Effect of Fe3O4 Nanoparticles on Structure and Activity of Brain Microtubules

A. Dadrasa, Gh. Riazia, A. Naghshineha, Sh. Ahmadiana

a Institute of Biochemistry and Biophysics (IBB), the University of Tehran, Tehran, Iran

Microtubules are the most important proteins existing in eukaryotic cells specially neurons in which microtubules array parallel. Microtubules are composed of tubulin dimers which have rather high dipole moment. Probable vibrations in such electric dipoles could lead to electromagnetic field around microtubules. So it is interesting to know the effect of low frequency magnetic field on microtubule structure and activities.In this paper, it has been tried to use Fe3O4 nanomagnets to investigate microtubules dynamic and function by turbidimetry methods and TEM, therefore analyze probable changes in tubulin structure by the methods of fluorescence spectrophotometer. We found the increase of tubulin activities and conformational changes by the usage of these nanomagnets in vitro. Also microtubule orientation toward nanomagnets has been observed.

Abstract

Microtubules perform a variety of key cellular functions such as chromosome segregation, cellular organization, axonal transport and cell motility [1, 2]. Microtubules are hollow cylindrical rods with an outer diameter roughly 25 nm composed of α and β tubulin dimers which can assemble or disassemble [2]. tubulin heterodimers have an electric dipole moment about 1714 debye [3] and highly negative charges in the physiologic pH [4]. Probable vibrations in such electric dipoles could lead to electromagnetic field generation around microtubules [5, 9]. It is considerable that microtubule disfunction is involved in neurodegenerative diseases such as Alzheimer [6]. Magnetites (Fe3O4) are the ferromagnetic iron oxides that are composed of alternative networks of Fe2+ and Fe3+ which antiferromagnetically coupled. Relative unpaired electron spins result in strong magnetization of them [7]. It is interesting to investigate effects of magnetic field generating around Fe3O4 nanomagnets on microtubule organization.

Introduction

Tubulin, containing αβ subunits, was freshly purified from sheep brain after two cycles of assembly and disassembly. Detection of tubulin assembly was carried out in presence of Mg2+GTP 1mM at 37°C by UV/Visible spectrophotometer equipped with a temperature control accessory. The assembly process was detected via the absorbance at 350 nm for 30 min. Intrinsic fluorescence emission of tubulin was measured to survey tubulin conformation in presence of Fe3O4 nanoparticles. The assembled microtubules were placed on a formvar-coated EM grid to survey microtubule organization in presence of magnetites. Uranyl acetate 1% was placed on grid for negative staining to observe microtubules.

Materials and Methods

It seems that the magnetic nanoparticles could affect on tubulin dipole-dipole interactions through the magnetic fields generating around them. It is implicated that the natural and cylindrical shape of microtubule proteins is a consequence of their own dipole-dipole interactions [8]. Collision plausibility of tubulin dimers with each other would be increased by increasing Fe3O4 nanoparticles concentration. Microtubules could generate local electromagnetic fields around themselves via their own vibrations [5, 9]. We proposed that magnetic fields generated around Fe3O4 could interfere with microtubule organization and could increase microtubule oscillation. Tubulin conformation would be changed in presence of Fe3O4 nanoparticles as indicated by intrinsic fluorescence.

Results and Discussion

a b

c

Tubulin assembly in presence of various concentrations of Fe3O4 nanoparticles:Tubulin assembly was carried out in presence of Mg2+GTP (1mM) at 37ᵒC. The absorbance of 350 nm was monitored for 30 min. The slope of elongation phase of polymerization process was increased as indicated.

Intrinsic fluorescence spectra of tubulins in presence of Fe3O4 nanoparticles following excitation at 295 nm. Maximum intensity of fluorescence was decreased by increasing Fe3O4 nanoparticles concentration as indicated.

TEM images of microtubule filaments: a) Microtubules polymerized normally. b, c) Microtubule polymers in presence of Fe3O4 nanoparticles: Increases of microtubule density around Fe3O4 nanoparticles was observed.

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References