 2014; Zanic et al., 2009).

The localization of proteins along different regions of the microtubule is central to the role of microtubules in cell migration, intracellular transport, and cell division. EB1 is a key cellular protein that autonomously localizes to the growing ends of microtubules (‘tip tracks’) and recruits other important proteins that have little or no affinity to growing microtubule ends (Bieling et al., 2007; Dixit et al., 2009; Morrison et al., 1998; Mustyatsa et al., 2017). It has been shown that improper localization of EB1 at growing microtubule plus-ends can lead to disruptions in both cell division and cell migration (Dema et al., 2023; Dong et al., 2010; Honoré et al., 2008; Mustyatsa et al., 2017; Rogers et al., 2002; van Haren et al., 2018).

EB1 binds a small pocket within the microtubule lattice that is created by four tubulin dimers. It has been shown that EB1 binds with a higher affinity to GTP-tubulin subunits as compared to GDP-tubulin subunits. (Maurer et al., 2011; Maurer et al., 2012; Maurer et al., 2014; Zanic et al., 2009; Zhang et al., 2015). This difference in affinity likely increases the enrichment of EB1 within the GTP-cap at growing microtubule plus-ends.

Recent work has demonstrated that EB1 can bind to a partial binding pocket composed of 2–3 tubulin subunits, either at the tip of a protofilament, along the side of an exposed protofilament, or at lattice openings within the microtubule (Reid et al., 2019). We describe these exposed, partial binding pockets as ‘protofilament-edge’ sites. Specifically, we use the term ‘protofilament-edge’ to describe any partial EB1 binding site on the microtubule lattice, as opposed to closed (4-tubulin) binding sites. Importantly, we recently reported that the arrival rate of EB1 to 2-tubulin protofilament-edge sites was ~70 fold faster than to closed 4-tubulin pockets, due to a reduced diffusional steric hindrance to binding (Reid et al., 2019). Here, a partial EB1 binding site on the microtubule lattice led to a dramatic reduction in the diffusional steric hindrance that EB1 encounters in order to become properly oriented and then to slide into a closed, 4-tubulin binding pocket. In other words, the expanded physical access that is afforded by EB1 binding to a partial, 2-tubulin binding pocket (as compared to a closed 4-tubulin binding pocket) led to a ~70 fold increase in the EB1 on-rate. Because protofilament-edge sites are present at growing microtubule plus-ends (Atherton et al., 2018; Gudimchuk et al., 2020; Guesdon et al., 2016), we hypothesized that this large difference in EB1 arrival rates could have important repercussions for the efficiency of EB1 tip tracking at growing microtubule plus-ends. We thus predicted that the rapid binding of EB1 to protofilament-edge sites at the growing microtubule plus-end could increase the efficiency of EB1 plus-end tip tracking.

In this work, we generated a single-molecule stochastic simulation that incorporated the assembly and hydrolysis of individual tubulin subunits, as well as the binding and unbinding of EB1 molecules. Importantly, in our simulation, EB1 bound rapidly to protofilament-edge sites, and bound more slowly to closed-lattice sites. In addition, consistent with previous affinity measurements, the off-rate of EB1 from GTP-tubulin sites was low, with higher EB1 off-rates from GDP-tubulin sites. The simulation predicted that rapid binding to protofilament-edge sites increased the efficiency of EB1 tip-tracking at growing microtubule plus-ends. To test this prediction, we used cell-free biophysical assays, as well as live-cell imaging, in combination with a DARPin that binds exclusively to protofilament-edge sites, and whose binding site partially overlaps with the EB1 binding site (Pecqueur et al., 2012). We found that DARPin suppressed EB1 protofilament-edge binding on stabilized microtubules, and led to a disruption of EB1 tip tracking on dynamic microtubules plus-ends, both in cell-free experiments and in cells. Together, our work predicts that protofilament-edge binding, along with a differential EB1 binding affinity for GTP vs GDP tubulin, facilitates efficient EB1 tip tracking.

In this work, we developed a molecular-scale computational simulation that incorporated both tubulin assembly dynamics, and EB1 on-off dynamics. Our simulation predicted that the binding of EB1 to protofilament-edge sites contributes to efficient tip-tracking of EB1 at growing microtubule plus-ends. To test this prediction, we used DARPin, a synthetic peptide, which binds to protofilament-edge sites on microtubules and partially overlaps the EB1 binding site. We found that DARPin blocks EB1 binding at protofilament-edge sites on stabilized microtubules, and importantly, this blocking of EB1 binding to protofilament-edge sites led to a disruption of EB1 tip tracking in dynamic microtubule cell-free assays, and in cells. We conclude that the rapid binding of EB1 to protofilament-edge sites facilitates the tip tracking of EB1 at growing microtubule ends. We note that, while we found that DARPin is not likely to indirectly suppress EB1 tip tracking by altering the plus-end tip conformation or the GTP-tubulin hydrolysis rate (Figure 5C), it remains possible that DARPin treatment could alter the conformation of the tubulin that composes the microtubule tip to indirectly suppress EB1 tip tracking. Furthermore, while the model leads to robust tip tracking by leveraging our previous experimental and simulation results that demonstrate the on-rate of EB1 molecules is rapid to protofilament-edge sites (Reid et al., 2019), we cannot exclude that another end-specific feature, that was not considered here, may be possible.

Previously, a model was developed to explain the peak position of EB1 on the growing microtubule tip, which is slightly distal from the tip of the growing microtubule (Figure 1C; Maurer et al., 2014). Because both our currently described model and the previously described model were able to reproduce the localization of EB1 on the microtubule, we sought to compare and contrast the described mechanisms in each of the two models.

In the previously described work by Maurer et al., 2014, a model was developed that relied on a constant length microtubule template with three EB1 binding ‘zones.’ Here, explicit tubulin assembly dynamics were not included in the model, but rather a constant length microtubule template was employed, in which there was an EB1 binding ‘exclusion zone’ at the tip of the microtubule. A tubulin subunit maturation rate was included in the model, which led to a second zone, slightly distal from the tip of the microtubule, in which EB1 binding was allowed. Finally, a second tubulin subunit maturation rate was employed, which led to a third zone, far from the tip and along the microtubule lattice, in which EB1 disassociation was allowed. The second tubulin subunit maturation rate likely corresponds to GTP-tubulin to GDP-tubulin hydrolysis, which is similar both in the magnitude of the hydrolysis rates employed, and in the EB1 off-rates employed, between the Maurer model and our newly described model (Supplementary file 3).

Thus, the primary difference between the two models was in the binding of EB1 to the microtubule lattice. Here, we predict that the key features of the Maurer model that involved exclusion of EB1 binding to the tip of the microtubule, along with a binding zone just distal to the microtubule tip, are incorporated into our newly described model by the ability of newly arriving tubulin subunits to ‘lock in’ protofilament-edge bound EB1 molecules into a stable 4-tubulin pocket, and by binding of EB1 to protofilament-edge sites along exposed protofilament sides that are distal from the tip of the microtubule. Specifically, in our new model, EB1 molecules arrive rapidly to easily accessible protofilament-edge sites at the growing tip of the microtubule, and to exposed protofilament sides (Figure 7, step 1). Then, upon new tubulin subunit addition, protofilament-edge bound EB1 molecules at the tip of the microtubules are ‘locked in’ to a stable, 4-GTP-tubulin pocket (Figure 7, step 2), leading to a low EB1 off-rate. Thus, in our new model, EB1 accumulates on the GTP-cap at the growing microtubule end (Maurer et al., 2014; Roth et al., 2019). Finally, upon the hydrolysis of GTP-tubulin to GDP-tubulin, the affinity of EB1 for the GDP-tubulin subunits is reduced, and EB1 dissociates from the microtubule (Figure 7, step 3). Thus, we predict that the primary difference between our new model, and the previous Maurer et al model, may be in (1) the inclusion of tubulin assembly dynamics, and (2) rapid EB1 binding to protofilament-edge sites. These features eliminate the requirement for an explicit EB1 binding ‘exclusion zone’ at the tip of the microtubule, and naturally lead to a decrease in signal at the tip of the microtubule.

Figure 7

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The tubulin assembly portion of the model was built on earlier work, in which individual tubulin subunits were allowed to arrive and depart from the growing microtubule plus-end (Margolin et al., 2011; Margolin et al., 2012) (see Methods). Future work will involve examining the effects of a slower tubulin association rate on EB1-occupied protofilament edge sites, and whether EB1 binding to protofilament edge sites could assist in neighboring protofilament zippering on flared microtubule tips. Furthermore, microtubule targeting drugs that suppress the kinetics of tubulin assembly at the growing microtubule plus-end, such as Taxol (Castle et al., 2017), could potentially disrupt EB1 tip tracking by slowing the capture and ‘lock in’ of EB1 to 4-tubulin pocket binding sites (Figure 7, step 2), an idea that could be explored in future work.

As described above, our model predicts that rapid protofilament-edge binding increases the efficiency of EB1 tip tracking. In the simulation, the peak EB1 location is slightly distal from the tip of the growing microtubule (Figure 1C), similar to previous reports (Maurer et al., 2014). We surmise that the peak EB1 location in the simulation is heavily influenced by EB1 molecules that are stably bound to GTP-tubulin closed-lattice sites on the growing microtubule tip. Indeed, by reporting the fraction of EB1 molecules that are bound to GTP-Tubulin protofilament-edge sites as compared to GTP-Tubulin closed lattice sites, we found that there are ~twofold more EB1 molecules bound to closed-lattice GTP-tubulin sites, as compared to protofilament-edge sites, at any one time in the simulation (Figure 1—figure supplement 1H). Furthermore, the number of EB1 binding sites at the tip of each protofilament is explicitly limited by the number of protofilaments in the microtubule (13 binding sites). Thus, EB1 binding to numerous protofilament-edge sites along exposed protofilament sides that are distal from the tip of the microtubule may also contribute to the peak EB1 location. This idea is consistent with results from the ‘split comet’ simulations (Figure 3A and B). Here, by substantially increasing the taper at the tip of the simulated growing microtubule (≤~3 μm), the EB1 comet was greatly extended in length, and altered in configuration, thus shifting the location of EB1 binding (Figure 3B). However, the location of the simulated EB1 peak position was insensitive to small changes in tip taper (Figure 1—figure supplement 1B).

A key aspect of the simulation is that EB1 molecules arrive rapidly to protofilament-edge sites at the tip of the growing microtubule. We propose that, because the on-rate of new tubulin molecules is also rapid (simulated arrival rate for tubulin: ~85 s^–1 at 10 μM tubulin), the simulated EB1 molecules that bind to protofilament-edge sites at the tip of the growing microtubule are quickly ‘locked in’ to a closed-lattice GTP-tubulin binding configuration (Figure 7). Thus, while most of the EB1 molecules at the microtubule tip are indeed bound to closed-lattice GTP-tubulin sites (Figure 1—figure supplement 1H), many of these EB1 molecules likely originated as arrivals to protofilament-edge sites (Figure 1—figure supplement 1A). To test this idea, we ran simulations in which we recorded the initial binding location of EB1, to determine the fraction of EB1 molecules that initially bound to protofilament-edges as compared to closed-lattice positions (Figure 1—figure supplement 1I). We found that ~50% of all EB1 binding events occurred at protofilament-edge sites (Figure 1—figure supplement 1I). However, importantly, the EB1 molecules that initially bound to protofilament-edge sites were heavily concentrated at the growing microtubule tip, while EB1 molecules that bound to closed-lattice sites were more uniformly distributed throughout the microtubule (Figure 1—figure supplement 1J and A; Video 2). This is because closed-lattice binding occurs throughout the microtubule, rather than specifically near to the growing microtubule plus-end.

While protofilament-edge binding is a key aspect of our model, it is important to emphasize that both rapid binding of EB1 to protofilament-edges (50-100:1 edge:lattice), as well as a differential GDP- to GTP-tubulin off-rate (6-12:1 GDP:GTP), were critical to produce robust tip tracking in the model. Because both of these factors contribute to tip tracking, this leads to a highly robust model, that does not require a narrow set of parameter values for either effect, in order to reproduce experimental results (Figure 2G–I). Thus, based on the experimentally measured EB1 on and off rates, a differential GDP- to GTP-tubulin off-rate, together with rapid protofilament-edge binding, was required for EB1 tip tracking in the model. Importantly, we observed robust tip tracking in the model without requiring narrow parameter sets, or by establishing an EB1 binding exclusion zone on the microtubule, as has been previously hypothesized.

Recent work has demonstrated that growing microtubule tips are less homogeneous than previously thought, such that they exhibit a wide range of protofilament lengths between the leading and lagging protofilaments, both in cells and in cell-free experiments (Atherton et al., 2018; Cleary and Hancock, 2021; Coombes et al., 2013; Gudimchuk et al., 2020; Guesdon et al., 2016; Igaev and Grubmüller, 2022). Here, a wide range of protofilament lengths at the growing microtubule end would lead to increased numbers of protofilament-edge sites on the exposed protofilament sides, which, in our model, would increase the EB1 on-rate to the tip. In addition, in our model, other tip configurations, such as partial curvature (Bechstedt et al., 2014; Farmer et al., 2021), or flaring of the growing microtubule plus-end (McIntosh et al., 2018), would also contribute to EB1 tip tracking (Figure 1—figure supplement 1C). Here, partial curvature or flaring requires opening of the closed microtubule tube – indicating that protofilaments or groups of protofilaments are separated from each other. Importantly, separation between protofilaments means that the number of protofilament-edge sites would be enriched, as new protofilament sides would be exposed. Thus, the role of protofilament-edge sites in facilitating EB1 tip tracking could apply to a wide range of growing microtubule tip configurations.

Recently published work provides support for the importance of EB1 protofilament-edge site binding in the efficiency of EB1 tip tracking. Specifically, by using the microtubule polymerase protein XMAP215 in cell-free experiments, the range of protofilament lengths between the leading and lagging protofilaments at the growing microtubule plus-end was increased (Farmer et al., 2021). Importantly, an increase in EB1-GFP intensity at the growing microtubule tip was observed with increased XMAP215-induced tip taper (Farmer et al., 2021). We note that increased tip taper would likely correspond to an increase in the number of protofilament-edge sites along exposed protofilament sides, similar to our split comet phenotype (Figure 3). Thus, XMAP215 could increase the efficiency of EB1 tip tracking by adding new protofilament-edge sites to the growing microtubule plus-end. This suggests that EB1 recruitment, and by extension the recruitment of the +Tip Complex, could be sensitive to the number of protofilament-edge sites at the tip of the growing microtubule. Correspondingly, a recent report found that EB1, and thus CLASP2, is redistributed from the plus-end onto the microtubule lattice in cells subjected to stretch and compression cycles (Li et al., 2023). This result is consistent with the idea that microtubule bending could cause openings and holes in the lattice, leading to the creation of new protofilament-edge sites along the lattice, which in turn causes a redistribution of EB1 from the plus-end tip to the lattice (Figure 4C, blue).

In conclusion, we find that protofilament-edge sites are an important contributing factor for proper EB1 tip tracking along growing microtubule ends, and that EB1 tip tracking is suppressed by blocking the protofilament-edge sites at growing microtubule ends. Therefore, altering the number of exposed protofilament-edge sites at the growing microtubule tip, or along the microtubule lattice, may provide a new mechanism to regulate EB1 localization in cells.

All data generated or analyzed during this study are included in this manuscript and supporting files. Source data files have been provided for Figures 1—6.

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Author details

1. Samuel J Gonzalez

   Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States

   Contribution

   Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5372-8068

2. Julia M Heckel

   Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States

   Contribution

   Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5953-1717

3. Rebecca R Goldblum

   1. Department of Biophysics, Molecular Biology, and Biochemistry, University of Minnesota, Minneapolis, United States
   2. Medical Scientist Training Program, University of Minnesota, Minneapolis, United States

   Contribution

   Software, Writing – review and editing

   Competing interests

   No competing interests declared

4. Taylor A Reid

   Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States

   Contribution

   Software, Writing – review and editing

   Competing interests

   No competing interests declared

5. Mark McClellan

   Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States

   Contribution

   Resources, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

6. Melissa K Gardner

   Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Project administration, Writing – review and editing

   For correspondence

   klei0091@umn.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5906-7363

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