Microtubule Buckling Affects Cargo Transportation By Molecular Motor
Authors: Nasrin Syeda Rubaiya , Afrin Tanjina , Kabir Arif Md. Rashedul , Inoue Daisuke , Torisawa Takayuki , Sada Kazuki , Kakugo Akira 
Published: February 2020

A cell can be best resembled by a metropolitan city where all its functions are driven by the interactions of densely packed proteins. For the traffic system in this ‘city’, the expressway consists of actin and microtubules (MTs). On the other hand, the stepping motor proteins serve as ‘vehicles’ and transport cargo from the nucleus (minus end) to the periphery (plus end) and vice versa [1]. Kinesin-1 and cytoplasmic dynein are the two major cytoplasmic motors that transport cargos towards the plus and minus end of the MT respectively. Stimulated or spontaneous cell contraction, or constrained MT polymerization at the cell periphery give rise to buckling deformation of MT in cells, that may result into buckling with short wavelengths (e.g., several µm) [2-5]. While significant advancement is made in understanding the role of kinesin in transporting cargo individually or as team along undeformed MT [6-8], how the deformity in the MT due to buckling may affect the motility of the motor is overlooked. Kinesin takes regular steps of 8 nm in a hand-over-hand fashion [9] whereas dynein shows variable step sizes ranging from 8 – 32 nm uncoordinatedly [10]. Advanced studies on the structure and mechanism still do not provide clear idea on how the uncoordinated stepping mechanism in dynein responds to the buckled MT. Therefore, the effect of buckling of MT on the cargo transportation has a large scope to explore. Previously, we developed an experimental set-up, micro-stretcher, to control buckling of the MT filaments on a 2D elastic medium [11]. Present study exploits the knowledge from the system of MT buckling to further explore its contribution on the dynein based cargo transportation in vitro. For this, we fixed MTs on a pre-stretched elastic substrate, polydimethylsiloxane (PDMS), through interaction with kinesins. Relaxation of the pre-stretched PDMS consequently developed axial compression stress on the MTs, resulting into their buckling. We controlled the release of tensile strain of the PDMS to tune the buckling extent of MTs. Next, we immobilized the MTs with mild glutaraldehyde treatment and allowed the transportation of Qdots by motors along the undeformed and deformed MTs. We analyzed different parameters, e.g. velocities, pauses and run lengths from the observation of the cargo transportation. With increasing MT buckling we observed changes in the cargo transportation behavior of the motors. For kinesins, we observed that, the cargo velocities decreased with increasing strain in the MTs and finally, cargo transportation was halted at high strain region. While for dynein, cargo transportation prevailed with increased overall velocity with two peaks, one occurring in the low and the other at high strain region. Our goal is to explore the mechanism of how the buckling deformation of MT is affecting the observed phenomena in motor protein-based cargo transportation. Structural deformation in MTs, particularly, have showed to implicate several neurodegenerative diseases such as Alzheimer's disease, cardiovascular syndromes, neurological disorders and cancers etc. [12]. Therefore in vitro study of motor proteins in transporting cargo along deformed MTs will contribute not only in understanding the mechanism of motor motility in cells but also to target specific pathways for various neurodegenerative diseases.

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