Stefan Lakämper

Directionality of individual kinesin‐5 Cin8 motors is modulated by loop 8, ionic strength and microtubule geometry

Kinesin‐5 motors fulfil essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT) plus‐end directed motors. The Saccharomyces cerevisiae kinesin‐5 Cin8 was found, surprisingly, to switch directionality. Here, we have examined directionality using single‐molecule fluorescence motility assays and live‐cell microscopy. On spindles, Cin8 motors mostly moved slowly (∼25 nm/s) towards the midzone, but occasionally also faster (∼55 nm/s) towards the spindle poles. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid (380 nm/s) and processive minus‐end to slow plus‐end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel MTs, while driving steady plus‐end relative sliding. Between parallel MTs, plus‐end motion was only occasionally observed. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus‐end motility and affected the ionic strength‐dependent directional switching of Cin8 in vitro. The deletion mutant cells exhibited reduced midzone‐directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8.

Single fungal kinesin motor molecules move processively along microtubules.

Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in allowing some single-headed kinesins to move processively. Neurospora kinesin (NKin) is a phylogenetically distant, dimeric kinesin from Neurospora crassa with high gliding speed and an unusual neck domain. We quantified the processivity of NKin and compared it to human kinesin, HKin, using gliding and fluorescence-based processivity assays. Our data show that NKin is a processive motor. Single NKin molecules translocated microtubules in gliding assays on average 2.14 micro m (N = 46). When we tracked single, fluorescently labeled NKin motors, they moved on average 1.75 micro m (N = 182) before detaching from the microtubule, whereas HKin motors moved shorter distances (0.83 micro m, N = 229) under identical conditions. NKin is therefore at least twice as processive as HKin. These studies, together with biochemical work, provide a basis for experiments to dissect the molecular mechanisms of processive movement.

Kinesin-5 Kip1 is a bi-directional motor that stabilizes microtubules and tracks their plus-ends in vivo

Summary In this study, we examined the anaphase functions of the S. cerevisiae kinesin-5 homolog Kip1. We show that Kip1 is attached to the mitotic spindle midzone during late anaphase. This attachment is essential to stabilize interpolar microtubule (iMTs) plus-ends. By detailed examination of iMT dynamics we show that at the end of anaphase, iMTs depolymerize in two stages: during the first stage, one pair of anti-parallel iMTs depolymerizes at a velocity of 7.7 µm/minute; during the second stage, ∼90 seconds later, the remaining pair of iMTs depolymerizes at a slower velocity of 5.4 µm/minute. We show that upon the second depolymerization stage, which coincides with spindle breakdown, Kip1 follows the plus-ends of depolymerizing iMTs and translocates toward the spindle poles. This movement is independent of mitotic microtubule motor proteins or the major plus-end binding or tracking proteins. In addition, we show that Kip1 processively tracks the plus-ends of growing and shrinking MTs, both inside and outside the nucleus. The plus-end tracking activity of Kip1 requires its catalytic motor function, because a rigor mutant of Kip1 does not exhibit this activity. Finally, we show that Kip1 is a bi-directional motor: in vitro, at high ionic strength conditions, single Kip1 molecules move processively in the minus-end direction of the MTs, whereas in a multi-motor gliding assay, Kip1 is plus-end directed. The bi-directionality and plus-end tracking activity of Kip1, properties revealed here for the first time, allow Kip1 to perform its multiple functions in mitotic spindle dynamics and to partition the 2-micron plasmid.

The Effect of Monastrol on the Processive Motility of a Dimeric Kinesin-5 Head/Kinesin-1 Stalk Chimera

Controlled activity of several kinesin motors is required for the proper assembly of the mitotic spindle. Eg5, a homotetrameric bipolar kinesin-5 from Xenopus laevis, can cross-link and slide anti-parallel microtubules apart by a motility mechanism comprising diffusional and directional modes. How this mechanism is regulated, possibly by the tail domains of the opposing motors, is poorly understood. In order to explore the basic unregulated kinesin-5 motor activity, we generated a stably dimeric kinesin-5 construct, Eg5Kin, consisting of the motor domain and neck linker of Eg5 and the neck coiled coil of Drosophila melanogaster kinesin-1 (DmKHC). In single-molecule motility assays, we found this chimera to be highly processive. In addition, we studied the effect of the kinesin-5-specific inhibitor monastrol using single-molecule fluorescence assays. We found that monastrol reduced the length of processive runs, but strikingly did not affect velocity. Quantitative analysis of monastrol dose dependence suggests that two bound monastrol molecules are required to be bound to an Eg5Kin dimer to terminate a run.

Back on track - on the role of the microtubule for kinesin motility and cellular function.

The evolution of cytoskeletal filaments (actin- and intermediate-filaments, and the microtubules) and their associated motor- and non-motor-proteins has enabled the eukaryotic cell to achieve complex organizational and structural tasks. This ability to control cellular transport processes and structures allowed for the development of such complex cellular organelles like cilia or flagella in single-cell organisms and made possible the development and differentiation of multi-cellular organisms with highly specialized, polarized cells. Also, the faithful segregation of large amounts of genetic information during cell division relies crucially on the reorganization and control of the cytoskeleton, making the cytoskeleton a key prerequisite for the development of highly complex genomes. Therefore, it is not surprising that the eukaryotic cell continuously invests considerable resources in the establishment, maintenance, modification and rearrangement of the cytoskeletal filaments and the regulation of its interaction with accessory proteins. Here we review the literature on the interaction between microtubules and motor-proteins of the kinesin-family. Our particular interest is the role of the microtubule in the regulation of kinesin motility and cellular function. After an introduction of the kinesin–microtubule interaction we focus on two interrelated aspects: (1) the active allosteric participation of the microtubule during the interaction with kinesins in general and (2) the possible regulatory role of post-translational modifications of the microtubule in the kinesin–microtubule interaction.

A Chimeric Kinesin-1 Head/Kinesin-5 Tail Motor Switches between Diffusive and Processive Motility

Homotetrameric kinesin-5 motors are essential for chromosome separation and assembly of the mitotic spindle. These kinesins bind between two microtubules (MTs) and slide them apart, toward the spindle poles. This process must be tightly regulated in mitosis. In in vitro assays, Eg5 moves diffusively on single MTs and switches to a directed mode between MTs. How allosteric communication between opposing motor domains works remains unclear, but kinesin-5 tail domains may be involved. Here we present a single-molecule fluorescence study of a tetrameric kinesin-1 head/kinesin-5 tail chimera, DK4mer. This motor exhibited fast processive motility on single MTs interrupted by pauses. Like Eg5, DK4mer diffused along MTs with ADP, and slid antiparallel MTs apart with ATP. In contrast to Eg5, diffusive and processive periods were clearly distinguishable. This allowed us to measure transition rates among states and for unbinding as a function of buffer ionic strength. These data, together with results from controls using tail-less dimers, indicate that there are two modes of interaction with MTs, separated by an energy barrier. This result suggests a scheme of motor regulation that involves switching between two bound states, possibly allosterically controlled by the opposing tetramer end. Such a scheme is likely to be relevant for the regulation of native kinesin-5 motors.

The natural diterpene tonantzitlolone A and its synthetic enantiomer inhibit cell proliferation and kinesin-5 function.

Tonantzitlolone A, a diterpene isolated from the Mexican plant Stillingia sanguinolenta, shows cytostatic activity. Both the natural product tonantzitlolone A and its synthetic enantiomer induce monoastral spindle formation in cell experiments which indicates inhibitory activity on kinesin-5 mitotic motor molecules. These inhibitory effects on kinesin-5 could be verified in in vitro single-molecule motility assays, where both tonantzitlolones interfered with kinesin-5 binding to its cellular interaction partner microtubules in a concentration-dependent manner, yet with a larger effect of the synthetic enantiomer. In contrast to kinesin-5 inhibition, both tonantzitlolone A enantiomers did not affect conventional kinesin-1 function; hence tonantzitlolones are not unspecific kinesin inhibitors. The observed stronger inhibitory effect of the synthetic enantiomer demonstrates the possibility to enhance the overall moderate anti-proliferative effect of the lead compound tonantzitlolon A by chemical modification.

Neck-Linker-Length Dependence of Processive Kinesin-5 Motility

To explore the basic motor activity of the mitotic Kinesin-5, we previously constructed a stable dimeric Kinesin-5 head/Kinesin-1 stalk chimera (Eg5Kin), which contains the motor domain and 14 amino acids of the neck linker of Xenopus leavis Eg5 fused to the neck coiled coil of Drosophila melanogaster Kinesin-1. In contrast to truncated dimeric Eg5-513 (Valentine and Block, 2009, Biophys. J. 97:1671), Eg5Kin is a highly processive motor (Lakamper et al., 2010, J. Mol. Biol. 399:1).We have here investigated the effect of varying neck-linker length on the motile properties of Eg5Kin. As truncated versions of Eg5 contain the native 18 amino acids of the neck linker, we generated six Eg5Kin constructs comprising of 13 to up to the 18 amino acids of the native Eg5 neck linker, possibly providing a physiological context.Using single-molecule fluorescence, we found that all six constructs are active motor molecules capable of processive motility. In a first set of experiments, we found that the neck-linker length influences the run length, but not the velocity of the motor. We thus confirm the findings of Shastry and Hancock (2010, Curr. Biol. 20:939) with a different motor. In addition we used optical-trap assays to investigate the change in the average force the motor constructs generated and found only a small variation. Our data thus suggest that the neck-linker length of Eg5 is at least not the sole determinant for speed and force generation.

The Motility of Monomeric and Dimeric Variants of Eg5 studied in the Presence of the Kinesin-5-specific Inhibitor Monastrol

The homo-tetrameric motor-protein Eg5 from Xenopus laevis drives relative sliding of anti-parallel microtubules, most likely by the processive action of its two sets of dimeric motor domains at each end. As recently shown by Kwok et al. (NCB 2006) and Kapitein et al. (JCB 2008) , tetrameric motors move on a single microtubule in a fashion including diffusional and directional episodes, while motors moving between anti-parallel microtubules act in a highly directional and processive fashion. We have studied the processive behavior of a dimeric chimera (Eg5Kin) carrying the Eg5-motor and neck-linker and the Kinesin-1 neck and stalk. While Eg5Kin displays essentially the same motile properties as a truncated Eg5 (Eg5-513 his, Krysziak et al., JBC 2006, Valentine et al., NCB, 2006) its processivity is 40x increased to about 240 consecutive 8nm-steps on average, at a velocity of 95 nm/s. With increasing monastrol concentrations we find a dose-dependent and cooperative reduction in run length, but not in speed, indicating that two monastrol molecules are required to terminate a processive run. To further study the allosteric effect of monastrol on the motility of Eg5-motors, we generated monomeric and dimeric Eg5-constructs and compared their surface gliding-velocities in the presence of increasing concentrations monastrol.

Manipulating and imaging molecular motors with optical traps, single-molecule fluorescence and atomic force microscopy

Motor proteins move cargos in cells and are involved in coordinated large-scale force generation e.g. in cell locomotion, cell division or muscle contraction. Optical traps in conjunction with high-resolution light microscopy has been a tool of choice to analyze dynamic properties of motors, such as power strokes, step lengths or unitary forces. Much detail about microscopic function has also been learned by direct imaging.