Microtubule Ends Research Articles

Human CLASP2 specifically regulates microtubule catastrophe and rescue.

Cytoplasmic linker-associated proteins (CLASPs) are microtubule-associated proteins essential for microtubule regulation in many cellular processes. However, the molecular mechanisms underlying CLASP activity are not understood. Here, we use purified protein components and total internal reflection fluorescence microscopy to investigate the effects of human CLASP2 on microtubule dynamics in vitro. We demonstrate that CLASP2 suppresses microtubule catastrophe and promotes rescue without affecting the rates of microtubule growth or shrinkage. Strikingly, when CLASP2 is combined with EB1, a known binding partner, the effects on microtubule dynamics are strongly enhanced. We show that synergy between CLASP2 and EB1 is dependent on a direct interaction, since a truncated EB1 protein that lacks the CLASP2-binding domain does not enhance CLASP2 activity. Further, we find that EB1 targets CLASP2 to microtubules and increases the dwell time of CLASP2 at microtubule tips. Although the temporally averaged microtubule growth rates are unaffected by CLASP2, we find that microtubules grown with CLASP2 display greater variability in growth rates. Our results provide insight into the regulation of microtubule dynamics by CLASP proteins and highlight the importance of the functional interplay between regulatory proteins at dynamic microtubule ends.

Kinesin expands and stabilizes the GDP-microtubule lattice.

Kinesin-1 is a nanoscale molecular motor that walks towards the fast growing (plus) ends of microtubules, hauling molecular cargo to specific reaction sites in cells. Kinesin-driven transport is central to the self-organisation of eukaryotic cells and shows great promise as a tool for nano-engineering1. Recent work hints that kinesin may also play a role in modulating the stability of its microtubule track, both in vitro2,3 and in vivo4, but results are conflicting5–7 and mechanisms are unclear. Here we report a new dimension to the kinesin-microtubule interaction, whereby strong-binding state (ATP-bound and apo) kinesin-1 motor domains inhibit the shrinkage of GDP-microtubules by up to 2 orders of magnitude and expand their lattice spacing by ~1.6%. Our data reveal an unexpected mechanism by which the mechanochemical cycles of kinesin and tubulin interlock, allowing motile kinesins to influence the structure, stability and mechanics of their microtubule track.

Lateral attachment of kinetochores to microtubules is enriched in prometaphase rosette and facilitates chromosome alignment and bi-orientation establishment

Faithful chromosome segregation is ensured by the establishment of bi-orientation; the attachment of sister kinetochores to the end of microtubules extending from opposite spindle poles. In addition, kinetochores can also attach to lateral surfaces of microtubules; called lateral attachment, which plays a role in chromosome capture and transport. However, molecular basis and biological significance of lateral attachment are not fully understood. We have addressed these questions by focusing on the prometaphase rosette, a typical chromosome configuration in early prometaphase. We found that kinetochores form uniform lateral attachments in the prometaphase rosette. Many transient kinetochore components are maximally enriched, in an Aurora B activity-dependent manner, when the prometaphase rosette is formed. We revealed that rosette formation is driven by rapid poleward motion of dynein, but can occur even in its absence, through slow kinetochore movements caused by microtubule depolymerization that is supposedly dependent on kinetochore tethering at microtubule ends by CENP-E. We also found that chromosome connection to microtubules is extensively lost when lateral attachment is perturbed in cells defective in end-on attachment. Our findings demonstrate that lateral attachment is an important intermediate in bi-orientation establishment and chromosome alignment, playing a crucial role in incorporating chromosomes into the nascent spindle.

Human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore–microtubule attachments

Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska complex and Ndc80 complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore-microtubule attachments in vivo. While purified Ska complex tracks with disassembling microtubule tips, it remains unclear whether the Ska complex-microtubule interaction is sufficiently strong to make a significant contribution to kinetochore-microtubule coupling. Alternatively, Ska complex might affect kinetochore coupling indirectly, through recruitment of phosphoregulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Cross-linking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex microtubule-binding domain or the Ska3 C terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together, our results indicate that the Ska complex can directly strengthen the kinetochore-microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the microtubule.

Molecular Requirements for the Transition from Lateral to End-on Microtubule Binding and Dynamic Coupling

Accurate chromosome segregation relies on the ill-understood ability of kinetochores to convert their lateral microtubule attachment into the microtubule plus-end association, capable of the processive motion with tubulin dynamics. Here we report that this transition can be recapitulated in vitro using only two components: the plus end-directed kinesin CENP-E and the microtubule wall-binding Ndc80 protein. CENP-E's primary role is to establish the end configurations for Ndc80, while Ndc80 mediates the maintenance of end attachment. To gain insights into the molecular requirements for end-conversion we paired CENP-E with other microtubule-binding proteins. Ska1, CENP-E Tail, EB1 and CLASP2 differed in their ability to retain the microtubule ends, and none performed as robustly as Ndc80. Likewise, a non-mitotic transporter Kinesin-1 failed to support the Ndc80-mediated end-conversion, implying that the pair of CENP-E kinesin and Ndc80 is optimally suited for this activity. To investigate the underlying mechanistic differences between these motors and MAPs, we developed a Brownian dynamics model for the molecular ensembles of proteins engaged stochastically in walking and diffusion on the microtubule wall. Modeling demonstrates that the observed differences in the end-retention by different MAPs can be largely attributed to their different residency times and rates of diffusion on the microtubule wall. The model also recapitulates the strikingly different behavior of Kinesin-1 and CENP-E, suggesting that it is rooted in their distinct force-detachment characteristics. Following a model prediction, we were able to achieve robust end-conversion with Kinesin-1 by amending its dynamic response via the reduced ATP concentration. Together, our results argue strongly that microtubule end-conversion is an emergent property of the ensemble of transporting motors and diffusing MAPs, limited by the microtubule end boundary. We propose that similar mechanism ensures microtubule end conversion at mitotic kinetochores.

Microtubule Structural State Recognition by End Binding Protein 1

Microtubules are structural polymers that participate in a wide range of cellular functions and are the site of localization and activity for a host of proteins. End binding protein 1 (EB1) localizes primarily to GTP-bound tubulin subunits at the growing ends of microtubules, where it facilitates interactions with other key cellular proteins. However, reports of sublocalization of EB1 within the GTP-rich region at growing microtubule ends suggest that there may be an additional layer of regulation for EB1 binding to the microtubule. Using both bulk and sub-microtubule correlation TIRF microscopy experiments, we found that, independently of nucleotide state, EB1 exhibits preferential binding for non-lattice structures such as those found at microtubule ends or defects. To predict the mechanism for this preference, we performed 3D molecular diffusion simulations, and found that the unique binding location of EB1 at the pocket-like interface between four adjacent tubulin dimers results in a form of structural state recognition due to a high steric hindrance to binding within the lattice, which is reduced at microtubule ends or lattice defects. Additionally, our experiments and simulations using a tubulin face binding protein resulted in elimination of microtubule structure recognition, suggesting a general principle for protein association to cellular polymers based on the location and conformation of the binding interface.

Binding Interactions with Tubulin's C-terminal Tail as Studied by Solution NMR

Microtubules, formed by the polymerization of alpha and beta tubulin heterodimers, play an essential role in cell division and motility. The disordered C-terminal tails of each tubulin monomer are major sites of tubulin regulation. The post-translational modifications of the C-terminal tails enable microtubules to perform a wide range of functions, including the ability to control the dynamics and flexibility of microtubules as well as their ability to bind to microtubule-associated proteins (MAPs). Despite the importance of these C-terminal tails and their modifications, little structural information is available on how they affect binding to MAPs. Moreover, the functionality of the C-terminal tails may depend on whether the tail is attached to the ordered tubulin body or it is present as a free peptide. To address these limitations, we developed techniques to use solution nuclear magnetic resonance (NMR) as a probe of the C-terminal tails (Wall et al, ACS Chem Bio, 2016). Using this approach, we study interactions between the disordered C-terminal tails and different MAPs with a particular emphasis on how post-translational modifications may affect these binding interactions. Of particular interest to us are the +TIP families of MAPs, which localize to the growing end of microtubules.

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

Every time a cell divides, it needs to duplicate its DNA and evenly distribute it between the two new ‘daughter’ cells. To move and distribute DNA, the cell builds a large machine called a spindle, which is made of stiff cables called microtubules. Many proteins, including a motor called dynein, help to organize the spindle’s microtubules. One of dynein’s jobs is to cluster all microtubules at the two tips of the spindle, pulling them into shape. Without this clustering, spindles have the wrong shape and structure and can make mistakes when moving DNA. Microtubules have a ‘plus’ end and a ‘minus’ end, and motor proteins usually only travel in one specified direction. Dynein, for example, moves towards the minus end of microtubules, which is where most of the clustering happens. It can form a complex with other proteins that help clustering, one of which is called NuMA. Until now, it was thought that dynein transports NuMA to the minus ends. To test this model, Hueschen et al. studied dividing human cells under a microscope and isolated minus ends with the help of a laser. The experiments showed that, in fact, NuMA gets to minus ends independently of dynein. Once it is on the minus ends, NuMA grabs hold of another protein complex called dynactin, which then gathers dynein. Dynein then pulls the spindles into shape from the minus ends. When NuMA was experimentally removed from the cells, dynein-dynactin complexes were scattered along the entire length of the microtubule instead of pulling specifically on minus-ends, which resulted in disorganized spindles. Thus, where dynein complexes pull determines what spindle shape they build. Hueschen et al. also showed that dynein complexes only pull on minus-ends while the cell divides, which makes sense, because NuMA remains hidden in the cell nucleus for the rest of the time. Together, these results suggest that NuMA makes sure dynein pulls specifically on the minus-ends of the microtubules to tighten the spindle at the right time. A next step will be to test how the mechanical properties of the spindle are changed without dynein pulling on minus-ends. A better knowledge of how different proteins work together to build the spindle and help cells divide can help us understand what goes wrong when cells divide abnormally, as in the case of cancer cells.

A Combretastatin Analogue C12 Binds to Colchicine Site in Tubulin, Inhibits Spindle Microtubule Dynamics, Activates Mitotic Checkpoint and Induces Apoptosis in Cancer Cells

Several combretastatin derivatives are currently undergoing clinical trials for various types of cancer. In this work, we elucidated the antiproliferative mechanism of action of a highly potentcombretastatin analogue, C12 (5-Quinolin-3-yl and 4-(3,4,5-trimethoxyphenyl) substituted imidazol-2-amine). The compound displayed much stronger activity than CA-4 against different types of cancer cells. Further, C12 inhibited the proliferation of different cancer cells more effectively compared to their non-cancerous counterparts. The binding of C12 to tubulin was examined using surface plasma resonance and the intrinsic fluorescence of C12. Using modified Dixon plot, C12 was found to competitively inhibit the binding of podophyllotoxin, a colchicine-site binding agent, to tubulin. A molecular docking analysis also supported the finding that C12 shares its binding site in tubulin with colchicine. C12 displayed preferential binding to GTP-tubulin than GDP-tubulin indicating that it may bind to the end of microtubules. Consistent with this idea, C12 inhibited the binding of a microtubule plus-end tracking protein, EB1, to microtubules. C12 depolymerized microtubules both in in-vitro and in cells. Using fluorescence recovery after photobleaching technique, C12 was found to strongly suppress the dynamics of spindle microtubules in live HeLa cells. C12 treatment induced multipolar spindle formation, increased the chromosome compactness index, activated mitotic checkpoint protein BubR1 and ultimately induced apoptotic cell death in HeLa cells.

A Minimal System for Microtubule-Based Cell Polarity

Establishment of cell polarity is essential for processes such as growth and division. In fission yeast, polarity factors travel at the tips of microtubules to the cell ends where they associate with the membrane and subsequently maintain a polarized distribution. While many molecular components have been shown to play a role in this polarization process, it remains unknown which molecular functionalities are minimally required. We show that a chimera protein that combines a membrane-binding motif with microtubule tip affinity is able to transiently concentrate at cell ends in wild type fission yeast cells. However, unlike natural polarity factors, these chimera proteins loose their polarized distribution as soon as microtubules disappear. In parallel, we established an in vitro system that allows us to verify the minimal requirements for microtubule-based cell polarity. We use micro-fabrication techniques combined with surface functionalization to create rigid chambers with affinity for proteins, and microfluidic techniques to create elongated emulsion droplets with functionalized lipid boundaries. We demonstrate that proteins that accumulate at the ends of growing microtubules can be transferred to the boundaries of these confined spaces. Interestingly, only proteins that travel in clusters at microtubule ends remain associated with these boundaries after microtubules disappear. This hints to a role of microtubule-end induced protein-protein interactions in the establishment of robust cell polarity patterns.

Dynamics of Microtubule Minus Ends

Dynamic organization of microtubule minus ends is essential in many cellular contexts, including focused poles of a mitotic spindle, or non-centrosomal arrays in neurons and epithelial cells. Recent identification of proteins that specifically recognize and modulate microtubule minus ends suggests that minus-end dynamics are actively regulated in cells. In vitro, both microtubule ends switch between phases of assembly and disassembly, behavior known as microtubule dynamic instability. However, while the minus ends have characteristically slower growth rates, the frequency at which they transition from the growing to the shrinking state (known as ‘catastrophe frequency’) is similar to that at the plus ends. Thus, microtubule minus ends provide a unique perspective on the intrinsic mechanisms underlying microtubule dynamics. Here, we use an in vitro reconstitution approach with total-internal reflection fluorescence microscopy to investigate the molecular mechanisms of minus end dynamics and its regulation.

Kinesin-8 Depolymerizes Microtubules with a Force-Dependent Mechanism

Microtubules are highly dynamic cellular filaments with dramatic structural rearrangements and length changes during the cell cycle. Therefore, accurate control of their length is key to many essential processes like cell division. Apart from the intrinsic dynamic instability of microtubules, one destabilizing, active factor are motor proteins of the kinesin-8 family. They can depolymerize microtubules in a collective and length-dependent manner by interacting with their ends. However, how kinesin-8 depolymerizes microtubules is unclear. Here, we tracked the microtubule end-binding activity of yeast kinesin-8s, Kip3, with high precision under varying loads using optical tweezers. We observed that single Kip3 motors stayed dynamically attached to the microtubule end for up to 200 s, making several 8-nm forward and backward steps. Interestingly, while small hindering loads led to backward slipping away from the end, assisting loads suppress such diffusive stepping and decrease the end-residence time of Kip3 exponentially. We hypothesize that Kip3 is searching for the weakest bound tubulin dimer and that force either applied by consecutive motors or external load accelerates tubulin removal. Understanding the mechanics of kinesin-8's end activity will provide important insight for cell division with implications for cancer research.

Local control of intracellular microtubule dynamics by EB1 photodissociation.

End-binding proteins, EBs, are adaptors that recruit functionally diverse +TIP proteins to growing microtubule plus ends. To test with high spatial and temporal accuracy how, when and where +TIP protein complexes contribute to dynamic cell biology, we developed a photo-inactivated EB1 variant (π-EB1) by inserting a blue light-sensitive protein-protein interaction module between the microtubule- and +TIP-binding domains of EB1. π-EB1 replaces endogenous EB1 function in the absence of blue light. In contrast, blue light-mediated π-EB1 photo-dissociation results in rapid +TIP complex disassembly, and acutely and reversibly attenuates microtubule growth independent of microtubule end association of the microtubule polymerase CKAP5 (ch-TOG, XMAP215). Local π-EB1 photo-dissociation allows subcellular microtubule dynamics control at the second and micrometre scale, and elicits aversive turning of migrating cancer cells. Importantly, light-mediated domain splitting can serve as template to optically control other intracellular protein activities.

Insights into cortical microtubule nucleation and dynamics in Arabidopsis leaf cells.

Plant microtubules (MTs) are nucleated from the γ-tubulin-containing ring complex (γTuRC). In cortical MT arrays of interphase plant cells, γTuRC is preferentially recruited to the lattice of preexisting MTs, where it initiates MT nucleation in either a branch- or bundle-forming manner, or dissociates without mediating nucleation. In this study, we analyzed how γTuRCs influence MT nucleation and dynamics in cotyledon pavement cells of Arabidopsis thaliana We found that γTuRC nucleated MTs at angles of ∼40° toward the plus-ends of existing MTs, or in predominantly antiparallel bundles. A small fraction of γTuRCs was motile and tracked MT ends. When γTuRCs decorated the depolymerizing MT end, they reduced the depolymerization rate. Non-nucleating γTuRCs associated with the MT lattice promoted MT regrowth after a depolymerization phase. These results suggest that γTuRCs not only nucleate MT growth but also regulate MT dynamics by stabilizing MT ends. On rare occasions, a non-MT-associated γTuRC was pushed in the direction of the MT minus-end, while nucleating a new MT, suggesting that the polymerizing plus-end is anchored to the plasma membrane.

SPIRAL2 Stabilises Endoplasmic Microtubule Minus Ends in the Moss Physcomitrella patens.

Stabilisation of minus ends of microtubules (MTs) is critical for organising MT networks in land plant cells, in which all MTs are nucleated independent of centrosomes. Recently, Arabidopsis SPIRAL2 (SPR2) protein was shown to localise to plus and minus ends of cortical MTs, and increase stability of both ends. Here, we report molecular and functional characterisation of SPR2 of the basal land plant, the moss Physcomitrella patens. In protonemal cells of P. patens, where non-cortical, endoplasmic MT network is organised, we observed SPR2 at minus ends, but not plus ends, of endoplasmic MTs and likely also of phragmoplast MTs. Minus end decoration was reconstituted in vitro using purified SPR2, suggesting that moss SPR2 is a minus end-specific binding protein (-TIP). We generated a loss-of-function mutant of SPR2, in which frameshift-causing deletions/insertions were introduced into all four paralogous SPR2 genes by means of CRISPR/Cas9. Protonemal cells of the mutant showed instability of endoplasmic MT minus ends. These results indicate that moss SPR2 is a MT minus end stabilising factor.Key words: acentrosomal microtubule network, microtubule minus end, P. patens, CAMSAP/Nezha/Patronin.

Structural analyses of key features in the KANK1·KIF21A complex yield mechanistic insights into the cross-talk between microtubules and the cell cortex

The cross-talk between dynamic microtubules and the cell cortex plays important roles in cell division, polarity, and migration. A critical adaptor that links the plus ends of microtubules with the cell cortex is the KANK N-terminal motif and ankyrin repeat domains 1 (KANK1)/kinesin family member 21A (KIF21A) complex. Genetic defects in these two proteins are associated with various cancers and developmental diseases, such as congenital fibrosis of the extraocular muscles type 1. However, the molecular mechanism governing the KANK1/KIF21A interaction and the role of the conserved ankyrin (ANK) repeats in this interaction are still unclear. In this study, we present the crystal structure of the KANK1·KIF21A complex at 2.1 Å resolution. The structure, together with biochemical studies, revealed that a five-helix-bundle-capping domain immediately preceding the ANK repeats of KANK1 forms a structural and functional supramodule with its ANK repeats in binding to an evolutionarily conserved peptide located in the middle of KIF21A. We also show that several missense mutations present in cancer patients are located at the interface of the KANK1·KIF21A complex and destabilize its formation. In conclusion, our study elucidates the molecular basis underlying the KANK1/KIF21A interaction and also provides possible mechanistic explanations for the diseases caused by mutations in KANK1 and KIF21A.

In vitro reconstitution of lateral to end-on conversion of kinetochore-microtubule attachments.

During mitosis, kinetochores often bind to the walls of spindle microtubules, but these lateral interactions are then converted into a different binding mode in which microtubule plus-ends are embedded at kinetochores, forming dynamic "end-on" attachments. This remarkable configuration allows continuous addition or loss of tubulin subunits from the kinetochore-bound microtubule ends, concomitant with movement of the chromosomes. Here, we describe novel experimental assays for investigating this phenomenon using a well-defined in vitro reconstitution system visualized by fluorescence microscopy. Our assays take advantage of the kinetochore kinesin CENP-E, which assists in microtubule end conversion in vertebrate cells. In the experimental setup, CENP-E is conjugated to coverslip-immobilized microbeads coated with selected kinetochore components, creating conditions suitable for microtubule gliding and formation of either static or dynamic end-on microtubule attachment. This system makes it possible to analyze, in a systematic and rigorous manner, the molecular friction generated by the microtubule wall-binding proteins during lateral transport, as well as the ability of these proteins to establish and maintain association with microtubule plus-end, providing unique insights into the specific activities of various kinetochore components.

A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition.

The microtubule depolymerising kinesin-13, MCAK, is phosphorylated at residue T537 by Cdk1. This is the only known phosphorylation site within MCAK’s motor domain. To understand the impact of phosphorylation by Cdk1 on microtubule depolymerisation activity, we have investigated the molecular mechanism of the phosphomimic mutant T537E. This mutant significantly impairs microtubule depolymerisation activity and when transfected into cells causes metaphase arrest and misaligned chromosomes. We show that the molecular mechanism underlying the reduced depolymerisation activity of this phosphomimic mutant is an inability to recognise the microtubule end. The microtubule-end residence time is reduced relative to wild-type MCAK, whereas the lattice residence time is unchanged by the phosphomimic mutation. Further, the microtubule-end specific stimulation of ADP dissociation, characteristic of MCAK, is abolished by this mutation. Our data shows that T537E is unable to distinguish between the microtubule end and the microtubule lattice.

Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces

The macromolecular kinetochore functions to generate interactions between chromosomal DNA and spindle microtubules [1]. To facilitate chromosome movement and segregation, kinetochores must maintain associations with both growing and shrinking microtubule ends. It is critical to define the proteins and their properties that allow kinetochores to associate with dynamic microtubules. The kinetochore-localized human Ska1 complex binds to microtubules and tracks with depolymerizing microtubule ends [2]. We now demonstrate that the Ska1 complex also autonomously tracks with growing microtubule ends invitro, a key property that would allow this complex to act at kinetochores to mediate persistent associations with dynamic microtubules. To define the basis for Ska1 complex interactions with dynamic microtubules, we investigated the tubulin-binding properties of the Ska1 microtubule binding domain. In addition to binding to the microtubule lattice and dolastatin-induced protofilament-like structures, we demonstrate that the Ska1 microtubule binding domain can associate with soluble tubulin heterodimers and promote assembly of oligomeric ring-like tubulin structures. We generated mutations on distinct surfaces of the Ska1 microtubule binding domain that disrupt binding to soluble tubulin but do not prevent microtubule binding. These mutants display compromised microtubule tracking activity invitro and result in defective chromosome alignment and mitotic progression in cells using a CRISPR/Cas9-based replacement assay. Our work supports a model in which multiple surfaces of Ska1 interact with diverse tubulin substrates to associate with dynamic microtubule polymers and facilitate optimal chromosome segregation.

EXTH-33. TO ACHIEVE GREATER EFFICACY IN TUMOR-TREATMENT FIELDS: MODELING BASED ON THE INTRACELLULAR MECHANISMS

AbstractDespite invasive surgical resection of glioblastoma followed by radiotherapy and/or chemotherapy with concomitant side effects, most patients survive just 1 to 2 years, with no improvement in survival rates in over a decade. In contrast, low-intensity, alternating electric fields delivered non-invasively via scalp electrodes (tumor-treating fields, ‘TTFields’) lack side effects and combined with chemotherapy significantly increase glioblastoma patients’ survival without affecting quality of life. TTFields are in clinical trials for non-small lung cell, pancreatic, and ovarian cancers. To identify TTFields’ mechanisms, improve efficacy, and expand side-effect-free application to more cancer cell types we have built multi-purpose finite element models in COMSOL of TTFields’ effect on critical sub-cellular structures. It is well-established that TTFields interfere with rapidly-dividing cells’ mitotic processes, causing abnormal chromosome segregation, aneuploidy, and apoptosis, probably through multiple mechanisms. The sub-cellular targets of TTFields are likely high dipole-moment and polarizable molecules such as microtubules (MT) and septin, whose unperturbed functioning are critical to silencing cell cycle checkpoints and normal progression of mitosis. Our models allow quantitative investigation of physical interactions between the electric field and these structures, thereby gaining deep insight into the biophysics of TTFields. The models have revealed electric field amplification at MT ends, high current and high energy density in the counter-ion layers surrounding MT C-termini thought to be the critical MT signaling mechanism. These findings support TTFields’ reported mechanisms of action. We are currently testing mechanistic hypotheses posed by TTFields research teams, including: 1. Dislocalization of septins causing aberrant mitotic exit; 2. Mitotic spindle disruption leading to improper chromosome segregation and mitotic catastrophe; 3. Disruption of MT polymerization homeostasis. We encourage researchers to send us their hypotheses to test in our modeling.