Inferring Latent States and Refining Force Estimates via Hierarchical Dirichlet Process Modeling in Single Particle Tracking Experiments

Optical microscopy provides rich spatio-temporal information characterizing in vivo molecular motion. However, effective forces and other parameters used to summarize molecular motion change over time in live cells due to latent state changes, e.g., changes induced by dynamic micro-environments, photobleaching, and other heterogeneity inherent in biological processes. This study focuses on techniques for analyzing Single Particle Tracking (SPT) data experiencing abrupt state changes. We demonstrate the approach on GFP tagged chromatids experiencing metaphase in yeast cells and probe the effective forces resulting from dynamic interactions that reflect the sum of a number of physical phenomena. State changes are induced by factors such as microtubule dynamics exerting force through the centromere, thermal polymer fluctuations, etc. Simulations are used to demonstrate the relevance of the approach in more general SPT data analyses. Refined force estimates are obtained by adopting and modifying a nonparametric Bayesian modeling technique, the Hierarchical Dirichlet Process Switching Linear Dynamical System (HDP-SLDS), for SPT applications. The HDP-SLDS method shows promise in systematically identifying dynamical regime changes induced by unobserved state changes when the number of underlying states is unknown in advance (a common problem in SPT applications). We expand on the relevance of the HDP-SLDS approach, review the relevant background of Hierarchical Dirichlet Processes, show how to map discrete time HDP-SLDS models to classic SPT models, and discuss limitations of the approach. In addition, we demonstrate new computational techniques for tuning hyperparameters and for checking the statistical consistency of model assumptions directly against individual experimental trajectories; the techniques circumvent the need for "ground-truth" and subjective information.Comment: 25 pages, 6 figures. Differs only typographically from PLoS One publication available freely as an open-access article at http://journals.plos.org/plosone/article?id=10.1371/journal.pone.013763

Anomalous transport in the crowded world of biological cells

A ubiquitous observation in cell biology is that diffusion of macromolecules and organelles is anomalous, and a description simply based on the conventional diffusion equation with diffusion constants measured in dilute solution fails. This is commonly attributed to macromolecular crowding in the interior of cells and in cellular membranes, summarising their densely packed and heterogeneous structures. The most familiar phenomenon is a power-law increase of the MSD, but there are other manifestations like strongly reduced and time-dependent diffusion coefficients, persistent correlations, non-gaussian distributions of the displacements, heterogeneous diffusion, and immobile particles. After a general introduction to the statistical description of slow, anomalous transport, we summarise some widely used theoretical models: gaussian models like FBM and Langevin equations for visco-elastic media, the CTRW model, and the Lorentz model describing obstructed transport in a heterogeneous environment. Emphasis is put on the spatio-temporal properties of the transport in terms of 2-point correlation functions, dynamic scaling behaviour, and how the models are distinguished by their propagators even for identical MSDs. Then, we review the theory underlying common experimental techniques in the presence of anomalous transport: single-particle tracking, FCS, and FRAP. We report on the large body of recent experimental evidence for anomalous transport in crowded biological media: in cyto- and nucleoplasm as well as in cellular membranes, complemented by in vitro experiments where model systems mimic physiological crowding conditions. Finally, computer simulations play an important role in testing the theoretical models and corroborating the experimental findings. The review is completed by a synthesis of the theoretical and experimental progress identifying open questions for future investigation.Comment: review article, to appear in Rep. Prog. Phy

On the Phase Behaviour of Soft Matter: Understanding Complex Interactions via Quantitative Imaging

The effect of microscopic colloid interactions on the resultant macroscopic phase behaviour is a frequently studied topic in soft matter resarch, and lies at the heart of this thesis. Key structural and dynamic properties of colloidal model systems across liquid-solid transitions are tracked using optical imaging techniques. The first studied system comprises of thermosensitive microgels. These are soft, crosslinked polymer networks of colloidal size, which have been used as model systems to investigate various phase transitions. They display a rich phase behaviour due to their soft potential and internal core-corona structure. Especially, their thermosensitivity allows us to use temperature as an external control to tune particle size, volume fraction and effective interaction potential in situ. However, a thorough understanding of the effective interactions between microgels is lacking, and constitutes a key research question in this thesis. We therefore quantitatively compare experimental and numerical pair correlation functions (g(r)s) across the phase diagram, obtained from confocal microscopy and simulations. We find that neutral, swollen microgel interactions are temperature-dependent, but also hinge on whether the core or corona of the microgel is explored. This approach is repeated for ionic microgels with varying crosslinker density, where the introduction of acrylic acid complicates the resultant swelling behaviour. For this reason, we start by decoupling the core and corona swelling response to various charge regimes via light scattering experiments, and found that dangling polymer strands can extend up to several 100 nm outside of the network. Dangling ends had a pronounced effect on the interactions and phase behaviour of ionic microgels, but their contribution is missing within the current theoretical framework. Finally, liquid-solid transitions in concentrated protein solutions are investigated. Two well studied globular proteins, lysozyme and γB-crystallin, were used as model systems with completely different interactions. No unambiguous experimental demonstration of the existence of an arrested glassy state had been published so far for either protein. A combination of two passive microrheology techniques now allowed us to confirm the formation of a glass phase at concentrations above a critical arrest concentration, and to obtain quantitative insight into the concentration dependence of the zero shear viscosity prior to arrest

Structure-Dynamics Relationships in Complex Fluids and Disordered Porous Solids Assessed using NMR: Structure-Dynamics Relationships in Complex Fluidsand Disordered Porous Solids Assessed using NMR

A NMR study of the structure-dynamics relationships in heterogeneous materials is presented. In the first part, transport in soft-matter systems is studied using the pulsed field gradient NMR technique (PFG NMR). The molecular crowding effect in biological matter has been addressed using polymer solutions as model systems. By performing ensemble-based diffusion studies, the earlier obtained data on anomalous diffusion have been complemented. The transition to normal diffusion on a larger time scale has been shown. Taking advantages of the NMR approach, transport properties of microemulsions consisting of micellar colloids dissolved in liquid crystals have been investigated. The self-diffusivities measured under equilibrium conditions have shown weak correlations with microscopic ordering and macroscopic phase transitions occurring in the systems under study. The formation of micelles is shown to be decisive for macroscopic separation at the isotropic-nematic transition. The second part of the thesis covers heterogeneous effects in diffusion for fluids in porous solids, as probed using a combination of NMR diffusometry and structure characterization methods. Ionic liquids have been investigated, revealing a complex behavior under confinement. The attempts to correlate the observed characteristics of the ionic liquids with their internal chemical structure were undertaken. Finally, the series of nanoporous glasses with tunable pore structure characteristics were studied. Strong correlations between their structure and the preparation conditions as well as between the resulting transport properties have been shown

SINGLE-MOLECULE STUDIES OF RAD4-RAD23 REVEAL A DYNAMIC DNA DAMAGE RECOGNITION PROCESS

Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. As the first step towards productive repair, the human NER damage sensor XPC-RAD23B needs to efficiently locate sites of damage among billons of base pairs of undamaged DNA. In this dissertation, we investigated the dynamic protein-DNA interactions during the damage recognition step using a combination of fluorescence-based single-molecule DNA tightrope assays, atomic force microscopy, as well as cell survival and in vivo repair kinetics assays. We observed that quantum dot-labeled Rad4-Rad23, the yeast homolog of human XPC-RAD23B, formed nonmotile complexes on DNA or conducted a one-dimensional search via either random diffusion or constrained motion along DNA. Using atomic force microscopy, we studied binding of Rad4 lacking the β-hairpin domain 3 (BHD3) to damage-containing DNA and found that this structural motif is non-essential for damage-specific binding or DNA bending. Furthermore, we demonstrated that deletion of seven residues in the tip of β-hairpin in BHD3 increased Rad4-Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance. Finally, we explore existing physical models and examples of subdiffusive motion, and discuss a model in which constrained motion by Rad4-Rad23 on DNA may be driven by conformational changes of the protein

Realizing and probing driven quantum systems with ultracold gases

Ultracold quantum gases offer a versatile platform to study a wide range of open questions in condensed matter physics and beyond. In particular, their controllability, isolation from noisy thermal environments, and evolution on experimentally-accessible timescales make them a natural choice to probe the effects of driving on time evolution and energy. This thesis details the construction of two cold-atom apparatuses, a lithium machine and a strontium machine, for quantum emulation experiments studying driven systems. Initial numerical simulations along two experimental lines are briefly discussed, and results from the first two experiments on the strontium machine are then presented. In the first, a strontium Bose-Einstein condensate in an optical trap is strongly driven to emulate ultrafast photoionization processes; in a series of proof-of-principle experiments measuring the momenta and energy of particles ejected from the trap, we demonstrate the viability of this technique to study open questions in strong-field physics. The second experiment realizes a tunable quasicrystal, the energy structure for which is described by the multifractal Hofstadter butterfly. Quasiperiodic structures host not only phonons, but also a higher-dimension analogue called phasons. In the experiment, we demonstrate phasonic spectroscopy for the first time by directly driving one of these modes; we characterize the coupling to the resulting excitations, and directly map a slice of the Hofstadter energy spectrum