Effect Of Tubulin Research Articles

Modeling neuron growth using isogeometric collocation based phase field method

We present a new computational framework of neuron growth based on the phase field method and develop an open-source software package called “NeuronGrowth_IGAcollocation”. Neurons consist of a cell body, dendrites, and axons. Axons and dendrites are long processes extending from the cell body and enabling information transfer to and from other neurons. There is high variation in neuron morphology based on their location and function, thus increasing the complexity in mathematical modeling of neuron growth. In this paper, we propose a novel phase field model with isogeometric collocation to simulate different stages of neuron growth by considering the effect of tubulin. The stages modeled include lamellipodia formation, initial neurite outgrowth, axon differentiation, and dendrite formation considering the effect of intracellular transport of tubulin on neurite outgrowth. Through comparison with experimental observations, we can demonstrate qualitatively and quantitatively similar reproduction of neuron morphologies at different stages of growth and allow extension towards the formation of neurite networks.

Abstract A88: Openers of voltage dependent anion channels are anti-Warburg agents that enhance mitochondrial metabolism, decrease glycolysis, activate JNK and induce ROS-dependent killing of cancer cells

Abstract Background: Warburg metabolism is characterized by enhanced aerobic glycolysis and suppressed mitochondrial metabolism. Voltage dependent anion channels (VDAC) located in the mitochondrial outer membrane control flux of metabolites into mitochondria. Free α,β-tubulin closes VDAC in planar lipid bilayers (Rostovtseva et al., PNAS 105:18746), and high free tubulin in cancer cells decreases mitochondrial membrane potential (ΔΨ) by limiting ingress of respiratory substrates and ATP that supportΔΨ formation (Maldonado et al., Cancer Res. 70:10192). The small molecule erastin opens VDAC by antagonizing the inhibitory effect of tubulin on VDAC (Maldonado et al., JBC 288:11920). Here, we hypothesized that erastin and erastin-like antagonists of VDAC-tubulin interaction increase mitochondrial redox state, decrease glycolysis, increase mitochondrial formation of reactive oxygen species (ROS) and activate c-jun N-terminal kinase (JNK), culminating in mitochondrial dysfunction and death of cancer cells. Our AIM was to evaluate the effects of erastin/erastin-like compounds on mitochondrialΔΨ, NAD(P)H+ and ROS, lactate generation, JNK activation and cell killing in HepG2 and Huh7 hepatocarcinoma cells. Methods: Confocal/multiphoton fluorescence microscopy assessedΔΨ (tetramethylrhodamine methylester), ROS (chloromethyldichlorofluorescein [cmDCF]; MitoSOX Red) and NAD(P)H (autofluorescence). JNK was assessed by Western blotting and cell killing by propidium iodide fluorometry. Results: In both HepG2 and Huh7, erastin and small molecules X1 and X2 identified in a high-throughput screen increasedΔΨ and NAD(P)H. The three compounds also prevented mitochondrial depolarization by cytosolic high free tubulin induced by nocodazole (microtubule depolymerizing agent). Initial increases ofΔΨ were followed by mitochondrial depolarization occurring 1-2 h after X1 and X2 and 3-4 h after erastin. Lactate generation tested after X1 decreased by 60%. cmDCF and MitoSOX fluorescence increased 30 min after X1 and 60 min after X2 and erastin. The mitochondrially targeted antioxidant MitoQ blocked this increase of ROS. Additionally, erastin caused JNK activation with maximal phosphorylation within 1 h. Both X1 and X2 caused killing of cancer cells (~93% and ~76% respectively), which the antioxidant N-acetylcysteine (100µM) blocked. By contrast, X1 and X2 caused <25% cell death in primary rat hepatocytes. Conclusion: Antagonists of VDAC-tubulin interaction promote mitochondrial metabolism and inhibit glycolysis. These anti-Warburg drugs also cause mitochondrial generation of ROS, which in turn leads to JNK activation, mitochondrial dysfunction and selective death of cancer cells that is prevented by antioxidants. Grants: T32DK083262 to DND; DK073336, DK037034 and 14.Z50.31.0028 to JJL; ACS 13-043-01 and COBRE Pilot Project GM103542 to ENM. Citation Format: David N. DeHart, Monika Gooz, John J. Lemasters, John J. Lemasters, Eduardo N. Maldonado. Openers of voltage dependent anion channels are anti-Warburg agents that enhance mitochondrial metabolism, decrease glycolysis, activate JNK and induce ROS-dependent killing of cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A88.

The dynamic behaviour of mitochondria in living zygotes during maturation and meiosis in Chlamydomonas reinhardtii

To understand fully the function of mitochondria during the development of cells and organs, it is important to elucidate the dynamics of their morphology. However, the detailed morphology of mitochondria during meiosis has not yet been studied in algae. We examined the mitochondrial morphology of Chlamydomonas reinhardtii and classified zygotes into seven types by mitochondrial morphology in order to analyse the morphological change in mature and meiotic zygotes. We also investigated the oxygen consumption of living zygotes and the effects of tubulin and actin polymerization inhibitors on mitochondria, using fluorescence microscopy and oxygen electrodes. During zygote maturation, mitochondria fragmented into small particles, with a large decrease in oxygen consumption. When mature zygotes were exposed to light, mitochondria became tubular and formed a network, and oxygen consumption gradually recovered. At the same time, particle-like mitochondrial nucleoids became stringy and produced new nucleoid particles. Tubular mitochondria accumulated around the cell nucleus and then spread throughout the cell. Cell division followed (first and second rounds), and the resultant daughter cells had tubular mitochondria in a mesh-like arrangement. An inhibitor of tubulin polymerization, demecolcine, inhibited the assembly of mitochondria around the cell nucleus, whereas an inhibitor of actin polymerization, latrunculin B, inhibited the formation of tubular mitochondria. These results suggest that microtubules are probably involved in mitochondrial accumulation around the cell nucleus, whereas microfilaments may maintain the tubular network of mitochondria.

Brain plasma membrane Na+,K+-ATPase is inhibited by acetylated tubulin.

Membranes from brain tissue contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. The hydrophobic behavior of this tubulin is due to the formation of a complex with the alpha-subunit of Na+,K+-ATPase. In the present work we show that the interaction of tubulin with Na+K+-ATPase inhibits the enzyme activity. We found that the magnitude of the inhibition is correlated with: (1) concentration of the acetylated tubulin isoform present in the tubulin preparation used, and (2) amount of acetylated tubulin isoform isolated as a hydrophobic compound. In addition, some compounds involved in the catalytic action of Na+K+-ATPase were assayed to determine their effects on the inhibitory capability of tubulin on this enzyme. The inhibitory effect of tubulin was only slightly decreased by ATP at relatively low nucleotide concentration (0.06 mM). NaCl (1-160 mM) and KCl (0.2-10 mM) showed no effect whereas inorganic phosphate abolished the inhibitory effect of tubulin in a concentration-dependent manner.

Participation of Tubulin in the Stimulatory Regulation of Adenylyl Cyclase in Rat Cerebral Cortex Membranes

This study examined effects of tubulin on the activation of adenylyl cyclase in rat cerebral cortex membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of the enzyme by approximately 156% under conditions in which stimulation rather than inhibition of the enzyme was favored. Tubulin-GppNHp activated isoproterenol-sensitive adenylyl cyclase, potentiated forskolin-stimulated activity of the enzyme, and reduced agonist binding affinity for beta-adrenergic receptors. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to Gs alpha as well as Gi alpha. These results suggest that, in rat cerebral cortex membranes, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to Gs alpha, as well as affecting the Gi-mediated inhibitory pathway.

Effect of Tubulin on the Activity of the Muscle Isoenzyme of Lactate-Dehydrogenase

The interaction between muscle-type lactate dehydrogenase (LDH m) and tubulin was investigated by monitoring the combined effect of NADH and tubulin on steady-state kinetics and the combined effect of NADH and pH on complex formation between tubulin and the enzyme. Steady-state kinetics showed that LDH m is inhibited by tubulin. Experiments with heart-type lactate dehydrogenase (LDH h) showed that the inhibition is unique to the muscle-type enzyme. The magnitude of the inhibition is dependent upon the concentration of NADH as well as the pH of the buffer medium. The enzyme was less sensitive to inhibition at 50μM NADH than at 10μM NADH. Since this effect of NADH is not due to an ionic strength contribution, it is deemed to be specific. In contrast to the absence of tubulin, its presence induced a modification of the kinetic behavior of LDH m; i.e., the velocity dependence on NADH concentration displayed a marked sigmoid response. The inhibition of LDH m by tubulin is more pronounced at lower pH values than at higher pH values. The pH-dependent inhibitory profile is shifted to the left (i.e., p K a is decreased) with increasing concentrations of NADH. This pattern is remarkably similar to that observed for the binding of the enzyme on Sepharose immobilized tubulin and is consistent with the premise that inhibition is a result of interaction between these proteins. NAD + was much less effective than NADH in dissociating LDH m from immobilized tubulin. Results from these in vitro studies are consistent with similar observations dealing with other glycolytic enzymes and cytoskeleton proteins, which show that enzyme catalytic properties are modified upon binding.

Effect of tubulin upon chronic indolent ulcers.

Effect of tubulin upon chronic indolent ulcers.