XMAP215 is a long thin molecule that does not increase microtubule stiffness

Abstract

XMAP215 is a microtubule associated protein that speeds microtubule plus end growth by seven- to tenfold and protects these ends from destabilization by the Kin I kinesin, XKCM1. To understand the mechanisms responsible for these activities, it is necessary to know the structure of XMAP215. By unidirectional shadowing and electron microscopy, XMAP215 appeared as an elongate molecule of 60±18 nm, suggesting that XMAP215 could span up to seven to eight tubulin dimers along a protofilament. Most XMAP215 molecules were straight but a subset were bent suggesting that XMAP215 is flexible. Antibodies to the C terminus labeled one end of XMAP215 with no evidence for XMAP215 dimerization. Incubation of XMAP215 and tubulin at 4°C resulted in assembly of curved protofilaments, which appeared to be incomplete tubulin rings. Measurements from rotary shadowed samples showed that tubulin/XMAP215 partial rings had an average width of 8.8±1.8 nm compared with 5.6±1.1 nm for rings assembled from tubulin dimers alone, suggesting that XMAP215 adds a width of approximately 3.2 nm to the curved tubulin protofilament. XMAP215 did not change the radius of curvature of these partial tubulin rings. Measurements of microtubule flexural rigidity by thermal fluctuations showed that XMAP215 did not change microtubule rigidity. Finally,sequence analysis shows that the N-terminal half of XMAP215 contains four repeats, each composed of multiple HEAT repeats.

Microtubules soften due to cross-sectional flattening

“…However, there is a considerable disagreement between different reported values of MT elastic moduli [46], the dependence of the elastic moduli on the presence of stabilizing agents (taxol and GMPCPP), as well as a possible length-dependence of flexural rigidity [39,50]. In principle, the properties of microtubules can be measured by either visualizing their intrinsic thermal fluctuations [4,8,21,31,46,50] or by applying an external force through optical trapping or hydrodynamic flow experiments [15,17,38,41,64,65,67]. Because of significant rigidity of MTs these two types of measurements typically probe different deformation (strain) regimes.…”

Control of molecular shuttles by designing electrical and mechanical properties of microtubules

“…Here, we additionally manipulate the mechanical stiffness of the MTs measured as flexural rigidity (k) or persistence length (L p ). In vivo, MTs control their stiffness depending on the intracellular roles of the filament; that is, stiffer MTs are needed in the axon to support its long structure, whereas more flexible MTs are needed in a proliferating cell to enable rapid redistribution (20). Many factors altering MT stiffness have been reported, including MT stabilizing agents, nucleotides, MT-associated proteins (MAPs), and growth rates.…”

“…Many factors altering MT stiffness have been reported, including MT stabilizing agents, nucleotides, MT-associated proteins (MAPs), and growth rates. However, there are still controversies on stiffening and softening factors, and the measured stiffness has not been used to manipulate MT gliding directions (17,(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). The cantilever beam model suggests that MT stiffness is proportional to the persistence of the gliding trajectory (19), so we proposed to design both the electrical and the mechanical properties simultaneously to improve controllability of the gliding directions.…”

Enhanced Mechanical Stability of Microtubules Polymerized with a Slowly Hydrolyzable Nucleotide Analogue

“…Most of these studies consider the microtubule as a uniform hollow cylinder. 4,[10][11][12][13][14][15][16][17] However, recent studies of paclitaxel-stabilized microtubules by intentional deformation of the microtubule cylinder using AFM have begun to probe mechanical properties of microtubules on a local level and have used models of the microtubule as a cylindrical shell with realistic molecular-level structure. [18][19][20][21] Although paclitaxel stabilizes microtubules from depolymerization and allows for in vitro studies of microtubule mechanical and structural properties, the structural rigidity of paclitaxel-stabilized microtubules has been found to vary significantly in GDP-containing, GMPCPP-containing, and MAP-stabilized microtubules in bending experiments, 4 chanical stability.…”

“…Paclitaxel, microtubule-associated proteins (MAPs), and GMPCPP have been used to stabilize microtubules in these experiments. Most of these studies consider the microtubule as a uniform hollow cylinder. ,− However, recent studies of paclitaxel-stabilized microtubules by intentional deformation of the microtubule cylinder using AFM have begun to probe mechanical properties of microtubules on a local level and have used models of the microtubule as a cylindrical shell with realistic molecular-level structure. − Although paclitaxel stabilizes microtubules from depolymerization and allows for in vitro studies of microtubule mechanical and structural properties, the structural rigidity of paclitaxel-stabilized microtubules has been found to vary significantly in GDP-containing, GMPCPP-containing, and MAP-stabilized microtubules in bending experiments, limiting the applicability of paclitaxel in studies of microtubule mechanical stability. In this study, the local mechanical and structural properties of GMPCPP-polymerized microtubules were probed using AFM in order to better understand the stabilizing effects of unhydrolyzed GTP and to relate this to models of the GTP cap.…”