Zaburdaev, V. ; Biais, N. ; Schmiedeberg, M. ; Eriksson, J. ; Jonsson, A. - B. ; Sheetz, M.  P. ; Weitz, D.  A. Uncovering the Mechanism of Trapping and Cell Orientation during Neisseria gonorrhoeae Twitching Motility. 2014, 107, 1523 - 1531. Publisher's VersionAbstract

AbstractNeisseria gonorrheae bacteria are the causative agent of the second most common sexually transmitted infection in the world. The bacteria move on a surface by means of twitching motility. Their movement is mediated by multiple long and flexible filaments, called type IV pili, that extend from the cell body, attach to the surface, and retract, thus generating a pulling force. Moving cells also use pili to aggregate and form microcolonies. However, the mechanism by which the pili surrounding the cell body work together to propel bacteria remains unclear. Understanding this process will help describe the motility of N. gonorrheae bacteria, and thus the dissemination of the disease which they cause. In this article we track individual twitching cells and observe that their trajectories consist of alternating moving and pausing intervals, while the cell body is preferably oriented with its wide side toward the direction of motion. Based on these data, we propose a model for the collective pili operation of N. gonorrheae bacteria that explains the experimentally observed behavior. Individual pili function independently but can lead to coordinated motion or pausing via the force balance. The geometry of the cell defines its orientation during motion. We show that by changing pili substrate interactions, the motility pattern can be altered in a predictable way. Although the model proposed is tangibly simple, it still has sufficient robustness to incorporate further advanced pili features and various cell geometries to describe other bacteria that employ pili to move on surfaces.

Larsen, R. J. ; Kim, J. - W. ; Zukoski, C. F. ; Weitz, D. A. Fluctuations in flow produced by competition between apparent wall slip and dilatancy. Rheologica Acta 2014, 53, 333-347. Publisher's VersionAbstract

Dense suspensions can exhibit a dramatic stress-induced transition from liquid-like to solid-like behavior. In many materials, the solid-like flow state is characterized by large flow fluctuations and instabilities. Although various experiments have been performed to characterize flow fluctuations, the mechanisms that govern the flow instabilities remain poorly understood. To elucidate these mechanisms, we characterize a system that rapidly fluctuates between two flow states. One of the flow states is dominated by apparent wall slip, and the other is dominated by dilatancy. The dilatant regime occurs at elevated stresses and is associated with reduced wall slip, whereas the wall slip-dominated regime occurs at lower stresses. At stresses that are intermediate between these two regimes, the material fluctuates between the two regimes in a semi-regular fashion. Our analysis of the fluctuations at millisecond timescales shows that fluctuations occur because neither regime is capable of supporting a constant stress in a stable manner. We rationalize our results in terms of the differences in the shear-induced particle pressure between regions that are particle-rich and regions of slip that are particle-depleted.

Jarosz, D.  F. ; Brown, J.  C. S. ; Walker, G.  A. ; Datta, M.  S. ; Ung, W.  L. ; Lancaster, A.  K. ; Rotem, A. ; Chang, A. ; Newby, G.  A. ; Weitz, D.  A. ; et al. Cross-Kingdom Chemical Communication Drives a Heritable, Mutually Beneficial Prion-Based Transformation of Metabolism. Cell 2014, 158, 1083 - 1093. Publisher's VersionAbstract

Summary In experimental science, organisms are usually studied in isolation, but in the wild, they compete and cooperate in complex communities. We report a system for cross-kingdom communication by which bacteria heritably transform yeast metabolism. An ancient biological circuit blocks yeast from using other carbon sources in the presence of glucose. [GAR+], a protein-based epigenetic element, allows yeast to circumvent this “glucose repression” and use multiple carbon sources in the presence of glucose. Some bacteria secrete a chemical factor that induces [GAR+]. [GAR+] is advantageous to bacteria because yeast cells make less ethanol and is advantageous to yeast because their growth and long-term viability is improved in complex carbon sources. This cross-kingdom communication is broadly conserved, providing a compelling argument for its adaptive value. By heritably transforming growth and survival strategies in response to the selective pressures of life in a biological community, [GAR+] presents a unique example of Lamarckian inheritance.

Wang, B. L. ; Ghaderi, A. ; Zhou, H. ; Agresti, J. ; Weitz, D. A. ; Fink, G. R. ; Stephanopoulos, G. Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption. Nat Biotech 2014, 32, 473 - 478. Publisher's VersionAbstract

Phenotyping single cells based on the products they secrete or consume is a key bottleneck in many biotechnology applications, such as combinatorial metabolic engineering for the overproduction of secreted metabolites. Here we present a flexible high-throughput approach that uses microfluidics to compartmentalize individual cells for growth and analysis in monodisperse nanoliter aqueous droplets surrounded by an immiscible fluorinated oil phase. We use this system to identify xylose-overconsuming Saccharomyces cerevisiae cells from a population containing one such cell per 104 cells and to screen a genomic library to identify multiple copies of the xylose isomerase gene as a genomic change contributing to high xylose consumption, a trait important for lignocellulosic feedstock utilization. We also enriched L-lactate-producing Escherichia coli clones 5,800[times] from a population containing one L-lactate producer per 104D-lactate producers. Our approach has broad applications for single-cell analyses, such as in strain selection for the overproduction of fuels, chemicals and pharmaceuticals.

Pessi, J. ; Santos, H. A. ; Miroshnyk, I. ; JoukoYliruusi, ; Weitz, D. A. ; Mirza, S. Microfluidics-assisted engineering of polymeric microcapsules with high encapsulation efficiency for protein drug delivery. International Journal of Pharmaceutics 2014, 472, 82 - 87. Publisher's VersionAbstract

Abstract In this study, microfluidic technology was employed to develop protein formulations. The microcapsules were produced with a biphasic flow to create water–oil–water (W/O/W) double emulsion droplets with ultrathin shells. Optimized microcapsule formulations containing 1% (w/w) bovine serum albumin (BSA) in the inner phase were prepared with poly(vinyl alcohol), polycaprolactone and polyethylene glycol. All the particles were found to be intact and with a particle size of 23–47 μm. Furthermore, the particles were monodisperse, non-porous and stable up to 4 weeks. The encapsulation efficiency of \{BSA\} in the microcapsules was 84%. The microcapsules released 30% of their content within 168 h. This study demonstrates that microfluidics is a powerful technique for engineering formulations for therapeutic proteins.

Guo, M. ; Ehrlicher, A.  J. ; Jensen, M.  H. ; Renz, M. ; Moore, J.  R. ; Goldman, R.  D. ; Lippincott-Schwartz, J. ; Mackintosh, F.  C. ; Weitz, D.  A. Probing the Stochastic, Motor-Driven Properties of the Cytoplasm Using Force Spectrum Microscopy. Cell 2014, 158, 822 - 832. Publisher's VersionAbstract

Molecular motors in cells typically produce highly directed motion; however, the aggregate, incoherent effect of all active processes also creates randomly fluctuating forces, which drive diffusive-like, nonthermal motion. Here, we introduce force-spectrum-microscopy (FSM) to directly quantify random forces within the cytoplasm of cells and thereby probe stochastic motor activity. This technique combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm to quantify the spectrum of force fluctuations. Using FSM, we show that force fluctuations substantially enhance intracellular movement of small and large components. The fluctuations are three times larger in malignant cells than in their benign counterparts. We further demonstrate that vimentin acts globally to anchor organelles against randomly fluctuating forces in the cytoplasm, with no effect on their magnitude. Thus, FSM has broad applications for understanding the cytoplasm and its intracellular processes in relation to cell physiology in healthy and diseased states.

Datta, S. S. ; Abbaspourrad, A. ; Weitz, D. A. Expansion and rupture of charged microcapsules. Mater. Horiz. 2014, 1 92-95. Publisher's VersionAbstract

We study the deformations of pH-responsive spherical microcapsules - micrometer-scale liquid drops surrounded by thin{,} solid shells - under the influence of electrostatic forces. When exposed to a large concentration of NaOH{,} the microcapsules become highly charged{,} and expand isotropically. We find that the extent of this expansion can be understood by coupling electrostatics with shell theory; moreover{,} the expansion dynamics is well described by Darcy{'}s law for fluid flow through the microcapsule shell. Unexpectedly{,} however{,} below a threshold NaOH concentration{,} the microcapsules begin to disintegrate{,} and eventually rupture; they then expand non-uniformly{,} ultimately forming large{,} jellyfish-like structures. Our results highlight the fascinating range of behaviors exhibited by pH-responsive microcapsules{,} driven by the interplay between electrostatic and mechanical forces.

Carroll, N. J. ; Jensen, K. H. ; Parsa, S. ; Holbrook, M. N. ; Weitz, D. A. Measurement of Flow Velocity and Inference of Liquid Viscosity in a Microfluidic Channel by Fluorescence Photobleaching. Langmuir 2014, 30, 4868-4874. Publisher's VersionAbstract

We present a simple, noninvasive method for simultaneous measurement of flow velocity and inference of liquid viscosity in a microfluidic channel. We track the dynamics of a sharp front of photobleached fluorescent dye using a confocal microscope and measure the intensity at a single point downstream of the initial front position. We fit an exact solution of the advection diffusion equation to the fluorescence intensity recovery curve to determine the average flow velocity and the diffusion coefficient of the tracer dye. The dye diffusivity is correlated to solute concentration to infer rheological properties of the liquid. This technique provides a simple method for simultaneous elucidation of flow velocity and liquid viscosity in microchannels.

Ostafe, R. ; Prodanovic, R. ; Ung, W. L. ; Weitz, D. A. ; Fischer, R. A high-throughput cellulase screening system based on droplet microfluidics. Biomicrofluidics 2014, 8 041102. Publisher's VersionAbstract

A new ultra-high-throughput screening assay for the detection of cellulase activity was developed based on microfluidic sorting. Cellulase activity is detected using a series of coupled enzymes leading to the formation of a fluorescent product that can be detected on a chip. Using this method, we have achieved up to 300-fold enrichments of the active population of cells and greater than 90% purity after just one sorting round. In addition, we proved that we can sort the cellulase-expressing cells from mixtures containing less than 1% active cells.

Datta, S. S. ; Dupin, J. - B. ; Weitz, D. A. Fluid breakup during simultaneous two-phase flow through a three-dimensional porous medium. Physics of Fluids 2014, 26, 062004. Publisher's VersionAbstract

We use confocal microscopy to directly visualize the simultaneous flow of both a wetting and a non-wetting fluid through a model three-dimensional (3D) porous medium. We find that, for small flow rates, both fluids flow through unchanging, distinct, connected 3D pathways; in stark contrast, at sufficiently large flow rates, the non-wetting fluid is broken up into discrete ganglia. By performing experiments over a range of flow rates, using fluids of different viscosities, and with porous media having different geometries, we show that this transition can be characterized by a state diagram that depends on the capillary numbers of both fluids, suggesting that it is controlled by the competition between the viscous forces exerted on the flowing oil and the capillary forces at the pore scale. Our results thus help elucidate the diverse range of behaviors that arise in two-phase flow through a 3D porous medium.

Chen, L. ; Li, Y. ; Fan, J. ; Bisoyi, H. K. ; Weitz, D. A. ; Li, Q. Photoresponsive Monodisperse Cholesteric Liquid Crystalline Microshells for Tunable Omnidirectional Lasing Enabled by a Visible Light-Driven Chiral Molecular Switch. Advanced Optical Materials 2014, 2 845-848. Publisher's VersionAbstract

A self‐organized, phototunable 3D photonic superstructure is fabricated from a photoresponsive 1D liquid crystal using microfluidics. The resulting microshells are water–oil–water double emulsions, in which the oil phase consists of the photoresponsive liquid crystals. The cholesteric microshells exhibit band‐edge lasing in all directions, and the wavelength of the resultant band‐edge laser is tuned via the pumping laser, causing photoisomerization of the chiral molecular switch.

Arriaga, L. R. ; Datta, S. S. ; Kim, S. - H. ; Amstad, E. ; Kodger, T. E. ; Monroy, F. ; Weitz, D. A. Ultrathin Shell Double Emulsion Templated Giant Unilamellar Lipid Vesicles with Controlled Microdomain Formation. Small 2014, 10, 950–956. Publisher's VersionAbstract

A microfluidic approach is reported for the high-throughput, continuous production of giant unilamellar vesicles (GUVs) using water-in-oil-in-water double emulsion drops as templates. Importantly, these emulsion drops have ultrathin shells; this minimizes the amount of residual solvent that remains trapped within the GUV membrane, overcoming a major limitation of typical microfluidic approaches for GUV fabrication. This approach enables the formation of microdomains, characterized by different lipid compositions and structures within the GUV membranes. This work therefore demonstrates a straightforward and versatile approach to GUV fabrication with precise control over the GUV size, lipid composition and the formation of microdomains within the GUV membrane.

Cohen, S. I. A. ; Rajah, L. ; Yoon, C. H. ; Buell, A. K. ; White, D. A. ; Sperling, R. A. ; Vendruscolo, M. ; Terentjev, E. M. ; Dobson, C. M. ; Weitz, D. A. ; et al. Spatial Propagation of Protein Polymerization. Physical Review Letters 2014, 112, 098101. Publisher's VersionAbstract

We consider the spatial dependence of filamentous protein self-assembly. Through studying the cases where the spreading of aggregated material is dominated either by diffusion or by growth, we derive analytical results for the spatial evolution of filamentous protein aggregation, which we validate against Monte Carlo simulations. Moreover, we compare the predictions of our theory with experimental measurements of two systems for which we identify the propagation as either growth or diffusion controlled. Our results connect the macroscopic observables that characterize the spatial propagation of protein self-assembly with the underlying microscopic processes and provide physical limits on spatial propagation and prionlike behavior associated with protein aggregation.

    Jensen, M.  H. ; Morris, E.  J. ; Gallant, C.  M. ; Morgan, K.  G. ; Weitz, D.  A. ; Moore, J.  R. Mechanism of Calponin Stabilization of Cross-Linked Actin Networks. Biophysical Journal 2014, 106, 793 - 800. Publisher's VersionAbstract

    The actin-binding protein calponin has been previously implicated in actin cytoskeletal regulation and is thought to act as an actin stabilizer, but the mechanism of its function is poorly understood. To investigate this underlying physical mechanism, we studied an in vitro model system of cross-linked actin using bulk rheology. Networks with basic calponin exhibited a delayed onset of strain stiffening (10.0% without calponin, 14.9% with calponin) and were able to withstand a higher maximal strain before failing (35% without calponin, 56% with calponin). Using fluorescence microscopy to study the mechanics of single actin filaments, we found that calponin increased the flexibility of actin filaments, evident as a decrease in persistence length from 17.6 μm without to 7.7 μm with calponin. Our data are consistent with current models of affine strain behavior in semiflexible polymer networks, and suggest that calponin stabilization of actin networks can be explained purely by changes in single-filament mechanics. We propose a model in which calponin stabilizes actin networks against shear through a reduction of persistence length of individual filaments.

      Datta, S. S. ; Ramakrishnan, T. S. ; Weitz, D. A. Mobilization of a trapped non-wetting fluid from a three-dimensional porous medium. Physics of Fluids 2014, 26, 022002. Publisher's VersionAbstract

      We use confocal microscopy to directly visualize the formation and complex morphologies of trapped non-wetting fluid ganglia within a model 3D porous medium. The wetting fluid continues to flow around the ganglia after they form; this flow is characterized by a capillary number, Ca. We find that the ganglia configurations do not vary for small Ca; by contrast, as Ca is increased above a threshold value, the largest ganglia start to become mobilized and are ultimately removed from the medium. By combining our 3D visualization with measurements of the bulk transport, we show that this behavior can be quantitatively understood by balancing the viscous forces exerted on the ganglia with the pore-scale capillary forces that keep them trapped within the medium. Our work thus helps elucidate the fluid dynamics underlying the mobilization of a trapped non-wetting fluid from a 3D porous medium.

      Comunian, T. A. ; Abbaspourrad, A. ; Favaro-Trindade, C. S. ; Weitz, D. A. Fabrication of solid lipid microcapsules containing ascorbic acid using a microfluidic technique. Food Chemistry 2014, 152, 271 - 275. Publisher's VersionAbstract

      The importance of ascorbic acid (AA) in the human diet has motivated food researchers to develop AA-fortified food products. However, this compound is very unstable. The aim of this work was to produce solid lipid microcapsules (SLMs) loaded with AA using microfluidic technology. The morphology of the SLMs was analysed by optical, scanning electron and confocal microscopy. We determined the encapsulation efficiency, particle size and stability of the encapsulated material. Two different means of enhancing the encapsulation efficiency and stability of AA were demonstrated: a pore blocking method and a micromolecule-chelating agent within the core. The results indicated the enormous potential of the designed vehicle to prevent AA degradation in a food product; additionally, this vehicle could mask the acidic taste of AA.

        Datta, S. S. ; Abbaspourrad, A. ; Amstad, E. ; Fan, J. ; Kim, S. - H. ; Romanowsky, M. ; Shum, H. C. ; Sun, B. J. ; Utada, A. S. ; Windbergs, M. ; et al. Double Emulsion Templated Solid Microcapsules: Mechanics And Controlled Release. Advanced Materials 2014, 26, 2205-2218. Publisher's VersionAbstract
        We describe how droplet microfluidics can be used to fabricate solid-shelled microcapsules having precisely controlled release behavior. Glass capillary devices enable the production of monodisperse double emulsion drops, which can then be used as templates for microcapsule formation. The exquisite control afforded by microfluidics can be used to tune the compositions and geometrical characteristics of the microcapsules with exceptional precision. We review the use of this approach to fabricate microcapsules that only release their contents when exposed to a specific stimulus – such as a change in temperature, exposure to light, a change in the chemical environment, or an external stress – only after a prescribed time delay, and at a prescribed rate.
        Röding, M. ; Guo, M. ; Weitz, D. A. ; Rudemo, M. ; Särkkä, A. Identifying directional persistence in intracellular particle motion using Hidden Markov Models. Mathematical Biosciences 2014, 248, 140 - 145. Publisher's VersionAbstract

        Particle tracking is a widely used and promising technique for elucidating complex dynamics of the living cell. The cytoplasm is an active material, in which the kinetics of intracellular structures are highly heterogeneous. Tracer particles typically undergo a combination of random motion and various types of directed motion caused by the activity of molecular motors and other non-equilibrium processes. Random switching between more and less directional persistence of motion generally occurs.

        We present a method for identifying states of motion with different directional persistence in individual particle trajectories. Our analysis is based on a multi-scale turning angle model to characterize motion locally, together with a Hidden Markov Model with two states representing different directional persistence. We define one of the states by the motion of particles in a reference data set where some active processes have been inhibited.

        We illustrate the usefulness of the method by studying transport of vesicles along microtubules and transport of nanospheres activated by myosin. We study the results using mean square displacements, durations, and particle speeds within each state. We conclude that the method provides accurate identification of states of motion with different directional persistence, with very good agreement in terms of mean-squared displacement between the reference data set and one of the states in the two-state model.

        Herranz-Blanco, B. ; Arriaga, L. R. ; Makila, E. ; Correia, A. ; Shrestha, N. ; Mirza, S. ; Weitz, D. A. ; Salonen, J. ; Hirvonen, J. ; Santos, H. A. Microfluidic assembly of multistage porous silicon-lipid vesicles for controlled drug release. Lab on a Chip 2014, 14, 1083 - 1086. Publisher's VersionAbstract

        A reliable microfluidic platform for the generation of stable and monodisperse multistage drug delivery systems is reported. A glass-capillary flow-focusing droplet generation device was used to encapsulate thermally hydrocarbonized porous silicon (PSi) microparticles into the aqueous cores of double emulsion drops, yielding the formation of a multistage PSi-lipid vesicle. This composite system enables a large loading capacity for hydrophobic drugs.

        Zhang, W. ; Seminara, A. ; Suaris, M. ; Brenner, M. P. ; Weitz, D. A. ; Angelini, T. E. Nutrient depletion in Bacillus subtilis biofilms triggers matrix production. New Journal of Physics 2014, 16, 015028. Publisher's VersionAbstract

        Many types of bacteria form colonies that grow into physically robust and strongly adhesiveaggregates known as biofilms. A distinguishing characteristic of bacterial biofilms is an extracellular polymeric substance (EPS) matrix that encases the cells and provides physical integrity to the colony. The EPS matrix consists of a large amount of polysaccharide, as well as protein filaments, DNA and degraded cellular materials. The genetic pathways that control the transformation of a colony into a biofilm have been widely studied, and yield a spatiotemporal heterogeneity in EPS production. Spatial gradients in metabolites parallel this heterogeneity in EPS, but nutrient concentration as an underlying physiological initiator of EPS production has not been explored. Here, we study the role of nutrient depletion in EPS production in Bacillus subtilis biofilms. By monitoring simultaneously biofilm size and matrix production, we find that EPS production increases at a critical colony thickness that depends on the initial amount of carbon sources in the medium. Through studies of individual cells in liquid culture we find that EPS production can be triggered at the single-cell level by reducing nutrient concentration. To connect the single-cell assays with conditions in the biofilm, we calculate carbon concentration with a model for the reaction and diffusion of nutrients in the biofilm. This model predicts the relationship between the initial concentration of carbon and the thickness of the colony at the point of internal nutrient deprivation.