Estrogen exerts cardioprotective effects via microtubule regulation.

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.

The female-predominate sex hormone 17β-estradiol exerts cardioprotective effects via multiple mechanisms. Available data demonstrate 17β-estradiol modulates microtubule dynamics in vitro, but its effects on pathogenic microtubule remodeling in pressure-overloaded cardiomyocytes are unexplored. Here, we show 17β-estradiol directly blunts microtubule polymerization in vitro, counteracts endothelin-mediated microtubule remodeling in iPSC-cardiomyocytes, and mitigates microtubule stabilization in pulmonary artery banded right ventricular cardiomyocytes. 17β-estradiol treatment blunts cardiomyocyte and nuclear hypertrophy, restores t-tubule architecture, and prevents mislocalization of connexin-43 in RV cardiomyocytes of pulmonary artery banded rats. These cellular phenotypes are paired with significant improvements in RV function. Thus, we propose 17β-estradiol exerts cardioprotective effects via direct modulation of microtubules in addition to its well ascribed signaling functions.

EB1 is a key cellular protein that delivers regulatory molecules throughout the cell via the tip-tracking of growing microtubule plus-ends. Thus, it is important to understand the mechanism for how EB1 efficiently tracks growing microtubule plus-ends. It is widely accepted that EB1 binds with higher affinity to GTP-tubulin subunits at the growing microtubule tip, relative to GDP-tubulin along the microtubule length. However, it is unclear whether this difference in affinity alone is sufficient to explain the tip-tracking of EB1 at growing microtubule tips. Previously, we found that EB1 binds to exposed microtubule protofilament-edge sites at a ~70 fold faster rate than to closed-lattice sites, due to diffusional steric hindrance to binding. Thus, we asked whether rapid protofilament-edge binding could contribute to efficient EB1 tip tracking. A computational simulation with differential EB1 on-rates based on closed-lattice or protofilament-edge binding, and with EB1 off-rates that were dependent on the tubulin hydrolysis state, robustly recapitulated experimental EB1 tip tracking. To test this model, we used cell-free biophysical assays, as well as live-cell imaging, in combination with a Designed Ankyrin Repeat Protein (DARPin) that binds exclusively to protofilament-edge sites, and whose binding site partially overlaps with the EB1 binding site. We found that DARPin blocked EB1 protofilament-edge binding, which led to a decrease in EB1 tip tracking on dynamic microtubules. We conclude that rapid EB1 binding to microtubule protofilament-edge sites contributes to robust EB1 tip tracking at the growing microtubule plus-end.

Accurate genome segregation in mitosis requires that all chromosomes are bioriented on the spindle. Cells monitor biorientation by sensing tension across sister centromeres. Chromosomes that are not bioriented have low centromere tension, which allows Aurora B (yeast Ipl1) to perform error correction that locally loosens kinetochore-microtubule attachments to allow detachment of microtubules and fresh attempts at achieving biorientation. However, it is not known whether low tension recruits Aurora B to centromeres or, alternatively, whether low tension directly activates Aurora B already localized at centromeres. In this work, we experimentally induced low tension in metaphase Saccharomyces cerevisiae yeast cells, then monitored Ipl1 localization. We find low tension recruits Ipl1 to centromeres. Furthermore, low tension-induced Ipl1 recruitment depended on Bub1, which is known to provide a binding site for Ipl1. In contrast, Top2, which can also recruit Ipl1 to centromeres, was not required. Our results demonstrate cells are sensitive to low tension at centromeres and respond by actively recruiting Ip1l for error correction.

During mitosis, sister chromatids are stretched apart at their centromeres via their attachment to oppositely oriented kinetochore microtubules. This stretching generates inwardly directed tension across the separated sister centromeres. The cell leverages this tension signal to detect and then correct potential errors in chromosome segregation, via a mechanical tension signaling pathway that detaches improperly attached kinetochores from their microtubules. However, the sequence of events leading up to these detachment events remains unknown. In this study, we used microfluidics to sustain and observe low-tension budding yeast metaphase spindles over multiple hours, allowing us to elucidate the tension history prior to a detachment event. We found that, under conditions in which kinetochore phosphorylation weakens low-tension kinetochore-microtubule connections, the mechanical forces produced via the dynamic growth and shortening of microtubules is required to efficiently facilitate detachment events. Our findings underscore the critical role of robust kinetochore microtubule dynamics in ensuring the fidelity of chromosome segregation during mitosis.

Significance Kinesin-14 motors represent an essential class of molecular motors that bind to microtubules and then walk toward the microtubule minus-end. However, whether these motors can interact with growing plus-ends of microtubules to impact the lengthening of microtubules remains unknown. We found that Kinesin-14 motors could bind to a protein that resides at growing microtubule plus-ends and then pull this protein away from the growing end. This interaction acted to disrupt microtubule growth and decrease microtubule lengths in cells, likely by exerting minus-end–directed forces at the microtubule tip to alter the configuration of the growing microtubule plus-end. This work demonstrates general principles for the diverse roles that force-generating molecular motors can play in regulating cellular processes.

During budding yeast mitosis, duplicated chromosomes are aligned at the center of the metaphase mitotic spindle, and the centromeres are stretched by forces generated within the mitotic spindle. In response to these stretching forces, mechanical tension builds up in the centromeric chromatin. The magnitude of this tension is detected by the cell to signal the attachment configuration of the sister chromosomes: a high tension signal would indicate that sister chromosomes are properly attached to opposite spindle poles, while a low tension signal could indicate the lack of a bipolar attachment. A low tension signal drives the cell to correct improper attachments in metaphase, thus preventing potential errors in anaphase chromosome segregation. In this paper, we describe a microscopy-based method to directly measure the magnitude of centromere tension in budding yeast metaphase spindles. The advantage of this method is that quantitative tension estimates are obtained without perturbing spindle and/or chromosome structure and as cells progress normally through mitosis.

In this issue, Ayukawa, Iwata, Imai, and colleagues (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202007033) use rapid temporal and high-spatial-resolution electron microscopy imaging to examine the earliest stages of new microtubule nucleation. They discover that straightening of curved tubulin oligomers increases the efficiency of microtubule nucleation.

In invertebrates, UNC-45 regulates myosin stability and functions. Vertebrates have two distinct isoforms of the protein: UNC-45B, expressed in muscle cells only, and UNC-45A, expressed in all cells and implicated in regulating both non-muscle myosin II (NMII)- and microtubule (MT)-associated functions. Here, we show that, in vitro and in human and rat cells, UNC-45A binds to the MT lattice, leading to MT bending, breakage and depolymerization. Furthermore, we show that UNC-45A destabilizes MTs independent of its C-terminal NMII-binding domain and even in the presence of the NMII inhibitor blebbistatin. These findings identified UNC-45A as a novel type of MT-severing protein with a dual non-mutually exclusive role in regulating NMII activity and MT stability. Because many human diseases, from cancer to neurodegenerative diseases, are caused by or associated with deregulation of MT stability, our findings have profound implications in the biology of MTs, as well as the biology of human diseases and possible therapeutic implications for their treatment.This article has an associated First Person interview with the joint first authors of the paper.

In the failing heart, the cardiac myocyte microtubule network is remodeled, which contributes to cellular contractile failure and patient death. However, the origins of this deleterious cytoskeletal reorganization are unknown. We now find that oxidative stress, a condition characteristic of heart failure, leads to cysteine oxidation of microtubules. Our electron and fluorescence microscopy experiments revealed regions of structural damage within the microtubule lattice that occurred at locations of oxidized tubulin. The incorporation of GTP-tubulin into these damaged, oxidized regions led to stabilized “hot spots” within the microtubule lattice, which suppressed the shortening of dynamic microtubules. Thus, oxidative stress may act inside of cardiac myocytes to facilitate a pathogenic shift from a sparse microtubule network into a dense, aligned network. Our results demonstrate how a disease condition characterized by oxidative stress can trigger a molecular oxidation event, which likely contributes to a toxic cellular-scale transformation of the cardiac myocyte microtubule network.

EB1 is a protein that tracks growing microtubule plus-ends, manifesting as a comet-like structure slightly behind the microtubule tip (‘EB1 tip tracking’). EB1 localizes proteins to growing microtubule plus-ends that normally have little or no affinity for microtubules, and thus EB1 plays a central role in multiple cellular processes. The canonical model for EB1 tip tracking suggests that EB1 binds a complete pocket between four GTP-tubulin heterodimers, and then dissociates as GTP hydrolyzes to GDP. Recent work, however, suggests that EB1 may preferentially bind to incomplete binding pockets, which have less than four tubulin heterodimers comprising the binding pocket. Here, EB1 has a dramatically increased binding rate onto incomplete binding pockets (edge sites) as compared to complete binding pockets (lattice sites), due to steric hindrance to binding. This finding suggests that increased microtubule ‘tip tapering’, which is characterized by a larger difference between the shortest and longest microtubule protofilaments, could enhance the efficiency of EB1 tip tracking due to increased availability of edge versus lattice sites. Thus, to examine the degree to which microtubule tip tapering could predict EB1 tip tracking, we created a stochastic model that allowed us to quantitatively compare the effect of characteristics, such as the degree of microtubule tip tapering, EB1 on-rates, and EB1 off-rates, on the efficiency of EB1 tip tracking. Our model predicts that variations in microtubule tip tapering, as well as EB1 on and off rates, all alter the net amount of EB1 binding to microtubules. However, microtubule tip tapering uniquely controls the EB1 localization profile relative to the microtubule tip, thus providing a testable model prediction. Our model therefore suggests that the degree of microtubule tapering can uniquely alter the binding profile of EB1 along the microtubule plus end.

Microtubules are dynamic, filamentous polymers of α/β-tubulin heterodimers that form a complex network throughout the cytoplasm. In cells, the microtubule cytoskeleton is subject to numerous post-translational modifications that confer functional specialization. Unlike most post-translational modifications that occur at the tubulin C-terminus, the acetylation of -tubulin (Lys40) occurs within the hollow microtubule lumen. Recent work suggests that structural defects, or ‘holes’ within the microtubule lattice, allow α-tubulin acetyltransferase 1 (αTAT1), the enzyme responsible for microtubule acetylation, to enter the hollow microtubule lumen, where it catalyzes tubulin acetylation. Using cell-free reconstitution experiments, fluorescence microscopy, and transmission electron microscopy, we found that pathological oxidative stress alters microtubule structure by introducing holes and structural defects within the lattice. Further, we found that oxidative stress accelerates acetylation of tubulin subunits within the microtubule lumen, but has little effect on the rate of microtubule detyrosination, which occurs on the external microtubule surface. Our cell-free reconstitution experiments and computational simulations together suggest that it is the location of aTAT1 binding within the microtubule lumen, and the lattice damage caused by oxidative stress, that leads to increased acetylation in oxidative stress-treated microtubules. Consistent with these results, oxidative stress promotes microtubule acetylation, but not detyrosination, in cells. This work provides insight into mechanisms through which pathological oxidative stress biochemically modifies and remodels the microtubule network in cells.

Neuronal axons terminate as synaptic boutons that form stable yet plastic connections with their targets. Synaptic bouton development relies on an underlying network of both long-lived and dynamic microtubules that provide structural stability for the boutons while also allowing for their growth and remodeling. However, a molecular-scale mechanism that explains how neurons appropriately balance these two microtubule populations remains a mystery. We hypothesized that α-tubulin acetyltransferase (αTAT), which both stabilizes long-lived microtubules against mechanical stress via acetylation and has been implicated in promoting microtubule dynamics, could play a role in this process. Using the Drosophila neuromuscular junction as a model, we found that non-enzymatic dαTAT activity limits the growth of synaptic boutons by affecting dynamic, but not stable, microtubules. Loss of dαTAT results in the formation of ectopic boutons. These ectopic boutons can be similarly suppressed by resupplying enzyme-inactive dαTAT or by treatment with a low concentration of the microtubule-targeting agent vinblastine, which acts to suppress microtubule dynamics. Biophysical reconstitution experiments revealed that non-enzymatic αTAT1 activity destabilizes dynamic microtubules but does not substantially impact the stability of long-lived microtubules. Further, during microtubule growth, non-enzymatic αTAT1 activity results in increasingly extended tip structures, consistent with an increased rate of acceleration of catastrophe frequency with microtubule age, perhaps via tip structure remodeling. Through these mechanisms, αTAT enriches for stable microtubules at the expense of dynamic ones. We propose that the specific suppression of dynamic microtubules by non-enzymatic αTAT activity regulates the remodeling of microtubule networks during synaptic bouton development.

Introduction: Female sex is associated with better right ventricular (RV) function in pulmonary hypertension (PH). The female sex hormone 17-beta-estradiol is postulated to mediate these differences, but the molecular mechanisms underlying these observations are incompletely defined. Interestingly, 17-beta-estradiol induces microtubule depolymerization in cell culture, which may be relevant to RV dysfunction because microtubule remodeling promotes RV dysfunction via dysregulation of junctophilin-2 (MT-JPH2 pathway). We speculate that 17-beta-estradiol modulates the MT-JPH2 pathway and preserves RV function in PH. Methods: Pressure-volume assessments quantified RV function in monocrotaline (MCT) and pulmonary artery-banded (PAB) rats. Immunoblots quantified the tubulin and junctophilin-2 protein content in RV extracts. Echocardiography quantified RV function by RV fractional area change for 379 human PH patients. Sedimentation experiments, fluorescence-based polymerization assessments, and total internal reflection fluorescence (TIRF) microscopy examined the effects of 17-beta-estradiol on microtubules. Results: Female sex results in better RV function and less dysregulation of the MT-JPH2 pathway in both MCT and PAB rats. Moreover, in human PH, female sex was associated with better RV function, which persisted after adjusting for afterload. 17-beta-estradiol inhibited microtubule polymerization in vitro and TIRF microscopy showed 17-beta-estradiol localized to microtubule tips and prevented further microtubule polymerization. Conclusions: Preclinical and human studies show that females are better able to tolerate RV pressure overload. There are blunted microtubule-mediated t-tubule remodeling and preserved RV function in female MCT and PAB rats. In human PH, females have better RV function. These findings may be due to 17-beta-estradiol directly regulating microtubule dynamics as shown by sedimentation and polymerization assays and TIRF microscopy. These results provide additional insights into sex-differences in RV function in PH.

During mitosis, tension develops across the centromere as a result of spindle-based forces. Metaphase tension may be critical in preventing mitotic chromosome segregation errors, however, the nature of force transmission at the centromere and the role of centromere mechanics in controlling metaphase tension remains unknown. We combined quantitative, biophysical microscopy with computational analysis to elucidate the mechanics of the centromere in unperturbed, mitotic human cells. We discovered that the mechanical stiffness of the human centromere matures during mitotic progression, which leads to amplified centromere tension specifically at metaphase. Centromere mechanical maturation is disrupted across multiple aneuploid cell lines, leading to a weak metaphase tension signal. Further, increasing deficiencies in centromere mechanical maturation are correlated with rising frequencies of lagging, merotelic chromosomes in anaphase, leading to segregation defects at telophase. Thus, we reveal a centromere maturation process that may be critical to the fidelity of chromosome segregation during mitosis.

Microtubules are cylindrical cytoskeletal polymers composed of a/β-tubulin heterodimers that make up an ordered tubulin lattice. Microtubules display dynamic length changes, termed “dynamic instability”, in which slow elongation phases are followed by rapid shortening. In cells, microtubules form a network that is a key component of the cellular cytoskeleton. Under pathological conditions of oxidative stress, we and others have found that cardiomyocytes display a denser microtubule cytoskeleton, which may lead to the progressive structural and functional cellular changes associated with myocardial ischemia and systolic dysfunction. This reorganization of the microtubule network occurs despite only small increases in tubulin expression, suggesting that alterations to microtubule length regulation are involved. Using biophysical reconstitution experiments and live-cell imaging, we found that oxidative stress directly provokes microtubule ‘rescue,’ the transition from rapid microtubule shortening to slow elongation, while it has little effect on other microtubule dynamic instability parameters or the microtubule nucleation rate. To explore a mechanism for this observation, we used electron microscopy, and observed structural damage, consisting of holes and sheet-like structures, under conditions of moderate oxidative stress. Further, using a quantitative two-color tubulin “repair” assay (Reid et al, 2017), we found that repair of structural defects within the microtubule lattice via the incorporation of free tubulin was 133% higher under conditions of oxidative stress compared to controls. Such repaired regions have been termed ‘rescue islands’ because they facilitate rescue events, and thus promote net microtubule elongation (Dimitrov et al., 2008). We conclude that microtubule structural damage may explain our observations of oxidative stress-mediated increases in microtubule density in cardiomyocytes, potentially providing insight into the progressive myocardial changes that accompany Ischemic Heart Disease.

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high-affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a ~ 6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the blunting of growing microtubule plus-ends by Vinblastine was correlated with reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.

During mitosis, motor proteins associate with microtubules to exert pushing forces that establish a mitotic spindle. These pushing forces generate opposing tension in the chromatin that connects oppositely attached sister chromatids, which may then act as a mechanical signal to ensure the fidelity of chromosome segregation during mitosis. However, the role of tension in mitotic cellular signaling remains controversial. In this study, we generated a gradient in tension over multiple isogenic budding yeast cell lines by genetically altering the magnitude of motor-based spindle forces. We found that a decreasing gradient in tension led to an increasing gradient in the rates of kinetochore detachment and anaphase chromosome mis-segregration, and in metaphase time. Simulations and experiments indicated that these tension responses originate from a tension-dependent kinetochore phosphorylation gradient. We conclude that the cell is exquisitely tuned to the magnitude of tension as a signal to detect potential chromosome segregation errors during mitosis.