Chemical tools for dissecting cell division.

Components of the cell division machinery typically function at varying cell cycle stages and intracellular locations. To dissect cellular mechanisms during the rapid division process, small-molecule probes act as complementary approaches to genetic manipulations, with advantages of temporal and in some cases spatial control and applicability to multiple model systems. This Review focuses on recent advances in chemical probes and applications to address select questions in cell division. We discuss uses of both enzyme inhibitors and chemical inducers of dimerization, as well as emerging techniques to promote future investigations. Overall, these concepts may open new research directions for applying chemical probes to advance cell biology.

Dual inhibition of Kif15 by oxindole and quinazolinedione chemical probes.

The mitotic spindle is a microtubule-based machine that segregates a replicated set of chromosomes during cell division. Many cancer drugs alter or disrupt the microtubules that form the mitotic spindle. Microtubule-dependent molecular motors that function during mitosis are logical alternative mitotic targets for drug development. Eg5 (Kinesin-5) and Kif15 (Kinesin-12), in particular, are an attractive pair of motor proteins, as they work in concert to drive centrosome separation and promote spindle bipolarity. Furthermore, we hypothesize that the clinical failure of Eg5 inhibitors may be (in part) due to compensation by Kif15. In order to test this idea, we screened a small library of kinase inhibitors and identified GW108X, an oxindole that inhibits Kif15 in vitro. We show that GW108X has a distinct mechanism of action compared with a commercially available Kif15 inhibitor, Kif15-IN-1 and may serve as a lead with which to further develop Kif15 inhibitors as clinically relevant agents.

Motor Reattachment Kinetics Play a Dominant Role in Multimotor-Driven Cargo Transport.

Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple kinesins coordinate their activity during cargo transport are poorly understood. To understand these multimotor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be fourfold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of a gold-nanoparticle-labeled motor with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than does kinesin-1. Finally, compared to cargo transported by two kinesin-1, cargo transported by two kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multimotor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads whereas kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.

The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State.

Single-molecule microscopy and stopped-flow kinetics assays were carried out to understand the microtubule polymerase activity of kinesin-5 (Eg5). Four lines of evidence argue that the motor primarily resides in a two-heads-bound (2HB) state. First, upon microtubule binding, dimeric Eg5 releases both bound ADPs. Second, microtubule dissociation in saturating ADP is 20-fold slower for the dimer than for the monomer. Third, ATP-triggered mant-ADP release is 5-fold faster than the stepping rate. Fourth, ATP binding is relatively fast when the motor is locked in a 2HB state. Shortening the neck-linker does not facilitate rear-head detachment, suggesting a minimal role for rear-head-gating. This 2HB state may enable Eg5 to stabilize incoming tubulin at the growing microtubule plus-end. The finding that slowly hydrolyzable ATP analogs trigger slower nucleotide release than ATP suggests that ATP hydrolysis in the bound head precedes stepping by the tethered head, leading to a mechanochemical cycle in which processivity is determined by the race between unbinding of the bound head and attachment of the tethered head.

Processivity of the kinesin-2 KIF3A results from rear head gating and not front head gating.

The kinesin-2 family motor KIF3A/B works together with dynein to bidirectionally transport intraflagellar particles, melanosomes, and neuronal vesicles. Compared with kinesin-1, kinesin-2 is less processive, and its processivity is more sensitive to load, suggesting that processivity may be controlled by different gating mechanisms. We used stopped-flow and steady-state kinetics experiments, along with single-molecule and multimotor assays to characterize the entire kinetic cycle of a KIF3A homodimer that exhibits motility similar to that of full-length KIF3A/B. Upon first encounter with a microtubule, the motor rapidly exchanges both mADP and mATP. When adenosine 5'-[(ß,γ)-imido]triphosphate was used to entrap the motor in a two-head-bound state, exchange kinetics were unchanged, indicating that rearward strain in the two-head-bound state does not alter nucleotide binding to the front head. A similar lack of front head gating was found when intramolecular strain was enhanced by shortening the neck linker domain from 17 to 14 residues. In single-molecule assays in ADP, the motor dissociates at 2.1 s(-1), 20-fold slower than the stepping rate, demonstrating the presence of rear head gating. In microtubule pelleting assays, the KD(Mt) is similar in ADP and ATP. The data and accompanying simulations suggest that, rather than KIF3A processivity resulting from strain-dependent regulation of nucleotide binding (front head gating), the motor spends a significant fraction of its hydrolysis cycle in a low affinity state but dissociates only slowly from this state. This work provides a mechanism to explain differences in the load-dependent properties of kinesin-1 and kinesin-2.

Exploring the inhibitory activity of short-chain phospholipids against amyloid fibrillogenesis of hen egg-white lysozyme.

Amyloid fibrillogenesis is an important pathological feature of a group of degenerative human diseases. The 129-residue enzyme hen egg-white lysozyme has been shown to form fibrils in vitro at pH 2.0 and 55°C. In this research, using various spectroscopic techniques, light scattering, and transmission electron microscopy, we first examined the influence of short-chain phospholipids on the amyloid fibrillogenesis and the structural changes derived from hen lysozyme in vitro. Both model short-chain phospholipids were observed to mitigate the fibrillogenesis of hen lysozyme. Also, urea-induced unfolding results suggested that the susceptibility of hen lysozyme to conformational changes elicited by the denaturant was observed to decrease upon addition of short-chain phospholipids. Moreover, our molecular dynamics simulations results demonstrated that the observed inhibitory action of short-chain phosoholipids against hen lysozyme fibrillogenesis might be attributable to the interference of ß-strand extension by the binding of phospholipids to lysozyme's ß-sheet-rich region. We believe that the outcome from this study may contribute to a better understanding the molecular factors affecting amyloid fibrillogenesis and the molecular mechanism(s) of the interactions between phospholipids/lipids and amyloid-forming proteins.

Tension promotes kinetochore-microtubule release by Aurora B kinase.

To ensure accurate chromosome segregation, interactions between kinetochores and microtubules are regulated by a combination of mechanics and biochemistry. Tension provides a signal to discriminate attachment errors from bi-oriented kinetochores with sisters correctly attached to opposite spindle poles. Biochemically, Aurora B kinase phosphorylates kinetochores to destabilize interactions with microtubules. To link mechanics and biochemistry, current models regard tension as an input signal to locally regulate Aurora B activity. Here, we show that the outcome of kinetochore phosphorylation depends on tension. Using optogenetics to manipulate Aurora B at individual kinetochores, we find that kinase activity promotes microtubule release when tension is high. Conversely, when tension is low, Aurora B activity promotes depolymerization of kinetochore-microtubules while maintaining attachment. Thus, phosphorylation converts a catch-bond, in which tension stabilizes attachments, to a slip-bond, which releases microtubules under tension. We propose that tension is a signal inducing distinct error-correction pathways, with release or depolymerization being advantageous for typical errors characterized by high or low tension, respectively.

Kinesin-5 Promotes Microtubule Nucleation and Assembly by Stabilizing a Lattice-Competent Conformation of Tubulin.

Besides sliding apart antiparallel microtubules during spindle elongation, the mitotic kinesin-5, Eg5, promotes microtubule polymerization, emphasizing its importance in mitotic spindle length control. Here, we characterize the Eg5 microtubule polymerase mechanism by assessing motor-induced changes in the longitudinal and lateral tubulin-tubulin bonds that form the microtubule lattice. Isolated Eg5 motor domains promote microtubule nucleation, growth, and stability; thus, crosslinking tubulin by pairs of motor heads is not necessary for polymerase activity. Eg5 binds preferentially to microtubules over free tubulin, which contrasts with microtubule-depolymerizing kinesins that preferentially bind free tubulin over microtubules. Colchicine-like inhibitors that stabilize the bent conformation of tubulin allosterically inhibit Eg5 binding, consistent with a model in which Eg5 induces a curved-to-straight transition in tubulin. Domain swap experiments establish that the family-specific loop11-helix 4 junction, which resides near the nucleotide-sensing switch-II domain, is necessary and sufficient for the polymerase activity of Eg5. Thus, we propose a microtubule polymerase mechanism in which Eg5 at the plus-end promotes a curved-to-straight transition in tubulin that enhances lateral bond formation and thereby promotes microtubule growth and stability. One implication is that regulation of Eg5 motile properties by regulatory proteins or small molecule inhibitors could also have effects on intracellular microtubule dynamics.

Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity.

To uncover their contrasting mechanisms, antimitotic drugs that inhibit Eg5 (kinesin-5) were analyzed in mixed-motor gliding assays of kinesin-1 and Eg5 motors in which Eg5 "braking" dominates motility. Loop-5 inhibitors (monastrol, STLC, ispinesib, and filanesib) increased gliding speeds, consistent with inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, consistent with locking Eg5 in a rigor state. Biochemical and single-molecule assays demonstrated that BRD9876 acts as an ATP- and ADP-competitive inhibitor with 4 nM KI. Consistent with its microtubule polymerase activity, Eg5 was shown to stabilize microtubules against depolymerization. This stabilization activity was eliminated in monastrol but was enhanced by BRD9876. Finally, in metaphase-arrested RPE-1 cells, STLC promoted spindle collapse, whereas BRD9876 did not. Thus, different Eg5 inhibitors impact spindle assembly and architecture through contrasting mechanisms, and rigor inhibitors may paradoxically have the capacity to stabilize microtubule arrays in cells.

A Tubulin Binding Switch Underlies Kip3/Kinesin-8 Depolymerase Activity.

Kinesin-8 motors regulate the size of microtubule structures, using length-dependent accumulation at the plus end to preferentially disassemble long microtubules. Despite extensive study, the kinesin-8 depolymerase mechanism remains under debate. Here, we provide evidence for an alternative, tubulin curvature-sensing model of microtubule depolymerization by the budding yeast kinesin-8, Kip3. Kinesin-8/Kip3 uses ATP hydrolysis, like other kinesins, for stepping on the microtubule lattice, but at the plus end Kip3 undergoes a switch: its ATPase activity is suppressed when it binds tightly to the curved conformation of tubulin. This prolongs plus-end binding, stabilizes protofilament curvature, and ultimately promotes microtubule disassembly. The tubulin curvature-sensing model is supported by our identification of Kip3 structural elements necessary and sufficient for plus-end binding and depolymerase activity, as well as by the identification of an α-tubulin residue specifically required for the Kip3-curved tubulin interaction. Together, these findings elucidate a major regulatory mechanism controlling the size of cellular microtubule structures.

Crystal structure of Zen4 in the apo state reveals a missing conformation of kinesin.

Kinesins hydrolyse ATP to transport intracellular cargoes along microtubules. Kinesin neck linker (NL) functions as the central mechano-chemical coupling element by changing its conformation through the ATPase cycle. Here we report the crystal structure of kinesin-6 Zen4 in a nucleotide-free, apo state, with the NL initial segment (NIS) adopting a backward-docked conformation and the preceding α6 helix partially melted. Single-molecule fluorescence resonance energy transfer (smFRET) analyses indicate the NIS of kinesin-1 undergoes similar conformational changes under tension in the two-head bound (2HB) state, whereas it is largely disordered without tension. The backward-docked structure of NIS is essential for motility of the motor. Our findings reveal a key missing conformation of kinesins, which provides the structural basis of the stable 2HB state and offers a tension-based rationale for an optimal NL length to ensure processivity of the motor.

Examining kinesin processivity within a general gating framework.

Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ~1 µm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.