Ehssan Nazockdast

C. elegans chromosomes connect to centrosomes by anchoring into the spindle network.

The mitotic spindle ensures the faithful segregation of chromosomes. Here we combine the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging to reconstruct all microtubules in 3D and identify their plus- and minus-ends. We classify them as kinetochore (KMTs), spindle (SMTs) or astral microtubules (AMTs) according to their positions, and quantify distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.

Pair-particle dynamics and microstructure in sheared colloidal suspensions: Simulation and Smoluchowski theory

The Smoluchowski equation (SE) approach reduced to pair level provides an accepted method for analysis of the pair microstructure, i.e., the pair distribution function g(r), in sheared colloidal suspensions. Under dilute conditions, the resulting problem is well-defined, but for concentrated suspensions the coefficients of the pair SE are unclear. This work outlines a recently developed theoretical approach for analytical and numerical study of the pair SE for concentrated colloidal suspensions of spheres in shear flow, and then focuses upon evaluation of coefficients and related properties of the problem from Stokesian Dynamics simulation, over a wide range of particle volume fraction, ϕ, and Peclet number (ratio of shear to Brownian motion). The pair distribution function determined from the SE theory is in generally good agreement with Stokesian Dynamics, as are the computed viscosity and normal stresses of the material. The primary focus of the work is to consider the pair relative velocity predicted ...

Effect of repulsive interactions on structure and rheology of sheared colloidal dispersions

A previously developed Smoluchowski theory for concentrated hard-sphere suspensions in shear flow is extended to study structure and rheology of colloidal suspensions with soft repulsive interactions. Accelerated Stokesian Dynamics simulations are carried out to provide insight and to enable direct comparison with theoretical predictions. The effect of extended range repulsive interactions is studied by considering repulsive interactions with different steepness, using identical potentials in simulation and theory, for varying shear rates characterized in dimensionless form as 0.1 ≤ Pe ≤ 100; here, Pe = 6πηa3/kbT is the ratio of hydrodynamic to Brownian forces and η is the fluid viscosity, is the shear rate, a is the particle radius and kbT is the thermal energy. Examples of predicted microstructures and the equivalent simulated results for hard-sphere suspensions at ϕ = 0.40 are also presented for comparison. The predicted pair distribution function is in good agreement with simulations before the onset of a shear-induced ordering transition in simulations of the soft colloids. The calculations of shear viscosity based on the predicted microstructure were also in general agreement with simulation results. The role of hydrodynamic interactions on flow-induced structures is discussed in the context of the proposed theory.

A fast platform for simulating semi-flexible fiber suspensions applied to cell mechanics

Abstract We present a novel platform for the large-scale simulation of three-dimensional fibrous structures immersed in a Stokesian fluid and evolving under confinement or in free-space in three dimensions. One of the main motivations for this work is to study the dynamics of fiber assemblies within biological cells. For this, we also incorporate the key biophysical elements that determine the dynamics of these assemblies, which include the polymerization and depolymerization kinetics of fibers, their interactions with molecular motors and other objects, their flexibility, and hydrodynamic coupling. This work, to our knowledge, is the first technique to include many-body hydrodynamic interactions (HIs), and the resulting fluid flows, in cellular assemblies of flexible fibers. We use non-local slender body theory to compute the fluid–structure interactions of the fibers and a second-kind boundary integral formulation for other rigid bodies and the confining boundary. A kernel-independent implementation of the fast multipole method is utilized for efficient evaluation of HIs. The deformation of the fibers is described by nonlinear Euler–Bernoulli beam theory and their polymerization is modeled by the reparametrization of the dynamic equations in the appropriate non-Lagrangian frame. We use a pseudo-spectral representation of fiber positions and implicit time-stepping to resolve large fiber deformations, and to allow time-steps not excessively constrained by temporal stiffness or fiber–fiber interactions. The entire computational scheme is parallelized, which enables simulating assemblies of thousands of fibers. We use our method to investigate two important questions in the mechanics of cell division: (i) the effect of confinement on the hydrodynamic mobility of microtubule asters; and (ii) the dynamics of the positioning of mitotic spindle in complex cell geometries. Finally to demonstrate the general applicability of the method, we simulate the sedimentation of a cloud of semi-flexible fibers.

Cytoplasmic flows as signatures for the mechanics of mitotic positioning

Interactions of astral microtubules (MTs), the pronuclear complex, and the cell cortex with the cytoplasm during pronuclear migration in the first cell division of Caenorhabditis elegans have two key consequences: cytoplasm-filled astral MTs behave as a porous medium, and different mechanisms result in different cytoplasmic flows.

Forces positioning the mitotic spindle: Theories, and now experiments

The position of the spindle determines the position of the cleavage plane, and is thus crucial for cell division. Although spindle positioning has been extensively studied, the underlying forces ultimately responsible for moving the spindle remain poorly understood. A recent pioneering study by Garzon‐Coral et al. uses magnetic tweezers to perform the first direct measurements of the forces involved in positioning the mitotic spindle. Combining this with molecular perturbations and geometrical effects, they use their data to argue that the forces that keep the spindle in its proper position for cell division arise from astral microtubules growing and pushing against the cells cortex. Here, we review these ground‐breaking experiments, the various biomechanical models for spindle positioning that they seek to differentiate, and discuss new questions raised by these measurements.

Active microrheology of colloidal suspensions: Simulation and microstructural theory

Accelerated Stokesian Dynamics (ASD) simulation and a microstructural theory are applied to study structure and the viscosity of hard-sphere Brownian suspensions in active microrheology (MR).We consider moderate to dense suspensions, from near to far from equilibrium conditions.The theory explicitly considers many-body hydrodynamic interactions (HIs) in active MR, and is compared with ASD.Two conditions of moving the probe with constant force (CF) and constant velocity (CV) are considered.The structure is quantified using the probability distribution of colloidal particles around the probe, g(r), which is computed as a solution to the pair Smoluchowski equation (SE) for 0.2 >1 for CF while it continuously grows in CV.This contrast in behavior is related to the dispersion in the motion of the probe under CF conditions, while CV motion has no dispersion.This effect is incorporated in the theory as a force-induced hydrodynamic diffusion flux in the pair SE. We also demonstrate that, despite this difference of structure in CF and CV, g(r) near the probe is set by Pe, for both CF and CV resulting in similar values for their viscosity.Using the theory, the structural anisotropy and Brownian viscosity near equilibrium are shown to be quantitatively similar in both CF and CV motions, which is in contrast with the dilute theory which predict distortions and Brownian viscosities twice as large in CV, relative to CF.This difference arises due to the many-body interactions associated with the equilibrium structure in the moderate to dense regime.

Kinetochore Microtubules indirectly link Chromosomes and Centrosomes in C. elegans Mitosis

The mitotic spindle is a dynamic microtubule-based apparatus that ensures the faithful segregation of chromosomes by connecting chromosomes to spindle poles. How this pivotal connection is established and maintained during mitosis is currently debated. Here we combined large-scale serial electron tomography with live-cell imaging to uncover the spatial and dynamic organization of microtubules in the mitotic spindles in C. elegans. With this we quantified the position of microtubule minus and plus-ends as well as distinguished the different classes of microtubules, such as kinetochore, astral and spindle microtubules with their distinct properties. Although microtubules are nucleated from the centrosomes, we find only a few, if any, kinetochore microtubules directly connected to the spindle poles, suggesting an indirect pole to chromosome connection. We propose a model of kinetochore microtubule assembly and disassembly, in which microtubules undergo minus-end depolymerisation, resulting in a detachment from the centrosome. Our reconstructions and analyses of complete spindles expand our understanding of spindle architecture beyond the light microscopic limit.The mitotic spindle ensures the faithful segregation of chromosomes. To discover the nature of the crucial centrosome-to-chromosome connection during mitosis, we combined the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging. Using tomography, we reconstructed the positions of all microtubules in 3D, and identified their plus- and minus-ends. We classified them as kinetochore (KMTs), spindle (SMTs), or astral microtubules (AMTs) according to their positions, and quantified distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs are directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.