Mobility of Taxol in Microtubule Bundles

Ross, Jennifer L.; Fygenson, Deborah Kuchnir

doi:10.1016/s0006-3495(03)75123-6

Cited by 26 publications

(35 citation statements)

References 43 publications

(57 reference statements)

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“…where S, the maximum stoichiometry of botax binding to the MTs, and K D are parameters of the fit (13,37,38 (13). Briefly, 5 mg͞ml tubulin in GPEM-dex buffer was incubated for 20 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Tau was added to a final molar ratio with tubulin of 1:50, 1:10, or 1:1, and the sample was equilibrated for another 20 min at 37°C before being inserted into the flow cell. The botax concentration in solution was measured in situ by comparing the fluorescence intensity of the background to a set of standards (13). We were limited to [Tax] free Յ 10 M by the insolubility of botax and the detrimental effects of high DMSO concentrations on MTs (39).…”

Section: Methodsmentioning

confidence: 99%

“…The experimental procedures used here were the same as in a previous study (13). Briefly, samples were inserted into a flow cell, and the MTs were bundled and aligned by flow around Sephadex beads fixed to the slide.…”

Section: Methodsmentioning

confidence: 99%

“…FRAP data were analyzed as described (13). Briefly, intensity profiles of fluorescent bundles were taken from each video frame and were fit with Gaussians after subtracting a baseline to eliminate permanent background variations.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, Taxol affects MT structure by decreasing protofilament number (8) and increasing MT flexibility (9,10). The Taxolbinding site has been localized to the luminal face of ␤ tubulin and is accessible through 2-nm pores in the MT wall (11)(12)(13).…”

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confidence: 99%

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Tau induces cooperative Taxol binding to microtubules

Ross

Santangelo

Makrides

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Taxol and tau are two ligands that stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds ␤ tubulin in the MT interior. Tau is a MT-associated protein that binds both ␣ and ␤ tubulin on the MT exterior. Both Taxol and tau reduce MT dynamics and promote tubulin polymerization. Tau alone also acts to bundle, stiffen, and space MTs. A structural study recently suggested that Taxol and tau may interact by binding to the same site. Using fluorescence recovery after photobleaching, we find that tau induces Taxol to bind MTs cooperatively depending on the tau concentration. We develop a model that correctly fits the data in the absence of tau, yields the equilibrium dissociation constant of Ϸ2 M, and determines the escape rate of Taxol through one pore to be 1.7 ؋ 10 3 (M⅐s) ؊1 . Extension of the model yields a measure of Taxol cooperativity with a Hill coefficient of at least 15 when tau is present at a 1:1 molar ratio with tubulin.B ecause of their essential role in cell division, microtubules (MTs) are a major target for antimitotic cancer therapeutics such as paclitaxel (Taxol) (for reviews, see refs. 1 and 2). Taxol stabilizes the intrinsically labile MT polymer, promotes MT assembly, and suppresses MT dynamics (3-5), possibly by strengthening lateral contacts between tubulin dimers (6, 7). In addition, Taxol affects MT structure by decreasing protofilament number (8) and increasing MT flexibility (9, 10). The Taxolbinding site has been localized to the luminal face of ␤ tubulin and is accessible through 2-nm pores in the MT wall (11-13).In postmitotic differentiated neuronal cells, MTs form a major component of axons and dendrites and are thus essential for nervous system development and function. In this context, MT dynamics and functions are regulated by MT-associated proteins (MAPs) such as tau (14). In vitro, as well as in vivo, tau stabilizes and promotes MT assembly, as well as stiffens, bundles, and spaces MTs (9, 14-21). Tau is a natively unstructured protein that is localized primarily to neuronal cell bodies and axons (15,22). Tau expression is important for the establishment of normal axonal morphology and function (23-26). Furthermore, pathological tau function is implicated in the etiology of neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, and frontotemporal dementia with Parkinsonism linked to chromosome 17 (27).In the central nervous system, as a result of alternative splicing of two N-terminal exons and one C-terminal exon, tau is expressed as six major isoforms. Structurally, tau consists of two major ''domains.'' The N terminus, or ''projection domain,'' is involved in bundling and spacing MTs (18,19). The C terminus, the ''MTbinding domain,'' is composed of either three or four repeated motifs. This region binds to the exterior of preassembled MTs (28-31).A recent, intriguing cryoelectron microscopy study found that the MT-binding region can bind inside the MT lumen near the Taxolbinding site (32). Before this report, tau had been thought to bi...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Tau induces cooperative Taxol binding to microtubules

Ross

Santangelo

Makrides

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Supported phospholipid bilayers as a platform for neural progenitor cell culture

Thid

Holm

Eriksson

et al. 2007

J Biomedical Materials Res

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Supported phospholipid bilayers constitute a biomimetic platform for cell behavior studies and a new approach to the design of cell culture substrates. Phosphocholine bilayers are resistant to cell attachment, but can be functionalized with bioactive molecules to promote specific cell interactions. Here, we explore phosphocholine bilayers, functionalized with the laminin-derived IKVAV pentamer, as substrates for attachment, growth, and differentiation of neural progenitor cells (AHPs). By varying peptide concentration (0-10%), we discovered a strongly nonlinear relationship between cell attachment and IKVAV concentration, with a threshold of 1% IKVAV required for attachment, and saturation in cell binding at 3% IKVAV. This behavior, together with the 10-fold reduction in cell attachment when using a jumbled peptide sequence, gives evidence for a specific interaction between IKVAV and its AHP cell-surface receptor. After 8 days in culture, the peptide-functionalized bilayers promoted a high degree of cell cluster formation. This is in contrast to the predominant monolayer growth, observed for these cells on the standard laminin coated growth substrates. The peptide-functionalized bilayer did not induce differentiation levels over those observed for the laminin coated substrates. These results are promising in that peptide-functionalized bilayers can allow attachment and growth of stem cells without induction of differentiation.

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