[36] Preparation of tubulin from brain

Williams, Robley C.; Lee, James C.

doi:10.1016/0076-6879(82)85038-6

Cited by 391 publications

(192 citation statements)

References 11 publications

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“…Two batches of kinesin motors were prepared and used in this study. Bovine brain tubulin (Williams and Lee, 1982) was purified and rhodamine labeled as described previously (Hyman et al, 1991) MTs were polymerized by mixing 32 M rhodamine-labeled tubulin, 4 mM MgCl 2 , 1 mM guanosine triphosphate, and 5% DMSO in BRB80 buffer [80 mM piperazine-N,NЈ-bis(2-ethanesulfonic acid), 1 mM EGTA, and 1 mM MgCl 2 , pH 6.9 with KOH] and incubating at 37°C for 20 min. Polymerized MTs were stabilized with 10 M paclitaxel.…”

Section: Kinesins and In Vitro Microtubulesmentioning

confidence: 99%

Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

Bicek

Tüzel

Demtchouk

et al. 2009

MBoC

103

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Microtubules (MTs) have been proposed to act mechanically as compressive struts that resist both actomyosin contractile forces and their own polymerization forces to mechanically stabilize cell shape. To identify the origin of MT bending, we directly observed MT bending and F-actin transport dynamics in the periphery of LLC-PK1 epithelial cells. We found that F-actin is nearly stationary in these cells even as MTs are deformed, demonstrating that MT bending is not driven by actomyosin contractility. Furthermore, the inhibition of myosin II activity through the use of blebbistatin results in microtubules that are still dynamically bending. In addition, as determined by fluorescent speckle microscopy, MT polymerization rarely results, if ever, in bending. We suppressed dynamic instability using nocodazole, and we observed no qualitative change in the MT bending dynamics. Bending most often results from anterograde transport of proximal portions of the MT toward a nearly stationary distal tip. Interestingly, we found that in an in vitro kinesin-MT gliding assay, MTs buckle in a similar manner. To make quantitative comparisons, we measured curvature distributions of observed MTs and found that the in vivo and in vitro curvature distributions agree quantitatively. In addition, the measured MT curvature distribution is not Gaussian, as expected for a thermally driven semiflexible polymer, indicating that thermal forces play a minor role in MT bending. We conclude that many of the known mechanisms of MT deformation, such as polymerization and acto-myosin contractility, play an inconsequential role in mediating MT bending in LLC-PK1 cells and that MT-based molecular motors likely generate most of the strain energy stored in the MT lattice. The results argue against models in which MTs play a major mechanical role in LLC-PK1 cells and instead favor a model in which mechanical forces control the spatial distribution of the MT array.

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Section: Kinesins and In Vitro Microtubulesmentioning

confidence: 99%

Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

Bicek

Tüzel

Demtchouk

et al. 2009

MBoC

103

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“…All motors were expressed in bacteria and purified by Ni column chromatography as previously described (Hancock and Howard, 1998;Coy et al, 1999). Tubulin was purified from bovine brains and labeled with rhodamine as previously described (Williams and Lee, 1982;Hyman et al, 1991). Microtubules were polymerized by mixing 32 µM rhodamine-labeled tubulin, 4 mM MgCl 2 , 1 mM GTP and 5% DMSO in BRB80 buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl 2 , pH 6.9 with KOH), incubating at 37 • C for 20 min, and then diluting into a solution containing 10 µM paclitaxel.…”

Section: Kinesin and Microtubulesmentioning

confidence: 99%

Microtubule transport, concentration and alignment in enclosed microfluidic channels

Huang

Uppalapati

Hancock

et al. 2006

Biomed Microdevices

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The kinesin-microtubule system has emerged as a versatile model system for biologically-derived microscale transport. While kinesin motors in cells transport cargo along static microtubule tracks, for in vitro transport applications it is preferable to invert the system and transport cargofunctionalized microtubules along immobilized kinesin motors. However, for efficient cargo transport and to enable this novel transport system to be interfaced with traditional microfluidics, it is important to fabricate enclosed microchannels that are compatible with kinesin motors and microtubules, that enable fluorescence imaging of microtubule movement, and that provide fluidic connections for sample introduction. Here we construct a three-tier hierarchical system of microfluidic channels that links microscale transport channels to macroscopic fluid connections. Shallow microchannels (5 µm wide and 1 µm deep) are etched in a glass substrate and bonded to a cover glass using PMMA as an adhesive, while intermediate channels ( ∼ 100 µm wide) serve as reservoirs and connect to 250 µm deep microchannels that hold fine gauge tubing for fluid injection. To demonstrate Ying-Ming Huang and Maruti Uppalapati contributed equally to this work. the utility of this device, we first show the performance of a directional rectifier that redirects 96% of moving microtubules and, because any microtubules that detach rapidly rebind to the motor-coated surface, suffers no microtubule loss over time. Second, we develop an approach, using a headless kinesin construct, to eliminate gradients in motor adsorption and microtubule binding in the enclosed channels, which enables precise control of kinesin density in the microchannels. Finally, we show that a 60 µm diameter circular ring functionalized with motors concentrates and aligns bundles of ∼ 3000 uniformly oriented microtubules, while suffering negligible ATP depletion. These aligned isopolar microtubules are an important tool for microscale transport applications and can be employed as a model in vitro system for studying kinesin-driven microtubule organization in cells. Electronic Supplementary Material

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“…Tubulin was purified by the reversible assembly disassembly method with minor modifications (Williams and Lee 1982). Each partially purified tubulin solution was submitted to carboxylmethylation in the presence of 2 lM recombinant PIMT with or without 1 mM DSIP-isoD, and proteins were separated by 16-BAC acidic gel electrophoresis as described above.…”

Section: Preparation Of Tubulin From Human Hippocampus Tissuesmentioning

confidence: 99%

“…7) co-migrated with visible proteins in Coomassie blue-stained gels (data not shown), suggesting that these abundant proteins in brain correspond to tubulin. We therefore isolated tubulin by a microtubule assembly disassembly assay (Williams and Lee 1982) from epileptic and control hippocampus in order to measure the levels of abnormal aspartyl residues by PIMT carboxyl methylation and 16-BAC acidic gel electrophoresis. Identification of b-tubulin was confirmed by Q-Tof mass spectrometry.…”

Section: Loss Of Pimt In Human Epileptic Hippocampus 585mentioning

confidence: 99%

Down‐regulation of protein l‐isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

Lanthier

Bouthillier

Lapointe

et al. 2002

Journal of Neurochemistry

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Protein L-isoaspartyl methyltransferase (PIMT) repairs the damaged proteins which have accumulated abnormal aspartyl residues during cell aging. Gene targeting has elucidated a physiological role for PIMT by showing that mice lacking PIMT died prematurely from fatal epileptic seizures. Here we investigated the role of PIMT in human mesial temporal lobe epilepsy. Using surgical specimens of hippocampus and neocortex from controls and epileptic patients, we showed that PIMT activity and expression were 50% lower in epileptic hippocampus than in controls but were unchanged in neocortex. Although the protein was down-regulated, PIMT mRNA expression was unchanged in epileptic hippocampus, suggesting post-translational regulation of the PIMT level. Moreover, several proteins with abnormal aspartyl residues accumulate in epileptic hippocampus. Microtubules component b-tubulin, one of the major PIMT substrates, had an increased amount (two-fold) of L-isoaspartyl residues in the epileptic hippocampus. These results demonstrate that the down-regulation of PIMT in epileptic hippocampus leads to a significant accumulation of damaged tubulin that could contribute to neuron dysfunction in human mesial temporal lobe epilepsy.

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[36] Preparation of tubulin from brain

Cited by 391 publications

References 11 publications

Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

Microtubule transport, concentration and alignment in enclosed microfluidic channels

Down‐regulation of protein l‐isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

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