Dual Impact of a Benzimidazole Resistant β-Tubulin on Microtubule Behavior in Fission Yeast

The cytoskeleton microtubule consists of polymerized αβ-tubulin dimers and plays essential roles in many cellular events. Reagents that inhibit microtubule behaviors have been developed as antifungal, antiparasitic, and anticancer drugs. Benzimidazole compounds, including thiabendazole (TBZ), carbendazim (MBC), and nocodazole, are prevailing microtubule poisons that target β-tubulin and inhibit microtubule polymerization. The molecular basis, however, as to how the drug acts on β-tubulin remains controversial. Here, we characterize the S. pombe β-tubulin mutant nda3-TB101, which was previously isolated as a mutant resistance to benzimidazole. The mutation site tyrosine at position 50 is located in the interface of two lateral β-tubulin proteins and at the gate of a putative binging pocket for benzimidazole. Our observation revealed two properties of the mutant tubulin. First, the dynamics of cellular microtubules comprising the mutant β-tubulin were stabilized in the absence of benzimidazole. Second, the mutant protein reduced the affinity to benzimidazole in vitro. We therefore conclude that the mutant β-tubulin Nda3-TB101 exerts a dual effect on microtubule behaviors: the mutant β-tubulin stabilizes microtubules and is insensitive to benzimidazole drugs. This notion fine-tunes the current elusive molecular model regarding binding of benzimidazole to β-tubulin.

Expression and Clinical Significance of Cytoskeleton microtubule-associated - Tau in gastric carcinoma

Background and Objectives: Microtubules, the main components of spindles in the mitotic phase, can provide the suitable conditions for unlimited proliferation of tumor cells. Cytoskeleton microtubule-associated-Tau accelerates the progress of malignant tumor through microtubules. Currently, the expression of Cytoskeleton microtubule-associated-Tau in gastric cancer and the relationship between clinical pathological factors research has not yet been reported, so the report aims to elucidate the features of Cytoskeleton microtubule-associated-Tau expression in normal gastric tissue and gastric carcinoma tissue.Materials and Methods: The expressions of Cytoskeleton microtubule-associated protein-Tau protein and Cytoskeleton microtubule-associated protein-Tau mRNA were investigated in 60 cases of gastric carcinoma (cases) and 10 cases of normal gastric tissues (controls). Immunohistochemistry and RT-PCR were respectively used to detect the expression of Cytoskeleton microtubule-associated protein-Tau protein and Tau mRNA in the normal and carcinomatous gastric tissues.Results: The expressions of both cytoskeleton microtubule-associated protein-Tau protein and Tau mRNA in the carcinomatous gastric tissue were higher than in the normal gastric tissue (P<0.05). Likewise, both biomarkers were expressed significantly lower in the well-differentiated and moderately-differentiated gastric carcinoma was than in the poorly-differentiated one (P<0.05). Moreover, their expressions in the gastric carcinoma without lymph node metastasis was lower than in the gastric carcinoma with lymph node metastasis (P<0.05).Conclusion: The expressions of Cytoskeleton microtubule-associated-protein, Tau-protein and Cytoskeleton microtubule-associated-protein, Tau-mRNA were significantly different between the normal gastric tissue and the gastric carcinoma tissue, and were correlated with the degree differentiation and lymph node status.JCMS Nepal. 2015;11(3):6-11.

AT1 receptor-mediated accumulation of extracellular angiotensin II in proximal tubule cells: role of cytoskeleton microtubules and tyrosine phosphatases

Long-term angiotensin II (ANG II) administration is associated with increased ANG II accumulation in the kidney, but intrarenal compartment(s) involved in this response remains to be determined. We tested the hypothesis that 1) extracellular ANG II is taken up by proximal tubule cells (PTCs) through AT1 receptor-mediated endocytosis, 2) this process is regulated by cytoskeleton microtubule- and tyrosine phosphatase-dependent mechanisms, and 3) AT1 receptor-mediated endocytosis of ANG II has a functional relevance by modulating intracellular cAMP signaling. In cultured PTCs, [125I]Tyr-labeled ANG II and fluorescein labeled-ANG II were internalized in a time-dependent manner and colocalized with the endosome marker Alexa Fluor 594-transferrin. Endocytosis of extracellular ANG II was inhibited by the AT1 receptor blocker losartan (16.5 ± 4.6%, P < 0.01 vs. ANG II, 78.3 ± 6.2%) and by the tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 30.0 ± 3.5%, P < 0.05 vs. ANG II). Intracellular ANG II levels were increased by ∼58% (basal, 229.8 ± 11.4 vs. ANG II, 361.3 ± 11.8 pg ANG II/mg protein, P < 0.01), and the responses were blocked by losartan ( P < 0.01), the cytoskeleton microtubule inhibitor colchicine ( P < 0.05), and PAO ( P < 0.01), whereas depletion of clathrin-coated pits with hyperosmotic sucrose had no effect (356.1 ± 25.5 pg ANG II/mg protein, not significant). ANG II accumulation was associated with significant inhibition of both basal (control, 15.5 ± 2.8 vs. ANG II, 9.1 ± 2.4 pmol/mg protein, P < 0.05) and forskolin-stimulated cAMP signaling (forskolin, 68.7 ± 8.6 vs. forskolin + ANG II, 42.8 ± 13.8 pmol/mg protein, P < 0.01). These effects were blocked by losartan and PAO. We conclude that extracellular ANG II is internalized in PTCs through AT1 receptor-mediated endocytosis and that internalized ANG II may play a functional role in proximal tubule cells by inhibiting intracellular cAMP signaling.