Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine

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Microtubules (MTs) are hollow cytoskeletal filaments assembled from αβ-tubulin heterodimers. Tau, an unstructured protein found in neuronal axons, binds to MTs and regulates their dynamics. Aberrant Tau behavior is associated with neurodegenerative dementias, including Alzheimer's. Here, we report on a direct force measurement between paclitaxel-stabilized MTs coated with distinct Tau isoforms by synchrotron small-angle X-ray scattering (SAXS) of MT-Tau mixtures under osmotic pressure (P). In going from bare MTs to MTs with Tau coverage near the physiological submonolayer regime (Tau/tubulin-dimer molar ratio; Φ Tau = 1/ 10), isoforms with longer N-terminal tails (NTTs) sterically stabilized MTs, preventing bundling up to P B ∼ 10,000-20,000 Pa, an order of magnitude larger than bare MTs. Tau with short NTTs showed little additional effect in suppressing the bundling pressure (P B ∼ 1,000-2,000 Pa) over the same range. Remarkably, the abrupt increase in P B observed for longer isoforms suggests a mushroom to brush transition occurring at 1/13 < Φ Tau < 1/10, which corresponds to MT-bound Tau with NTTs that are considerably more extended than SAXS data for Tau in solution indicate. Modeling of Tau-mediated MT-MT interactions supports the hypothesis that longer NTTs transition to a polyelectrolyte brush at higher coverages. Higher pressures resulted in isoform-independent irreversible bundling because the polyampholytic nature of Tau leads to short-range attractions. These findings suggest an isoform-dependent biological role for regulation by Tau, with longer isoforms conferring MT steric stabilization against aggregation either with other biomacromolecules or into tight bundles, preventing loss of function in the crowded axon environment.Tau | intrinsically disordered proteins | microtubule | SAXS | force measurement

“…Tubulin was purified as described (74,75). Briefly, MTassociated protein-rich tubulin was isolated from bovine brain through successive in vitro polymerization/depolymerization cycles.…”

Section: Methodsmentioning

confidence: 99%

Direct force measurements reveal that protein Tau confers short-range attractions and isoform-dependent steric stabilization to microtubules

Chung

Choi

Miller

et al. 2015

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“…Purified tau was stored at -70°C. Bovine brain MT protein (Ϸ70% tubulin and 30% MTassociated proteins) was prepared by three cycles of assembly and disassembly (31). Tubulin was purified from the MT protein by elution through a Whatman P-11 phosphocellulose column (32) and equilibrated in 50 mM Pipes͞1 mM MgSO 4 ͞1 mM EGTA͞0.1 mM GTP.…”

Section: Methodsmentioning

confidence: 99%

Differential regulation of microtubule dynamics by three- and four-repeat tau: Implications for the onset of neurodegenerative disease

Panda

Samuel

Massie

et al. 2003

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232

233

The microtubule (MT)-associated protein tau is important in neuronal development and in Alzheimer's and other neurodegenerative diseases. Genetic analyses have established a cause-and-effect relationship between tau dysfunction͞misregulation and neuronal cell death and dementia in frontotemporal dementia and parkinsonism associated with chromosome 17; several mutations causing this dementia lead to increased ratios of four-repeat (4R) to three-repeat (3R) wild-type tau, and an attractive hypothesis is that the abnormally high ratio of 4R to 3R tau might lead to neuronal cell death by altering normal tau functions in adult neurons. Thus, we tested whether 3R and 4R tau might differentially modulate the dynamic instability of MTs in vitro using video microscopy. Although both isoforms promoted MT polymerization and decreased the tubulin critical subunit concentration to approximately similar extents, 4R tau stabilized MTs significantly more strongly that 3R tau. For example, 4R tau suppressed the shortening rate, whereas 3R tau had little or no detectable effect. Similarly, 3R tau had no effect on the length shortened during a shortening event, whereas 4R tau strongly reduced this parameter. Further, when MTs were diluted into buffer containing 4R tau, the MTs were stabilized and shortened slowly. In contrast, when diluted into 3R tau, the MTs were unstable and shortened rapidly. Thus, 4R tau stabilizes MTs differently and significantly more strongly than 3R tau. We suggest a ''dosage effect'' or haploinsufficiency model in which both tau alleles must be active and properly regulated to produce appropriate amounts of each tau isoform to maintain MT dynamics within a tolerable window of activity.M any neurodegenerative diseases exhibit abnormal pathological fibers composed primarily of the microtubule (MT)-associated protein, tau (for a recent review, see ref. 1). These disorders, termed ''tauopathies,'' include Alzheimer's disease, frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), Pick's disease, and progressive supranuclear palsy. In 1998, several groups reported a direct genetic linkage between mutations in the tau gene and FTDP-17 (2-5). Although some tau mutations are structural and others are regulatory, all exhibit dominant phenotypes. Thus, both dysfunction and misregulation of tau are causally related to neuronal cell death, neurodegenerative disease, and dementia.Tau is normally present predominantly in the cell bodies and axons of neuronal cells and is necessary for the establishment of neuronal cell polarity and axon outgrowth, axonal transport, and maintenance of axonal morphology (6-10). Tau is also expressed in glial cells (11). Mechanistically, tau stimulates MT polymerization, stabilizes MTs, and suppresses MT dynamics (12-15). Because MT dynamics must be tightly regulated for cells to function and remain viable (e.g., ref. 16), it follows that the action of tau must also be finely regulated.Although there is only a single tau gene, alternative splicing of tau mRNA p...

“…Taxol resistance arising from mutations of tubulin genes has been observed previously, although the effects on dynamic instability have not been determined (18,28,29). Several MAPs and other microtubule regulatory proteins including MAP4, TOG (tumor overexpressed gene), and stathmin can up-or down-regulate microtubule dynamics, often depending on their phosphorylation states (30)(31)(32)(33)(34).…”

Section: Why Are Microtubules Of A549-t12 and -T24 Cells Highly Dynamic?mentioning

confidence: 99%

Resistance to Taxol in lung cancer cells associated with increased microtubule dynamics

Gonçalvès

Braguer

Kamath

et al. 2001

228

172

Microtubule dynamics are crucial for mitotic spindle assembly and chromosome movement. Suppression of dynamics by Taxol appears responsible for the drug's potent ability to inhibit mitosis and cell proliferation. Although Taxol is an important chemotherapeutic agent, development of resistance limits its efficacy. To examine the role of microtubule dynamics in Taxol resistance, we measured the dynamic instability of individual rhodamine-labeled microtubules in Taxol-sensitive and -resistant living human cancer cells. Taxol-resistant A549-T12 and -T24 cell lines were selected from a human lung carcinoma cell line, A549. They are, respectively, 9-and 17-fold resistant to Taxol and require low concentrations of Taxol for proliferation. We found that microtubule dynamic instability was significantly increased in the Taxol-resistant cells. For example, with A549-T12 cells in the absence of added Taxol, microtubule dynamicity increased 57% as compared with A549 cells. The length and rate of shortening excursions increased 75 and 59%, respectively. These parameters were further increased in A549-T24 cells, with overall dynamicity increasing by 167% compared with parental cells. Thus, the decreased Taxol-sensitivity of these cells can be explained by their increased microtubule dynamics. When grown without Taxol, A549-T12 cells were blocked at the metaphase͞anaphase transition and displayed abnormal mitotic spindles with uncongressed chromosomes. In the presence of 2-12 nM Taxol, the cells grew normally, suggesting that mitotic block resulted from excessive microtubule dynamics. These results indicate that microtubule dynamics play an important role in Taxol resistance, and that both excessively rapid dynamics and suppressed dynamics impair mitotic spindle function and inhibit proliferation.