The mechanical properties of kinesin-1: a holistic approach

During the last 25 years, a vast amount of research has gone into understanding the mechanochemical cycle of kinesin-1 and similar processive motor proteins. An experimental method that has been widely used to this effect is the in vitro study of kinesin-1 molecules moving along microtubules while pulling a bead, the position of which is monitored optically while trapped in a laser focus. Analysing results from such experiments, in which thermally excited water molecules are violently buffeting the system components, can be quite difficult. At low loads, the effect of the mechanical properties of the entire molecule must be taken into account, as stalk compliance means the bead position recorded is only weakly coupled to the movement of the motor domains, the sites of ATP hydrolysis and microtubule binding. In the present review, findings on the mechanical and functional properties of the various domains of full-length kinesin-1 molecules are summarized and a computer model is presented that uses this information to simulate the motion of a bead carried by a kinesin molecule along a microtubule, with and without a weak optical trap present. A video sequence made from individual steps of the simulation gives a three-dimensional visual insight into these types of experiment at the molecular level.

KINESIN BYPASSING BLOCKAGES ON MICROTUBULE RAILS

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.

An isoform of kinesin light chain specific for the Golgi complex

Conventional kinesin is a motor protein implicated in the transport of a variety of cytoplasmic organelles along microtubules. The kinesin molecule consists of two heavy chains with motor domains at their amino termini and two light chains, which, together with the carboxyl termini of the heavy chains, are proposed to mediate binding to cargoes. Since the light chains are represented by multiple isoforms diverging at their carboxyl termini they are presumed to specify kinesin targeting to organelles. Previously, we isolated five cDNAs, encoding hamster kinesin light chain isoforms, and found that one of them (B or C) preferentially associated with mitochondria. To obtain additional evidence proving the specific location of various kinesin light chain isoforms on organelles, we made an antibody against a 56 amino-acid sequence found at the carboxyl-terminal regions of the hamster D and E isoforms. By indirect immunofluorescence, this antibody specifically labeled the Golgi complex in cultured cells. In western blots of total cell homogenates, it recognized two close polypeptides, one of which co-purified with the Golgi membranes. Thus, the results of this and previous studies demonstrate that different kinesin light chains are associated with different organelles in cells.

The role of thermal activation in motion and force generation by molecular motors

The currently accepted mechanism for ATP–driven motion of kinesin is called the hand–over–hand model, where some chemical transition during the ATP hydrolysis cycle stretches a spring, and motion and force production result from the subsequent relaxation. It is essential in this mechanism for the moving head of kinesin to dissociate, while the other head remains firmly attached to the microtubule. Here we propose an alternative Brownian motor model where the action of ATP modulates the interaction potential between kinesin and the microtubule rather than a spring internal to the kinesin molecule alone. In this model neither head need dissociate (which predicts that under some circumstances a single–headed kinesin can display processive motion) and the transitions by which the motor moves are best described as thermally activated steps. This model is consistent with a wide range of experimental data on the force–velocity curves, the one ATP to one–step stoichiometry observed at small load, and the stochastic properties of the stepping.

Processivity of the Motor Protein Kinesin Requires Two Heads

A single kinesin molecule can move for hundreds of steps along a microtubule without dissociating. One hypothesis to account for this processive movement is that the binding of kinesin's two heads is coordinated so that at least one head is always bound to the microtubule. To test this hypothesis, the motility of a full-length single-headed kinesin heterodimer was examined in the in vitro microtubule gliding assay. As the surface density of single-headed kinesin was lowered, there was a steep fall both in the rate at which microtubules landed and moved over the surface, and in the distance that microtubules moved, indicating that individual single-headed kinesin motors are not processive and that some four to six single-headed kinesin molecules are necessary and sufficient to move a microtubule continuously. At high ATP concentration, individual single-headed kinesin molecules detached from microtubules very slowly (at a rate less than one per second), 100-fold slower than the detachment during two-headed motility. This slow detachment directly supports a coordinated, hand-over-hand model in which the rapid detachment of one head in the dimer is contingent on the binding of the second head.