Microtubule Dynamics In Cells Research Articles

Profilin affects microtubule dynamics via actin.

Profilin binds microtubules in vitro. However, a new study by Vitriol and colleagues (https://doi.org/10.1083/jcb.202309097) now suggests that effects of profilin on microtubule dynamics in cells are indirect and result from its impact on actin dynamics rather than its direct binding to microtubules.

Abstract 513: Intra-tubular damage is targeted by maytansinoids and rescued by NF1: Revisiting mechanism and biomarkers of an established ADC payload

Abstract There is great interest in the identification of biomarkers to guide development of antibody-drug conjugates (ADC). We previously showed that loss of Neurofibromatosis 1 (NF1), a gene frequently mutated across cancers, enhances the activity of DM1, the maytansinoid payload of T-DM1, through a novel function in regulating microtubule (MT) dynamics. Maytansinoids are puzzlingly more effective in cells (in the nanomolar range) vs in vitro (in the micromolar range). Since maytansinoids bind at the interface between tubulin dimers, they are thought to only bind soluble tubulin dimers or MT ends, which would suggest very few binding sites available for pharmacological interaction in vivo, at odds with data. Here we investigated the interaction of DM1 with NF1 and MTs, using cellular and reductionist in vitro systems. To measure in vivo MT dynamics, we transiently transfected the MT end-binding protein EB3-GFP and reconstructed MT trajectories by live-cell imaging. Upon DM1 treatment, KO cells showed a highly significant reduction in MT speed, demonstrating a direct role for NF1 on MT dynamics in cells. In turbidity-based tubulin polymerization assays, recombinant NF1 greatly accelerated polymerization, and completely rescued DM1-induced inhibition. Visual inspection of fluorescent MTs showed that NF1 induced significant MT bundling, a defining feature of many MT-associated proteins, which generates signal indistinguishable from true MT polymerization in turbidity assays. To follow the dynamics of individual microtubules, we applied Total Internal Reflection (TIRF) microscopy on glass-immobilized MTs. As expected, polymerization in the presence of NF1 led to a significant increase in MT dynamics (elongation speed, rescue and catastrophe rate). Expectedly, DM1 led to significant reduction in the fraction of elongating MTs and speed, but these defects were completely or partially rescued by NF1. Importantly, DM1 did not only lead to MT shortening (as proposed by the current model), but also to clear and frequent MT fracturing, indicating that the drug is not only engaging MT ends but also intra-tubular binding sites. This is consistent with recent models of MT formation which incorporate the frequent presence of areas of discontinuity or damage induced by mechanical stress, exposing intra-tubular DM1 binding sites. Interestingly, adding NF1 to DM1-treated MTs generated areas of de novo intra-tubular tubulin insertion, coincident with damaged sites, suggesting an entirely novel role for NF1 in MT repair.In conclusion, we provide evidence for a model in which maytansinoids bind not only to soluble tubulin dimers and MT ends, but also to intra-tubular damaged sites. Thus, the number of binding sites in cells would be proportional to MT damage, suggesting a mechanism for differential efficacy across tumor types and a potential avenue for combinatorial drug development. Citation Format: Eleonora Messuti, Bruno Achutti Duso, Alessia Castiglioni, Giulia Tini, Emanuele Bonetti, Giuseppe Ciossani, Silvia Monzani, Daria Khuntsariya, Zdeněk Lánský, Marcus Braun, Luigi Scietti, Luca Mazzarella. Intra-tubular damage is targeted by maytansinoids and rescued by NF1: Revisiting mechanism and biomarkers of an established ADC payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 513.

Synthesis and characterization of bis-amide SSE1917 as a microtubule-stabilizing anticancer agent

Microtubule dynamics are critical for spindle assembly and chromosome segregation during cell division. Pharmacological inhibition of microtubule dynamics in cells causes prolonged mitotic arrest, resulting in apoptosis, an approach extensively employed in treating different types of cancers. The present study reports the synthesis of thirty-two novel bis-amides (SSE1901-SSE1932) and the evaluation of their antiproliferative activities. N-(1-oxo-3-phenyl-1-(phenylamino)propan-2-yl)benzamide (SSE1917) exhibited the most potent activity with GI50 values of 0.331 ± 0.01 µM in HCT116 colorectal and 0.48 ± 0.27 µM in BT-549 breast cancer cells. SSE1917 stabilized microtubules in biochemical and cellular assays, bound to taxol site in docking studies, and caused aberrant mitosis and G2/M arrest in cells. Prolonged treatment of cells with the compound increased p53 expression and triggered apoptotic cell death. Furthermore, SSE1917 suppressed the growth of both mouse and patient-derived human colon cancer organoids, highlighting its potential therapeutic value as an anticancer agent.

In Silico Exploration of Microtubule Agent Griseofulvin and Its Derivatives Interactions with Different Human β-Tubulin Isotypes

Tubulin isotypes are known to regulate microtubule stability and dynamics, as well as to play a role in the development of resistance to microtubule-targeted cancer drugs. Griseofulvin is known to disrupt cell microtubule dynamics and cause cell death in cancer cells through binding to tubulin protein at the taxol site. However, the detailed binding mode involved molecular interactions, and binding affinities with different human β-tubulin isotypes are not well understood. Here, the binding affinities of human β-tubulin isotypes with griseofulvin and its derivatives were investigated using molecular docking, molecular dynamics simulation, and binding energy calculations. Multiple sequence analysis shows that the amino acid sequences are different in the griseofulvin binding pocket of βI isotypes. However, no differences were observed at the griseofulvin binding pocket of other β-tubulin isotypes. Our molecular docking results show the favorable interaction and significant affinity of griseofulvin and its derivatives toward human β-tubulin isotypes. Further, molecular dynamics simulation results show the structural stability of most β-tubulin isotypes upon binding to the G1 derivative. Taxol is an effective drug in breast cancer, but resistance to it is known. Modern anticancer treatments use a combination of multiple drugs to alleviate the problem of cancer cells resistance to chemotherapy. Our study provides a significant understanding of the involved molecular interactions of griseofulvin and its derivatives with β-tubulin isotypes, which may help to design potent griseofulvin analogues for specific tubulin isotypes in multidrug-resistance cancer cells in future.

Regulation of actin and microtubules by TDP-43 and profilin.

TDP-43 is a classic DNA/RNA binding protein important for the normal function of numerous cellular activities that directly and indirectly impinge on the actin and microtubule cytoskeletons. When TDP-43 levels are reduced, neurons display phenotypes consistent with the loss of actin-microtubule crosstalk functions including aberrant morphology, fewer dendritic branches, and diminished area of growth cones. TDP-43 is direct binding partner of profilin (kD = 115.2 nM), which also regulates actin and microtubule dynamics, and variants of either protein are considered causative agents in several neurodegenerative diseases. First, we explored whether TDP-43 directly interacts with the actin or microtubules cytoskeletons and whether TDP-43 could mediate cytoskeletal crosstalk by forming phase-separated biomolecular condensates. We used TIRF microscopy assays to measure the impact of purified TDP-43 on actin and microtubule assembly. TDP-43 bound and sequestered both actin and tubulin (kD = 22.7 nM). inhibiting the assembly of either polymer. We also explored how interactions between profilin and TDP-43 modulated the dynamics of either cytoskeleton or phase separation. The addition of profilin increased TDP-43 condensate formation (number and size), further inhibited actin assembly, but stabilized microtubule assembly. To investigate this in a more physiological context, we imaged the cytoskeleton and TDP-43 in Neuroblastoma-2a cells. Cells lacking profilin displayed less robust actin and microtubule arrays compared to endogenous or rescue controls. In addition, TDP-43 is mislocalized to the cytoplasm, mirroring the disease state of amyotrophic lateral sclerosis (ALS). Rescue experiments expressing wild type profilin on plasmids restored actin and microtubule arrays and the nuclear localization of TDP-43, whereas profilin plasmids harboring ALS-related mutations did not. Thus, profilin and TDP-43 are important regulators of actin and microtubule dynamics in cells and in the context of ALS.

CBIO-07. ST-401: A BRAIN-PENETRANT MICROTUBULE-TARGETING AGENT THAT KILLS CANCER CELLS IN INTERPHASE

Abstract Microtubule targeting agents (MTAs) are indispensable medicines to treat a wide range of cancers, and evidence suggests that glioblastoma is sensitive to disruptions in microtubule (MT) functions; but most MTAs do not cross the blood brain barrier. To address this limitation, we developed a brain-penetrant MTA, ST-401, that inhibited the growth of human glioblastoma in culture at nanomolar concentrations (Horne et al. (2020) Neuro-Onc Adv. 3(1): vdaa165). Of note, ST-401 binds to the colchicine site, inhibits tubulin assembly and reversibly reduces MT dynamics in cells with a milder activity compared to the reference MTA, nocodazole. Pharmacokinetic analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 demonstrated significant antitumor and radiosensitizing activities. Testing of ST-401 on the NCI 60 cancer cell panel indicated that its anti-tumor activity 1) did not correlate with any of the compounds tested thus far and 2) only weakly correlated with MTAs that act through alternate mechanisms [Taxol and Vinblastine]. Thus, ST-401 may kill cancer cells through a novel mechanism related to disruption of MT function that is different from MTAs tested thus far on the NCI 60 cancer cell panel. As initial step to discover this novel mechanism of action, we studied the anti-tumor activity of ST-401 and nocodazole in HCT116 cells in culture. As expected, low concentrations (30 and 100 nM) of nocodazole killed HCT116 cells in mitosis and increased p-P53; by contrast these low concentrations of ST-401 killed HCT116 cells in interphase and did not increase p-P53. Our study shows that the brain-penetrant MTA, ST-401, kills cancers in interphase, a response that contrasts with the activity of the reference MTA nocodazole. ST-401 represents a promising chemical scaffold to develop brain-penetrant therapeutics for the treatment of patients diagnosed with glioblastoma.

Directionally biased sidestepping of Kip3/kinesin-8 is regulated by ATP waiting time and motor–microtubule interaction strength

Kinesin-8 motors, which move in a highly processive manner toward microtubule plus ends where they act as depolymerases, are essential regulators of microtubule dynamics in cells. To understand their navigation strategy on the microtubule lattice, we studied the 3D motion of single yeast kinesin-8 motors, Kip3, on freely suspended microtubules in vitro. We observed short-pitch, left-handed helical trajectories indicating that kinesin-8 motors frequently switch protofilaments in a directionally biased manner. Intriguingly, sidestepping was not directly coupled to forward stepping but rather depended on the average dwell time per forward step under limiting ATP concentrations. Based on our experimental findings and numerical simulations we propose that effective sidestepping toward the left is regulated by a bifurcation in the Kip3 step cycle, involving a transition from a two-head-bound to a one-head-bound conformation in the ATP-waiting state. Results from a kinesin-1 mutant with extended neck linker hint toward a generic sidestepping mechanism for processive kinesins, facilitating the circumvention of intracellular obstacles on the microtubule surface.

Interaction of microtubule depolymerizing agent indanocine with different human αβ tubulin isotypes.

Tubulin isotypes are known to regulate the stability and dynamics of microtubules, and are also involved in the development of resistance against microtubule-targeted cancer drugs. Indanocine, a potent microtubule depolymerizing agent, is highly active against multidrug-resistant (MDR) cancer cells without affecting normal cells. It is known to disrupt microtubule dynamics in cells and induce apoptotic cell death. Indanocine is reported to bind to tubulin at the colchicine site i.e. at the interface of αβ tubulin heterodimer. However, it’s precise binding mode, involved molecular interactions and the binding affinities with different αβ-tubulin isotypes present in MDR cells are not well understood. Here, the binding affinities of human αβ-tubulin isotypes with indanocine were examined, employing the molecular modeling approach i.e. docking, molecular dynamics simulation and binding energy calculations. Multiple sequence analysis suggests that the amino acid sequences are different in the indanocine binding pockets of βI, βIIa, βIII and βVI isotypes. However, such differences are not observed in the amino acid sequences of βIVa, βIVb, and βV tubulin isotypes at indanocine binding pockets. Docking and molecular dynamics simulation results show that indanocine prefers the interface binding pocket of αβIIa, αβIII, αβIVb, αβV, and αβVI tubulin isotypes; whereas it is expelled from the interface binding pocket of αβIVa and αβI-tubulin isotypes. Further, binding free energy calculations show that αβVI has the highest binding affinity and αβI has the lowest binding affinity for indanocine among all β-tubulin isotypes. The binding free energy decreases in the order of αβVI > αβIVb > αβIIa > αβIII > αβV > αβIVa > αβI. Thus, our study provides a significant understanding of involved molecular interactions of indanocine with tubulin isotypes, which may help to design potent indanocine analogues for specific tubulin isotypes in MDR cells in future.

A small-molecule activator of kinesin-1 drives remodeling of the microtubule network

The microtubule motor kinesin-1 interacts via its cargo-binding domain with both microtubules and organelles, and hence plays an important role in controlling organelle transport and microtubule dynamics. In the absence of cargo, kinesin-1 is found in an autoinhibited conformation. The molecular basis of how cargo engagement affects the balance between kinesin-1's active and inactive conformations and roles in microtubule dynamics and organelle transport is not well understood. Here we describe the discovery of kinesore, a small molecule that in vitro inhibits kinesin-1 interactions with short linear peptide motifs found in organelle-specific cargo adaptors, yet activates kinesin-1's function of controlling microtubule dynamics in cells, demonstrating that these functions are mechanistically coupled. We establish a proof-of-concept that a microtubule motor-cargo interface and associated autoregulatory mechanism can be manipulated using a small molecule, and define a target for the modulation of microtubule dynamics.

Reflective imaging improves spatiotemporal resolution and collection efficiency in light sheet microscopy

Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, and gentle imaging of live specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. We also report a modified deconvolution algorithm that removes associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to <300 nm in all three dimensions) by applying our method to single-view LSFM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture. We demonstrate the broad applicability of our method in a variety of samples, studying mitochondrial, membrane, Golgi, and microtubule dynamics in cells and calcium activity in nematode embryos.

Strong fascin expression promotes metastasis independent of its F-actin bundling activity.

High expression of the actin bundling protein Fascin increases the malignancy of tumor cells. Here we show that fascin expression is up-regulated in more malignant sub-cell lines of MDA-MB-231 cells as compared to parental cells. Since also parental MDA-MB-231 cells exhibit high fascin levels, increased fascin expression was termed as “hyperexpression”. To examine the effect of fascin hyperexpression, fascin was hyperexpressed in parental MDA-MB-231 cells and metastasis was analyzed in NOD scid gamma (NSG) mice. In addition, the effect of fascin mutants with inactive or constitutively active actin bundling activity was examined. Unexpectedly, we found that hyperexpression of both, wildtype (wt) and mutant fascin strongly increased metastasis in vivo, showing that the effect of fascin hyperexpression did not depend on its actin bundling activity. Cellular assays revealed that hyperexpression of wt and mutant fascin increased adhesion of MDA-MB-231 cells while transmigration and proliferation were not affected. Since it has been shown that fascin controls adhesion by directly interacting with microtubules (MTs), we analyzed if fascin hyperexpression affects MT dynamics. We found that at high concentrations fascin significantly increased MT dynamics in cells and in cell-free approaches. In summary our data show that strong expression of fascin in breast cancer cells increases metastasis independent of its actin bundling activity. Thus, it seems that the mechanism of fascin-stimulated metastasis depends on its concentration.

Profilin Directly Promotes Microtubule Growth through Residues Mutated in Amyotrophic Lateral Sclerosis

Profilin is an abundant actin monomer-binding protein with critical actin regulatory roles invivo [1, 2]. However, profilin also influences microtubule dynamics in cells, which may be mediated in part through its interactions with formins that in turn bind microtubules [3, 4]. Specific residues on human profilin-1 (PFN1) are mutated in patients with amyotrophic lateral sclerosis (ALS) [5, 6]. However, the observation that some ALS-linked PFN1 mutants fail to alter cellular actin organization or dynamics [5-8] or invitro actin-monomer affinity [9] has been perplexing, given that profilin is best understood as an actin regulator. Here, we investigated direct effects of profilin on microtubule dynamics and whether ALS-linked mutations in PFN1 disrupt such functions. We found that human, fly, and yeast profilin homologs all directly enhance microtubule growth rate by several-fold invitro. Microtubule stimulatory effects were unaffected by mutations in the canonical actin- or poly-proline-binding sites of profilin. Instead, microtubule activities depended on specific surface residues on profilin mutated in ALS patients. Furthermore, microtubule effects were attenuated by increasing concentrations of actin monomers, suggesting competition between actin and microtubules for binding profilin. Consistent with these biochemical observations, a 2-fold increase in the expression level of wild-type PFN1, but not the ALS-linked PFN1 mutants, increased microtubule growth rates in cells. Together, these results demonstrate that profilin directly enhances the growth rate of microtubules. They further suggest that ALS-linked mutations in PFN1 may perturb cellular microtubule dynamics and/or the coordination between the actin and microtubule cytoskeletons, leading to motor neuron degeneration.

MCAK-mediated regulation of endothelial cell microtubule dynamics is mechanosensitive to myosin-II contractility.

Compliance and dimensionality mechanosensing, the processes by which cells sense the physical attributes of the extracellular matrix (ECM), are known to drive cell branching and shape change largely through a myosin-II-mediated reorganization of the actin and microtubule (MT) cytoskeletons. Subcellular regulation of MT dynamics is spatially controlled through a Rac1-Aurora-A kinase pathway that locally inhibits the MT depolymerizing activity of mitotic centromere-associated kinesin (MCAK), thereby promoting leading-edge MT growth and cell polarization. These results suggest that the regulation of MT growth dynamics is intimately linked to physical engagement of the cell with the ECM. Here, we tested the hypothesis that MCAK contributes to compliance and dimensionality mechanosensing-mediated regulation of MT growth dynamics through a myosin-II-dependent signaling pathway. We cultured endothelial cells (ECs) on collagen-coupled stiff or compliant polyacrylamide ECMs to examine the effects of MCAK expression on MT growth dynamics and EC branching morphology. Our results identify that MCAK promotes fast MT growth speeds in ECs cultured on compliant 2D ECMs but promotes slow MT growth speeds in ECs cultured on compliant 3D ECMs, and these effects are myosin-II dependent. Furthermore, we find that 3D ECM engagement uncouples MCAK-mediated regulation of MT growth persistence from myosin-II-mediated regulation of growth persistence specifically within EC branched protrusions.

Invited review: Microtubule severing enzymes couple atpase activity with tubulin GTPase spring loading.

Microtubules are amazing filaments made of GTPase enzymes that store energy used for their own self-destruction to cause a stochastically driven dynamics called dynamic instability. Dynamic instability can be reproduced in vitro with purified tubulin, but the dynamics do not mimic that observed in cells. This is because stabilizers and destabilizers act to alter microtubule dynamics. One interesting and understudied class of destabilizers consists of the microtubule-severing enzymes from the ATPases Associated with various cellular Activities (AAA+) family of ATP-enzymes. Here we review current knowledge about GTP-driven microtubule dynamics and how that couples to ATP-driven destabilization by severing enzymes. We present a list of challenges regarding the mechanism of severing, which require development of experimental and modeling approaches to shed light as to how severing enzymes can act to regulate microtubule dynamics in cells. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 547-556, 2016.

Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails

Microtubule dynamics in cells are regulated by associated proteins that can be either stabilizers or destabilizers. A class of destabilizers that is important in a large number of cellular activities is the microtubule-severing enzymes, yet little is known about how they function. Katanin p60 was the first ATPase associated with microtubule severing. Here, we investigate the activity of katanin severing using a GFP-labeled human version. We quantify the effect of katanin concentration on katanin binding and severing activity. We find that free tubulin can inhibit severing activity by interfering with katanin binding to microtubules. The inhibition is mediated by the sequence of the tubulin and specifically depends on the carboxy-terminal tails. We directly investigate the inhibition effect of tubulin carboxy-terminal tails using peptide sequences of α-, β-, or detyrosinated α-tubulin tails that have been covalently linked to bovine serum albumin. Our results show that β-tubulin tails are the most effective at inhibiting severing, and that detyrosinated α-tubulin tails are the least effective. These results are distinct from those for other severing enzymes and suggest a scheme for regulation of katanin activity in cells dependent on free tubulin concentration and the modification state of the tubulin.

Effect of microtubule-associated protein-4 on epidermal cell migration under different oxygen concentrations.

After skin trauma, regional epidermal cell migration mediates the re-epithelialization of the wound surface, which is an important step for wound healing, yet the underlying molecular regulatory mechanism is unclear. In the current study, HaCaT cells were maintained under different oxygen concentrations (1%, 21%, 40% and 65%). Technologies including immunofluorescence staining, wound scratch, transwell invasion, western blot and low-expression lentiviral vector were utilized to observe the changes in microtubule dynamics and the microtubule-associated protein (MAP)4 expression. MAP4's effect on cell migration under different oxygen concentrations was also studied. The results showed that under hyperoxic (40% and 65%) and hypoxic (1%) conditions, HaCaT cells were able to regulate cell microtubule dynamics by MAP4, thus promoting cell migration. On the other hand, MAP4 silencing through targeted shRNA attenuated HaCaT cell migration under the above oxygen concentrations. These results imply that MAP4 plays an important role in epidermal cell migration under different oxygen concentrations.

Lessons from in vitro reconstitution analyses of plant microtubule-associated proteins.

Plant microtubules, composed of tubulin GTPase, are irreplaceable cellular components that regulate the directions of cell expansion and cell division, chromosome segregation and cell plate formation. To accomplish these functions, plant cells organize microtubule structures by regulating microtubule dynamics. Each microtubule localizes to the proper position with repeated growth and shortening. Although it is possible to reconstitute microtubule dynamics with pure tubulin solution in vitro, many microtubule-associated proteins (MAPs) govern microtubule dynamics in cells. In plants, major MAPs are identified as microtubule stabilizers (CLASP and MAP65 etc.), microtubule destabilizers (kinesin-13, katanin, MAP18 and MDP25), and microtubule dynamics promoters (EB1, MAP215, MOR1, MAP200, SPR2). Mutant analyses with forward and reverse genetics have shown the importance of microtubules and individual MAPs in plants. However, it is difficult to understand how each MAP regulates microtubule dynamics, such as growth and shortening, through mutant analyses. In vitro reconstitution analyses with individual purified MAPs and tubulin are powerful tools to reveal how each MAP regulates microtubule dynamics at the molecular level. In this review, I summarize the results of in vitro reconstitution analyses and introduce current models of how each MAP regulates microtubule dynamic instability.

Association between an alternative promoter polymorphism and sperm deformity rate is due to modulation of the expression of KATNAL1 transcripts in Chinese Holstein bulls.

Katanin p60 subunit A-like 1 (KATNAL1) is an ATPase that regulates Sertoli cell microtubule dynamics and sperm retention. We evaluated one novel splice variant and characterized the promoter and a functional single nucleotide polymorphism (SNP) of the bovine KATNAL1 gene to explore its expression pattern, possible regulatory mechanism and relationship with semen traits in Chinese Holstein bulls. A novel splice variant, KATNAL1 transcript variant 2 (KATNAL1-TV2) of the retained 68bp in intron 2, was identified by RT-PCR and compared with KATNAL1 transcript variant 1 (KATNAL1-TV1, NM 001192918.1) in various tissues. Bioinformatics analyses predicted that KATNAL1 transcription was regulated by two promoters: P1 in KATNAL1-TV1 and P2 in KATNAL1-TV2. Results of qRT-PCR revealed that KATNAL1-TV1 had higher expression than did KATNAL1-TV2 in testes of adult bulls (P<0.05). Promoter luciferase activity analysis suggested that the core sequences of P1 and P2 were mapped to the region of c.-575˜c.-180 and c.163-40˜c.333+59 respectively. One novel SNP (c.163-210T>C, ss836312085) located in intron 1 was found using sequence alignment. The SNP in P2 resulted in the presence of the DeltaE binding site, improving its basal promoter activity (P<0.05); and we observed a greater sperm deformity rate in bulls with the genotype CC than in those with the genotype TT (P<0.05), which indicated that different genotypes were associated with the bovine semen traits. Bioinformatics analysis of the KATNAL1 protein sequence predicted that the loss of the MIT domain in the KATNAL1-TV2 transcript resulted in protein dysfunction. These findings help us to understand that a functional SNP in P2 and subsequent triggering of expression diversity of KATNAL1 transcripts are likely to play an important role with regard to semen traits in bull breeding programs.

DHTP is an allosteric inhibitor of the kinesin-13 family of microtubule depolymerases.

The kinesin-13 family of microtubule depolymerases is a major regulator of microtubule dynamics. RNA interference-induced knockdown studies have highlighted their importance in many cell division processes including spindle assembly and chromosome segregation. Since microtubule turnovers and most mitotic events are relatively rapid (in minutes or seconds), developing tools that offer faster control over protein functions is therefore essential to more effectively interrogate kinesin-13 activities in living cells. Here, we report the identification and characterization of a selective allosteric kinesin-13 inhibitor, DHTP. Using high resolution microscopy, we show that DHTP is cell permeable and can modulate microtubule dynamics in cells.

PM060184, a new tubulin binding agent with potent antitumor activity including P-glycoprotein over-expressing tumors

PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total synthesis and is under evaluation in clinical studies in patients with advanced cancer diseases. It was recently published that PM060184 presents the highest known affinities among tubulin-binding agents, and that it targets tubulin dimers at a new binding site. Here, we show that PM060184 has a potent antitumor activity in a panel of different tumor xenograft models. Moreover, PM060184 is able to overcome P-gp mediated resistance in vivo, an effect that could be related to its high binding affinity for tubulin. To gain insight into the mechanism responsible of the observed antitumor activity, we have characterized its molecular and cellular effects. We have observed that PM060184 is an inhibitor of tubulin polymerization that reduces microtubule dynamicity in cells by 59%. Interestingly, PM060184 suppresses microtubule shortening and growing at a similar extent. This action affects cells in interphase and mitosis. In the first case, the compound induces a disorganization and fragmentation of the microtubule network and the inhibition of cell migration. In the second case, it induces the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. These effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. Taken together, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent.