Tubulin Synthesis Research Articles

Effects and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats

Effects and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats

The Amazing Evolutionary Complexity of Eukaryotic Tubulins: Lessons from Naegleria and the Multi-tubulin Hypothesis.

The multi-tubulin hypothesis proposed in 1976 was motivated by finding that the tubulin to build the flagellar apparatus was synthesized de novo during the optional differentiation of Naegleria from walking amoebae to swimming flagellates. In the next decade, with the tools of cloning and sequencing, we were able to establish that the rate of flagellar tubulin synthesis in Naegleria is determined by the abundance of flagellar α- and β-tubulin mRNAs. These experiments also established that the tubulins for Naegleria mitosis were encoded by separate, divergent genes, candidates for which remain incompletely characterized. Meanwhile an unanticipated abundance of tubulin isotypes has been discovered by other researchers. Together with the surprises of genome complexity, these tubulin isotypes require us to rethink how we might utilize the opportunities and challenges offered by the evolutionary diversity of eukaryotes.

A rice tubulin tyrosine ligase like 12 regulates phospholipase D activity and tubulin synthesis

All plant α-tubulins encode a C-terminal tyrosine. An elusive tubulin tyrosine carboxypeptidase can cleave off, and a tubulin tyrosine ligase (TTL) re-ligate this tyrosine. The biological function of this cycle remains unclear but may correlate with microtubule stability. To get insight into the functional context of this phenomenon, we used cold-induced elimination of microtubules as experimental model. In previous work, we had analysed a rice TTL-like 12 (OsTTLL12), the only potential candidate of plant TTL. To follow the effect of OsTTLL12 upon microtubule responses in vivo, we expressed OsTTLL12-RFP into tobacco BY-2 cells stably overexpressing NtTUA3-GFP. We found that overexpression of OsTTLL12-RFP made microtubules disappear faster in response to cold stress, accompanied with more rapid Ca2+ influx, culminating in reduced cold tolerance. Treatment with different butanols indicated that α-tubulin detyrosination/tyrosination differently interacts with phospholipase D (PLD) dependent signalling. In fact, rice PLDα1 decorated microtubules and increased detyrosinated α-tubulin. Unexpectedly, overexpression of the two proteins (OsTTLL12-RFP, NtTUA3-GFP) mutually regulated the accumulation of their transcripts, leading us to a model, where tubulin detyrosination feeds back upon tubulin transcripts and defines a subset of microtubules for interaction with PLD dependent stress signalling.

Inhibition of microtubule assembly competent tubulin synthesis leads to accumulation of phosphorylated tau in neuronal cell bodies

Neurofibrillary tangles, a pathological hallmark of Alzheimer’s disease (AD), are somatodendritic filamentous inclusions composed of hyperphosphorylated tau. Microtubule loss is also a common feature of affected neurons in AD. However, whether and how the disruptions of microtubules and the microtubule-associated proteins occur in the pathogenesis of AD remain unclear. Recent evidence indicates that reduced expression of tubulin by knocking down a tubulin chaperon can cause tau neurotoxicity. Thus, the disruption of tubulin homeostasis may result in the acquisition of tau pathogenesis and ultimately cause tauopathy. To investigate whether the disruption of tubulin maintenance induces tau abnormalities in mammalian neurons, we developed a miRNA-mediated knockdown system of tubulin-specific chaperon E (Tbce), which is a factor required for the de novo synthesis of tubulin. Tbce knockdown in mouse primary cultured neurons induced an increase in tubulin in the cell body at 14 days in vitro. Accumulated tubulin was not acetylated or incorporated in microtubules, indicating that they were functionally inert. Concomitantly, tau also accumulated in neuronal cell bodies. The mis-localized tau was phosphorylated at Ser202/Thr205 and Ser396/Ser404. These results indicate that Tbce knockdown in mammalian neurons induces not only a reduction in properly folded tubulins, which are microtubule assembly competent, but also an accumulation of phosphorylated tau in the cell body of mammalian neurons. These findings suggest that disruption of the homeostatic mechanism for maintaining tubulin biosynthesis and/or microtubules can cause tau accumulation in the cell body, which is commonly observed in tauopathies.

Inhibition of Tubulin β-chain May Play a Regulatory Role in the Development of Rheumatoid Arthritis

AbstractPathological characteristics of Rheumatoid arthritis (RA), a systemic autoimmune disease, includes abnormal proliferation of synovial fibroblasts and increased angiogenesis.The study assessed the effect of tubulin β-chain gene (TBB) gene knockdown on the behaviour of rheumatoid arthritis (RA) fibroblast-like synoviocytes (SFs), which is significantly affected by the synthesis of tubulin. Cultured SFs were isolated from the mixed knee-joint synovial tissue of five RA patients and treated with siRNA targeting TBB. Knockdown efficiency and expression of relevant genes were detected by RT-PCR following 24 h and 36 h of transfection. MTT, Transwell, and wound scratch assays and flow cytometric analysis were used to assess cell proliferation and invasive and migratory capacity following TBB knockdown. The expression of TBB was detected after inhibition of the ERK, STAT3 and NF-kB pathways. The expression of IL-17, TNF-α, IL-1α, IL1-β, and IL-6 was quantified following TBB knockdown. The proliferation, invasion, and migration of RASFs were significantly decreased following TBB knockdown. ELISA showed a significant decrease in interleukin 1β (IL-1β) secretion and insignificantly reduced secretion of TNF-α, IL-17, and IL-1α after TBB knockdown. The expression of TBB in RASFs decreased with the increasing concentration of a STAT3 pathway inhibitor while TBB expression increased after down regulation of the ERK pathway. These results support a protective role for TBB knockdown in suppressing RASFs, laying a foundation for further studies on the pathogenic mechanisms of RA.

Elevated Levels of Selenium Species in Cerebrospinal Fluid of Amyotrophic Lateral Sclerosis Patients with Disease-Associated Gene Mutations

Background: Although an increasing role of genetic susceptibility has been recognized, the role of environmental risk factors in amyotrophic lateral sclerosis (ALS) etiology is largely uncertain; among neurotoxic chemicals, epidemiological and biological plausibility has been provided for pesticides, the heavy metal lead, the metalloid selenium, and other persistent organic pollutants. Selenium involvement in ALS has been suggested on the basis of epidemiological studies, in vitro investigations, and veterinary studies in which selenium induced a selective toxicity against motor neurons. Objective: Hypothesizing a multistep pathogenic mechanism (genetic susceptibility and environmental exposure), we aimed to study selenium species in ALS patients carrying disease-associated gene mutations as compared to a series of hospital controls. Methods: Using advanced analytical techniques, we determined selenium species in cerebrospinal fluid sampled at diagnosis in 9 ALS patients carrying different gene mutations (C9ORF72, SOD1, FUS, TARDBP, ATXN2, and TUBA4A) compared to 42 controls. Results: In a patient with the tubulin-related TUBA4A mutation, we found highly elevated levels (in μg/L) of glutathione-peroxidase-bound selenium (32.8 vs. 1.0) as well as increased levels of selenoprotein-P-bound selenium (2.4 vs. 0.8), selenite (1.8 vs. 0.1), and selenate (0.9 vs. 0.1). In the remaining ALS patients, we detected elevated selenomethionine-bound selenium levels (0.38 vs. 0.06). Conclusions: Selenium compounds can impair tubulin synthesis and the cytoskeleton structure, as do tubulin-related gene mutations. The elevated selenium species levels in the TUBA4A patient may have a genetic etiology and/or represent a pathogenic pathway through which this mutation favors disease onset, though unmeasured confounding cannot be excluded. The elevated selenomethionine levels in the other patients are also of interest due to the toxicity of this nonphysiological selenium species. Our study is the first to assess selenium exposure in genetic ALS, suggesting an interaction between this environmental factor and genetics in triggering disease onset.

Divergent microtubule assembly rates after short- versus long-term loss of end-modulating kinesins.

Depletion of microtubule (MT) regulators can initiate stable alterations in MT assembly rates that affect chromosome instability and mitotic spindle function, but the manner by which cellular MT assembly rates can stably increase or decrease is not understood. To investigate this phenomenon, we measured the response of microtubule assembly to both rapid and long-term loss of MT regulators MCAK/Kif2C and Kif18A. Depletion of MCAK/Kif2C by siRNA stably decreases MT assembly rates in mitotic spindles, whereas depletion of Kif18A stably increases rates of assembly. Surprisingly, this is not phenocopied by rapid rapamycin-dependent relocalization of MCAK/Kif2C and Kif18A to the plasma membrane. Instead, this treatment yields opposite affects on MT assembly. Rapidly increased MT assembly rates are balanced by a decrease in nucleated microtubules, whereas nucleation appears to be maximal and limiting for decreased MT assembly rates and also for long-term treatments. We measured amplified tubulin synthesis during long-term depletion of MT regulators and hypothesize that this is the basis for different phenotypes arising from long-term versus rapid depletion of MT regulators.

In vitro sensitivity of Fusarium graminearum isolates to fungicides

Head blight of wheat is a disease of global importance. In Brazil, it can cause damage of up to 27%. As resistant cultivars are not available yet, short-term disease control relies on the use of fungicides. The first step to reach effective management is to identify potent fungicides. In vitro experiments were conducted to determine the inhibitory concentration 50% (IC50) for mycelial growth or conidial germination, according to the chemical group of fungicides, of five Fusarium graminearum isolates of different origins. The following demethylation inhibitor (DMI) fungicides were tested: epoxiconazole, cyproconazole, metconazole, prochloraz, protioconazole and tebuconazole. In addition, azoxystrobin, kresoxim-methyl, pyraclostrobin and trifloxystrobin were included in the study, representing Quinone outside inhibitor fungicides (QoI), as well as a tubulin synthesis inhibitor, carbendazim and two ready mixtures, trifloxystrobin + tebuconazole or trifloxistrobin + prothioconazole. DMI's showed lower IC50 values compared to the QoI's. For the five tested isolates, in the overall mean, IC50 considering mycelial growth ranged for DMI's from 0.01 mg/L (metconazole, prochloraz and prothioconazole) to 0.12 mg/L (cyproconazole) and considering conidial germination for QoI's from 0.21 mg/L (azoxystrobin) to 1.33 mg/L (trifloxystrobin). The IC50 for carbendazim was 0.07 mg/L. All tested isolates can be considered sensitive to the studied DMI's, although certain differences in sensitivity could be detected between the isolates originating from one same state.

Microtubules as a Critical Target for Arsenic Toxicity in Lung Cells in Vitro and in Vivo

To understand mechanisms for arsenic toxicity in the lung, we examined effects of sodium m-arsenite (As3+) on microtubule (MT) assembly in vitro (0–40 µM), in cultured rat lung fibroblasts (RFL6, 0–20 µM for 24 h) and in the rat animal model (intratracheal instillation of 2.02 mg As/kg body weight, once a week for 5 weeks). As3+ induced a dose-dependent disassembly of cellular MTs and enhancement of the free tubulin pool, initiating an autoregulation of tubulin synthesis manifest as inhibition of steady-state mRNA levels of βI-tubulin in dosed lung cells and tissues. Spindle MT injuries by As3+ were concomitant with chromosomal disorientations. As3+ reduced the binding to tubulin of [3H]N-ethylmaleimide (NEM), an -SH group reagent, resulting in inhibition of MT polymerization in vitro with bovine brain tubulins which was abolished by addition of dithiothreitol (DTT) suggesting As3+ action upon tubulin through -SH groups. In response to As3+, cells elevated cellular thiols such as metallothionein. Taxol, a tubulin polymerization agent, antagonized both As3+ and NEM induced MT depolymerization. MT–associated proteins (MAPs) essential for the MT stability were markedly suppressed in As3+-treated cells. Thus, tubulin sulfhydryls and MAPs are major molecular targets for As3+ damage to the lung triggering MT disassembly cascades.

MAP4 Mechanism that Stabilizes Mitochondrial Permeability Transition in Hypoxia: Microtubule Enhancement and DYNLT1 Interaction with VDAC1

Mitochondrial membrane permeability has received considerable attention recently because of its key role in apoptosis and necrosis induced by physiological events such as hypoxia. The manner in which mitochondria interact with other molecules to regulate mitochondrial permeability and cell destiny remains elusive. Previously we verified that hypoxia-induced phosphorylation of microtubule-associated protein 4 (MAP4) could lead to microtubules (MTs) disruption. In this study, we established the hypoxic (1% O2) cell models of rat cardiomyocytes, H9c2 and HeLa cells to further test MAP4 function. We demonstrated that increase in the pool of MAP4 could promote the stabilization of MT networks by increasing the synthesis and polymerization of tubulin in hypoxia. Results showed MAP4 overexpression could enhance cell viability and ATP content under hypoxic conditions. Subsequently we employed a yeast two-hybrid system to tag a protein interacting with mitochondria, dynein light chain Tctex-type 1 (DYNLT1), by hVDAC1 bait. We confirmed that DYNLT1 had protein-protein interactions with voltage-dependent anion channel 1 (VDAC1) using co-immunoprecipitation; and immunofluorescence technique showed that DYNLT1 was closely associated with MTs and VDAC1. Furthermore, DYNLT1 interactions with MAP4 were explored using a knockdown technique. We thus propose two possible mechanisms triggered by MAP4: (1) stabilization of MT networks, (2) DYNLT1 modulation, which is connected with VDAC1, and inhibition of hypoxia-induced mitochondrial permeabilization.

Polyalkoxybenzenes from Plants. 5. Parsley Seed Extract in Synthesis of Azapodophyllotoxins Featuring Strong Tubulin Destabilizing Activity in the Sea Urchin Embryo and Cell Culture Assays

A series of 4-azapodophyllotoxin derivatives with modified rings B and E have been synthesized using allylpolyalkoxybenzenes from parsley seed oil. The targeted molecules were evaluated in vivo in a phenotypic sea urchin embryo assay for antimitotic and tubulin destabilizing activity. The most active compounds identified by the in vivo sea urchin embryo assay featured myristicin-derived ring E. These molecules were determined to be more potent than podophyllotoxin. Cytotoxic effects of selected molecules were further confirmed and evaluated by conventional assays with A549 and Jurkat human leukemic T-cell lines including cell growth inhibition, cell cycle arrest, cellular microtubule disruption, and induction of apoptosis. The ring B modification yielded 6-OMe substituted molecule as the most active compound. Finally, in Jurkat cells, compound induced caspase-dependent apoptosis mediated by the apical caspases-2 and -9 and not caspase-8, implying the involvement of the intrinsic caspase-9-dependent apoptotic pathway.

Alterations in the expression of genes for tubulin and stress in two Arabidopsis auxin mutants during simulated microgravity

The effect of microgravity on the expression of genes for tubulin, stress and aging in Arabidopsis seedlings from wild-type, and the auxinic mutants aux 1 and eir/pin2 is reported. Simulated microgravity was produced through the Random Positioning Machine (RPM). The results showed that the tubulin genes TUA4, TUA6 and TUB2, in plants subjected to μg, showed a peak of expression at 3h in the wild-type, whereas in the mutants it was delayed to 6h. The microgravity treatment tested on the expression of stress genes FeSOD1 and CAT3 showed ony a significant increase of senescence SAG12 showed increased expression in the wild-type starting at 3h, whereas in the mutant a significant increase was seen only at 24h. From the collected data we conclude that auxin transport and action should be involved in tubulin synthesis, stress, and the senescence process.

Short synthesis of noscapine, bicuculline, egenine, capnoidine, and corytensine alkaloids through the addition of 1-siloxy-isobenzofurans to imines

A concise diastereotioselective strategy for the synthesis of noscapine, bicuculline, and egenine (1a–c), as well as capnoidine and corytensine (2a,b), was developed using diastereoselective addition of 1-siloxy-isobenzofurans 4a and 4b to iminium ion 5 in a one-pot approach. The synthesis features the use of imine 13 obtained through Bischler–Napieralsky reaction from amine 11. The addition of ionic liquids as addictives in the reactions afforded erythro configuration in major adduct compounds. The synthetic route can also be applied in the total synthesis of promising tubulin binding agent EM105 (3).

TBCD links centriologenesis, spindle microtubule dynamics, and midbody abscission in human cells.

Microtubule-organizing centers recruit α- and β-tubulin polypeptides for microtubule nucleation. Tubulin synthesis is complex, requiring five specific cofactors, designated tubulin cofactors (TBCs) A–E, which contribute to various aspects of microtubule dynamics in vivo. Here, we show that tubulin cofactor D (TBCD) is concentrated at the centrosome and midbody, where it participates in centriologenesis, spindle organization, and cell abscission. TBCD exhibits a cell-cycle-specific pattern, localizing on the daughter centriole at G1 and on procentrioles by S, and disappearing from older centrioles at telophase as the protein is recruited to the midbody. Our data show that TBCD overexpression results in microtubule release from the centrosome and G1 arrest, whereas its depletion produces mitotic aberrations and incomplete microtubule retraction at the midbody during cytokinesis. TBCD is recruited to the centriole replication site at the onset of the centrosome duplication cycle. A role in centriologenesis is further supported in differentiating ciliated cells, where TBCD is organized into “centriolar rosettes”. These data suggest that TBCD participates in both canonical and de novo centriolar assembly pathways.

Chlorpyrifos-oxon interferes with differentiation of N2a neuroblastoma cells.

Event Abstract Back to Event Chlorpyrifos-oxon interferes with differentiation of N2a neuroblastoma cells. E Nikolaidis1, J Flaskos2, M Sachana2*, W Harris3 and A J. Hargreaves3 1 Aristotle University of Thessaloniki, Laboratory of Pharmacology, Greece 2 Aristotle University of Thessaloniki, Laboratory of Biochemistry and Toxicology, Greece 3 Nottingham Trent University, Interdisciplinary Biomedical Research Centre, United Kingdom Introduction. A rapidly growing body of data over the last decade indicates that two of the organophosphate (OP) ester pesticides, chlorpyrifos (CPF) and diazinon (DZN), are capable of inducing developmental neurotoxicity (DNT) (1). Our previous research shows that both DZN (2) and its main in vivo metabolite, diazinon-oxon (DZO) (3), inhibit neurite outgrowth, a well established in vitro biomarker of DNT (4) in N2a cells. Furthermore, we have shown that CPF also impairs neurite outgrowth in the same cellular model (5, 6). The aims of the present study were to assess the ability of the oxon in vivo metabolite of CPF, chlorpyrifos-oxon (CPO) to interfere with neurite outgrowth and to investigate the biochemical nature of any morphological effects. Methods. After 24 h exposure and induction of differentiation, N2a cells were either stained for morphological analysis or processed for SDS-PAGE and Western blotting. The blots were probed with monoclonal antibodies recognizing tubulin (B512) and GAP-43 (GAP-7B10). Results. Compared to the control, CPO, at a concentration of 10 μM, inhibited the outgrowth of neurites within 24 h. Immunoblot analysis revealed a significant decrease in reactivity with the anti-GAP-43 antibody in N2a cells treated with CPO, whereas there was no change in reactivity with the anti-α-tubulin antibody. Discussion. The results indicate that CPO causes a significant reduction in the number of neurites similarly to its parent compound (5, 6). In addition, exposure of N2a cells to CPO decreases the levels of GAP-43, an important protein in the maintenance of axons that is known to be increased during axon development. In contrast, the levels of α-tubulin are unaffected by CPO, since tubulin synthesis shows small or no specific increase during axon growth. It is inferred that the DNT which CPF causes in vivo may be partly attributed to its oxon metabolite.

695 Sulforaphane Sensitizes Colorectal Cancer Cells to Oxaliplatin Induced Cell Growth Inhibition: Key Role of p38 MAPK

Background: Microtubule-targeting agents (MTAs) are cancer drugs that act primarily by altering microtubule dynamics and interfering with mitosis. While MTAs are in widespread clinical use, innate and acquired resistance is an ongoing problem. Resistance is thought to stem primarily from drug efflux and expression of alternate tubulin isoforms with MTAresistant microtubule dynamics. We found that the compound T0070907 reduced tumor growth in a murine xenograft model of colon cancer. In human colorectal cancer (CRC) cell lines, T0070907 caused near-total loss of the α and β tubulin proteins, leading to collapse of visible microtubule filaments and eventual cell death. Dramatic tubulin loss is a novel phenotype not reported for any of the standard MTAs. This observation suggests the possible development of MTAs that work by a mechanism less susceptible to escape via expression of alternate tubulin isoforms. As a first step toward determining the molecular mechanisms of tubulin loss, we determined whether T0070907 affects degradation and activity of the microtubule associated proteins that control microtubule polymerization. Methods: The effects of T0070907 on tubulin levels and phosphorylation of the microtubuleassociated protein stathmin/Op18 and its upstream kinase MARK kinase were examined using Western blot in the CRC cell lines HT-29, SW620, and DLD-1. Tubulin protein halflives were measured after addition of the protein synthesis inhibitor cycloheximide. Results: T0070907 caused loss of α and β, but not γ, δ, or e tubulin. The magnitude of the tubulin reduction was much greater than that found when microtubule destabilization leads to tubulin synthesis inhibition. Tubulin half lives decreased from >12h to 6h, suggesting increased degradation. Consistent with this idea, treatments that sequestered tubulin in polymerized microtubules (paclitaxel), leaving the tubulin unavailable for degradation, reduced T0070907-induced tubulin loss. Conversely, the microtubule-destabilizing agents nocodazole and vinblastine increased the rate of tubulin loss. T0070907 did not strongly affect microtubule polymerization In Vitro, suggesting that it does not directly bind to either microtubules or tubulin. However, T0070907 led to increases in MARK kinase activation as well as phosphorylation and inactivation of its downstream target, the microtubulesequestering protein stathmin/Op18. Conclusion: T0070907 promotes tubulin degradation and stathmin inactivation. As knockdown of stathmin in Drosophila leads to a drastic reduction in tubulin levels, we propose that T0070907 reduces tubulin in part by targeting stathmin.

696 Resveratrol Sensitizes Colorectal Cancer Cells to Oxaliplatin-Induced Cell Death via Intracellular Polyamine Depletion

Background: Microtubule-targeting agents (MTAs) are cancer drugs that act primarily by altering microtubule dynamics and interfering with mitosis. While MTAs are in widespread clinical use, innate and acquired resistance is an ongoing problem. Resistance is thought to stem primarily from drug efflux and expression of alternate tubulin isoforms with MTAresistant microtubule dynamics. We found that the compound T0070907 reduced tumor growth in a murine xenograft model of colon cancer. In human colorectal cancer (CRC) cell lines, T0070907 caused near-total loss of the α and β tubulin proteins, leading to collapse of visible microtubule filaments and eventual cell death. Dramatic tubulin loss is a novel phenotype not reported for any of the standard MTAs. This observation suggests the possible development of MTAs that work by a mechanism less susceptible to escape via expression of alternate tubulin isoforms. As a first step toward determining the molecular mechanisms of tubulin loss, we determined whether T0070907 affects degradation and activity of the microtubule associated proteins that control microtubule polymerization. Methods: The effects of T0070907 on tubulin levels and phosphorylation of the microtubuleassociated protein stathmin/Op18 and its upstream kinase MARK kinase were examined using Western blot in the CRC cell lines HT-29, SW620, and DLD-1. Tubulin protein halflives were measured after addition of the protein synthesis inhibitor cycloheximide. Results: T0070907 caused loss of α and β, but not γ, δ, or e tubulin. The magnitude of the tubulin reduction was much greater than that found when microtubule destabilization leads to tubulin synthesis inhibition. Tubulin half lives decreased from >12h to 6h, suggesting increased degradation. Consistent with this idea, treatments that sequestered tubulin in polymerized microtubules (paclitaxel), leaving the tubulin unavailable for degradation, reduced T0070907-induced tubulin loss. Conversely, the microtubule-destabilizing agents nocodazole and vinblastine increased the rate of tubulin loss. T0070907 did not strongly affect microtubule polymerization In Vitro, suggesting that it does not directly bind to either microtubules or tubulin. However, T0070907 led to increases in MARK kinase activation as well as phosphorylation and inactivation of its downstream target, the microtubulesequestering protein stathmin/Op18. Conclusion: T0070907 promotes tubulin degradation and stathmin inactivation. As knockdown of stathmin in Drosophila leads to a drastic reduction in tubulin levels, we propose that T0070907 reduces tubulin in part by targeting stathmin.

694 The Tubulin-Targeting Agent T0070907 Promotes Tubulin Degradation and Inactivates the Tubulin-Sequestering Protein Stathmin/Op18

Background: Microtubule-targeting agents (MTAs) are cancer drugs that act primarily by altering microtubule dynamics and interfering with mitosis. While MTAs are in widespread clinical use, innate and acquired resistance is an ongoing problem. Resistance is thought to stem primarily from drug efflux and expression of alternate tubulin isoforms with MTAresistant microtubule dynamics. We found that the compound T0070907 reduced tumor growth in a murine xenograft model of colon cancer. In human colorectal cancer (CRC) cell lines, T0070907 caused near-total loss of the α and β tubulin proteins, leading to collapse of visible microtubule filaments and eventual cell death. Dramatic tubulin loss is a novel phenotype not reported for any of the standard MTAs. This observation suggests the possible development of MTAs that work by a mechanism less susceptible to escape via expression of alternate tubulin isoforms. As a first step toward determining the molecular mechanisms of tubulin loss, we determined whether T0070907 affects degradation and activity of the microtubule associated proteins that control microtubule polymerization. Methods: The effects of T0070907 on tubulin levels and phosphorylation of the microtubuleassociated protein stathmin/Op18 and its upstream kinase MARK kinase were examined using Western blot in the CRC cell lines HT-29, SW620, and DLD-1. Tubulin protein halflives were measured after addition of the protein synthesis inhibitor cycloheximide. Results: T0070907 caused loss of α and β, but not γ, δ, or e tubulin. The magnitude of the tubulin reduction was much greater than that found when microtubule destabilization leads to tubulin synthesis inhibition. Tubulin half lives decreased from >12h to 6h, suggesting increased degradation. Consistent with this idea, treatments that sequestered tubulin in polymerized microtubules (paclitaxel), leaving the tubulin unavailable for degradation, reduced T0070907-induced tubulin loss. Conversely, the microtubule-destabilizing agents nocodazole and vinblastine increased the rate of tubulin loss. T0070907 did not strongly affect microtubule polymerization In Vitro, suggesting that it does not directly bind to either microtubules or tubulin. However, T0070907 led to increases in MARK kinase activation as well as phosphorylation and inactivation of its downstream target, the microtubulesequestering protein stathmin/Op18. Conclusion: T0070907 promotes tubulin degradation and stathmin inactivation. As knockdown of stathmin in Drosophila leads to a drastic reduction in tubulin levels, we propose that T0070907 reduces tubulin in part by targeting stathmin.

P2-135: Mechanisms of neuronal traffic inhibition by tau protein

The missorting of Tau protein from the axonal to the somatodendritic compartment and loss of synapses are hallmarks of Alzheimer's disease which precede neuronal loss and the pathological aggregation of tau protein into neurofibrillary tangles. We are interested in the mechanisms by which Tau can affect the integrity of synapses. One major factor is the inhibition of movement of mitochondria by Tau which leads to mitochondrial dysfunction and energy deprivation (loss of ATP production). We have now studied the pathway of Tau's interference with mitochondrial transport. Primary hippocampal neurons were transfected with CFP-Tau, mitochondrial traffic and synaptic integrity was observed by time-resolved confocal microscopy, followed by thin-sectioning electron microscopy to visualize the state of mitochondria, synapses, and the cytoskeleton. The results show that Tau operates on two distinct levels. One is a direct inhibition mechanism whereby bound Tau covers the microtubule surface and consequently interferes with the attachment of motor proteins. The second is an indirect inhibition mechanism which is based on the higher stability of microtubules when Tau is increased. This leads to a reduced level of tubulin subunits in the cytosol, which in turn causes the upregulation of tubulin synthesis (Cleveland et al., Nature 1983). This causes an increase in microtubule density, the microtubules become tightly bundled so that mitochondria can no longer move freely along axons and dendrites. The example illustrates how an improvement of microtubule stability can cause traffic deficits in neurons, rather than curing them. Research supported by MPG and DFG.

Temperature-shock induction of multiple flagella induces additional synthesis of flagellum specific mRNAs and tubulin

Four mRNAs (α- and β-tubulin, flagellar calmodulin and Class-I), specifically expressed when Naegleria amebae differentiate into flagellates, were followed at 5–10 min intervals during the temperature-shock induction of multiple flagella in order to better understand how basal body and flagellum number are regulated. Surprisingly, tubulin synthesis continued during the 37 min temperature shock. An initial rapid decline in α- and β-tubulin and flagellar calmodulin mRNAs was followed by a rapid re-accumulation of mRNAs before the temperature was lowered. mRNA levels continued to increase until they exceeded control levels by 4–21%. Temperature shock delayed flagella formation 37 min, produced twice as much tubulin protein synthesis and three fold more flagella. Labeling with an antibody against Naegleria centrin suggested that basal body formation was also delayed 30–40 min. An extended temperature shock demonstrated that lowering the temperature was not required for return of mRNAs to near control levels suggesting that induction of multiple flagella and the formation of flagella per se are affected in different ways. We suggest that temperature-shock induction of multiple flagella reflects increased mRNA accumulation combined with interference with the regulation of the recently reported microtubule-nucleating complex needed for basal body formation.