Jeremy Cooper

The Ndc80 Kinetochore Complex Forms Load-Bearing Attachments to Dynamic Microtubule Tips via Biased Diffusion

Kinetochores couple chromosomes to the assembling and disassembling tips of microtubules, a dynamic behavior that is fundamental to mitosis in all eukaryotes but poorly understood. Genetic, biochemical, and structural studies implicate the Ndc80 complex as a direct point of contact between kinetochores and microtubules, but these approaches provide only a static view. Here, using techniques for manipulating and tracking individual molecules in vitro, we demonstrate that the Ndc80 complex is capable of forming the dynamic, load-bearing attachments to assembling and disassembling tips required for coupling in vivo. We also establish that Ndc80-based coupling likely occurs through a biased diffusion mechanism and that this activity is conserved from yeast to humans. Our findings demonstrate how an ensemble of Ndc80 complexes may provide the combination of plasticity and strength that allows kinetochores to maintain load-bearing tip attachments during both microtubule assembly and disassembly.

Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B

The Dam1 complex, regulated by aurora B phosphorylation, confers a more stable microtubule association for the Ndc80 complex at kinetochores (see also related paper by Lampert et al. in this issue).

Phosphoregulation and depolymerization-driven movement of the Dam1 complex do not require ring formation

During mitosis, kinetochores form persistent attachments to microtubule tips and undergo corrective detachment in response to phosphorylation by Ipl1 (Aurora B) kinase. The Dam1 complex is required to establish and maintain bi-oriented attachment to microtubule tips in vivo, and it contains multiple sites phosphorylated by Ipl1 (Refs 2, 3, 4, 5, 6, 7, 8, 9, 10). Moreover, a number of kinetochore-like functions can be reconstituted in vitro with pure Dam1 complex. These functions are believed to derive from the ability of the complex to self-assemble into rings. Here we show that rings are not necessary for dynamic microtubule attachment, Ipl1-dependent modulation of microtubule affinity or the ability of Dam1 to move processively with disassembling microtubule tips. Using two fluorescence-based assays, we found that the complex exhibited a high affinity for microtubules (Kd of approximately 6 nM) that was reduced by phosphorylation at Ser 20, a single Ipl1 target residue in Dam1. Moreover, individual complexes underwent one-dimensional diffusion along microtubules and detached 2.5-fold more frequently after phosphorylation by Ipl1. Particles consisting of one to four Dam1 complexes — too few to surround a microtubule — were captured and carried by disassembling tips. Thus, even a small number of binding elements could provide a dynamic, phosphoregulated microtubule attachment and thereby facilitate accurate chromosome segregation.

Catalysis of the microtubule on-rate is the major parameter regulating the depolymerase activity of MCAK.

The kinesin-13, MCAK, is a critical regulator of microtubule dynamics in eukaryotic cells. We have functionally dissected the structural features responsible for MCAKs potent microtubule depolymerization activity. MCAKs positively charged neck enhances its delivery to microtubule ends not by tethering the molecule to microtubules during diffusion, as commonly thought, but by catalyzing the association of MCAK to microtubules. On the other hand, this same positively charged neck slightly diminishes MCAKs ability to remove tubulin subunits once at the microtubule end. Conversely, dimerization reduces MCAK delivery but improves MCAKs ability to remove tubulin subunits. The reported kinetics for these events predicts a nonspecific binding mechanism that may represent a paradigm for the diffusive interaction of many microtubule-binding proteins.

The diffusive interaction of microtubule binding proteins

Microtubule-based motility is often thought of as specifically referring to the directed stepping of microtubule-based motors such as kinesin or dynein. However, microtubule lattice diffusion (also known as diffusional motility) provides a second mode of transport that is shared by a much broader class of microtubule binding proteins. Microtubule lattice diffusion offers distinct advantages as a transport mechanism including speed, bidirectional microtubule end targeting, and no requirement for direct chemical energy (i.e. ATP). It remains to be seen whether a universal binding mechanism for this interaction will be identified but electrostatic interactions appear to play a significant role. In the meantime, the well-studied subject of DNA binding proteins that diffuse along the DNA backbone provides an insightful analog for understanding the nature of microtubule-based diffusional motility.

The role of the kinesin-13 neck in microtubule depolymerization.

To ensure genetic integrity, replicated chromosomes must be accurately distributed to daughter cells—a process that is accomplished on the microtubule spindle. Kinesin-13 motors play an essential role in this process by performing regulated microtubule depolymerization. We set out to dissect the depolymerization mechanism of these kinesins, and in particular, the role of their conserved neck sequence. We used a monomeric kinesin-13 MCAK, consisting of the neck and motor core, which has strong depolymerizing activity. In the presence of a non-hydrolysable ATP analogue, this construct induced formation of rings around microtubules. The rings are built from tubulin protofilaments that are bent by the kinesin-13 motor engaged at the ATP-binding step of its ATPase cycle. Our data suggest that the ring-microtubule interaction is mediated by the neck and support the idea of a role for the kinesin-13 neck in depolymerization efficiency, acting by optimising release of tubulin from microtubule ends.

A prospective study of intraoperative pulse oximetry failure

Since pulse oximetry is now an ASA standard for intraoperative monitoring, we sought to determine the intraoperative failure rate for this device. We prospectively evaluated the intraoperative failure rate of our pulse oximeters at the four University of Washington Hospitals (University of Washington Medical Center, Veterans Affairs Medical Center [VAMC], Childrens Hospital and Medical Center, and Harborview Medical Center [HMC]) recorded from April 1989 to August 1989. We defined failure as the inability to obtain any oximetry reading for a cumulative period of more than 30 minutes during any anesthetic procedure after all equipment malfunctions had been eliminated. Our pulse oximeters failed in 124 of 11,046 cases studied; this is a failure rate of 1.12%, which ranged from 0.56% at HMC to 4.24% at VAMC. The failure rate at VAMC (4.24%) was significantly higher than the other hospitals (p<0.001). Those cases associated with the pulse oximeter failure had the following characteristics: (1) an ASA status of 3 or higher, (2) lengthy operations, and (3) elderly patients. When the device did fail in a patient, it did not function for 32% of the mean anesthesia time. We conclude that the intraoperative use of the pulse oximetry can provide information about blood oxygen saturation in most patients. However, in approximately 1% of the patients we studied in the operating room, mechanically functioning pulse oximeters failed to provide readings of blood oxygen saturations during routine operative use.

A kinesin-13 mutant catalytically depolymerizes microtubules in ADP

The kinesin-13 motor protein family members drive the removal of tubulin from microtubules (MTs) to promote MT turnover. A point mutation of the kinesin-13 family member mitotic centromere-associated kinesin/Kif2C (E491A) isolates the tubulin-removal conformation of the motor, and appears distinct from all previously described kinesin-13 conformations derived from nucleotide analogues. The E491A mutant removes tubulin dimers from stabilized MTs stoichiometrically in adenosine triphosphate (ATP) but is unable to efficiently release from detached tubulin dimers to recycle catalytically. Only in adenosine diphosphate (ADP) can the mutant catalytically remove tubulin dimers from stabilized MTs because the affinity of the mutant for detached tubulin dimers in ADP is low relative to lattice-bound tubulin. Thus, the motor can regenerate for further cycles of disassembly. Using the mutant, we show that release of tubulin by kinesin-13 motors occurs at the transition state for ATP hydrolysis, which illustrates a significant divergence in their coupling to ATP turnover relative to motile kinesins.

Reconstitution and Functional Analysis of Kinetochore Subcomplexes

Kinetochores are multifunctional supercomplexes that link chromosomes to dynamic microtubule tips. Groups of proteins from the kinetochore are arranged into distinct subcomplexes that copurify under stringent conditions and cause similar phenotypes when mutated. By coexpressing all the components of a given subcomplex from a polycistronic plasmid in bacteria, many laboratories have had great success in purifying active subcomplexes. This has enabled the study of how the microtubule-binding subcomplexes of the kinetochore interact with both the microtubule lattice and dynamic microtubule tips. Here we outline methods for rapid cloning of polycistronic vectors for expression of kinetochore subcomplexes, their purification, and techniques for functional analysis using total internal reflection fluorescence microscopy (TIRFM).

State-dependent Block of CNG Channels by Dequalinium

Cyclic nucleotide–gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca2+. Not surprisingly, they are blocked by a number of Ca2+ channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca2+-activated K+ channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3–5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10–20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.