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In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ~8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Biml p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs.

Optical traps are routinely used for the manipulation of neutral particles. However, optical trap design is limited by the lack of an accurate theory. The generalized Lorenz-Mie theory (GLMT) solves the scattering problem for arbitrary particle size and predicts radial forces accurately. Here we show that the GLMT predicts the observed radial and axial forces in a variety of optical manipulators. We also present a dimensionless parameter [beta] for the prediction of axial forces. Coupled with our correlation for radial escape forces, we now have a set of two simple correlations for the practical design of radiation-force-based systems.

We developed a stochastic model of microtubule (MT) assembly dynamics that estimates tubulin-tubulin bond energies, mechanical energy stored in the lattice dimers, and the size of the tubulin-GTP cap at MT tips. First, a simple assembly/disassembly state model was used to screen possible combinations of lateral bond energy ([DELTA][G.sub.Lat]) and longitudinal bond energy ([DELTA][G.sub.Long]) plus the free energy of immobilizing a dimer in the MT lattice ([DELTA][G.sub.s]) for rates of MT growth and shortening measured experimentally. This analysis predicts [DELTA][G.sub.Lat] in the range of -3.2 to -5.7 [k.sub.B]T and [DELTA][G.sub.Long] plus [DELTA][G.sub.s] in the range of -6.8 to -9.4 [k.sub.B]T. Based on these estimates, the energy of conformational stress for a single tubulin-GDP dimer in the lattice is 2.1-2.5 [k.sub.B]T. Second, we studied how tubulin-GTP cap size fluctuates with different hydrolysis rules and show that a mechanism of directly coupling subunit addition to hydrolysis fails to support MT growth, whereas a finite hydrolysis rate allows growth. By adding rules to mimic the mechanical constraints present at the MT tip, the model generates tubulin-GTP caps similar in size to experimental estimates. Finally, by combining assembly/ disassembly and cap dynamics, we generate MT dynamic instability with rates and transition frequencies similar to those measured experimentally. Our model serves as a platform to examine GTP-cap dynamics and allows predictions of how MT-associated proteins and other effectors alter the energetics of MT assembly.

Remote manipulation of physical objects by means of radiation forces is discussed. The emphasis is on two radiation force-based systems: 1) laser trapping, which relies on a strongly convergent beam to form a focal point in which particles can be trapped and manipulated, and 2) laser guidance, which uses a weakly convergent beam to achieve radial confinement of particles about a beam axis, coupled with pushing along the beam axis.