Focal adhesions are controlled by microtubules through local contractility regulation.

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

SUN2 regulates mitotic duration in response to extracellular matrix rigidity.

How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here, we examined how extracellular matrix (ECM) rigidity affects cell division. We found that cells divide more rapidly when cultured on rigid substrates. While we observed no effect of ECM rigidity on rounding or postmitotic spreading duration, we found that changes in matrix stiffness impact mitosis progression. We noticed that ECM elasticity up-regulates the expression of the linker of nucleoskeleton and cytoskeleton (LINC) complex component SUN2, which in turn promotes metaphase-to-anaphase transition by acting on mitotic spindle formation, whereas when cells adhere to soft ECM, low levels of SUN2 expression perturb astral microtubule organization and delay the onset of anaphase.

Radiosensitization of Breast Cancer Cells with a 2-Methoxyestradiol Analogue Affects DNA Damage and Repair Signaling In Vitro.

Radiation resistance and radiation-related side effects warrant research into alternative strategies in the application of this modality to cancer treatment. Designed in silico to improve the pharmacokinetics and anti-cancer properties of 2-methoxyestradiol, 2-ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16) disrupts microtubule dynamics and induces apoptosis. Here, we investigated whether pre-exposure of breast cancer cells to low-dose ESE-16 would affect radiation-induced deoxyribonucleic acid (DNA) damage and the consequent repair pathways. MCF-7, MDA-MB-231, and BT-20 cells were exposed to sub-lethal doses of ESE-16 for 24 h before 8 Gy radiation. Flow cytometric quantification of Annexin V, clonogenic studies, micronuclei quantification, assessment of histone H2AX phosphorylation and Ku70 expression were performed to assess cell viability, DNA damage, and repair pathways, in both directly irradiated cells and cells treated with conditioned medium. A small increase in apoptosis was observed as an early consequence, with significant repercussions on long-term cell survival. Overall, a greater degree of DNA damage was detected. Moreover, initiation of the DNA-damage repair response was delayed, with a subsequent sustained elevation. Radiation-induced bystander effects induced similar pathways and were initiated via intercellular signaling. These results justify further investigation of ESE-16 as a radiation-sensitizing agent since pre-exposure appears to augment the response of tumor cells to radiation.

Marginal band microtubules are acetylated by αTAT1.

The discoid shape of resting platelets is maintained by a peripheral, circular bundle of microtubules called marginal band. Marginal band microtubules are acetylated on lysine 40 of the alpha-tubulin subunits. We have previously shown that the deacetylase HDAC6 is responsible for tubulin deacetylation in platelets and that the hyperacetylated state of the microtubules in HDAC6KO platelets correlates with faster activation/spreading kinetics, pointing to a regulatory role of this modification. So far, the question about the reverse enzyme, responsible for tubulin acetylation in platelets, has remained unanswered. Several enzymes have been described as having tubulin acetylation activity. Here we identify αTAT1 as the enzyme responsible for the acetylation of marginal band microtubules. We show that αTAT1 deficiency has only minor consequences for platelet production and function. A residual tubulin acetylation level in αTAT1 deficient platelet lysates suggests the presence of an additional tubulin-acetylating enzyme that is unable to acetylate marginal band microtubules.

Characterization of Signalling Pathways That Link Apoptosis and Autophagy to Cell Death Induced by Estrone Analogues Which Reversibly Depolymerize Microtubules.

The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds' effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.

Live imaging of single platelets at work.

Although live imaging of dynamic processes in platelets is a challenging task, several important observations have been published during the last 20 years. We will discuss the amazing insights that have been achieved, the difficulties that can be encountered as well as some questions still open and the future technical perspectives.

Specific Targeting of Plant and Apicomplexa Parasite Tubulin through Differential Screening Using In Silico and Assay-Based Approaches.

Dinitroanilines are chemical compounds with high selectivity for plant cell α-tubulin in which they promote microtubule depolymerization. They target α-tubulin regions that have diverged over evolution and show no effect on non-photosynthetic eukaryotes. Hence, they have been used as herbicides over decades. Interestingly, dinitroanilines proved active on microtubules of eukaryotes deriving from photosynthetic ancestors such as Toxoplasma gondii and Plasmodium falciparum, which are responsible for toxoplasmosis and malaria, respectively. By combining differential in silico screening of virtual chemical libraries on Arabidopsis thaliana and mammal tubulin structural models together with cell-based screening of chemical libraries, we have identified dinitroaniline related and non-related compounds. They inhibit plant, but not mammalian tubulin assembly in vitro, and accordingly arrest A. thaliana development. In addition, these compounds exhibit a moderate cytotoxic activity towards T. gondii and P. falciparum. These results highlight the potential of novel herbicidal scaffolds in the design of urgently needed anti-parasitic drugs.

Single assay-wide variance experimental (SAVE) design for high-throughput screening.

MOTIVATION: Advantages of statistical testing of high-throughput screens include P-values, which provide objective benchmarks of compound activity, and false discovery rate estimation. The cost of replication required for statistical testing, however, may often be prohibitive. We introduce the single assay-wide variance experimental (SAVE) design whereby a small replicated subset of an entire screen is used to derive empirical Bayes random error estimates, which are applied to the remaining majority of unreplicated measurements. RESULTS: The SAVE design is able to generate P-values comparable with those generated with full replication data. It performs almost as well as the random variance model t-test with duplicate data and outperforms the commonly used Z-scores with unreplicated data and the standard t-test. We illustrate the approach with simulated data and with experimental small molecule and small interfering RNA screens. The SAVE design provides substantial performance improvements over unreplicated screens with only slight increases in cost.

A novel inhibitor of the mitochondrial respiratory complex I with uncoupling properties exerts potent antitumor activity.

Cancer cells are highly dependent on bioenergetic processes to support their growth and survival. Disruption of metabolic pathways, particularly by targeting the mitochondrial electron transport chain complexes (ETC-I to V) has become an attractive therapeutic strategy. As a result, the search for clinically effective new respiratory chain inhibitors with minimized adverse effects is a major goal. Here, we characterize a new OXPHOS inhibitor compound called MS-L6, which behaves as an inhibitor of ETC-I, combining inhibition of NADH oxidation and uncoupling effect. MS-L6 is effective on both intact and sub-mitochondrial particles, indicating that its efficacy does not depend on its accumulation within the mitochondria. MS-L6 reduces ATP synthesis and induces a metabolic shift with increased glucose consumption and lactate production in cancer cell lines. MS-L6 either dose-dependently inhibits cell proliferation or induces cell death in a variety of cancer cell lines, including B-cell and T-cell lymphomas as well as pediatric sarcoma. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI-1) partially restores the viability of B-lymphoma cells treated with MS-L6, demonstrating that the inhibition of NADH oxidation is functionally linked to its cytotoxic effect. Furthermore, MS-L6 administration induces robust inhibition of lymphoma tumor growth in two murine xenograft models without toxicity. Thus, our data present MS-L6 as an inhibitor of OXPHOS, with a dual mechanism of action on the respiratory chain and with potent antitumor properties in preclinical models, positioning it as the pioneering member of a promising drug class to be evaluated for cancer therapy. MS-L6 exerts dual mitochondrial effects: ETC-I inhibition and uncoupling of OXPHOS. In cancer cells, MS-L6 inhibited ETC-I at least 5 times more than in isolated rat hepatocytes. These mitochondrial effects lead to energy collapse in cancer cells, resulting in proliferation arrest and cell death. In contrast, hepatocytes which completely and rapidly inactivated this molecule, restored their energy status and survived exposure to MS-L6 without apparent toxicity.

Intensity quantile estimation and mapping--a novel algorithm for the correction of image non-uniformity bias in HCS data.

MOTIVATION: Image non-uniformity (NU) refers to systematic, slowly varying spatial gradients in images that result in a bias that can affect all downstream image processing, quantification and statistical analysis steps. Image NU is poorly modeled in the field of high-content screening (HCS), however, such that current conventional correction algorithms may be either inappropriate for HCS or fail to take advantage of the information available in HCS image data. RESULTS: A novel image NU bias correction algorithm, termed intensity quantile estimation and mapping (IQEM), is described. The algorithm estimates the full non-linear form of the image NU bias by mapping pixel intensities to a reference intensity quantile function. IQEM accounts for the variation in NU bias over broad cell intensity ranges and data acquisition times, both of which are characteristic of HCS image datasets. Validation of the method, using simulated and HCS microtubule polymerization screen images, is presented. Two requirements of IQEM are that the dataset consists of large numbers of images acquired under identical conditions and that cells are distributed with no within-image spatial preference. AVAILABILITY AND IMPLEMENTATION: MATLAB function files are available at http://nadon-mugqic.mcgill.ca/.

Molecular crosstalk between apoptosis and autophagy induced by a novel 2-methoxyestradiol analogue in cervical adenocarcinoma cells.

BACKGROUND: 2-Methoxyestradiol has been shown to induce both autophagy and apoptosis in various carcinogenic cell lines. Although a promising anti-cancer agent, it has poor bioavailability and rapid in vivo metabolism which decreases its efficiency. In order to improve 2-methoxyestradiol's anti-proliferative properties, a novel 2-methoxyestradiol analogue, 2-ethyl-3-O-sulphamoyl-estra-1,3,5 (10)16-tetraene (ESE-16), was previously in silico-designed in our laboratory. This study investigated ESE-16 for its anti-proliferative potential on a cervical adenocarcinoma cell (HeLa) cell line. Additionally, the possible intracellular crosstalk mechanisms between the two types of cell death were investigated. METHODS AND RESULTS: HeLa cells exposed to 0.5 µM ESE-16 for 24 hours showed morphological evidence of both apoptotic and autophagic death pathways as assessed by polarization-optical transmitted light differential interference contrast microscopy, fluorescent microscopy and transmission electron microscopy. Flow cytometric cyclin B1 quantification revealed induction of programmed cell death after halting cell cycle progression in metaphase. Confocal microscopy demonstrated that ESE-16 caused microtubule fragmentation. Flow cytometric analysis of cell cycle progression and phosphatidylserine flip determination confirmed induction of apoptosis. Moreover, an increase in aggresome formation and microtubule-associated protein light chain, LC3, was demonstrated indicative of autophagy. Both caspase 8 and 3 were upregulated in a spectrophotometric analysis, indicating the involvement of the extrinsic pathway of apoptotic induction. CONCLUSIONS: We conclude that the novel in silico-designed compound, ESE-16, exerts its anti-proliferative effect on the tumorigenic human epithelial cervical (HeLa) cells by sequentially targeting microtubule integrity, resulting in a metaphase block, causing induction of both autophagic and apoptotic cell death via a crosstalk mechanism that involves the extrinsic pathway. Future investigations will expand on signal transduction pathways involved in both apoptosis and autophagy for assessment of ESE-16 effects on microtubule dynamic instability parameters.

[Phenotypic screens or one stone to kill two birds: discover the target and its pharmacological regulator]. / Les criblages phénotypiques ou comment faire d'une pierre deux coups: découvrir la cible et la molécule pharmacologique capable de la réguler.

Phenotypic screens, in which chemical libraries are assayed on cells with the aim to isolate compounds that interfere with a given cell function, are a risky but powerful strategy to discover, in the same approach, new therapeutic targets and the compounds able to regulate them. With a strong experience of nearly 10 years in the field, we present the advantages of such an approach, the possible troubles and technical solutions. We also present in this paper a french network which has been recently built and that gather all the competencies needed for screening approaches.

The fate of mitochondria during platelet activation.

Blood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam-scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction.

The microtubule cytoskeleton: An old validated target for novel therapeutic drugs.

Compounds targeting microtubules are widely used in cancer therapy with a proven efficacy. However, because they also target non-cancerous cells, their administration leads to numerous adverse effects. With the advancement of knowledge on the structure of tubulin, the regulation of microtubule dynamics and their deregulation in pathological processes, new therapeutic strategies are emerging, both for the treatment of cancer and for other diseases, such as neuronal or even heart diseases and parasite infections. In addition, a better understanding of the mechanism of action of well-known drugs such as colchicine or certain kinase inhibitors contributes to the development of these new therapeutic approaches. Nowadays, chemists and biologists are working jointly to select drugs which target the microtubule cytoskeleton and have improved properties. On the basis of a few examples this review attempts to depict the panorama of these recent advances.

The Role of LIM Kinases during Development: A Lens to Get a Glimpse of Their Implication in Pathologies.

The organization of cell populations within animal tissues is essential for the morphogenesis of organs during development. Cells recognize three-dimensional positions with respect to the whole organism and regulate their cell shape, motility, migration, polarization, growth, differentiation, gene expression and cell death according to extracellular signals. Remodeling of the actin filaments is essential to achieve these cell morphological changes. Cofilin is an important binding protein for these filaments; it increases their elasticity in terms of flexion and torsion and also severs them. The activity of cofilin is spatiotemporally inhibited via phosphorylation by the LIM domain kinases 1 and 2 (LIMK1 and LIMK2). Phylogenetic analysis indicates that the phospho-regulation of cofilin has evolved as a mechanism controlling the reorganization of the actin cytoskeleton during complex multicellular processes, such as those that occur during embryogenesis. In this context, the main objective of this review is to provide an update of the respective role of each of the LIM kinases during embryonic development.

Design, synthesis and biological evaluation of quinoline-2-carbonitrile-based hydroxamic acids as dual tubulin polymerization and histone deacetylases inhibitors.

A series of quinoline and quinazoline analogs were designed and synthesized as new tubulin polymerization (TP) and histone deacetylases (HDAC) inhibitors. Compounds 12a and 12d showed the best cytotoxicity activities against a panel of human cancer cell lines with an averaged IC50 value of 0.6 and 0.7 nM, respectively. Furthermore, these lead compounds showed good activities against CA-4-resistant colon-carcinoma and multidrug-resistant leukemia cells. In addition, compounds 12a and 12d induced HT29 cell cycle arrest in the G2/M phase and produced caspase-induced apoptosis of HT29 cells through mitochondrial dysfunction. Also, 12a and 12d inhibited HDAC8, 6, and 11 activities. Furthermore, lead compound 12a exhibited higher metabolic stability than isoCA-4 and was highly potent in suppressing tumor growth in the fibrosarcoma MCA205 tumor model. Collectively, these studies suggest that 12a represents a new dual inhibitor of TP and HDAC activities, which makes it a suitable candidate for further investigations in clinical development.

New potent dual inhibitors of CK2 and Pim kinases: discovery and structural insights.

Protein kinase casein kinase 2 (CK2) is a serine/threonine kinase with evidence of implication in growth dysregulation and apoptosis resistance, making it a relevant target for cancer therapy. Several CK2 inhibitors have been developed showing variable efficiency, emphasizing the need to expand the chemical diversity of those inhibitors. We report the identification and characterization of 2,8-difurandicarboxylic acid derivatives as a new class of nanomolar ATP-competitive inhibitors. Selectivity profiling pointed out proviral insertion Moloney virus kinases (Pim kinases) as the only other kinases that are significantly inhibited. By combining structure-activity relationship analysis with structural determination, we were able to determine the binding mode of these inhibitors for both kinases and to explain their strong inhibitory potency. Essential chemical features necessary for activity on both kinases were then identified. The described compounds are not cell permeable: however, they could provide a lead for developing novel inhibitors usable also in vivo. Given the similar but not redundant pathophysiological functions of CK2 and Pim family members, such inhibitors would provide new attractive leads for targeted cancer therapy. This work highlights that 2 functionally related kinases from different kinome branches display exquisite sensitivity to a common inhibitor.

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends.

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to alpha-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.

Characterization of Microtubule Destabilizing Drugs: A Quantitative Cell-Based Assay That Bridges the Gap between Tubulin Based- and Cytotoxicity Assays.

(1) Background: Microtubule depolymerizing agents (MDAs) are commonly used for cancer treatment. However, the therapeutic use of such microtubule inhibitors is limited by their toxicity and the emergence of resistance. Thus, there is still a sustained effort to develop new MDAs. During the characterization of such agents, mainly through in vitro analyses using purified tubulin and cytotoxicity assays, quantitative comparisons are mandatory. The relationship between the effect of the drugs on purified tubulin and on cell viability are not always direct. (2) Methods: We have recently developed a cell-based assay that quantifies the cellular microtubule content. In this study, we have conducted a systematic comparative analysis of the effect of four well-characterized MDAs on the kinetics of in vitro tubulin assembly, on the cellular microtubule content (using our recently developed assay) and on cell viability. (3) Conclusions: These assays gave complementary results. Additionally, we found that the drugs' effect on in vitro tubulin polymerization is not completely predictive of their relative cytotoxicity. Their effect on the cellular microtubule content, however, is closely related to their effect on cell viability. In conclusion, the assay we have recently developed can bridge the gap between in vitro tubulin assays and cell viability assays.

Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization.

Actin networks are dynamically regulated through constant depolymerization and polymerization cycles. Although the fundamental mechanisms that govern these processes have been identified, the nature and role of post-translational modifications (PTMs) of actin and actin regulatory proteins are not completely understood. Here, we employed Actin CytoFRET, a method that we developed for real time detection of fluorescence resonance energy transfer (FRET) signals generated by actin dynamics, to screen a small library of PTM-interfering compounds on a biosensor leukemic T cell line. This strategy led to the identification of small molecule inhibitors of deubiquitinating enzymes (DUBs) as potent inducers of actin polymerization and blockers of chemotactic cell migration. The examination of the underlying mechanism further revealed that the actin depolymerizing protein cofilin represents a major effector of DUB inhibitor (DUBi)-induced actin reorganization. We found that DUB blockade results in the accumulation of polyubiquitinated proteins and ROS production, associated with cofilin oxidation and dephosphorylation on serine 3, which provokes uncontrolled actin polymerization impairing cell migration. Together, our study highlights DUBs as novel regulators of actin dynamics through ROS-dependent cofilin modulation, and shows that DUBi represent attractive novel tools to impede leukemic cell migration.