Narrow Results

Kinesins are motor proteins which convert the chemical energy of ATP into mechanical energy to move along proteinaceous microtubule rails and to transport different cargoes to defined intracellular destinations. It is well documented that following the track of a single protofilament is the thermodynamically most effective mechanism of kinesin movement along microtubules. However, the question arises what happens when a kinesin molecule encounters a hindrance along the protofilament. The present study describes a simple, cell-free approach which enables to study the effects of structural blockages on kinesin-based transport. This experimental approach uses dimeric conventional kinesin moving nanometre-sized gold beads along immobilized microtubules whose surface has been irreversibly decorated by blocking proteins. We demonstrated that the continuous bead transport temporarily stopped at sites of blockages, but usually continued after a certain resting time. Our results suggest that single dimeric kinesin molecules are able to change to another protofilament if the next tubulin dimer where the second head should bind is blocked. A bypassing mechanism is discussed which is considered to be one fundamental prerequisite to realize a kinesin-mediated cargo-transport along microtubules over long distances, required for e.g., the fast axonal transport in motor neurons.

Kinesins, motor proteins moving along microtubules (MTs) in cells, can potentially be utilized as nano-scale transport systems with an inverted gliding assay, in which the MTs glide on a kinesin-coated surface. Although the key requirements include controls of the gliding direction of MTs, the details of motility properties of gliding MTs have not been elucidated. Here, the angular velocity of gliding MTs was quantitatively measured, particularly focusing on the effects of MT length and kinesin density. The gliding assay of MTs was performed on a substrate coated with kinesin densities of 7.5, 38, and 75 µg/ml that resulted in kinesin spacing of 7.8, 4.2, and 3.1 µm, respectively. The angular velocity for MTs shorter than kinesin spacing significantly decreased with increasing length, and that for MTs longer than kinesin spacing was not affected by their length. Moreover, the angular velocity for MTs longer than kinesin spacing was substantially higher at lower kinesin density. These results suggest that both the number of kinesins associated with MTs and the kinesin spacings may determine the gliding direction.