Leslie Wilson

Publications of Leslie Wilson

• Cooperative Stabilization of Microtubule Dynamics by EB1 and CLIP-170 Involves Displacement of Stably Bound P(i) at Microtubule Ends.

  Authors: Manu Lopus, Cristina Manatschal, Ruben M Buey, Saša Bjelić, Herbert P Miller, Michel O Steinmetz, Leslie Wilson

  Biochemistry. 03/2012;

  End binding protein 1 (EB1) and cytoplasmic linker protein of 170 kDa (CLIP-170) are two well-studied microtubule plus-end-tracking proteins (+TIPs) that target growing microtubule plus ends in theEnd binding protein 1 (EB1) and cytoplasmic linker protein of 170 kDa (CLIP-170) are two well-studied microtubule plus-end-tracking proteins (+TIPs) that target growing microtubule plus ends in the form of comet tails and regulate microtubule dynamics. However, the mechanism by which they regulate microtubule dynamics is not well understood. Using full-length EB1 and a minimal functional fragment of CLIP-170 (ClipCG12), we found that EB1 and CLIP-170 cooperatively regulate microtubule dynamic instability at concentrations below which neither protein is effective. By use of small-angle X-ray scattering and analytical ultracentrifugation, we found that ClipCG12 adopts a largely extended conformation with two noninteracting CAP-Gly domains and that it formed a complex in solution with EB1. Using a reconstituted steady-state mammalian microtubule system, we found that at a low concentration of 250 nM, neither EB1 nor ClipCG12 individually modulated plus-end dynamic instability. Higher concentrations (up to 2 μM) of the two proteins individually did modulate dynamic instability, perhaps by a combination of effects at the tips and along the microtubule lengths. However, when low concentrations (250 nM) of EB1 and ClipCG12 were present together, the mixture modulated dynamic instability considerably. Using a pulsing strategy with [γ(32)P]GTP, we further found that unlike EB1 or ClipCG12 alone, the EB1-ClipCG12 mixture partially depleted the microtubule ends of stably bound (32)P(i). Together, our results suggest that EB1 and ClipCG12 act cooperatively to regulate microtubule dynamics. They further indicate that stabilization of microtubule plus ends by the EB1-ClipCG12 mixture may involve modification of an aspect of the stabilizing cap.

• Characterization and detection of cellular and proteomic alterations in stable stathmin-overexpressing, taxol-resistant BT549 breast cancer cells using offgel IEF/PAGE difference gel electrophoresis.

  Authors: Manimalha Balasubramani, Chitose Nakao, Guy T Uechi, John Cardamone, Kathy Kamath, Kristen L Leslie, Raghavan Balachandran, Leslie Wilson, Billy W Day, Mary Ann Jordan

  Mutation research. 06/2011; 722(2):154-64.

  Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers.Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers. High stathmin levels have been associated with the development of resistance to the widely used anti-cancer drug taxol ((®)Taxol, paclitaxel). The mechanisms of stathmin-mediated taxol resistance are not well-understood at the molecular level. To better understand the role of stathmin in taxol resistance, we stably overexpressed stathmin twofold in BT549 human breast cancer cells and characterized several cell processes involved in the mechanism of action of taxol. After stable overexpression of stathmin, neither the cell doubling time nor the mitotic index was altered and the microtubule polymer mass was reduced only modestly (by 18%). Unexpectedly, microtubule dynamicity was reduced by 29% after stathmin overexpression, resulting primarily from reduction in the catastrophe frequency. Sensitivity to taxol was reduced significantly (by 44%) in a clonogenic assay, and stathmin appeared to protect the cells from the spindle-damaging effects of taxol. The results suggest that in the stably stathmin-overexpressing clones, compensatory gene expression occurred that resulted in normal rates of cell proliferation and prevented the increase in catastrophe frequency expected in response to stathmin. Stathmin overexpression protected the cells from taxol-induced abnormal mitoses, and thus induced taxol resistance. Using offgel IEF/PAGE difference gel electrophoresis, we identified a number of proteins whose expression is reduced in the taxol-resistant stathmin-overexpressing cell lines, including proteins involved in the cytoskeleton and cell structure, the stress response, protein folding, glycolysis, and catalysis.

• Nanoscale assembly in biological systems: from neuronal cytoskeletal proteins to curvature stabilizing lipids.

  Authors: Cyrus R Safinya, Uri Raviv, Daniel J Needleman, Alexandra Zidovska, Myung Chul Choi, Miguel A Ojeda-Lopez, Kai K Ewert, Youli Li, Herbert P Miller, Joel Quispe, Bridget Carragher, Clinton S Potter, Mahn Won Kim, Stuart C Feinstein, Leslie Wilson

  Advanced materials (Deerfield Beach, Fla.). 05/2011; 23(20):2260-70.

  The review will describe experiments inspired by the rich variety of bundles and networks of interacting microtubules (MT), neurofilaments, and filamentous-actin in neurons where the nature of theThe review will describe experiments inspired by the rich variety of bundles and networks of interacting microtubules (MT), neurofilaments, and filamentous-actin in neurons where the nature of the interactions, structures, and structure-function correlations remain poorly understood. We describe how three-dimensional (3D) MT bundles and 2D MT bundles may assemble, in cell free systems in the presence of counter-ions, revealing structures not predicted by polyelectrolyte theories. Interestingly, experiments reveal that the neuronal protein tau, an abundant MT-associated-protein in axons, modulates the MT diameter providing insight for the control of geometric parameters in bio- nanotechnology. In another set of experiments we describe lipid-protein-nanotubes, and lipid nano-tubes and rods, resulting from membrane shape evolution processes involving protein templates and curvature stabilizing lipids. Similar membrane shape changes, occurring in cells for the purpose of specific functions, are induced by interactions between membranes and proteins. The biological materials systems described have applications in bio-nanotechnology.

• Combinatorial Tau pseudophosphorylation: markedly different regulatory effects on microtubule assembly and dynamic instability than the sum of the individual parts.

  Authors: Erkan Kiris, Donovan Ventimiglia, Mehmet E Sargin, Michelle R Gaylord, Alphan Altinok, Kenneth Rose, B S Manjunath, Mary Ann Jordan, Leslie Wilson, Stuart C Feinstein

  The Journal of biological chemistry. 02/2011; 286(16):14257-70.

  Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias.Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias. A huge literature has led to the widely held notion that aberrant Tau hyperphosphorylation is central to these disorders. Unfortunately, our mechanistic understanding of the functional effects of combinatorial Tau phosphorylation remains minimal. Here, we generated four singly pseudophosphorylated Tau proteins (at Thr(231), Ser(262), Ser(396), and Ser(404)) and four doubly pseudophosphorylated Tau proteins using the same sites. Each Tau preparation was assayed for its abilities to promote microtubule assembly and to regulate microtubule dynamic instability in vitro. All four singly pseudophosphorylated Tau proteins exhibited loss-of-function effects. In marked contrast to the expectation that doubly pseudophosphorylated Tau would be less functional than either of its corresponding singly pseudophosphorylated forms, all of the doubly pseudophosphorylated Tau proteins possessed enhanced microtubule assembly activity and were more potent at regulating dynamic instability than their compromised singly pseudophosphorylated counterparts. Thus, the effects of multiple pseudophosphorylations were not simply the sum of the effects of the constituent single pseudophosphorylations; rather, they were generally opposite to the effects of singly pseudophosphorylated Tau. Further, despite being pseudophosphorylated at different sites, the four singly pseduophosphorylated Tau proteins often functioned similarly, as did the four doubly pseudophosphorylated proteins. These data lead us to reassess the conventional view of combinatorial phosphorylation in normal and pathological Tau action. They may also be relevant to the issue of combinatorial phosphorylation as a general regulatory mechanism.

• Tau isoform-specific modulation of kinesin-driven microtubule gliding rates and trajectories as determined with tau-stabilized microtubules.

  Authors: Austin Peck, M Emre Sargin, Nichole E LaPointe, Kenneth Rose, B S Manjunath, Stuart C Feinstein, Leslie Wilson

  Cytoskeleton (Hoboken, N.J.). 01/2011; 68(1):44-55.

  We have utilized tau-assembled and tau-stabilized microtubules (MTs), in the absence of taxol, to investigate the effects of tau isoforms with three and four MT binding repeats upon kinesin-driven MTWe have utilized tau-assembled and tau-stabilized microtubules (MTs), in the absence of taxol, to investigate the effects of tau isoforms with three and four MT binding repeats upon kinesin-driven MT gliding. MTs were assembled in the presence of either 3-repeat tau (3R tau) or 4-repeat tau (4R tau) at tau:tubulin dimer molar ratios that approximate those found in neurons. MTs assembled with 3R tau glided at 31.1 μm/min versus 25.8 μm/min for 4R tau, a statistically significant 17% difference. Importantly, the gliding rates for either isoform did not change over a fourfold range of tau concentrations. Further, tau-assembled MTs underwent minimal dynamic instability behavior while gliding and moved with linear trajectories. In contrast, MTs assembled with taxol in the absence of tau displayed curved gliding trajectories. Interestingly, addition of 4R tau to taxol-stabilized MTs restored linear gliding, while addition of 3R tau did not. The data are consistent with the ideas that (i) 3R and 4R tau-assembled MTs possess at least some isoform-specific features that impact upon kinesin translocation, (ii) tau-assembled MTs possess different structural features than do taxol-assembled MTs, and (iii) some features of tau-assembled MTs can be masked by prior assembly by taxol. The differences in kinesin-driven gliding between 3R and 4R tau suggest important features of tau function related to the normal shift in tau isoform composition that occurs during neural development as well as in neurodegeneration caused by altered expression ratios of otherwise normal tau isoforms.

• Maytansine and cellular metabolites of antibody-maytansinoid conjugates strongly suppress microtubule dynamics by binding to microtubules.

  Authors: Manu Lopus, Emin Oroudjev, Leslie Wilson, Sharon Wilhelm, Wayne Widdison, Ravi Chari, Mary Ann Jordan

  Molecular cancer therapeutics. 10/2010; 9(10):2689-99.

  Maytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity haveMaytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity have prevented successful clinical use. Recently, antibody-conjugated maytansine derivatives have been developed to overcome these drawbacks. Several conjugates show promising early clinical results. We evaluated the effects on microtubule polymerization and dynamic instability of maytansine and two cellular metabolites (S-methyl-DM1 and S-methyl-DM4) of antibody-maytansinoid conjugates that are potent in cells at picomolar levels and that are active in tumor-bearing mice. Although S-methyl-DM1 and S-methyl-DM4 inhibited polymerization more weakly than maytansine, at 100 nmol/L they suppressed dynamic instability more strongly than maytansine (by 84% and 73%, respectively, compared with 45% for maytansine). However, unlike maytansine, S-methyl-DM1 and S-methyl-DM4 induced tubulin aggregates detectable by electron microscopy at concentrations ≥2 μmol/L, with S-methyl-DM4 showing more extensive aggregate formation than S-methyl-DM1. Both maytansine and S-methyl-DM1 bound to tubulin with similar K(D) values (0.86 ± 0.2 and 0.93 ± 0.2 μmol/L, respectively). Tritiated S-methyl-DM1 bound to 37 high-affinity sites per microtubule (K(D), 0.1 ± 0.05 μmol/L). Thus, S-methyl-DM1 binds to high-affinity sites on microtubules 20-fold more strongly than vinblastine. The high-affinity binding is likely at microtubule ends and is responsible for suppression of microtubule dynamic instability. Also, at higher concentrations, S-methyl-DM1 showed low-affinity binding either to a larger number of sites on microtubules or to sedimentable tubulin aggregates. Overall, the maytansine derivatives that result from cellular metabolism of the antibody conjugates are themselves potent microtubule poisons, interacting with microtubules as effectively as or more effectively than the parent molecule.

• Maytansinoid-antibody conjugates induce mitotic arrest by suppressing microtubule dynamic instability.

  Authors: Emin Oroudjev, Manu Lopus, Leslie Wilson, Charlene Audette, Carmela Provenzano, Hans Erickson, Yelena Kovtun, Ravi Chari, Mary Ann Jordan

  Molecular cancer therapeutics. 10/2010; 9(10):2700-13.

  Maytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoidsMaytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoids (DM1 and DM4) attached to tumor-specific antibodies have shown promising clinical results. To determine the mechanism by which the antibody-DM1 conjugates inhibit cell proliferation, we examined the effects of the cleavable anti-EpCAM-SPP-DM1 and uncleavable anti-EpCAM-SMCC-DM1 conjugates on MCF7 human breast tumor cells. We also examined the effects of the free maytansinoids, maytansine and S-methyl DM1 (a version of DM1 that is stable in cell culture medium), for comparison. Both the conjugates and free maytansinoids potently inhibited MCF7 cell proliferation at nanomolar and subnanomolar concentrations, respectively, by arresting the cells in mitotic prometaphase/metaphase. Arrest occurred in concert with the internalization and intracellular processing of both conjugates under conditions that induced abnormal spindle organization and suppressed microtubule dynamic instability. Microtubule depolymerization occurred only at significantly higher drug concentrations. The results indicate that free maytansinoids, antibody-maytansinoid conjugates, and their metabolites exert their potent antimitotic effects through a common mechanism involving suppression of microtubule dynamic instability.

• Cytotoxic effects of bilberry extract on MCF7-GFP-tubulin breast cancer cells.

  Authors: Vy Nguyen, Jessica Tang, Emin Oroudjev, Choong Jae Lee, Cecilia Marasigan, Leslie Wilson, George Ayoub

  Journal of medicinal food. 04/2010; 13(2):278-85.

  Bilberry (European blueberry) has been reported to have many biological effects, including anticancer activity. In this study, we investigated the antiproliferative effects of bilberry extract inBilberry (European blueberry) has been reported to have many biological effects, including anticancer activity. In this study, we investigated the antiproliferative effects of bilberry extract in relation to its ability to induce apoptosis and affect microtubule assembly and organization in MCF7 human breast cancer cells. We observed that bilberry extract inhibited cell proliferation in a concentration-dependent fashion with a 50% inhibitory concentration of 0.3-0.4 mg/mL, in concert with induction of apoptotic cell death. At these concentrations there was no selective inhibition of mitosis or any other cell cycle stage, nor was there any apparent effect on the microtubule or actin cytoskeletons. However, somewhat higher extract concentrations (0.5-0.9 mg/mL) did cause an increase in the fraction of cells at the G(2)/M phase of the cell cycle, together with destruction of microtubules and formation of punctate tubulin aggregates in the cells. Bilberry extract at 0.3-0.4 mg/mL did not appreciably inhibit microtubule polymerization in vitro, but significant inhibition of polymerization (approximately 30%) did occur at higher extract concentrations (0.5-1 mg/mL). We conclude that bilberry extract as ingested by humans, not just the purified anthocyanins it contains, inhibits proliferation of and induces apoptosis in breast cancer cells at its lowest effective concentrations via a mechanism that does not involve action on microtubules or on mitosis. We further conclude that at somewhat higher concentrations the extract modifies microtubule organization in cells and causes accumulation of cells at mitosis by a direct action on microtubules.

• Eribulin binds at microtubule ends to a single site on tubulin to suppress dynamic instability.

  Authors: Jennifer A Smith, Leslie Wilson, Olga Azarenko, Xiaojie Zhu, Bryan M Lewis, Bruce A Littlefield, Mary Ann Jordan

  Biochemistry. 02/2010; 49(6):1331-7.

  Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressingEribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing dynamic instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086-1095]. Using [(3)H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall K(d) of 46 microM, but also showing a real or apparent very high affinity (K(d) = 0.4 microM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 +/- 1.3 molecules per microtubule (K(d) = 3.5 microM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that the binding of a single eribulin molecule at a microtubule end can potently inhibit its growth. Eribulin does not suppress dynamic instability at microtubule minus ends. Preincubation of microtubules with 2 or 4 microM vinblastine induced additional lower-affinity eribulin binding sites, most likely at splayed microtubule ends. Overall, our results indicate that eribulin binds with high affinity to microtubule plus ends and thereby suppresses dynamic instability.

• Preparation of microtubule protein and purified tubulin from bovine brain by cycles of assembly and disassembly and phosphocellulose chromatography.

  Authors: Herbert P Miller, Leslie Wilson

  Methods in cell biology. 01/2010; 95:3-15.

• Analysis of dynamic instability of steady-state microtubules in vitro by video-enhanced differential interference contrast microscopy with an appendix by Emin Oroudjev.

  Authors: Mythili Yenjerla, Manu Lopus, Leslie Wilson

  Methods in cell biology. 01/2010; 95:189-206.

  Microtubules are major constituents of the cytoskeleton which display dynamic properties. They exhibit dynamic instability which is defined as the stochastic switching between growing and shorteningMicrotubules are major constituents of the cytoskeleton which display dynamic properties. They exhibit dynamic instability which is defined as the stochastic switching between growing and shortening at microtubule ends. Dynamic instability plays an important role in diverse cellular functions including cell migration and mitosis. Many successful antimitotic drugs and microtubule-associated proteins (MAPs) are known to modulate microtubule dynamics, and it is important to analyze the in vitro dynamic instability of microtubules to study the mechanism of action of microtubule-targeted therapeutics and MAPs. In this chapter, we describe a method to analyze the in vitro dynamic instability of microtubules at steady state using video-enhanced differential contrast (VE-DIC) microscopy in detail. In this method, microtubules are assembled to steady state at 30 degrees C with MAP-free tubulin in a slide chamber in the presence of GTP, using sea urchin axonemes as nucleating seeds. Images of microtubules are enhanced and recorded in real time by a video camera and an image processor connected to a DIC microscope which is maintained at 30 degrees C. We use two software programs to track and analyze the growing and shortening of plus or minus ends of microtubules in the real-time images recorded using VE-DIC. In this chapter, we describe the instructions to use the tracking software Real Time Measurement II (RTM II) program. The instructions to use the analysis software Microtubule Life History Analysis Procedures (MT-LHAP) in Igor Pro software have been described in detail in an appendix (Oroudjev, 2010) following this chapter.

• The neuroprotective peptide NAP does not directly affect polymerization or dynamics of reconstituted neural microtubules.

  Authors: Mythili Yenjerla, Nichole E LaPointe, Manu Lopus, Corey Cox, Mary Ann Jordan, Stuart C Feinstein, Leslie Wilson

  Journal of Alzheimer's disease : JAD. 01/2010; 19(4):1377-86.

  NAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is a neuroprotective peptide that shows cognitive protection in patients with amnestic mild cognitive impairment, a precursor to Alzheimer's disease. NAPNAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is a neuroprotective peptide that shows cognitive protection in patients with amnestic mild cognitive impairment, a precursor to Alzheimer's disease. NAP exhibits potent neuroprotective properties in several in vivo and cellular models of neural injury. While NAP has been found in many studies to affect microtubule assembly and/or stability in neuronal and glial cells at fM concentrations, it has remained unclear whether NAP acts directly or indirectly on tubulin or microtubules. We analyzed the effects of NAP (1 fM-1 microM) on the assembly of reconstituted bovine brain microtubules in vitro and found that it did not significantly (p< 0.05) alter polymerization of either purified tubulin or of a mixture of tubulin and unfractionated microtubule-associated proteins. NAP also had no significant effect (p < 0.05) on the growing and shortening dynamics of steady-state microtubules at their plus ends, nor did it alter the polymerization or dynamics of microtubules assembled in the presence of 3-repeat or 4-repeat tau. Thus, the neuroprotective activity of NAP does not appear to involve a direct action on the polymerization or dynamics of purified tubulin or microtubules.

• Hesperidin suppressed proliferations of both Human breast cancer and androgen-dependent prostate cancer cells.

  Authors: Choong Jae Lee, Leslie Wilson, Mary Ann Jordan, Vy Nguyen, Jessica Tang, Gregory Smiyun

  Phytotherapy research : PTR. 06/2009;

  Hesperidin, a flavonoid derived from citrus fruits, has been reported to show various biological effects including anticancer activity. This study investigated whether hesperidin affected theHesperidin, a flavonoid derived from citrus fruits, has been reported to show various biological effects including anticancer activity. This study investigated whether hesperidin affected the proliferation of MCF-7 human breast cancer cells transfected with green fluorescent protein (GFP)/alpha-tubulin (MCF-7-GFP-Tubulin cells), androgen-independent PC-3 and DU-145 prostate cancer cells, and androgen-dependent LNCaP prostate cancer cells. The results were as follows. (1) Hesperidin inhibited the proliferation of MCF-7-GFP-Tubulin cells, probably not through an antimitotic mechanism. (2) Hesperidin also inhibited both basal and testosterone-induced proliferation of LNCaP cells. (3) However, hesperidin did not significantly affect the cell proliferation of two hormone-independent prostate cancer cells, PC-3 and DU-145. It is concluded that hesperidin can inhibit the proliferation of breast cancer cells through mechanisms other than antimitosis and it is suggested that hesperidin be further investigated for the possible interaction with androgenic receptors and involvement in signaling pathway after receptor binding in prostate cancer cells through future research. Copyright (c) 2009 John Wiley & Sons, Ltd.

• Regulation of microtubule dynamic instability in vitro by differentially phosphorylated stathmin.

  Authors: Tapas Manna, Douglas A Thrower, Srinivas Honnappa, Michel O Steinmetz, Leslie Wilson

  The Journal of biological chemistry. 05/2009;

  Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass throughStathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two a:ss tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser16, Ser25, Ser38 and Ser63) reduces its microtubule destabilizing activity. However, the effects of phosphorylation of stathmin's individual serine residues on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser16 or Ser63, and doubly phosphorylated at Ser25 and Ser38 on its ability to modulate microtubule dynamic instability at steady state in vitro. Phosphorylation at either Ser16 or Ser63 strongly reduced or abolished stathmin's ability to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser25, Ser38 did not affect stathmin's binding to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that stathmin's effects on dynamic instability are strongly but differently attenuated by phosphorylation at Ser16 and Ser63 and support the hypothesis that selective targeting by Ser16 -specific or Ser63 -specific kinases provides complimentary mechanisms for regulating microtubule function.

• Microtubule Dynamics

  Authors: Manu Lopus, Mythili Yenjerla, Leslie Wilson

  01/2009: pages 153-160;

• Carbendazim inhibits cancer cell proliferation by suppressing microtubule dynamics.

  Authors: Mythili Yenjerla, Corey Cox, Leslie Wilson, Mary Ann Jordan

  The Journal of pharmacology and experimental therapeutics. 12/2008;

  Carbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of humanCarbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of human cancer cells including drug- and multidrug-resistant and p53-deficient cell lines. Due to its promising preclinical antitumor activity, it has undergone Phase I clinical trials and is under further clinical development. While it weakly inhibits polymerization of brain microtubules and induces G2/M arrest in tumor cells, its mechanism of action in human cells has not been fully elucidated. We examined its mechanism of action in MCF7 human breast cancer cells and found that it inhibits proliferation (IC50: 10 microM) and half-maximally arrests mitosis at a similar concentration (8 microM), in concert with suppression of microtubule dynamic instability without appreciable microtubule depolymerization. It induces mitotic spindle abnormalities and reduces the metaphase intercentromere distance of sister chromatids, indicating reduction of tension on kinetochores, thus leading to metaphase arrest. With microtubules assembled in vitro from pure tubulin, carbendazim also suppresses dynamic instability, reducing the dynamicity by 50% at 10 microM with only minimal (21%) reduction of polymer mass. Carbendazim binds to mammalian tubulin (Kd, 42.8 +/- 4.0 microM). Unlike some benzimidazoles that bind to the colchicine-site in tubulin, carbendazim does not compete with colchicine nor does it compete with vinblastine for binding to brain tubulin. Thus carbendazim binds to an as yet unidentified site in tubulin and inhibits tumor cell proliferation by suppressing the growing and shortening phases of microtubule dynamic instability, thus inducing mitotic arrest.

• FTDP-17 mutations in tau alter the regulation of microtubule dynamics - an "Alternative Core" model for normal and pathological tau action.

  Authors: Adria C Leboeuf, Sasha F Levy, Michelle Gaylord, Arnab Bhattacharya, Ambuj K. Singh, Mary Ann Jordan, Leslie Wilson, Stuart C Feinstein

  The Journal of biological chemistry. 11/2008;

  Mutations affecting either the structure or regulation of the microtubule associated protein tau cause neuronal cell death and dementia. However, the molecular mechanisms mediating these deleteriousMutations affecting either the structure or regulation of the microtubule associated protein tau cause neuronal cell death and dementia. However, the molecular mechanisms mediating these deleterious effects remain unclear. Among tau's most characterized activities is the ability to regulate microtubule dynamics, known to be essential for proper cell function and viability. Here, we have tested the hypothesis that tau mutations causing neurodegeneration also alter the ability of tau to regulate the dynamic instability behaviors of microtubules. Using in vitro microtubule dynamics assays to assess average microtubule growth rates, microtubule growth rate distributions and catastrophe frequencies, we found that all tested mutants possessing amino acid substitutions or deletions mapping to either the repeat or inter-repeat regions of tau do indeed compromise its ability to regulate microtubule dynamics. Further mutational analyses suggest a novel mechanism of tau regulatory action based on an "alternative core" of microtubule binding and regulatory activities composed of two repeats and the inter-repeat between them. In this model, the inter-repeat serves as the primary regulator of microtubule dynamics while the flanking repeats serve as tethers to properly position the inter-repeat on the microtubule. Importantly, since there are multiple inter-repeats on each tau molecule, there are also multiple cores on each tau molecule, each with distinct mechanistic capabilities, thereby providing significant regulatory potential. Taken together, the data are consistent with a microtubule misregulation mechanism for tau mediated neuronal cell death and provide a novel mechanistic model for normal and pathological tau action.

• Suppression of microtubule dynamic instability and turnover in MCF7 breast cancer cells by sulforaphane.

  Authors: Olga Azarenko, Tatiana Okouneva, Keith W Singletary, Mary Ann Jordan, Leslie Wilson

  Carcinogenesis. 11/2008;

  Sulforaphane, a prominent isothiocyanate present in cruciferous vegetables, is believed to be responsible along with other isothiocyanates for the cancer preventive activity of such vegetables.Sulforaphane, a prominent isothiocyanate present in cruciferous vegetables, is believed to be responsible along with other isothiocyanates for the cancer preventive activity of such vegetables. Sulforaphane arrests mitosis, possibly by affecting spindle microtubule function. A critical property of microtubules is their rapid and time-sensitive growth and shortening dynamics (dynamic instability), and suppression of dynamics by antimitotic anticancer drugs (e.g., taxanes and the vinca alkaloids) is central to the anticancer mechanisms of such drugs. We found that at concentrations that inhibited proliferation and mitosis of MCF7-GFP-alpha-tubulin breast tumor cells by approximately 50% ( approximately 15 muM), sulforaphane significantly modified microtubule organization in arrested spindles without modulating the spindle microtubule mass, in a manner similar to that of much more powerful antimitotic drugs. By using quantitative fluorescence video microscopy, we determined that at its mitotic IC(50), sulforaphane suppressed the dynamic instability of the interphase microtubules in these cells, strongly reducing the rate and extent of growth and shortening and decreasing microtubule turnover, without affecting the polymer mass. Sulforaphane suppressed the dynamics of purified microtubules in a similar fashion at concentrations well below those required to depolymerize microtubules, indicating that the suppression of dynamic instability by sulforaphane in cells is due to a direct effect on the microtubules. The results indicate that sulforaphane arrests proliferation and mitosis by stabilizing microtubules in a manner weaker than but similar to more powerful clinically used antimitotic anticancer drugs, and strongly support the hypothesis that inhibition of mitosis by microtubule stabilization is important for sulforaphane's chemopreventive activity.

• Inhibition of centromere dynamics by eribulin (E7389) during mitotic metaphase.

  Authors: Tatiana Okouneva, Olga Azarenko, Leslie Wilson, Bruce A Littlefield, Mary Ann Jordan

  Molecular cancer therapeutics. 07/2008; 7(7):2003-11.

  Eribulin (E7389), a synthetic analogue of halichondrin B in phase III clinical trials for breast cancer, binds to tubulin and microtubules. At low concentrations, it suppresses the growth phase ofEribulin (E7389), a synthetic analogue of halichondrin B in phase III clinical trials for breast cancer, binds to tubulin and microtubules. At low concentrations, it suppresses the growth phase of microtubule dynamic instability in interphase cells, arrests mitosis, and induces apoptosis, suggesting that suppression of spindle microtubule dynamics induces mitotic arrest. To further test this hypothesis, we measured the effects of eribulin on dynamics of centromeres and their attached kinetochore microtubules by time-lapse confocal microscopy in living mitotic U-2 OS human osteosarcoma cells. Green fluorescent protein-labeled centromere-binding protein B marked centromeres and kinetochore-microtubule plus-ends. In control cells, sister chromatid centromere pairs alternated under tension between increasing and decreasing separation (stretching and relaxing). Eribulin suppressed centromere dynamics at concentrations that arrest mitosis. At 60 nmol/L eribulin (2 x mitotic IC(50)), the relaxation rate was suppressed 21%, the time spent paused increased 67%, and dynamicity decreased 35% (but without reduction in mean centromere separation), indicating that eribulin decreased normal microtubule-dependent spindle tension at the kinetochores, preventing the signal for mitotic checkpoint passage. We also examined a more potent, but in tumors less efficacious antiproliferative halichondrin derivative, ER-076349. At 2 x IC(50) (4 nmol/L), mitotic arrest also occurred in concert with suppressed centromere dynamics. Although media IC(50) values differed 15-fold between the two compounds, the intracellular concentrations were similar, indicating more extensive relative uptake of ER-076349 into cells compared with eribulin. The strong correlation between suppression of kinetochore-microtubule dynamics and mitotic arrest indicates that the primary mechanism by which eribulin blocks mitosis is suppression of spindle microtubule dynamics.

• A general modeling and visualization tool for comparing different members of a group: application to studying tau-mediated regulation of microtubule dynamics.

  Authors: Arnab Bhattacharya, Sasha Levy, Adria LeBoeuf, Michelle Gaylord, Leslie Wilson, Ambuj K. Singh, Stuart C Feinstein

  BMC bioinformatics. 02/2008; 9:339.

  ABSTRACT: BACKGROUND: Innumerable biological investigations require comparing collections of molecules, cells or organisms to one another with respect to one or more of their properties. Almost allABSTRACT: BACKGROUND: Innumerable biological investigations require comparing collections of molecules, cells or organisms to one another with respect to one or more of their properties. Almost all of these comparisons are performed manually, which can be susceptible to inadvertent bias as well as miss subtle effects. The development and application of computer-assisted analytical and interpretive tools could help address these issues and thereby dramatically improve these investigations. RESULTS: We have developed novel computer-assisted analytical and interpretive tools and applied them to recent studies examining the ability of 3-repeat and 4-repeat tau to regulate the dynamic behavior of microtubules in vitro. More specifically, we have developed an automated and objective method to define growth, shortening and attenuation events from real time videos of dynamic microtubules, and demonstrated its validity by comparing it to manually assessed data. Additionally, we have used the same data to develop a general strategy of building different models of interest, computing appropriate dissimilarity functions to compare them, and embedding them on a two-dimensional plot for visualization and easy comparison. Application of these methods to assess microtubule growth rates and growth rate distributions established the validity of the embedding procedure and revealed non-linearity in the relationship between the tau:tubulin molar ratio and growth rate distribution. CONCLUSION: This work addresses the need of the biological community for rigorously quantitative and generally applicable computational tools for comparative studies. The two-dimensional embedding method retains the inherent structure of the data, and yet markedly simplifies comparison between models and parameters of different samples. Most notably, even in cases where numerous parameters exist by which to compare the different samples, our embedding procedure provides a generally applicable computational strategy to detect subtle relationships between different molecules or conditions that might otherwise escape manual analyses.