Differential Effects of Vinblastine on Polymerization and Dynamics at Opposite Microtubule Ends

Panda, Dulal; Jordan, Mary Ann; Chu, Kevin; Wilson, Leslie

doi:10.1074/jbc.271.47.29807

Cited by 117 publications

(103 citation statements)

References 38 publications

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“…Growth of the brain microtubules occurred at both the plus and minus ends of the axonemes. The plus ends of brain microtubules were distinguished from the minus ends by their higher growth rates, greater excursion lengths, and larger number of microtubules per axoneme end as previously reported (5,27). In contrast, HeLa cell microtubule growth occurred predominantly at one end of the seeds.…”

Section: Methodssupporting

confidence: 50%

“…The plus end dynamics of individual HeLa cell microtubules were examined at steady state (35°C) by video-enhanced DIC microscopy (see "Experimental Procedures") using PMEM buffer, a buffer previously used for analysis of brain microtubule dynamics (27,31). Several life-history traces showing the changes in length of HeLa cell microtubules with time are shown in Fig.…”

Section: Purification and Characterization Of Hela Cell Tubulin-tomentioning

confidence: 99%

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Intrinsically Slow Dynamic Instability of HeLa Cell Microtubules in Vitro

Newton

DeLuca

Himes

et al. 2002

Journal of Biological Chemistry

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The dynamic behavior of mammalian microtubules has been extensively studied, both in living cells and with microtubules assembled from purified brain tubulin. To understand the intrinsic dynamic behavior of mammalian nonneural microtubules, we purified tubulin from cultured HeLa cells. We find that HeLa cell microtubules exhibit remarkably slow dynamic instability, spending most of their time in an attenuated state. The tempered dynamics contrast sharply with the dynamics of microtubules prepared from purified bovine brain tubulin under similar conditions. In accord with their minimal dynamic instability, assembled HeLa cell microtubules displayed a slow treadmilling rate and a low guanosine-5-triphosphate hydrolysis rate at steady state. We find that unlike brain tubulin, which consists of a heterogeneous mixture of ␤-tubulin isotypes (␤ II , ␤ III , and ␤ IV and a low level of ␤ I ), HeLa cell tubulin consists of ␤ I tubulin (ϳ80%) and a minor amount of ␤ IV tubulin (ϳ20%). The slow dynamic behavior of HeLa cell microtubules in vitro differs strikingly from the dynamic behavior of microtubules in living cultured mammalian cells, supporting the idea that accessory factors create the robust dynamics that occur in cells.

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Section: Methodssupporting

confidence: 50%

Section: Purification and Characterization Of Hela Cell Tubulin-tomentioning

confidence: 99%

Intrinsically Slow Dynamic Instability of HeLa Cell Microtubules in Vitro

Newton

DeLuca

Himes

et al. 2002

Journal of Biological Chemistry

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“…Preparation of Tubulin and Microtubules-Tubulin was isolated from porcine brain by two cycles of polymerization and depolymerization as described (31). Taxol (paclitaxel; SigmaAldrich) MTs (32), Taxol-MT seeds (33), rhodamine-labeled tubulin (34), and subtilisin-treated Taxol-MTs (35) were prepared as described previously.…”

Section: Methodsmentioning

confidence: 99%

Interactions between EB1 and Microtubules

Zhu

Gupta

Slabbekoorn

et al. 2009

Journal of Biological Chemistry

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Plus end tracking proteins (؉TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved ؉TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip. Microtubules (MTs)2 are a major component of the cytoskeleton, the network of proteinacious fibers that endow the cell with structural integrity, motile properties, and internal organization (1-4). MTs play a particularly important role in cell organization: they help to pull the chromosomes apart at mitosis, act as a "railway" for intracellular transport, and define the localization and structure of internal membrane systems (5-11).The cellular functions of MTs are highly dependent on their dynamic nature, which is regulated by a number of microtubule associated proteins (MAPs) (3, 9, 12, 13). The plus end tracking proteins (ϩTIPs) are an unusual group of MAPs that preferentially localize to growing MT plus ends (13)(14)(15). A large number of proteins have now been identified as ϩTIPs, but one of the most important is EB1 (end binding protein-1) (4,13,16,17). EB1 was initially discovered as a binding partner of the adenomatous polyposis coli protein, but it is becoming apparent that EB1 binds to an astonishing array of other proteins including other ϩTIPs (CLIP-170, p150, and CLASPs), molecular motors (Tea2 and kinesin), signal transduction proteins (Rho-GEF2), and cytoskeletal scaffolding proteins (spectraplakins and formins) (4, 13, 15). As might be expected from a protein with so many interactions, EB1 is strikingly well conserved across eukaryotes (18 -21). These observations suggest that EB1 is the structural and evolutionary core of a complex of proteins that regulates the behavior of MT plus ends (4, 13-15). However, its activities and the mechanisms of i...

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“…Taxol stabilizes microtubules at low concentration and promotes microtubule assembly at high concentration (Schiff et al, 1979;Xiao et al, 2006). Vinblastine inhibits microtubule dynamics at low concentration and disassembles microtubules and induces tubulin self-association into spiral aggregates at high concentration (Yu and Baas 1995;Panda et al, 1996;Gigant et al, 2005). We observed qualitative effects supporting these specific drug activities at the Drosophila NMJ (data not shown).…”

Section: Resultsmentioning

confidence: 49%

“…When vinblastine was used at higher concentration (≥ 5 μM), tubulin-GFP aggregates form in NMJ boutons. This was expected based on the known molecular mechanism of vinblastine (Panda et al, 1996;Gigant et al, 2005).…”

Section: Drug Treatmentsmentioning

confidence: 99%

In vivo assay of presynaptic microtubule cytoskeleton dynamics in Drosophila

Yan

Broadie

2007

Journal of Neuroscience Methods

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Disrupted microtubule dynamics in neuronal synapses has been suggested as an underlying cause for several devastating neurological diseases, including Hereditary Spastic Paraplegia (HSP) and Fragile X Syndrome (FXS). However, previous studies have been restricted to indirect assays of synaptic microtubules, i.e. immunocytochemistry of microtubule-associated proteins and posttranslationally modified tubulins characteristic of microtubules with different stabilities. Very little is known about synaptic microtubule dynamics in vivo, or how microtubule dynamics may be disrupted in disease states. In this study, we develop methods to analyze microtubule dynamics directly in living synaptic boutons in situ. We use fluorescence recovery after photobleaching (FRAP) of transgenic green fluorescent protein (GFP) tagged tubulin at the well-characterized Drosophila neuromuscular junction (NMJ) synapse. FRAP measurements of tubulin-GFP demonstrate biphasic recovery kinetics. Treatment with taxol to stabilize microtubules and promote microtubule assembly reduces both recovery phases. Treatment with vinblastine to disassemble microtubules increases the fast recovery phase and decreases the slow recovery phase. These data indicate that the fast recovery phase is generated by rapid diffusion of tubulin subunits and the slow phase is generated by the relatively slow turnover of microtubules. This study demonstrates that tubulin-GFP fluorescence recovery after photobleaching can be used to assay microtubule dynamics directly in living synapses.

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Differential Effects of Vinblastine on Polymerization and Dynamics at Opposite Microtubule Ends

Cited by 117 publications

References 38 publications

Intrinsically Slow Dynamic Instability of HeLa Cell Microtubules in Vitro

Intrinsically Slow Dynamic Instability of HeLa Cell Microtubules in Vitro

Interactions between EB1 and Microtubules

In vivo assay of presynaptic microtubule cytoskeleton dynamics in Drosophila

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