Raja Paul

The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly

Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.

Timing of centrosome separation is important for accurate chromosome segregation

Centrosome separation can be completed either before or after nuclear envelope breakdown (NEB). A combination of experimental and computational approaches shows that incomplete centrosome separation at NEB decreases the accuracy of chromosome segregation and thus represents a severe threat to genome stability.

Dynein Antagonizes Eg5 by Crosslinking and Sliding Antiparallel Microtubules

Mitotic spindle assembly requires the combined activity of various molecular motor proteins, including Eg5 and dynein. Together, these motors generate antagonistic forces during mammalian bipolar spindle assembly; what remains unknown, however, is how these motors are functionally coordinated such that antagonism is possible. Given that Eg5 generates an outward force by crosslinking and sliding apart antiparallel microtubules (MTs), we explored the possibility that dynein generates an inward force by likewise sliding antiparallel MTs. We reasoned that antiparallel overlap, and therefore the magnitude of a dynein-mediated force, would be inversely proportional to the initial distance between centrosomes. To capitalize on this relationship, we utilized a nocodazole washout assay to mimic spindle assembly. We found that Eg5 inhibition led to either monopolar or bipolar spindle formation, depending on whether centrosomes were initially separated by less than or greater than 5.5 microm, respectively. Mathematical modeling predicted this same spindle bistability in the absence of functional Eg5 and required dynein acting on antiparallel MTs to do so. Our results suggest that dynein functionally coordinates with Eg5 by crosslinking and sliding antiparallel MTs, a novel role for dynein within the framework of spindle assembly.

Computer simulations predict that chromosome movements and rotations accelerate mitotic spindle assembly without compromising accuracy

The mitotic spindle self-assembles in prometaphase by a combination of centrosomal pathway, in which dynamically unstable microtubules search in space until chromosomes are captured, and a chromosomal pathway, in which microtubules grow from chromosomes and focus to the spindle poles. Quantitative mechanistic understanding of how spindle assembly can be both fast and accurate is lacking. Specifically, it is unclear how, if at all, chromosome movements and combining the centrosomal and chromosomal pathways affect the assembly speed and accuracy. We used computer simulations and high-resolution microscopy to test plausible pathways of spindle assembly in realistic geometry. Our results suggest that an optimal combination of centrosomal and chromosomal pathways, spatially biased microtubule growth, and chromosome movements and rotations is needed to complete prometaphase in 10–20 min while keeping erroneous merotelic attachments down to a few percent. The simulations also provide kinetic constraints for alternative error correction mechanisms, shed light on the dual role of chromosome arm volume, and compare well with experimental data for bipolar and multipolar HT-29 colorectal cancer cells.

Concerted effort of centrosomal and Golgi-derived microtubules is required for proper Golgi complex assembly but not for maintenance.

Using computational modeling and laser microsurgery, we establish that neither the centrosomal microtubule array nor the Golgi-derived array is solely sufficient for correct Golgi assembly. Only the concerted effort of both MT arrays results in the integral, polarized Golgi complex necessary for polarized trafficking and cell motility.

Domain growth in random magnets

We study the kinetics of domain growth in ferromagnets with random exchange interactions. We present detailed Monte Carlo results for the nonconserved random-bond Ising model, which are consistent with power law growth with a variable exponent. These results are interpreted in the context of disorder barriers with a logarithmic dependence on the domain size. Further, we clarify the implications of logarithmic barriers for both nonconserved and conserved domain growth.

Mutual Diffusion of the Gas Pairs H2–Ne, H2–Ar, and H2–Xe at Different Temperatures

The mutual diffusion coefficients of H2 with Ne, Ar, and Xe have been determined by the two‐bulb technique of Ney and Armistead in the temperature range —30° to 68°C. Diffusion was allowed to take place through a precision capillary tube connecting the two diffusion bulbs and samples of the gas were analyzed at different times with the help of a previously calibrated thermal‐conductivity analyzer.A least‐square method was then followed to calculate the force constants on the Lennard‐Jones (12:6) potential model from the experimental values of the diffusion coefficients. Also, using experimental values of the mutual‐diffusion coefficient, thermal conductivity, and viscosity of pure components, the thermal conductivity of various mixtures were calculated and a good agreement with the experimental data was obtained.

Condensation phenomena in nanopores: A Monte Carlo study

The nonequilibrium dynamics of condensation phenomena in nanopores is studied via Monte Carlo simulations of a lattice-gas model. Hysteretic behavior of the particle density as a function of the density of a reservoir is obtained for various pore geometries in two and three dimensions. The shape of the hysteresis loops depend on the characteristics of the pore geometry. The evaporation of particles from a pore can be fitted to a stretched exponential decay of the particle density. Phase-separation dynamics inside the pore is effectively described by a random walk of the non-wetting phases. Domain evolution is significantly slowed down in the presence of a random wall-particle potential and gives rise to a temperature-dependent growth exponent. A geometric roughness of the pore wall only delays the onset of a pure domain growth.

Domain growth in Ising systems with quenched disorder

We present results from extensive Monte Carlo (MC) simulations of domain growth in ferromagnets and binary mixtures with quenched disorder. These are modeled by the random-bond Ising model and the dilute Ising model with either nonconserved (Glauber) spin-flip kinetics or conserved (Kawasaki) spin-exchange kinetics. In all cases, our MC results are consistent with power-law growth with an exponent theta(T, epsilon) which depends on the quench temperature T and the disorder amplitude epsilon. Such exponents arise naturally when the coarsening domains are trapped by energy barriers that grow logarithmically with the domain size. Our MC results show excellent agreement with the predicted dependence of theta(T, epsilon).

Universal aging properties at a disordered critical point.

We investigate, analytically near the dimension d(uc) =4 and numerically in d=3 , the nonequilibrium relaxational dynamics of the randomly diluted Ising model at criticality. Using the exact renormalization-group method to one loop, we compute the two times t, t(w) correlation function and fluctuation dissipation ratio (FDR) for any Fourier mode of the order parameter, of finite wave vector q . In the large time separation limit, the FDR is found to reach a nontrivial value X(infinity) independently of (small) q and coincide with the FDR associated to the total magnetization obtained previously. Explicit calculations in real space show that the FDR associated to the local magnetization converges, in the asymptotic limit, to this same value X(infinity). Through a Monte Carlo simulation, we compute the autocorrelation function in three dimensions, for different values of the dilution fraction p at T(c) (p) . Taking properly into account the corrections to scaling, we find, according to the renormalization-group predictions, that the autocorrelation exponent lambda(c) is independent of p . The analysis is complemented by a study of the nonequilibrium critical dynamics following a quench from a completely ordered state.