Mythili Yenjerla

Publications of Mythili Yenjerla

• 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.

• 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.

• Chapter 11 - 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.

  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°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°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.