Christina Thiede

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.

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.

Regulation of bi-directional movement of single kinesin-5 Cin8 molecules.

Kinesin-5 mechanoenzymes drive mitotic spindle dynamics as slow, processive microtubule (MT)-plus-end directed motors. Surprisingly, the Saccharomyces cerevisiae kinesin-5 Cin8 was recently found to be bi-directional: it can move processively in both directions on MTs. Two hypotheses have been suggested for the mechanism of the directionality switch: (1) single molecules of Cin8 are intrinsically minus-end directed, but mechanical coupling between two or more motors triggers the switch; (2) a single motor can switch direction, and “cargo binding” i.e., binding between two MTs triggers the switch to plus-end motility. Single-molecule fluorescence data we published recently, and augment here, favor hypothesis (2). In low-ionic-strength conditions, single molecules of Cin8 move in both minus- and plus-end directions. Fluorescence photo bleaching data rule out aggregation of Cin8 while they move in the plus and in the minus direction. The evidence thus points toward cargo regulation of directionality, which is likely to be related to cargo regulation in other kinesins. The molecular mechanisms of this regulation, however, remain to be elucidated.

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.

Endoplasmic reticulum sorting and kinesin-1 command the targeting of axonal GABAB receptors.

In neuronal cells the intracellular trafficking machinery controls the availability of neurotransmitter receptors at the plasma membrane, which is a critical determinant of synaptic strength. Metabotropic γ amino-butyric acid (GABA) type B receptors (GABABRs) are neurotransmitter receptors that modulate synaptic transmission by mediating the slow and prolonged responses to GABA. GABABRs are obligatory heteromers constituted by two subunits, GABABR1 and GABABR2. GABABR1a and GABABR1b are the most abundant subunit variants. GABABR1b is located in the somatodendritic domain whereas GABABR1a is additionally targeted to the axon. Sushi domains located at the N-terminus of GABABR1a constitute the only difference between both variants and are necessary and sufficient for axonal targeting. The precise targeting machinery and the organelles involved in sorting and transport have not been described. Here we demonstrate that GABABRs require the Golgi apparatus for plasma membrane delivery but that axonal sorting and targeting of GABABR1a operate in a pre-Golgi compartment. In the axon GABABR1a subunits are enriched in the endoplasmic reticulum (ER), and their dynamic behavior and colocalization with other secretory organelles like the ER-to-Golgi intermediate compartment (ERGIC) suggest that they employ a local secretory route. The transport of axonal GABABR1a is microtubule-dependent and kinesin-1, a molecular motor of the kinesin family, determines axonal localization. Considering that progression of GABABRs through the secretory pathway is regulated by an ER retention motif our data contribute to understand the role of the axonal ER in non-canonical sorting and targeting of neurotransmitter receptors.

Deletion of the tail domain of the kinesin-5 Cin8 affects its directionality

Background: Single molecules of the kinesin-5 Cin8 were previously demonstrated to be minus-end-directed under high-ionic-strength conditions. Results: Under high-ionic-strength conditions, Cin8 lacking the tail domain is bidirectional. Conclusion: The tail domain is one of the factors that regulate Cin8 directionality. Significance: An important structural element was identified that regulates the directionality of kinesin-5 motors. The bipolar kinesin-5 motors are one of the major players that govern mitotic spindle dynamics. Their bipolar structure enables them to cross-link and slide apart antiparallel microtubules (MTs) emanating from the opposing spindle poles. The budding yeast kinesin-5 Cin8 was shown to switch from fast minus-end- to slow plus-end-directed motility upon binding between antiparallel MTs. This unexpected finding revealed a new dimension of cellular control of transport, the mechanism of which is unknown. Here we have examined the role of the C-terminal tail domain of Cin8 in regulating directionality. We first constructed a stable dimeric Cin8/kinesin-1 chimera (Cin8Kin), consisting of head and neck linker of Cin8 fused to the stalk of kinesin-1. As a single dimeric motor, Cin8Kin switched frequently between plus and minus directionality along single MTs, demonstrating that the Cin8 head domains are inherently bidirectional, but control over directionality was lost. We next examined the activity of a tetrameric Cin8 lacking only the tail domains (Cin8Δtail). In contrast to wild-type Cin8, the motility of single molecules of Cin8Δtail in high ionic strength was slow and bidirectional, with almost no directionality switches. Cin8Δtail showed only a weak ability to cross-link MTs in vitro. In vivo, Cin8Δtail exhibited bias toward the plus-end of the MTs and was unable to support viability of cells as the sole kinesin-5 motor. We conclude that the tail of Cin8 is not necessary for bidirectional processive motion, but is controlling the switch between plus- and minus-end-directed motility.

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.