Dynamics of emerging actin networks

Life is an ensemble of countless emerging properties arising through self-assembly and self-organization phenomena, manifesting at the cellular, the tissue and the organismal level. The mechanical integrity of a cell is orchestrated by the cytoskeleton, a dynamic system comprised of three biopolymers, actin, microtubules and intermediate filaments, acting in symphony, facilitated by a plethora of accessory proteins. Understanding the cytoskeletal functionality and its relation to other cellular components and properties is a prominent question in biophysics. Actin, a dynamic and polymorphic component, forms a variety of structures such as filaments, bundles, and their networks. The unique viscoelastic properties shown by actin-based structures have been extensively probed via rheological means. On the contrary, the underlying microstructural dynamics remain mostly uncovered. Actin bundles are crucial for eukaryotic cells; they are involved in the intracellular transport, contractive forces, mechanical stability, cell motility and environment exploration. This thesis takes a step forward to fathom the rich dynamics and emergent properties exhibited by actin bundles within flow-free confinements, a prerequisite for the study. To study a reversible reaction sequence in a step-by-step manner, one needs an open system. As a result, there have been relatively few studies in this direction, as most of the experimental systems are closed, for instance, sealed coverslips or liposomes. We created a straightforward microfluidic system, consisting of quasi two-dimensional, cell-sized compartments, enclosing sub-picolitre volumes. These `microchambers' are connected to the controlling channel (the reservoir) via narrow connecting channels, allowing exclusive diffusive transport into and out of the microchambers. The system represents an ideal environment to form an entangled network of actin filaments in a steady-state and is manipulable in a step-by-step fashion. We induce bundling of actin filaments in three ways: counterion condensation aided by magnesium ions, depletion interactions mimicked by polyethylene glycol, acting as a crowding agent, and specific interactions with actin exhibited by filamin, an actin binding protein. Above the critical concentration of bundling agents, actin filaments transform into an emerging network of actin bundles, a process associated with percolation, leading to a single connected entity. Sharing of filaments is an important parameter for the observed behaviour, as reducing the actin filament length exclusively forms bundles without percolation. We encounter a hierarchical process of bundling: filaments coalesce into small bundles that further fuse to form bigger bundles. Disassembly involves a similar hierarchy, additionally involving peeling-off of single filaments. We explore the reactions using time-lapse image analyses and apply kinetic models. Counterion condensation forms a network comprising of straight, rigid bundles facilitated by a zipping process (v ~ 12 µm/s), generating tension within the network. Disassembly leads to the release of the stored energy, utilized in the buckling of bundles, enabling us to estimate ~ 100 - 200 kT of stored energy. Crowding agents force the actin filaments to form an intriguing spindle-like structure, consisting of poles with sets of aligned filaments shared and stretched between them, which further transforms into a network of bundles. The disassembly constitutes the reversal of the process. Filamin forms ring-like networks, containing intrinsically curved bundles. Owing to the highly specific interactions, the network does not disassemble, even after 12 hours. In essence, using a bottom-up approach, we explore the emerging properties of actin bundles, with an emphasis on their dynamics

Correlating LIBS Coal Data for Coal Property Prediction

This report presents results for correlations between coal data derived from laboratory analysis and Laser Induced Breakdown Spectroscopy analysis. LIBS data were used to predict higher order properties of coal using artificial neural network models. Higher order coal properties such as heating value and ash fusion temperature are predicted using LIBS analysis and compared against standard laboratory measurements. Selected formulas for the prediction of coal properties are also presented and compared against the neural network and laboratory results

Second messenger-mediated tactile response by a bacterial rotary motor

When bacteria encounter surfaces, they respond with surface colonization and virulence induction. The mechanisms of bacterial mechanosensation and downstream signaling remain poorly understood. Here, we describe a tactile sensing cascade in Caulobacter crescentus in which the flagellar motor acts as sensor. Surface-induced motor interference stimulated the production of the second messenger cyclic diguanylate by the motor-associated diguanylate cyclase DgcB. This led to the allosteric activation of the glycosyltransferase HfsJ to promote rapid synthesis of a polysaccharide adhesin and surface anchoring. Although the membrane-embedded motor unit was essential for surface sensing, mutants that lack external flagellar structures were hypersensitive to mechanical stimuli. Thus, the bacterial flagellar motor acts as a tetherless sensor reminiscent of mechanosensitive channels

(R)evolution-on-a-chip

Billions of years of Darwinian evolution has led to the emergence of highly sophisticated and diverse life forms on Earth. Inspired by natural evolution, similar principles have been adopted in laboratory evolution for the fast optimization of genes and proteins for specific applications. In this review, we highlight state-of-the-art laboratory evolution strategies for protein engineering, with a special emphasis on in vitro strategies. We further describe how recent progress in microfluidic technology has allowed the generation and manipulation of artificial compartments for high-throughput laboratory evolution experiments. Expectations for the future are high: we foresee a revolution on-a-chip

A self-filling microfluidic device for noninvasive and time-resolved single red blood cell experiments

Existing approaches to red blood cell (RBC) experiments on the single-cell level usually rely on chemical or physical manipulations that often cause difficulties with preserving the RBC's integrity in a controlled microenvironment. Here, we introduce a straightforward, self-filling microfluidic device that autonomously separates and isolates single RBCs directly from unprocessed human blood samples and confines them in diffusion-controlled microchambers by solely exploiting their unique intrinsic properties. We were able to study the photo-induced oxygenation cycle of single functional RBCs by Raman microscopy without the limitations typically observed in optical tweezers based methods. Using bright-field microscopy, our noninvasive approach further enabled the time-resolved analysis of RBC flickering during the reversible shape evolution from the discocyte to the echinocyte morphology. Due to its specialized geometry, our device is particularly suited for studying the temporal behavior of single RBCs under precise control of their environment that will provide important insights into the RBC's biomedical and biophysical properties