Estradiol pauses microtubule growth without increased incidence of catastrophe events

Ogren, Allison N.; Zimmerman, Noah J.; Stark, Simone; McClellan, Mark; Gonzalez, Samuel J.; Hartweck, Lynn M.; Prins, Kurt W.; Gardner, Melissa K.

doi:10.1038/s42003-025-08362-8

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Published: 18 June 2025

Estradiol pauses microtubule growth without increased incidence of catastrophe events

Communications Biology volume 8, Article number: 938 (2025) Cite this article

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Abstract

Microtubules are long filaments that control cellular structure and influence intracellular transport. The female hormone estrogen has been implicated in alterations to the microtubule network for a variety of cell types. However, the effects of estrogen on individual microtubules are unknown. In this work we systematically investigated a mechanism by which estrogen could alter the length dynamics of individual microtubules. Using multi-line cell assays, cell-free experiments, and computational modeling, we found that estradiol acts to frustrate and pause microtubule growth in cells, independent of estrogen receptor pathways. Specifically, estradiol acts as a switch in which dynamic, growing microtubules were transformed into paused, non-growing microtubules. Estradiol pauses microtubule growth without inducing an increased incidence of catastrophe events, similar to the widely used microtubule poison colchicine. We conclude that estrogen’s ability to limit excessive microtubule proliferation could have important implications for therapeutic approaches in heart disease and breast cancer.

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Introduction

Microtubules are key cellular organelles that are important for many cellular processes. They are stiff, hollow tubes comprised of heterodimers of α- and β-tubulin, arranged in a tube of parallel protofilaments. Throughout the life of a cell, microtubules grow and shorten dynamically and are rearranged according to cellular needs. For example, during cell division microtubules play a critical role in the structure and function of the mitotic spindle. Microtubules have been implicated in disease states, such as in heart failure, where microtubule density is dramatically increased, likely impairing proper cardiac contractility^1,2,3,4,5. For these reasons, microtubules are a key drug target for diseases such as cancers and heart disease.

Interestingly, the female hormone estrogen has been implicated in alterations to the microtubule network for a variety of cell types. Specifically, published work investigating the effects of estrogen on broad-scale microtubule networks suggests that estrogen disrupts microtubule assembly, independently of estrogen receptors, in breast cancer cells and neurons^6,7,8. However, the effects of estrogen on individual microtubules and the molecular mechanisms underlying this disruption have not yet been determined.

In this study, we investigated interactions between the naturally occurring estrogen, 17β-estradiol, and microtubules. We used cell-free biophysical assays as well as cell assays across multiple cell types. We found that estradiol promoted extended pausing of microtubule growth in cells and in cell-free assays, independent of estrogen receptor pathways. Further, estradiol blocks microtubule polymerization via a similar mechanism to the widely used microtubule poison colchicine, which binds in the same pocket on tubulin heterodimers as estradiol^{9,10,11,12,13}. Interestingly, colchicine has been shown to exert beneficial effects on both breast cancer cells and failing hearts by mitigating microtubule growth^5,6,14. Consistent with this result, we found that estradiol restricted microtubule growth in multiple cell types, including breast cancer cells and human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes.

Results

Estradiol treatment promoted extended pausing of microtubules in cell-free assays

Previously published work has suggested that estrogen disrupts microtubule assembly in cells, independently of estrogen receptors^6,7,8. Thus, we first asked whether estradiol could have a direct effect on the growth behavior of microtubules, independent of estrogen receptors or any other microtubule-associated proteins. To assess this possibility, we employed a cell-free assay consisting of reconstituted dynamic microtubules in the presence and absence of estradiol (Fig. 1A). In this assay, the growth behavior of the dynamic microtubule extensions (green) grown from stabilized microtubule seeds (red), was evaluated using total internal reflectance (TIRF) microscopy (Fig. 1A).

**Fig. 1: In cell-free assays and in cells, treatment with 17β-estradiol leads to pauses in microtubule growth.**

We found that estradiol led to extended pause events during microtubule growth, in which a growing microtubule would cease growing, and remain at one length for an extended period (Fig. 1B, center, white arrows). Interestingly, 2-Methoxyestradiol (2-ME), a metabolite of 17β-estradiol, also led to extended pause events during microtubule growth (Fig. 1B, right). The pause time, defined as the total time during a single microtubule growth event in which the microtubule ceased growing and remained at a stable microtubule length (Fig. 1B, white arrows), was significantly increased by both estradiol and 2-ME (Fig. 1C, left; p = 8 × 10⁻⁶ (ANOVA)). Importantly, the fraction of the total microtubule growth time spent in a paused state was increased by ~52% in estradiol, and by ~61% in 2-ME (Fig. 1C, right; p = 1 × 10⁻⁵ (ANOVA)). Thus, estradiol and 2-ME reduced the total microtubule growth time in the cell-free assays, which would be predicted to ultimately diminish microtubule abundance in cells.

Prior to pausing, the control and estradiol-treated microtubules grew at similar rates (Supplementary Fig. 1A, left). This suggested that the size of the “GTP-cap” at the tip of the growing microtubules, which allows the microtubule to persist in a growing state, was similar in the presence and absence of estradiol. Because catastrophe events are typically preceded by a brief “pause” event, leading to hydrolysis of the GTP-tubulin cap¹⁵, we compared the frequency of catastrophe events in the presence and absence of estradiol. Surprisingly, estradiol and 2-ME did not alter catastrophe rates (Supplementary Fig. 1B, left; p = 0.22 (ANOVA)). Thus, estradiol acted as a switch, in which dynamic, growing microtubules were transformed into non-growing microtubules, without an increase in catastrophe frequency.

Estradiol promoted extended pausing of microtubules in LLC-Pk1 cells

To test whether the changes in microtubule growth behavior in the cell-free assays were observed in live cells, we examined how estradiol impacted microtubule dynamics in LLC-Pk1 cells. To visualize microtubule growth behavior, we used a transgenic LLC-Pk1 cell line expressing GFP–tubulin (Fig. 1D, top; Supplementary Movie 1)¹⁶. The duration of pause times was measured (Fig. 1D, bottom, black lines). Increasing concentrations of estradiol led to longer durations of pause times (Fig. 1E, left; p = 2 × 10⁻⁸ (ANOVA); Supplementary Movie 2). We note that, in cells, a large range of estradiol concentrations supports microtubule growth, perhaps due to microtubule-associated proteins that act to buffer the drug effect inside of cells. Importantly, the fraction of microtubule growth time spent in a paused state increased ~5.5-fold in the presence of 200 nM estradiol, which resulted in ~48% of the total microtubule growth time spent in a paused state (Fig. 1E, right). Thus, as in the cell-free assays, estradiol treatment transformed dynamic, growing microtubules into non-growing, paused microtubules.

Colchicine-induced extended microtubule pausing in cell-free assays and in LLC-Pk1 cells

Colchicine and estradiol are predicted to bind in the same pocket between the alpha and beta tubulin monomers (Fig. 1F; Supplementary Movie 3)^{11,17,18,19,20}, as residues within this “colchicine pocket” are sensitive to estradiols (Fig. 1F, magenta residues)^21,22. Thus, we next asked whether estradiol acted similarly to colchicine in altering microtubule dynamics. We first repeated our cell-free assays with colchicine treatment (Fig. 1G). For this assay, the concentration of colchicine was lower than for estradiol, as this was necessary to preserve comparable growth rates of microtubules between control and drug conditions. Microtubule pause time was significantly increased with colchicine treatment (Fig. 1H, left; p = 0.0035 (ANOVA)). Correspondingly, the fraction of the total microtubule growth time spent in a paused state was increased by 79% with colchicine, similar to the 52% increase in estradiol, albeit at a lower drug concentration (Fig. 1H, right; p = 3 × 10⁻⁸ (ANOVA)). As with estradiol, the catastrophe frequency was unchanged by colchicine (Supplementary Fig. 1B, right; p = 0.36 (ANOVA)), and the growth rates prior to pausing were similar (Supplementary Fig. 1A, right). Thus, estradiol and colchicine similarly paused microtubule growth without increasing catastrophe rates, although colchicine appears to be more potent in its effect.

Finally, microtubules in LLC-Pk1 cells were observed for growth behavior with colchicine treatment (Fig. 1I). As with estradiol, a larger range of colchicine concentrations supported microtubule growth in cells as compared to the cell-free assay, perhaps due to microtubule-associated proteins that act to buffer the drug effect inside of cells. Increasing concentrations of colchicine increased the microtubule pause time in cells, similar to estradiol (Fig. 1J, left). In addition, estradiol and colchicine had a similarly profound effect on the fraction of total microtubule growth time spent in a paused state (Fig. 1J, right; p = 0.6 linear regression fraction pause time vs. concentration, comparing the slope for estradiol vs. slope for colchicine). The culmination of these data suggested that estradiol and colchicine act through a similar mechanism to pause microtubule growth in cells.

Estradiol binds to free tubulin prior to polymerization

Previously published work suggests that the mechanism for colchicine-based “poisoning” of a growing microtubule is as follows: (1) colchicine binds to free tubulin, (2) the colchicine–tubulin complex incorporates into the growing microtubule tip, and (3) once incorporated at the tip of a protofilament, the colchicine–tubulin complex “poisons” the growth of the complex-incorporated protofilament, thus stagnating growth^10,13,23. To test whether estradiol acts to pause microtubule growth via a similar mechanism to colchicine, we examined each of these steps.

We first designed a TIRF microscopy experiment to measure the relative binding of estradiol to free tubulin or to polymerized microtubules (Fig. 2A). In this experiment, the binding of fluorescently labeled free tubulin or, alternatively, microtubules, to an anti-estradiol coated coverslip was assessed, in the presence and absence of estradiol (Fig. 2B and C; see the “Methods” section and Supplementary Fig. 1C). Specifically, this experiment was performed by incubating excess free tubulin (or microtubules) together with estradiol, and then this mixture was introduced to an imaging chamber with the anti-estradiol antibody pre-bound to the coverslip. By comparing the resulting images to control images without estradiol, we compared the likelihood of tubulin (or microtubules) binding to the anti-estradiol antibody, in the absence and presence of estradiol (Fig. 2B and C). Thus, we detected whether estradiol was able to form a complex with tubulin (Fig. 2B) (or microtubules (Fig. 2C)), which would then bind to the anti-estradiol antibody that was affixed to the coverslip (Fig. 2A). Experiments with increasing concentrations of anti-estradiol antibody were used to ensure that the concentration of the coverslip-attached antibody was not limiting in the experiment (Supplementary Fig. 1D). Thus, in this experiment, fluorescent tubulin binding to the coverslip represents estradiol–tubulin (or estradiol–microtubule) complexes that were bound to anti-estradiol antibody (Fig. 2A, right).

**Fig. 2: 17β-estradiol preferentially binds to free tubulin heterodimers and this complex is incorporated into the growing microtubule tip.**

The total area of bound fluorescent tubulin or microtubules in each image was normalized to the results for a corresponding matched control (with no estradiol) to account for coverslip quality or other imaging variations (Fig. 2B and C). The normalized area of binding was measured for increasing concentrations of estradiol in the initial incubation with tubulin (or microtubules). There was a significant increase in tubulin coverslip binding in response to increasing concentrations of estradiol (Fig. 2D, left; p < 0.0001 (ANOVA)). Analysis of the total number of binding events per image produced similar results as the area analysis (Supplementary Fig. 1E, right, p < 0.0001 (ANOVA)). In contrast, there was no relationship between estradiol concentration and the binding of polymerized microtubules to anti-estradiol antibody on the coverslip (Fig. 2D, right; p = 0.90 (ANOVA)). Thus, estradiol is preferentially bound to free tubulin over polymerized microtubules.

Estradiol–tubulin molecular complex binds to the growing microtubule tip

We next asked whether the estradiol–tubulin complexes could bind to growing microtubule tips. We used a fluorescent estradiol-GLOW reagent with TIRF microscopy in a cell-free assay (Fig. 2E) and collected images of the stabilized seed templates (Fig. 2E, blue) and the growing microtubule tips (Fig. 2E, green) to evaluate the relative binding of estradiol-GLOW to newly growing microtubule tips, as compared to the pre-grown microtubule seeds (Fig. 2F, left). We found that the binding area fraction of estradiol-GLOW to the growing microtubule tips was ~4-fold higher than the binding area fraction of estradiol-GLOW to the stabilized microtubule seeds (Fig. 2F, right; p < 0.0001 (paired t-test), see the “Methods” section). This suggested that estradiol–tubulin complexes form in solution, and then preferentially interact with the growing end of microtubules. To confirm this finding in cells, we treated LLC-Pk1 cells with estradiol-GLOW. Punctate spots of estradiol-GLOW (Fig. 2G, red) were observed to associate with the tip of a paused microtubule (Fig. 2G, green) in LLC-Pk1 cells (Fig. 2G, cyan arrows; see Supplementary Movie 4). In this time sequence, estradiol-GLOW ultimately incorporates into the lattice after associating at the tip during microtubule assembly (Fig. 2G, yellow arrows).

Computational modeling: growth stalling of a single protofilament results in extended microtubule pausing events

We found that estradiol binds to free tubulin and that this estradiol–tubulin complex then incorporates into the growing microtubule tip, similar to colchicine. It has been suggested that, once incorporated at the tip of an individual protofilament, colchicine–tubulin complexes act to “poison” the growth of that protofilament, thus stagnating microtubule growth^10,13,23. To test this idea for estradiol, we used a computational simulation to predict how microtubule growth behavior and structure may be altered if an estradiol–tubulin complex was stably incorporated at the tip of a single protofilament. We adapted a previously described three-dimensional molecular-scale computational model for microtubule assembly to include the stalling of a single protofilament by the stable incorporation of an estradiol–tubulin complex at the growing microtubule tip (Fig. 3A)^24,25,26,27. This model explicitly considers the contribution of individual protofilament growth and shortening to the overall microtubule growth behavior, via lateral and longitudinal bonds between tubulin subunits^24,26,27. In the simulation, each newly arriving tubulin subunit is stabilized within the growing microtubule lattice by immediately bonding to the lateral neighbors on either side of it, and by establishing a longitudinal bond with the tubulin subunit below it.

**Fig. 3: Simulations predict that growth stagnation of a single protofilament leads to extended microtubule pausing.**

We simulated the incorporation of an estradiol–tubulin complex at the growing microtubule tip by selecting a random protofilament for incorporation (Fig. 3A, center). Once a protofilament was selected for incorporation of the estradiol–tubulin complex, the growth rate for that protofilament was reduced by 99%, thus dramatically slowing an increase in its length (Fig. 3A, right).

Prior to the start of each simulation, a random time point was selected for the incorporation event to occur (T_{incorporation}), and a random protofilament was selected for the incorporation of the estradiol–tubulin complex. Once the simulation started, tubulin subunits arrived, departed, and were stochastically hydrolyzed. When the incorporation event occurred, growth of the selected protofilament was reduced by 99% for the duration of the incorporation event (Fig. 3A, right) while stochastic hydrolysis continued. The estradiol–tubulin complex incorporation event lasted for 60 ± 1 s (mean ± SD) in the simulation, after which growth on the targeted protofilament(s) proceeded as normal. Each simulation ran until a catastrophe event occurred, at which time the simulation would end (Fig. 3B, left, red).

A representative trace showing simulated microtubule length vs time is shown in Fig. 3B (left). The time point at which an estradiol–tubulin complex was incorporated into the tip of a single protofilament (T_{incorporation}) is noted in the trace (black vertical line). We observed a clear difference between the microtubule growth rate prior to the incorporation event (Fig. 3B, left, blue), as compared to the post-incorporation growth rate (Fig. 3B, left, magenta). Quantitatively, we observed a ~4.4-fold decrease in average microtubule growth rate after incorporation, relative to the growth rate prior to incorporation (Fig. 3B, right, p < 0.0001, paired t-test). Thus, the simulation results suggest that the incorporation of an estradiol–tubulin complex at the tip of a single protofilament could efficiently pause overall microtubule growth by inhibiting the growth of the affected protofilament.

In examining our simulation results, we noted that the stagnation in growth of a single protofilament also resulted in an increase in the difference between the lengths of the longest and shortest protofilaments at the microtubule tip (“tip extension length”; Fig. 3A, right; Fig. 3C, left (purple)). In the simulation, we found that the median tip extension length was increased after the estradiol complex incorporation event, relative to pre-incorporation (Fig. 3C, right; 23% increase, p < 0.0001, paired t-test).

Estradiol treatment leads to increased tip extension lengths in cell-free experiments and in cells

The simulation-predicted elongation of the growing microtubule tip was indicative of a mechanism in which the estradiol–tubulin complex prevented the growth of its incorporated protofilament, thus leading to the observed pausing of microtubule growth (Fig. 3A, right). Thus, to test this tip elongation prediction, we measured microtubule tip extension lengths in cell-free and cell-based assays.

First, we turned to transmission electron microscopy (TEM) to directly visualize microtubule tips from our cell-free, purified microtubule assay (Fig. 3D, see the “Methods” section). In this experiment, we used Taxol-stabilized microtubules to prevent depolymerization upon drying on the copper mesh grids, and we pre-treated the tubulin with very high concentrations of estradiol or colchicine to induce frequent tubulin–estradiol interactions, and to limit microtubule length for imaging in the electron microscopy grids. Estradiol treatment during growth increased microtubule tip extension lengths by ~62% (Fig. 3D, right, p = 0.0004, t-test). Similarly, microtubules treated with colchicine had tip extension lengths that were ~50% longer than controls (Fig. 3D, right; p = 0.56, colchicine vs. estradiol-treated microtubules). In agreement with our simulation, the binding of an estradiol- or colchicine-bound tubulin complex likely stalled the growth of the complex-incorporated protofilament, leading to increased tip extension lengths (Fig. 3A, right).

Second, we turned to live LLC-Pk1 cells to determine whether tip extension lengths were increased after estradiol–tubulin complex incorporation. Thus, we revisited our live-cell LLC-Pk1 videos and measured the length of the fluorescent signal drop-off at the tips of microtubules as a read-out for tip extension length (“tip standard deviation”, Fig. 3E, left; see the “Methods” section)²⁸. Here, a larger tip standard deviation (or slower drop off in fluorescence intensity at the microtubule tip) reflects a larger difference in protofilament lengths at the tip of the microtubule, which is consistent with a longer tip extension length (Fig. 3E, left-bottom, blue vs. magenta lines). To examine microtubule tip structure in cells, we selected the highest concentration of estradiol and colchicine from our cell experiments (Fig. 1J). The median microtubule tip standard deviation in cells treated with estradiol was ~56% longer than in controls (Fig. 3E, right, p < 0.0001, t-test). Similarly, cells treated with colchicine had median microtubule tip standard deviations that were ~43% longer than control cells (Fig. 3E, right; p = 0.45 colchicine vs. estradiol-treated cells).

Lastly, to further evaluate how estradiol may impact microtubule growth, microtubules were grown in the presence of the slowly hydrolyzing GTP analog GMPCPP, which allows for microtubule growth beyond the stalled growth of a single protofilament. We reasoned that, if a single protofilament with an estradiol–tubulin complex bound at its tip did not grow efficiently, this protofilament would not grow uniformly with the remainder of the microtubule. This would leave behind defects and holes in the GMPCPP microtubule lattice. Consistent with this idea, estradiol treatment led to defects and holes along the GMPCPP microtubule lattice (Supplementary Fig. 2A, p < 0.0001), and similar results were observed in colchicine (Supplementary Fig. 2B,C).

Estradiol pauses microtubule growth in iPSC-derived cardiomyocytes

To extend our results to distinct cell types with known microtubule network disruptions in disease states, we evaluated the effect of estradiol on microtubule length dynamics in hiPSC-derived cardiomyocytes (Fig. 4A, left; see the “Methods” section). In agreement with our LLC-Pk1 cells, increasing concentrations of estradiol led to longer durations of pause times in derived cardiomyocytes (Fig. 4A, center; p = 6 × 10⁻⁷ (ANOVA)). Further, the fraction of microtubule growth time spent in a paused state increased ~4.6-fold in the presence of 200 nM estradiol, such that ~41% of the total microtubule growth time was spent in a paused state (Fig. 4A, right; p < 0.0001 (ANOVA)), similar to colchicine (Fig. 4A, right, dark blue).

**Fig. 4: 17β-estradiol leads to microtubule pausing and tip elongation in human iPSC-derived cardiomyocytes.**

We then used estradiol-GLOW to examine whether the fluorescent estradiol could associate with microtubule tips in hiPSC-derived cardiomyocytes. We observed the interaction of estradiol-GLOW both with tubulin (Fig. 4B, white arrows), and with microtubules, including at the tips (Fig. 4C, cyan arrows), in derived cardiomyocytes.

Finally, we used fluorescence microscopy to analyze the tip structure in the hiPSC-derived cardiomyocytes. As above, a larger tip standard deviation (or slower drop-off in fluorescence intensity at the microtubule tip) reflects a larger difference in protofilament lengths at the tip of the microtubule, which is consistent with a longer tip extension length (Fig. 4D, left-bottom, blue vs. magenta lines). The median microtubule tip standard deviation in the hiPSC-derived cardiomyocytes treated with estradiol was ~54% longer than in the untreated controls (Fig. 4D, right, p < 0.0001, t-test), similar to colchicine (p = 0.54).

Taken together, these data suggest that, in hiPSC-derived cardiomyocytes, estradiol binds to a tubulin heterodimer in solution, which incorporates into a growing microtubule tip. This incorporation frustrates the growth of a single protofilament and ultimately leads to extended pausing of microtubule growth.

Estradiol-induced pausing is independent of estrogen receptor-α in hiPSC-derived cardiomyocytes

Human cardiac cells have estrogen receptors on the cell membrane, to which 17β-estradiol can bind, and thus trigger signaling pathways within the cell^29,30,31. Therefore, we asked whether the effects of estradiol on microtubules in the hiPSC-derived cardiomyocytes required the estrogen receptor-α. Differentiated cardiomyocytes were incubated with Vepdegestrant, an estrogen receptor-degrading compound (see Methods). Western blots verified the knockdown of the estrogen receptor-α with drug treatment (Supplementary Fig. 3, ~71% knockdown, p = 8 × 10⁻⁶ (ANOVA)), and the fraction of microtubule growth time spent in a paused state was evaluated. For the estrogen receptor-α knockdown cells, the fraction of microtubule growth time spent in a paused state increased ~2.7-fold after estradiol incubation (Fig. 4E, center; p < 0.0001 (t-test)), similar to colchicine (p = 0.58)). Furthermore, the median microtubule tip standard deviation in estrogen receptor-α knockdown cells treated with estradiol was ~18% longer than in the untreated controls (Fig. 4E, right, p = 0.011, t-test), similar to colchicine (p = 0.14). Taken together, these data suggest that estradiol’s effects on microtubules did not require estrogen receptor-α.

Estradiol-mediated microtubule pausing is independent of estrogen receptor activity in breast cancer cells

We next evaluated estradiol’s effect on microtubules in MCF-7 cells, a widely studied estrogen receptor positive (ER+), highly proliferative breast cancer cell line^32,33,34,35 (Fig. 5A, left). In line with other cell types, estradiol increased the fraction of microtubule growth time spent in a paused state by ~2.2-fold, with ~34% of the total microtubule growth time spent in a paused state (Fig. 5A, center, p = 1.4 × 10⁻⁷ (t-test)), similar to colchicine (dark blue). Further, the median microtubule tip standard deviation in the MCF-7 breast cancer cells treated with estradiol was ~62% longer than in the untreated controls (Fig. 5A, right, p = 0.0014, t-test), similar to colchicine (p = 0.39). Taken together, these data supported our estradiol model in a highly proliferative breast cancer cell line.

**Fig. 5: 17β-estradiol leads to microtubule growth pausing in breast cancer cells, independent of estrogen receptors.**

Finally, we evaluated estradiol’s effect on microtubules in MD-MBA-231, a breast cancer, estrogen receptor-negative, cell line^6,32,36. These experiments allowed us to both probe another type of breast cancer cell line and evaluate if estrogen receptor signaling had impacts on the estradiol–microtubule interaction. Again, and consistent with the MCF-7 cells, the MDA-MB-231 cells showed a ~5-fold increase in the fraction of microtubule growth time spent in a paused state in response to estradiol treatment (Fig. 5B, center; p < 0.0001, t-test), similar to colchicine (p = 0.86). Additionally, the median microtubule tip standard deviation in the MDA-MB-231 breast cancer cells treated with estradiol was ~2-fold larger than in the untreated controls (Fig. 5B, right, p = 0.00011 (t-test)), similar to colchicine (p = 0.28). These findings demonstrate that estrogen’s impact on microtubules was independent of estrogen receptors.

Discussion

In this manuscript, we found that estradiol binds to α- and β-tubulin heterodimers, and that this complex incorporates into growing microtubule tips (Fig. 6 steps 1 and 2). Following this incorporation event, the bound estradiol–tubulin complex slows the growth of its attached protofilament on the growing microtubule tip (Fig. 6, step 3). By slowing the growth of its attached protofilament, the estradiol–tubulin complex leads to an elongation of the tip extension length (Fig. 6, step 3), which frustrates and pauses the growth of the microtubule. In multiple cell lines, estradiol pauses microtubule growth, and this newly described property is independent of estrogen receptor activity. Interestingly, this mechanism phenocopies the known microtubule poison colchicine, and suggests that the two compounds exert similar effects on microtubules. Thus, our data describes a mechanism for estradiol-mediated regulation of microtubule polymerization and provides insights into why estradiol may reduce the microtubule network density in cells.

**Fig. 6: Working model for the mechanism of action of estradiol, leading to pausing of microtubule growth.**

The microtubule poison colchicine arrests microtubule polymerization by binding to tubulin, which results in disruption of mitotic spindle formation, thereby suppressing cell division and leading to apoptosis^9,12,37. Consistent with our results, a low concentration of colchicine suppresses microtubule dynamics³⁸. Importantly, at low but clinically relevant concentrations, microtubule targeting drugs such as colchicine and estradiol have the potential to block mitotic progression and induce apoptosis³⁹. Our recent work suggests that partial stabilization of microtubule dynamics during mitosis could inhibit the error correction process and thus stall mitotic progression⁴⁰. Thus, estradiol could act to inhibit cell division in cancer cells, similar to colchicine but perhaps without the potent toxicity to non-cancer cells that is present with colchicine treatment²⁰. Consistent with this idea, previous work suggests that estrogen can disrupt microtubule assembly independently of estrogen receptors in breast cancer cells and neurons^6,8,41.

Proper microtubule growth regulation is critical to mitosis, and thus for decades microtubules have been studied as a therapeutic target to stymie tumor expansion through cell division⁴². Previous studies show that estrogen is capable of disrupting microtubule networks in estrogen receptor-negative cancer cell lines⁶, and thus it is possible that estrogen could stymie growth in receptor-negative cells due to its direct effects on microtubules. However, given the complex tissue environments surrounding tumors, further study is needed to determine the effects of estrogen-mediated microtubule growth pausing in the tumor context.

Increased microtubule density is a hallmark of cardiac cell dysfunction, and there is a growing body of literature indicating that modulating microtubules may augment cardiac function^2,3. In rodents with stimulated pulmonary arterial hypertension, colchicine-mediated microtubule depolymerization exerts therapeutic effects in failing right heart ventricles⁵. Our study suggests that estradiol may have a similar therapeutic effect in failing heart cells.

Our study utilized acute concentrations of estradiol to induce the short-term, rapid effects shown herein with a relatively high frequency. Normal physiological concentrations of estradiol (less than 3 nM)⁴³ are lower than the experimental conditions used in this study. Thus, our experiments, both in the cell-free assays and in cells, were completed at acute/therapeutic concentrations⁴⁴, which are higher than normal physiological concentrations. Longer term cellular treatments at lower or physiological concentrations may contribute to a better understanding of how the direct interaction of estradiol with microtubules could potentially contribute to sex differences in cases such as cardiac function in heart failure⁴⁵.

However, pregnancy is associated with much higher concentrations of 17β-estradiol (~50 nM)⁴⁶, which is a concentration at which we observed significant effects in our study (Fig. 1J). Therefore, the short-term effects noted in our study could have direct relevance to further research into the physiological changes associated with estrogen increases in pregnancy⁴⁶. Specifically, there are cardiovascular changes associated with pregnancy that may be associated with the direct effects of estradiol on microtubules, and this would be an interesting avenue of future inquiry⁴⁷.

To the best of our knowledge, our study is the first to investigate the direct effects of naturally occurring 17β-estradiol on individual microtubules and to determine the mechanism for its effect on microtubule growth behavior. Future studies to determine the downstream cellular consequences of this direct interaction are needed, as well as study into the relative effects of other estrogens commonly present or clinically prescribed. Interestingly, there is evidence that some estrogen analogs that are commonly used as oral contraceptives have differing effects on cell proliferation and shape in estrogen-receptor-negative breast cancer cells, suggesting that there could be a differential effect on microtubules with different oral contraceptive use³⁶. Future studies are needed to determine which, if any, synthetic estrogen analogs directly bind to microtubules and frustrate their growth, similar to what we observed in this study.

Methods

Tubulin purification and labeling

Tubulin was purified from pig brain extract through repeated cycles of polymerization-depolymerization, and then labeled with rhodamine, Alexa-488, or Alexa-647 as previously described^48,49. Rhodamine-labeled tubulin was used for GMPCPP-stabilized microtubule seed preparation. Alexa-647-labeled tubulin was used for free tubulin in microtubule dynamics experiments.

Construction of stabilized GMPCPP microtubule seeds

Stable GMPCPP microtubule seeds were prepared from a mixture of 3.9 µM tubulin (25% rhodamine-labeled, 75% unlabeled), 1 mM GMPCPP, and 1.2 mM MgCl₂ in BRB80. This mixture was incubated first on ice for 5 min, followed by incubation for 2 h at 37 °C⁵⁰. Following incubation, GMPCPP microtubules were diluted into warm BRB80, spun via air-driven centrifuge (Airfuge, Beckman Coulter, 20 psi, 5 min), and resuspended in warm 10 µM Taxol (Sigma-Aldrich, catalog #T7191) in BRB80. These GMPCPP microtubules were stored at 37 °C and used for microtubule dynamics experiments up to 5 days after preparation.

Construction and preparation of flow chambers for TIRF microscopy imaging

Flow chambers were assembled for TIRF microscopy as described previously using brass housing brackets with secured glass coverslips⁴⁸ with the following modification: To create a ‘lane’ for the unidirectional flow of samples, two narrow strips of Parafilm were arranged parallel to each other in between two hydrophobic silanized coverslips. Chambers were subjected to heat to melt the Parafilm strips and create a seal between the coverslips. Before use, the chamber was treated at room temperature with rabbit anti-rhodamine antibody (Thermo Fisher A6397, diluted 1:50 in BRB80 (80 mM potassium PIPES buffer, pH 6.9, 1 mM MgCl₂, 1 mM EGTA, and 100 mM KOH)) for 30 min, followed by blocking with pluronic F127 for at least 20 min.

Dynamic microtubule assay and TIRF microscopy

Rhodamine-labeled GMPCPP microtubule ‘seeds’ were adhered to an anti-rhodamine antibody-coated flow chamber⁴ and washed with 80 µL pre-warmed Imaging Buffer (20 µg/mL glucose oxidase, 10 µg/mL catalase, 20 mM D-glucose, 10 mM DTT, 80 µg/mL casein, and 1% tween-20, in Brb80 (pH 6.9, composed of 80 mM PIPES, 1 mM EGTA, 1 mM MgCl₂, and 100 mM KOH)) to minimize photobleaching. A reaction mixture containing 12 µM tubulin (15.8% Alexa-488-labeled), 40 mM KCl, 1 mM GTP, 1X Imaging Buffer, and Brb80 to make up a total volume of 50 µL was prepared.

For 17β-estradiol (Sigma-Aldrich, catalog #E8875), colchicine (Sigma-Aldrich, catalog #C9754), and 2-methoxyestradiol (Sigma-Aldrich, catalog #M6383) experiments, 100 nM of 17β-estradiol, 40 nM of colchicine, 100 nM 2-methoxyestradiol, or equivalent DMSO was included in the reaction mixture and incubated at 37 °C for 30 min prior to introduction to the imaging chamber. The reaction mixture was centrifuged for 5 min at 4 °C to remove protein aggregates and the supernatant was introduced into the imaging chamber. Movies of dynamic microtubules were acquired at 30 °C for 30 min at 0.2 fps using 488 and 561 nm laser lines with a TIRF microscope (Nikon Eclipse Ti TIRF) fitted with an ×100 oil objective (Nikon CFI Apochromat TIRF 100XC Oil, 1.49 NA) and CCD camera (Andor, iXon3). This TIRF microscopy imaging system allowed for a 160 nm pixel size.

For the estradiol-GLOW experiments, estradiol-GLOW was obtained from Jena Biosciences (#PR958S). 300 nM estradiol-GLOW and 500 nM tubulin (20% Alexa-488-labeled) were mixed and incubated for 30 min at 37 °C to allow binding of estradiol-GLOW to tubulin prior to flowing into the imaging chamber and allowing this tubulin to polymerize onto stabilized seeds.

Dynamic microtubule assay image analysis

Kymographs for each dynamic microtubule extension were generated from movies using ImageJ, using line scans and the “reslice” function, as previously described²⁵. With this experimental protocol, multiple parameters of length dynamics (elongation rate, catastrophe time, and pausing time) were quantified using ImageJ. “Paused” microtubule growth events were defined as periods of time when the growth rate was <20 nm/s. Total growth time was measured from the first point of apparent green signal to the point of catastrophe, in seconds. A minimum of three biological replicates within each treatment group was obtained, and a minimum of 30 total microtubule growth events were analyzed.

Ribbon diagram for colchicine binding and estradiol-sensitive residues

A ribbon diagram for the tubulin heterodimer was modeled using 6xer (RCSB.org) for the tubulin heterodimer with colchicine-bound (blue) in the pocket between α- and β-tubulin. The residues that are sensitive to estradiols^21,22 are highlighted in magenta.

Cell lines and culture conditions

All cell lines were handled under standard culture conditions appropriate for the individual cell line.

The LLC-Pk1 cell line expressing EB1-GFP was a gift from Dr. Patricia Wadsworth⁵¹, and the cell line expressing GFP-Tubulin was a gift from Dr. Lynne Cassimeris¹⁶. The identities of the cell lines (non-human) were authenticated by microscopy observation and analysis. All cell experiments included a minimum of 3 biological replicates and 30 minimum analyzed microtubule growth tracking events.

The LLC-Pk1 cell lines were grown in Optimem media (Thermo Fisher #31985070), 10% fetal bovine sera + penicillin/streptomycin at 37 °C and 5% CO₂. Cells were grown in 14 mm glass bottom dishes for visualization by microscopy.

Human induced pluripotent stem cells (hiPSC) were obtained from the Allen Institute (Cell Line ID: AICS-0012 cl.105). The cells contained GFP-labeled α-tubulin and were cultured using mTeSR1 media on Matrigel-coated plates according to standard practices provided by the Allen Institute. These cells were differentiated to the cardiac myotube stage using standard protocols provided by the Allen Institute for Cell Science⁵². Once the cells were fully differentiated and were observed to be beating, the culture was maintained with new RPMI B27+ media with Penn/Strep every 48–72 h until use.

Human breast cancer cell lines were obtained from the laboratory of Dr. Lang at the University of Minnesota⁵³. Both the MCF-7 and MB-MDA-231 cell lines were cultured using DMEM media with Penn/Strep until use. Microtubules were labeled using Tubulin Tracker™ Deep Red (ThermoFisher, catalog #T34076) for one hour prior to imaging, concurrent with carbon dioxide stable media.

All cell lines were grown on 14 mm glass bottom dishes for visualization by microscopy and treated with carbon dioxide stable media at least 1 h prior to imaging to ensure stable pH conditions throughout the imaging session.

All cell lines tested negative for mycoplasma while in active culture use.

Confocal microscopy

Cells were imaged with a laser scanning confocal microscope (Nikon Ti2, 488 nm laser line) fitted with a ×100 oil objective (Nikon N2 Apochromat TIRF ×100 Oil, 1.49 NA), which allowed for a 0.16 μm pixel size. Time-lapse videos were taken for live-cell imaging with a time step of 3 s to allow for multichannel imaging and to minimize photobleaching in a single area during prolonged imaging sessions.

Western blotting

Human iPSC-derived cardiomyocytes were cultured for 30 h with and without Vepedegestrant treatment (TargetMol, catalog #T39710). A culture plate of these cells, along with MCF-7 (estrogen receptor positive) cells and MDA-MB-231 (estrogen receptor-negative) cells were disadhered with TrypLE, centrifuged, and the cell pellets lysed by boiling in denaturing, reducing electrophoresis sample buffer. Lysate proteins were separated on an acrylamide gel and transferred to PVDF membrane (Millipore #IPVH00010) and detected by western blotting with rabbit anti-estrogen receptor α antibody (Millipore #06-935), mouse anti-GAPDH (GeneTex #GTX627408), and anti-mouse and anti-rabbit HRP-secondary antibodies (CellSignal.com #7076P2, scbt.com #sc-2004) Protein bands were detected with SuperSignal West Femto chemiluminescent reagent (ThermoFisher #34095) by photography with a cooled CCD camera (Alpha Innotech, ChemiImager 5500). Relative band intensities on the images were quantified with ImageJ software (https://fiji.sc) and the estrogen receptor band of each sample was normalized to its corresponding GAPDH band.

Estradiol–tubulin binding assay

TIRF microscopy imaging chambers were constructed as above, except that two parallel chambers were created on one coverslip. Both chambers were treated for at least 30 min with rabbit anti-estradiol antibody (Rabbit monoclonal anti-estradiol, clone RM343: Sigma-Aldrich #SAB5600125) diluted in BRB80, followed by blocking with pluronic F127 for at least 20 min. In a microfuge tube, a tubulin reaction mixture containing 2 µM green labeled tubulin (50% fluorescently labeled) or GMPCPP-stabilized microtubules (as above) in imaging buffer, with and without estradiol at concentrations ranging from 0 to 200 nM, was incubated for 30 min at 37 °C. After blocking was completed, the slide with two chambers was placed on the TIRF microscope. Then, in the “experimental” imaging chamber, the mixture of labeled tubulin (or microtubules) together with estradiol was introduced to the coverslip-adhered anti-estradiol antibody. In a second “control” chamber, built on the exact same coverslip, labeled tubulin (or microtubules) (no estradiol) was introduced. Thus, the control (labeled tubulin/microtubules only) is representative of non-specific binding to the coverslip. The reaction mixtures were allowed to incubate in the imaging chamber for 10 min, at which point the reaction mixtures were gently washed out with BRB80. Three biological replicates with 120 images of each chamber reaction were collected and analyzed with an automated MATLAB object detection program. By normalizing each estradiol experiment to its corresponding no-estradiol control, we reported the total area of tubulin binding over and above non-specific binding to the coverslip.

Estradiol-GLOW in live cells

LLC-Pk1 cells were treated with Tubulin Tracker™ Deep Red (ThermoFisher, catalog #T34076) for one hour prior to live-cell imaging, and 13 nM estradiol-GLOW in DMSO 30 min prior to imaging. Videos were taken of individual cells where microtubules could be clearly seen in a single z-plane with a 3 s time step.

HiPSC-derived cardiomyocytes expressing GFP-tubulin were treated with 13 nM estradiol-GLOW in DMSO 30 min prior to confocal live-cell imaging. Videos were taken of individual cells where microtubules could be clearly seen in a single z-plane.

MATLAB simulation: microtubule assembly

A 3D microtubule assembly simulation was used, as previously described²⁵. Briefly, the 3D simulation accounts for (1) GTP-tubulin subunit arrivals at the tip of the microtubule, (2) GTP and GDP-tubulin subunit departures from the tip, and (3) stochastic GTP hydrolysis of GTP-tubulin subunits that are buried in the lattice. The GTP-tubulin subunit arrival rate is based on the simulated tubulin concentrations and is calculated as previously described⁵⁴. After binding to the lattice, a GTP-tubulin subunit is subject to first-order stochastic hydrolysis, once it is buried in the lattice by the addition of a single longitudinal neighbor on top of it. Hydrolysis of a buried subunit changes its preferred angle in the lattice and therefore results in mechanical strain between lateral and longitudinal neighbors²⁷. To explicitly account for mechanical and chemical energetic differences between tubulin subunits at the microtubule tip based on their lateral and longitudinal neighbor configurations, the subunit departure rate from a given protofilament, k_off,PF, depends on the equilibrium constant, where

$${K}_{{{e}}{{q}}}\equiv \frac{{k}_{{{o}}{{n}},{{P}}{{F}}}}{{k}_{{{o}}{{f}}{{f}},{{P}}{{F}}}}={e}^{-\varDelta {{G}^{0}}_{{{t}}{{o}}{{t}}{{a}}{{l}}}/{k}_{{{B}}}T}$$

(1)

Therefore, by rearrangement:

$${k}_{{off},{PF}}=\frac{{k}_{{on},{PF}}}{{e}^{-\varDelta {G}_{{total}}^{0}}/{k}_{B}T}$$

(2)

where k_off,PF is the off-rate per individual protofilament (s^-1), k_B is Boltzmann’s constant, T is absolute temperature, and ΔG⁰_total is the total free energy of the stabilizing bonds on a specific subunit, given by

$$\Delta {G}_{{total}}^{0}=\sum \Delta {G}_{{Longitudinal}}^{0* }+\sum \Delta {G}_{{Lateral}}^{0}$$

(3)

Here, ΔG^0*_Longitudinal is the total longitudinal bond energy for a given subunit, and ΔG⁰_Lateral is the total lateral bond energy for a given subunit. Both lateral and longitudinal bond energies are penalized by bending and stretching, which decreases the stability of both lateral and longitudinal bonds and thus increases the off-rate of the associated tubulin subunit from the lattice.

As the stochastic 3D model computer simulation proceeds, the bending and stretching energy penalty introduced by stochastic hydrolysis of buried subunits in the lattice produces a behavior in which slow microtubule growth is followed by periods of rapid shortening (catastrophe events). Each microtubule simulation ended when a catastrophe event happened.

Simulations were performed using MATLAB (Mathworks, Natick, MA), as described previously²⁷, with exceptions as described below. General simulation parameters are listed in Table 1.

Table 1 Microtubule dynamics simulation parameters

Full size table

To properly simulate our in vitro experiments, we used a “3-state” model in the simulation, as previously described²⁴. Here, all simulations started with a microtubule “seed” that was composed of GMPCPP-tubulin. Simulation parameters for the GMPCPP seeds are as shown in Table 1 and are similar to previous work⁵⁵. Briefly, subunits could dissociate from the tip of a GMPCPP-seed, however, the GMPCPP-tubulin subunits in the seed did not hydrolyze, and so the effective off-rate for GMPCPP-tubulin subunits from the seed was low relative to the GTP/GDP-tubulin extensions which grew from the seed. The inclusion of a stable GMPCPP seed in the simulation eliminated the requirement for starting the simulation with an arbitrary GTP-cap size and so allowed dynamic microtubules to naturally nucleate from the seed and thus develop a GTP-cap as part of the assembly process. The rules for GTP-tubulin assembly from the GMPCPP seed were similar to the previously described 3D model²⁷, where GTP-tubulin subunits were stochastically incorporated at the microtubule tip and were then stochastically hydrolyzed to GDP-tubulin once the subunits were buried in the lattice.

MATLAB simulation: protofilament targeting

At the start of each microtubule simulation, the following additional variables were assigned:

N_targeted = number of protofilaments to be shut off (targeted) during the simulation. In all simulations, 1 protofilament was targeted.

PF_targeted = individual protofilament number that is to be targeted. Protofilament numbers ranged from 1 to 13.

T_targeted = time at which the protofilament(s) would be targeted. A random protofilament targeting time was selected from 0 to 120 s.

D_targeted = mean duration of time that the protofilaments were targeted and thus prevented from growing.

The duration of targeting (D_targeted) was calculated for each simulation by selecting from a random Gaussian distribution with mean equal to 60 s, and a standard deviation of 1 s. The time was recorded for each simulation step. At T_targeted, the on-rate of GTP-tubulin subunits to the targeted protofilament (PF_targeted) was reduced by 99%. This on-rate adjustment was kept in place until the elapsed time was greater than or equal to T_targeted + D_targeted. The simulation then continued until the microtubule length was less than or equal to the stabilized GMPCPP seed length (e.g., a catastrophe event occurred).

Growing microtubule tip assay for transmission electron microscopy

After stabilized microtubules were grown and spun down to remove any excess free tubulin, a dynamic growth mixture containing 12 μM free unlabeled tubulin, 1 mM GTP, 40 mM KCl, Brb80 buffer, and one of the following: 500 nM estradiol, 250 nM colchicine, or equivalent concentration of DMSO was prepared and allowed to incubate for 30 min at 37 °C to allow for binding of estradiol or colchicine to the free tubulin in solution. Then, the stabilized microtubule seeds were added to this mixture and gently mixed, and 10 μL of this mixture was then applied to pre-warmed 300-mesh carbon-coated copper grids as previously described⁴. The microtubules were allowed to grow on the grids for 5 min prior to staining with 3 drops of 1% uranyl acetate for 1 min. Filter paper was then used to wick away the excess stain from the grid. The grid was left to dry by air for 10 min and stored. Experiments were repeated within each treatment group a minimum of 3 times.

Transmission electron microscopy imaging and analysis

To obtain images of growing microtubule tips stained on carbon-coated copper grids, a Thermo Fisher Talos F200X transmission electron microscope was used (200 keV acceleration voltage, University of Minnesota Shepherd Labs). Images were taken at various magnifications and transferred as PNG files for analysis.

Images were first processed using ImageJ to set appropriate scales, then relative tip lengths were measured from an assigned origin point. The length of the longest protofilament from this point was measured, followed by the length of the shortest protofilament, and the difference between these lengths was calculated and used to represent the tip extension length in nanometers.

Microtubule damage assay and TEM imaging

Stabilized microtubules were grown as above with 1 mM GMPCPP at 37 °C and 500 nM estradiol, 250 nM colchicine, or equivalent concentration of DMSO. These microtubules were then spun down and resuspended. These microtubules were then applied to copper TEM grids and imaged on a Tecnai T12 transmission electron microscope. Images were analyzed by manually measuring the length of damage sites relative to total length of microtubules. Experiments within each treatment group were repeated a minimum of 3 times.

Microtubule repair assay

Rhodamine labeled GMPCPP microtubules were spun down and resuspended. The microtubules ewre then exposed to a mixture of A649-tubulin (40% labeled), 100 mM MgCl₂, and 10 mM GTP in Brb80 buffer and incubated for 30 min at 37 °C to allow for “repair” in any growth damage sites left behind from being grown in the presence of estradiol or colchicine. Previously published MATLAB code was used to detect the area of green tubulin binding to red microtubules^24,50. Experiments within each treatment group were repeated 3 times, and a minimum of 30 images were used for analysis within each group.

Analysis of microtubule tip standard deviation

Videos recorded of live-cells with fluorescently labeled microtubules were converted to 16-bit stacks in ImageJ and shortened to eliminate periods of catastrophe or microtubule shortening. Then, these stacks were analyzed using previously published MATLAB code for measuring the standard deviation of fluorescence signal drop-off at growing microtubule tips, as previously described²⁸. A minimum of ten different cells within each treatment category for each cell type was analyzed, and the median and standard error for tip standard deviation was reported.

Statistics and reproducibility

All reported t-tests in the manuscript were either 2-tailed t-tests (2 samples assuming unequal variance), or single factor ANOVA tests. t-tests and ANOVA tests were performed using Microsoft Excel or GraphPad Prism. t-tests were used in all cases where sample data could be collected for individual microtubules or events. For each experiment, the number of replicates and sample sizes are noted and explained in each figure legend.

Box plots

For each box and whisker plot, the marker indicates the mean value, and the line across the center of the box indicates the median. The box limits show the lower and upper quartile of the data set. The whiskers show the minimum and maximum data values, excluding outliers.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All primary datasets (generated during the study) have been shared in Figshare.com. https://doi.org/10.6084/m9.figshare.29161676.v1⁵⁶.

Code availability

All code used in the manuscript will be provided upon request, with no access restrictions.

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Acknowledgements

We are grateful for the generous gift of the MCF-7 and MDA-MB-231 cell lines from the laboratory of Carol Lang, Ph.D. at the University of Minnesota. Additionally, we thank the University of Minnesota Characterization Facility (CharFac) in Shepherd Labs and especially Dr. Michael Odlyzko for extensive training and access to the Transmission Electron Microscopes. The Gardner laboratory is supported by a National Institutes of Health grant NIGMS R35-GM126974. K.W.P. is funded by NIH R01s HL158795 and HL162927. A.O. was supported by the National Institute of Health NIGMS Training Program in Muscle Research T32AR007612, and American Heart Association predoctoral training grant 23PRE1019410. We thank members of the Gardner, Courtemanche, von Diezmann, and Titus laboratories for helpful discussions.

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Authors and Affiliations

Department of Cell Biology and Genetics, University of Minnesota, Minneapolis, MN, USA
Allison N. Ogren, Noah J. Zimmerman, Simone Stark, Mark McClellan, Samuel J. Gonzalez & Melissa K. Gardner
University of Minnesota, Lillehei Heart Institute, Minneapolis, MN, USA
Lynn M. Hartweck & Kurt W. Prins

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Allison N. Ogren
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Noah J. Zimmerman
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Simone Stark
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Mark McClellan
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Samuel J. Gonzalez
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Lynn M. Hartweck
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Kurt W. Prins
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Melissa K. Gardner
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Contributions

A.O. conceived and performed experiments, performed analysis, drafted paper; N.Z. performed analysis; S.S. performed experiments; M.M. performed experiments and analysis; S.G. performed analysis; L.H. provided reagents; K.P. conceived work, supervised, and reviewed data and the manuscript; M.G. conceived work, supervised, and reviewed data and the manuscript.

Corresponding authors

Correspondence to Kurt W. Prins or Melissa K. Gardner.

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Ogren, A.N., Zimmerman, N.J., Stark, S. et al. Estradiol pauses microtubule growth without increased incidence of catastrophe events. Commun Biol 8, 938 (2025). https://doi.org/10.1038/s42003-025-08362-8

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Received: 19 February 2025
Accepted: 09 June 2025
Published: 18 June 2025
DOI: https://doi.org/10.1038/s42003-025-08362-8