Analysis of microtubule rigidity using hydrodynamic flow and thermal fluctuations.

P Venier,A.C Maggs,M.F Carlier,D Pantaloni

doi:10.1016/s0021-9258(17)36840-0

Abstract

We report the use of two independent new methods to measure the flexural rigidity of microtubules. Microtubules were grown off axonemal pieces adhering to a glass coverslip. In the first method, a hydrodynamic flow was applied to microtubules and the flexural rigidity was derived from the analysis of the bending shape of the microtubules at equilibrium in the flow. In the second method, the flexural rigidity was derived from the thermal fluctuations of the free end of axoneme-bound microtubules. With both methods, the flexural rigidity of standard GDP microtubules was estimated to be 0.85 +/- 0.2 x 10(-23) newtons x m2 which corresponded to a persistence length of 2 +/- 0.2 mm. Binding of ligands known to affect the biochemical properties of microtubules affected their rigidity. The structural analogs of inorganic phosphate AlF4- and [BeF3-, H2O], which bind to the site of the gamma-phosphate of GTP on GDP microtubule and reconstitute the GDP-Pi microtubule intermediate state of GTP hydrolysis, cause an approximately 3-fold increase in microtubule flexural rigidity and persistence length. Taxol and taxotere, antitumoral microtubule-stabilizing drugs, in contrast cause a decrease in flexural rigidity and appear to affect the three-dimensional superstructure of microtubules, which can no longer be considered as semi-flexible rods. The relationship between the mechanical properties of microtubules and their biological function is discussed.

Highlights

We report the use of two independent new methtodsbidimensional lattice within which tubulin subunits interact measure the flexural rigidityof microtubules
The structural analogosf inorganic phosphate AlF’;and [BeF;,01, which bind to the site of y-phosphate of GTP on GDP microtubule and reconstitute the GDP-Pi microtubule intermediate staotfeGTP hydrolysis,causean-%foldincrease in microtubule flexural rigidity and persistence lengtTha.x01 and taxocrotubules free insolution
The resultsof this analysisshowed that taxol-stabilized microtubules hada persistence length,L, of 5,200 pm anda Young‘s modulus, E, of 1.2 gigapascals, while thvpeareluliems i(nLapry data 1000 obtained in the absencoef taxol led to lower pm) for standard microtubules

Summary

MATERIALS AND METHODS

Chemicals-MES’ and PIPES were from Calbiochem,EGTA from Sigma, beryllium sulfatetetrahydrate and aluminum nitrate The breadthof the distribution represents +35% of the average velocity at 0.5 p m from the surface, which corresponds toa depth of focus of 0.35-0.40 p.We note fulfilled due to the large fluctuations in length of standard mi- that the value of the depth of focus derived from the velocity crotubules at steady state [3].To circumvent this difficulty, microtu- measurements is in good agreement with the calculated value bules can be stabilized by [BeF;, H,Ol or AlF; structural analogs of [24] corresponding to the high numerical aperture objective inorganic phosphate [13],which have been shown to bind to the siteof the y-phosphateof GTP and to reconstitute a stable GDP-P,-like microtubule, from which subunits dissociate at a very slow rate and which used here From these observations, we can considerthat microtubules attached to axonemes are constrained in a 0.4-pmhas lost its dynamic instability behavior. Analysis of the Bendingof Individual Microtubules ina Hydrodynamic Flow: Effect of BeFo,tnhFelexibility of

RESULTS

The value of the flexural rigidity could be derived from the

DISCUSSION

Methods