Shaping Giant Membrane Vesicles in 3D-Printed Protein Hydrogel Cages

Giant unilamellar phospholipid vesicles are attractive starting points for constructing minimal living cells from the bottom-up. Their membranes are compatible with many physiologically functional modules and act as selective barriers, while retaining a high morphological flexibility. However, their spherical shape renders them rather inappropriate to study phenomena that are based on distinct cell shape and polarity, such as cell division. Here, a microscale device based on 3D printed protein hydrogel is introduced to induce pH-stimulated reversible shape changes in trapped vesicles without compromising their free-standing membranes. Deformations of spheres to at least twice their aspect ratio, but also toward unusual quadratic or triangular shapes can be accomplished. Mechanical force induced by the cages to phase-separated membrane vesicles can lead to spontaneous shape deformations, from the recurrent formation of dumbbells with curved necks between domains to full budding of membrane domains as separate vesicles. Moreover, shape-tunable vesicles are particularly desirable when reconstituting geometry-sensitive protein networks, such as reaction-diffusion systems. In particular, vesicle shape changes allow to switch between different modes of self-organized protein oscillations within, and thus, to influence reaction networks directly by external mechanical cues

A doublet microlens array for imaging micron-sized objects

We present a high-numerical aperture, doublet microlens array for imaging micron-sized objects. The proposed doublet architecture consists of glass microspheres trapped on a predefined array of silicon microholes and covered with a thin polymer layer. A standard silicon microfabrication process and a novel fluidic assembly technique were combined to obtain an array of 56 µm diameter microlenses with a numerical aperture of ~0.5. Using such an array, we demonstrated brightfield and fluorescent image formation of objects directly on a CCD sensor without the use of intermediate lenses. The proposed technology is a significant advancement toward the unmet need of inexpensive, miniaturized optical modules which can be further integrated with lab-on-chip microfluidic devices and photonic chips for a variety of high-end imaging/detection applications.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90795/1/0960-1317_21_10_105024.pd

Microfluidic technologies for the synthesis and manipulation of biomimetic membranous nano-assemblies.

Microfluidics has been proposed as an attractive alternative to conventional bulk methods used in the generation of self-assembled biomimetic structures, particularly where there is a desire for more scalable production. The approach also allows for greater control over the self-assembly process, and parameters such as particle architecture, size, and composition can be finely tuned. Microfluidic techniques used in the generation of microscale assemblies (giant vesicles and higher-order multi-compartment assemblies) are fairly well established. These tend to rely on microdroplet templation, and the resulting structures have found use as comparmentalised motifs in artificial cells. Challenges in generating sub-micron droplets have meant that reconfiguring this approach to form nano-scale structures is not straightforward. This is beginning to change however, and recent technological advances have instigated the manufacture and manipulation of an increasingly diverse repertoire of biomimetic nano-assemblies, including liposomes, polymersomes, hybrid particles, multi-lamellar structures, cubosomes, hexosomes, nanodiscs, and virus-like particles. The following review will discuss these higher-order self-assembled nanostructures, including their biochemical and industrial applications, and techniques used in their production and analysis. We suggest ways in which existing technologies could be repurposed for the enhanced design, manufacture, and exploitation of these structures and discuss potential challenges and future research directions. By compiling recent advances in this area, it is hoped we will inspire future efforts toward establishing scalable microfluidic platforms for the generation of biomimetic nanoparticles of enhanced architectural and functional complexity

Magnetism, FeS colloids, and Origins of Life

A number of features of living systems: reversible interactions and weak bonds underlying motor-dynamics; gel-sol transitions; cellular connected fractal organization; asymmetry in interactions and organization; quantum coherent phenomena; to name some, can have a natural accounting via $physical$ interactions, which we therefore seek to incorporate by expanding the horizons of `chemistry-only' approaches to the origins of life. It is suggested that the magnetic 'face' of the minerals from the inorganic world, recognized to have played a pivotal role in initiating Life, may throw light on some of these issues. A magnetic environment in the form of rocks in the Hadean Ocean could have enabled the accretion and therefore an ordered confinement of super-paramagnetic colloids within a structured phase. A moderate H-field can help magnetic nano-particles to not only overcome thermal fluctuations but also harness them. Such controlled dynamics brings in the possibility of accessing quantum effects, which together with frustrations in magnetic ordering and hysteresis (a natural mechanism for a primitive memory) could throw light on the birth of biological information which, as Abel argues, requires a combination of order and complexity. This scenario gains strength from observations of scale-free framboidal forms of the greigite mineral, with a magnetic basis of assembly. And greigite's metabolic potential plays a key role in the mound scenario of Russell and coworkers-an expansion of which is suggested for including magnetism.Comment: 42 pages, 5 figures, to be published in A.R. Memorial volume, Ed Krishnaswami Alladi, Springer 201

Microstimulation and multicellular analysis: A neural interfacing system for spatiotemporal stimulation

Willfully controlling the focus of an extracellular stimulus remains a significant challenge in the development of neural prosthetics and therapeutic devices. In part, this challenge is due to the vast set of complex interactions between the electric fields induced by the microelectrodes and the complex morphologies and dynamics of the neural tissue. Overcoming such issues to produce methodologies for targeted neural stimulation requires a system that is capable of (1) delivering precise, localized stimuli a function of the stimulating electrodes and (2) recording the locations and magnitudes of the resulting evoked responses a function of the cell geometry and membrane dynamics. In order to improve stimulus delivery, we developed microfabrication technologies that could specify the electrode geometry and electrical properties. Specifically, we developed a closed-loop electroplating strategy to monitor and control the morphology of surface coatings during deposition, and we implemented pulse-plating techniques as a means to produce robust, resilient microelectrodes that could withstand rigorous handling and harsh environments. In order to evaluate the responses evoked by these stimulating electrodes, we developed microscopy techniques and signal processing algorithms that could automatically identify and evaluate the electrical response of each individual neuron. Finally, by applying this simultaneous stimulation and optical recording system to the study of dissociated cortical cultures in multielectode arrays, we could evaluate the efficacy of excitatory and inhibitory waveforms. Although we found that the proximity of the electrode is a poor predictor of individual neural excitation thresholds, we have shown that it is possible to use inhibitory waveforms to globally reduce excitability in the vicinity of the electrode. Thus, the developed system was able to provide very high resolution insight into the complex set of interactions between the stimulating electrodes and populations of individual neurons.Ph.D.Committee Chair: Stephen P. DeWeerth; Committee Member: Bruce Wheeler; Committee Member: Michelle LaPlaca; Committee Member: Robert Lee; Committee Member: Steve Potte

Responsive cell–material interfaces

Major design aspects for novel biomaterials are driven by the desire to mimic more varied and complex properties of a natural cellular environment with man-made materials. The development of stimulus responsive materials makes considerable contributions to the effort to incorporate dynamic and reversible elements into a biomaterial. This is particularly challenging for cell–material interactions that occur at an interface (biointerfaces); however, the design of responsive biointerfaces also presents opportunities in a variety of applications in biomedical research and regenerative medicine. This review will identify the requirements imposed on a responsive biointerface and use recent examples to demonstrate how some of these requirements have been met. Finally, the next steps in the development of more complex biomaterial interfaces, including multiple stimuli-responsive surfaces, surfaces of 3D objects and interactive biointerfaces will be discussed

Engineering 4D regulation toolbox to control spatiotemporal cell-free reconstitution

Bottom-up reconstituting well-characterized functional molecular entities, parts and modules towards a synthetic cell will give new insights into the general mechanisms and molecular origins of life. However, a remaining central challenge is how to organize cellular processes spatiotemporally from their component parts in vitro. To this end, we developed a 4D regulation toolbox to facilitate a bottom-up reconstitution in both time and space. The spatiotemporal regulation of the 4D toolbox covers the aspects from dynamic gene transcription & translation, reversible protein interaction, spatially protein positioning, sequential protein assembly, extends to defining geometrical membrane boundaries and mimicking cellular anisotropic microenvironment. Firstly, we developed a thermo-genetic regulation toolbox based on synthetic RNA thermometers, for temporally controlling protein expression in vitro. We validated RNA thermometers from in vivo to in vitro and tuned RNA thermometers through utilizing cell free protein synthesis system. Then we generated the thermo-sensitive protocell by encapsulating thermo-regulated transcription and translation machine in water-in-oil droplets. With the temperature sensing devices, the protocells can be operated with logic AND gates, differentially processing temperature stimuli into biological signals. Secondly, we engineered the PhyB-PIF6 system to spatiotemporally target proteins by light onto model membranes and thus sequentially guide protein pattern formation and structural assembly in vitro from the bottom up. We show that complex micrometer-sized protein patterns can be printed on timescales of seconds. Moreover, when printing self-assembling proteins such as the bacterial cytoskeleton protein FtsZ, the targeted assembly into filaments and large-scale structures such as artificial rings can be accomplished. To develop an artificial anisotropic membrane environment, we introduced a 3D printed protein hydrogel device to induce pH-stimulated reversible shape changes in trapped vesicles. Deformations towards unusual quadratic or triangular shapes can be accomplished. Mechanical force induced by the cages to phase-separated membrane vesicles can lead to spontaneous shape deformations. Moreover, the shape-tunable vesicle provides a spatially well-defined microenvironment for reconstituting shape-dependent protein systems, such as reaction-diffusion system that request explicitly non-spherical geometries. By taking advantages of the 3D printed hydrogel, we programmably engineered contractible scaffolds for actin-myosin motor reconstitution in 3D space. Nanoscale actomyosin motor as a bio-actuator could generate, transmit active contraction and then drive large-scale shape-morphing of complex 3D hydrogel scaffolds. In summary, by developing the spatiotemporal toolbox, this thesis introduces a promising step towards establishing bottom-up reconstitution in space and time, which could also guide future efforts in hierarchically building up the next level of complexity towards a minimal cell