Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes1,2. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules3. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B4,5. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules6. Moreover, tension increases the lifetimes of the reconstituted attachments directly, via a catch bond-like mechanism that does not require Aurora B7-10. Based on these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

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SUMMARY 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 a ‘slip clutch’ that allows the kinetochore to maintain a load-bearing tip attachment during both microtubule assembly and disassembly.

Kinetochores remain attached to microtubule (MT) tips during mitosis even as the tips assemble and disassemble under their grip, allowing filament dynamics to produce force and move chromosomes. The specific proteins that mediate tip attachment are uncertain, and the mechanism of MT-dependent force production is unknown. Recent work suggests that the Dam1 complex, an essential component of kinetochores in yeast, may contribute directly to kinetochore-MT attachment and force production, perhaps by forming a sliding ring encircling the MT. To test these hypotheses, we developed an in vitro motility assay where beads coated with pure recombinant Dam1 complex were bound to the tips of individual dynamic MTs. The Dam1-coated beads remained tip-bound and underwent assembly-and disassembly-driven movement over Ϸ3 m, comparable to chromosome displacements in vivo. Dam1-based attachments to assembling tips were robust, supporting 0.5-3 pN of tension applied with a feedback-controlled optical trap as the MTs lengthened Ϸ1 m. The attachments also harnessed energy from MT disassembly to generate movement against tension. Reversing the direction of force (i.e., switching to compressive force) caused the attachments to disengage the tip and slide over the filament, but sliding was blocked by areas where the MT was anchored to a coverslip, consistent with a coupling structure encircling the filament. Our findings demonstrate how the Dam1 complex may contribute directly to MT-driven chromosome movement.cytoskeleton ͉ DASH ͉ disassembly ͉ mitosis ͉ motility

Insufficient pancreatic β-cell mass or function results in diabetes mellitus. While significant progress has been made in regulating insulin secretion from β-cells in diabetic patients, no pharmacological agents have been described that increase β-cell replication in humans. Here we report aminopyrazine compounds that stimulate robust β-cell proliferation in adult primary islets, most likely as a result of combined inhibition of DYRK1A and GSK3B. Aminopyrazine-treated human islets retain functionality in vitro and after transplantation into diabetic mice. Oral dosing of these compounds in diabetic mice induces β-cell proliferation, increases β-cell mass and insulin content, and improves glycaemic control. Biochemical, genetic and cell biology data point to Dyrk1a as the key molecular target. This study supports the feasibility of treating diabetes with an oral therapy to restore β-cell mass, and highlights a tractable pathway for future drug discovery efforts.

In dividing cells, kinetochores couple chromosomes to the tips of growing and shortening microtubule (MT) fibers 1, 2 and tension at the kinetochore-MT interface promotes fiber elongation 3-6 . Tension-dependent MT fiber elongation is thought to be essential for coordinating chromosome alignment and separation 1, 3, 7-10 , but the mechanism underlying this effect is unknown. Using optical tweezers, we applied tension to a model of the kinetochore-microtubule interface composed of the yeast Dam1 complex 11-13 bound to individual dynamic microtubule tips 14 . Higher tension decreased the likelihood that growing tips would begin to shorten, slowed shortening, and increased the likelihood that shortening tips would resume growth. These effects are similar to the effects of tension on kinetochore-attached microtubule fibers in many cell types, suggesting that we have reconstituted a direct mechanism for microtubule length control in mitosis.For decades, a central problem for biologists has been to understand how MT lengths are controlled during mitosis 15, 16 . MTs are protein polymers that switch stochastically between phases of assembly and disassembly, during which tubulin subunits are added or lost from the filament tips 1 . This behavior, called 'dynamic instability', can be described by four parameters: the speeds of growth and shortening, and the rates of switching from growth to shortening and from shortening to growth, transitions known as 'catastrophes ' and 'rescues' 1, 17 In dividing cells, chromosomes are linked to the tips of MT fibers through specialized structures called kinetochores and their movements are coupled to fiber growth and shortening 1, 2 . Remarkably, kinetochores maintain persistent, load-bearing attachments to microtubule tips even as the filaments assemble and disassemble under their grip 1, 2, 18 . Classic micromanipulation experiments show that tension at the kinetochore-MT interface promotes MT fiber elongation 3-6 , and this effect is widely believed to be essential for controlling fiber lengths and thereby coordinating chromosome alignment and separation 1, 3, 7-10, 16 . Very little is known about the mechanism underlying this tension-dependent length control. However, the dynamic behavior of kinetochore-attached MT tips 18 implies that the mechanism underlying tension-dependent length control in vivo may act by altering one or more of the parameters of dynamic instability in response to load.A key unanswered question is whether tension promotes elongation via an indirect mechanism, where force transmitted through load-bearing kinetochore components regulates the activity of separate MT-modifying components, or by a direct mechanism, where the load-bearing Correspondence should be addressed to C.L.A.. * These authors contributed equally to this work. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2009 May 13. Published in final edited form as:Nat Cell Biol. 2007 July ; 9(7): 832-837. doi:10.1038/ncb1609. NIH-PA Author ManuscriptNIH-P...

We detail our use of computer-controlled optical traps to study interactions between kinetochore components and dynamic microtubules. Over the last two decades optical traps have helped uncover the working principles of conventional molecular motors, such as kinesin and dynein, but only recently have they been applied to study kinetochore function. The most useful traps combine sensitive position detectors and servo-control, allowing them to be operated as force clamps that maintain constant loads on objects as they move. Our instrument, which is among the simplest designs that permits force clamping, relies on a computer-controlled piezoelectric stage and a single laser for trapping and position detection. We apply it in motility assays where beads coated with pure microtubule-binding kinetochore components are attached to the tips of individual dynamic microtubules. Like kinetochores in vivo, the beads remain tip-attached, undergoing movements coupled to filament assembly and disassembly. The force clamp provides many benefits over instruments that lack feedback control. It allows tension to be applied continuously during both assembly-and disassembly-driven movement, providing a close match to the physiological situation. It also enables tracking with high resolution, and simplifies data interpretation by eliminating artifacts due to molecular compliance. The formation of persistent, load-bearing attachments to dynamic microtubule tips is fundamental to all kinetochore activities. Our direct, physical study of kinetochore-microtubule coupling may therefore furnish insights into many vital kinetochore functions, including correction of aberrant attachments and generation of the 'wait-anaphase' signals that delay mitosis until all kinetochores are properly attached.

Cardiovascular diseases, such as atherosclerosis and hypertension, are associated with arterial stiffening. Previous studies showed that ANG II exacerbated atherosclerosis and induced hypertension and aneurysm formation in apolipoprotein E-deficient (apoE-KO) mice. The aim of the present study was to examine the effects of chronic treatment of ANG II on the arterial elastic properties in apoE-KO mice. We hypothesized that ANG II will injure the arterial wall resulting in increased arterial stiffening. Male apoE-KO mice were infused with either ANG II (1.44 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) or vehicle (PBS) for 30 days. ANG II treatment accelerated atherosclerosis in the carotid artery by sixfold (P Ͻ 0.001) and increased blood pressure by 30% (P Ͻ 0.05). Additionally, our data demonstrated that ANG II increased arterial stiffening using both in vivo and in vitro methods. ANG II significantly increased pulse wave velocity by 36% (P Ͻ 0.01) and decreased arterial elasticity as demonstrated by a more than 900% increase in maximal stiffening (high strain Young's modulus) compared with vehicle (P Ͻ 0.05). These functional changes were correlated with morphological and biochemical changes as demonstrated by an increase in collagen content (60%), a decrease in elastin content (74%), and breaks in the internal elastic lamina in the aortic wall. In addition, endotheliumindependent vasorelaxation to sodium nitroprusside was impaired in the aortic rings of ANG II-treated mice compared with vehicle. Thus, the present data indicate that ANG II injures the artery wall in multiple ways and arterial stiffening may be a common outcome of ANG II-induced arterial damage.aneurysm; atherosclerosis; hypertension; vascular stiffness LARGE ARTERIES ARE CHARACTERIZED by their elastic properties and ability to synthesize many vasoactive substances. These properties enable the arterial wall to function as a modulator of blood pressure and more generally of cardiovascular hemodynamics (14). It is well recognized that the mechanical properties of large arteries are primarily determined by the composition of the arterial wall. The "passive" biomechanical properties of the arterial wall are influenced predominantly by the extracellular matrix proteins, collagen, and elastin, whereas the "active" properties depend on the activation of vascular smooth muscle cells. Aging, environmental and genetic factors are responsible for the functional (decreased release of vasodilators and increased synthesis of vasoconstrictors) and structural (smooth muscle cell hypertrophy, extracellular matrix accumulation, and degradation of elastin) (19) modifications of the arterial wall and the arterial endothelium. These modifications could lead to a diminution of elasticity and increased vascular stiffness.Atherosclerosis produces many pathological changes in the arterial wall, one of the most important being a progressive increase in arterial stiffening. It has also been demonstrated that hypertension can increase aortic stiffness (3,4,23,24,26,28,37). In addition, previous...