Microtubule Structure at 8 Å Resolution

Li, Huilin; DeRosier, David J.; Nicholson, William V.; Nogales, Eva; Downing, Kenneth H.

doi:10.1016/s0969-2126(02)00827-4

Cited by 386 publications

(421 citation statements)

References 35 publications

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“…Variability within a range of Ϯ0.2 nm in the relative position of tubulin dimers on adjacent PFs was also observed in these studies and interpreted as the result of thermally driven deformations of the lateral contacts between PFs (19,38). Electron crystallography maps obtained at 8-and 20-Å resolution give insight about the molecular origin of longitudinal and lateral interactions in MTs (41,42). One key structural element in determining lateral interactions between dimers is a long loop (the S7-H9 loop, also called ''M-loop''; Fig.…”

Section: Discussionsupporting

confidence: 66%

Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length

Pampaloni

Lattanzi²,

Jonáš

et al. 2006

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

328

376

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Microtubules are hollow cylindrical structures that constitute one of the three major classes of cytoskeletal filaments. On the mesoscopic length scale of a cell, their material properties are characterized by a single stiffness parameter, the persistence length ഞp. Its value, in general, depends on the microscopic interactions between the constituent tubulin dimers and the architecture of the microtubule. Here, we use single-particle tracking methods combined with a fluctuation analysis to systematically study the dependence of ഞ p on the total filament length L. Microtubules are grafted to a substrate with one end free to fluctuate in three dimensions. A fluorescent bead is attached proximally to the free tip and is used to record the thermal fluctuations of the microtubule's end. The position distribution functions obtained with this assay allow the precise measurement of ഞ p for microtubules of different contour length L. Upon varying L between 2.6 and 47.5 m, we find a systematic increase of ഞp from 110 to 5,035 m. At the same time we verify that, for a given filament length, the persistence length is constant over the filament within the experimental accuracy. We interpret this length dependence as a consequence of a nonnegligible shear deflection determined by subnanometer relative displacement of adjacent protofilaments. Our results may shine new light on the function of microtubules as sophisticated nanometer-sized molecular machines and give a unified explanation of seemingly uncorrelated spreading of microtubules' stiffness previously reported in literature.nanomechanics ͉ protofilaments ͉ single-particle tracking ͉ thermal fluctuation analysis T he mechanics of living cells is largely determined by the cytoskeleton, a self-organizing and highly dynamic network of filamentous proteins of different lengths and stiffnesses (1). Understanding the elastic response of purified cytoskeletal filaments is fundamental for the elucidation of the rheological behavior of the cytoskeleton. Microtubules (MTs) are hollow cylindrical filaments formed by, on average, 13 tubulin protofilaments (PFs) assembled in parallel. The MT outer and inner diameters are Ϸ25 and 15 nm, respectively. In cells, MTs are generally 1-10 m long, whereas in axons their length can be 50-100 m (2).The tubular structure of MTs implies a minimal crosssectional area, hence a high strength and stiffness combined with low density. In recent years, the mechanical properties of MTs have been investigated by several experimental approaches, such as thermal fluctuations (3-6), atomic force microscopy (AFM) (7-10), and optical tweezers (11-13).The standard reference model to describe the mechanical properties of a biopolymer on length scales much larger than any microscopic scale (the tube diameter for MTs) is the worm-like chain model (14,15). It is characterized in terms of a flexural rigidity (neglecting torsional rigidity). The combined effect of flexural rigidity and thermal fluctuations on the conformation of the filament is given by the ratio ...

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Section: Discussionsupporting

confidence: 66%

Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length

Pampaloni

Lattanzi²,

Jonáš

et al. 2006

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

328

376

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“…Structural studies have shown the pores to be large enough (1.5 nm ϫ 2.5 nm) to allow Taxol (Ϸ1 nm) to pass (11,12), but it has been suggested that the association rate of Taxol measured by using stopped-flow techniques is too fast to be explained by the pores (38,46). A recent study by Diaz et al (47) examined Taxol-binding kinetics to gluteraldehyde cross-linked MTs in the presence and absence of exterior-bound MAPs.…”

Section: Discussionmentioning

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).…”

mentioning

confidence: 99%

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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“…These loops coordinate lateral interactions between protofilaments to build a microtubule. Studies docking the tubulin dimer structure into high-resolution images of microtubules have established that M loops interact with N loops of laterally adjacent subunits (Li et al, 2002). Moreover, organisms with increased microtubule stability (such as arctic fish) have two amino acid substitutions in the ␣-tubulin M loop, Ala278Thr and Ser287Thr (Detrich et al, 2000).…”

Section: Some Mutations Localize To Domains That Coordinate Protofilamentioning

confidence: 99%

“…When oryzalin was inserted into the ␣-tubulin binding site within the microtubule structure, it was situated beneath the N loop between protofilaments in the microtubule ( Figure 5B). Studies fitting the tubulin dimer structure into high-resolution cryoelectron microscopy images of microtubules have established that protofilament-protofilament contact is mediated by N loops interacting with M loops of laterally adjacent subunits (Li et al, 2002). Oryzalin bound beneath the N loop may inhibit N loop interaction with the M loop of the adjacent protofilament ( Figure 5B, enlargement) with the consequence of microtubule disruption.…”

mentioning

confidence: 99%

Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

Morrissette

Mitra

Sept

et al. 2004

MBoC

139

153

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Protozoan parasites are remarkably sensitive to dinitroanilines such as oryzalin, which disrupt plant but not animal microtubules. To explore the basis of dinitroaniline action, we isolated 49 independent resistant Toxoplasma gondii lines after chemical mutagenesis. All 23 of the lines that we examined harbored single point mutations in ␣-tubulin. These point mutations were sufficient to confer resistance when transfected into wild-type parasites. Several mutations were in the M or N loops, which coordinate protofilament interactions in the microtubule, but most of the mutations were in the core of ␣-tubulin. Docking studies predict that oryzalin binds with an average affinity of 23 nM to a site located beneath the N loop of Toxoplasma ␣-tubulin. This binding site included residues that were mutated in several resistant lines. Moreover, parallel analysis of Bos taurus ␣-tubulin indicated that oryzalin did not interact with this site and had a significantly decreased, nonspecific affinity for vertebrate ␣-tubulin. We propose that the dinitroanilines act through a novel mechanism, by disrupting M-N loop contacts. These compounds also represent the first class of drugs that act on ␣-tubulin function. INTRODUCTIONMicrotubules are polymers constructed from ␣-␤-tubulin heterodimers (Downing and Nogales, 1998a,b). These structures are rapidly assembled and disassembled to create essential components of eukaryotic cells, such as spindles and flagella. The dynamic nature of microtubules makes them susceptible to pharmacological agents. Microtubule-disrupting and microtubule-stabilizing drugs have provided great insight into tubulin and microtubule function; they also have tremendous practical use. Compounds that perturb microtubule dynamics are currently some of the most effective drugs to treat medical conditions, including cancer, gout, and helminth infection (Jordan et al., 1998). Dinitroanilines (oryzalin, ethafluralin, and trifluralin) disrupt the microtubules of plants, ranging from the single-celled alga Chlamydomonas reinhardtii to higher plants such as the monocot Eleusine indica (James et al., 1993;Anthony et al., 1998;Zeng and Baird, 1999). Dinitroanilines also disrupt the microtubules of protozoa, including both free-living species such as Tetrahymena and protozoan parasites such as Trypanosoma spp., Leishmania spp., Entamoeba spp., Plasmodium falciparum, Cryptosporidium parvum, and Toxoplasma gondii (Chan and Fong, 1990;Chan et al., 1991;Gu et al., 1995;Edlind et al., 1996;Stokkermans et al., 1996;Armson et al., 1999; Makioka et al., 2000a,b;Traub-Cseko et al., 2001). Remarkably, the activity of dinitroanilines is restricted to plants and protozoa; these compounds are ineffective against vertebrate or fungal microtubules (Chan and Fong, 1990;Hugdahl and Morejohn, 1993;Murthy et al., 1994;Edlind et al., 1996).T. gondii is a member of the Apicomplexa, a phylum of parasites that includes several medically and agriculturally significant pathogens (Black and Boothroyd, 2000). Apicomplexans are obligate intracell...

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Microtubule Structure at 8 Å Resolution

Cited by 386 publications

References 35 publications

Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length

Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length

Tau induces cooperative Taxol binding to microtubules

Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

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