Live-cell super-resolution microscopy reveals a primary role for diffusion in polyglutamine-driven aggresome assembly.

The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed in vitro Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that the increasing surface area of the expanding aggresome increases the rate of accretion caused by diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies.

Solution to the two-body Smoluchowski equation with shear flow for charge-stabilized colloids at low to moderate Péclet numbers.

We developed an analytical theoretical method to determine the microscopical structure of weakly to moderately sheared colloidal suspensions in dilute conditions. The microstructure is described by the static structure factor, obtained by solving the stationary two-body Smoluchowski advection-diffusion equation. The singularly perturbed partial differential equation problem is solved by performing an angular averaging over the extensional and compressing sectors and by the rigorous application of boundary-layer theory (intermediate asymptotics). This allows us to expand the solution to a higher order in Péclet with respect to previous methods. The scheme is independent of the type of interaction potential. We apply it to the example of charge-stabilized colloidal particles interacting via the repulsive Yukawa potential and study the distortion of the structure factor. It is predicted that the distortion is larger at small wave vectors k and its dependence on Pe is a simple power law. At increasing Pe, the main peak of the structure factor displays a broadening and shift toward lower k in the extensional sectors, which indicates shear-induced spreading out of particle correlations and neighbor particles locally being dragged away from the reference one. In the compressing sectors, instead, a narrowing and shift toward high k is predicted, reflecting shear-induced ordering near contact and concomitant depletion in the medium range. An overall narrowing of the peak is also predicted for the structure factor averaged over the whole solid angle. Calculations are also performed for hard spheres, showing good overall agreement with experimental data. It is also shown that the shear-induced structure factor distortion is orders of magnitude larger for the Yukawa repulsion than for the hard spheres.

Microscopic theory for the pair correlation function of liquidlike colloidal suspensions under shear flow.

We present a theoretical framework to investigate the microscopic structure of concentrated hard-sphere colloidal suspensions under strong shear flows by fully taking into account the boundary-layer structure of convective diffusion. We solve the pair Smoluchowski equation with shear separately in the compressing and extensional sectors of the solid angle, by means of matched asymptotics. A proper, albeit approximate, treatment of the hydrodynamic interactions in the different sectors allows us to construct a potential of mean force containing the effect of the flow field on pair correlations. We insert the obtained pair potential in the Percus-Yevick relation and use the latter as a closure to solve the Ornstein-Zernike integral equation. For a wide range of either the packing fraction η and the Péclet (Pe) number, we compute the pair correlation function and extract scaling laws for its value at contact. For all the considered values of Pe, we observe a very good agreement between theoretical findings and numerical results from the literature, up to rather large values of η. The theory predicts a consistent enhancement of the structure factor S(k) at k→0, upon increasing the Pe number. We argue this behavior may signal the onset of a phase transition from the isotropic phase to a nonuniform one, induced by the external shear flow.

Direct observation of heterogeneous formation of amyloid spherulites in real-time by super-resolution microscopy.

Protein misfolding in the form of fibrils or spherulites is involved in a spectrum of pathological abnormalities. Our current understanding of protein aggregation mechanisms has primarily relied on the use of spectrometric methods to determine the average growth rates and diffraction-limited microscopes with low temporal resolution to observe the large-scale morphologies of intermediates. We developed a REal-time kinetics via binding and Photobleaching LOcalization Microscopy (REPLOM) super-resolution method to directly observe and quantify the existence and abundance of diverse aggregate morphologies of human insulin, below the diffraction limit and extract their heterogeneous growth kinetics. Our results revealed that even the growth of microscopically identical aggregates, e.g., amyloid spherulites, may follow distinct pathways. Specifically, spherulites do not exclusively grow isotropically but, surprisingly, may also grow anisotropically, following similar pathways as reported for minerals and polymers. Combining our technique with machine learning approaches, we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape at the level of single aggregate morphology. Our unifying framework for the detection and analysis of spherulite growth can be extended to other self-assembled systems characterized by a high degree of heterogeneity, disentangling the broad spectrum of diverse morphologies at the single-molecule level.

Radial distribution function of Lennard-Jones fluids in shear flows from intermediate asymptotics.

Determining the microstructure of colloidal suspensions under shear flows has been a challenge for theoretical and computational methods due to the singularly perturbed boundary-layer nature of the problem. Previous approaches have been limited to the case of hard-sphere systems and suffer from various limitations in their applicability. We present an alternative analytic scheme based on intermediate asymptotics which solves the Smoluchowski diffusion-convection equation including both intermolecular and hydrodynamic interactions. The method is able to recover previous results for the hard-sphere fluid and to predict the radial distribution function (rdf) of attractive fluids such as the Lennard-Jones (LJ) fluid. In particular, a new depletion effect is predicted in the rdf of the LJ fluid under shear. This method can be used for the theoretical modeling and understanding of real fluids subjected to flow, with applications ranging from chemical systems to colloids, rheology, plasmas, and atmospherical science.

Extended Charge-On-Particle Optimized Potentials for Liquid Simulation Acetone Model: The Case of Acetone-Water Mixtures.

It is well-known that classical molecular dynamics simulations of acetone-water mixtures lead to a strong phase separation when using most of the standard all-atom force fields, despite the well-known experimental fact that acetone is miscible with water in any proportion at room temperature. We describe here the use of a charge-on-particle model for accounting for the induced polarization effect in acetone-water mixtures which can solve the demixing problem at all acetone molar fractions. The polarizability effect is introduced by means of a virtual site (VS) on the carbonyl group of the acetone molecule, which increases its dipole moment and leads to a better affinity with water molecules. The VS parameter is set by fitting the density of the mixture at different acetone molar fractions. The main novelty of the VS approach lies on the transferability and universality of the model because the polarizability can be controlled without modifying the force field adopted, like previous efforts did. The results are satisfactory also in terms of the transport properties, that is, diffusivity and viscosity coefficients of the mixture.