Recording of cellular physiological histories along optically readable self-assembling protein chains.

Observing cellular physiological histories is key to understanding normal and disease-related processes. Here we describe expression recording islands-a fully genetically encoded approach that enables both continual digital recording of biological information within cells and subsequent high-throughput readout in fixed cells. The information is stored in growing intracellular protein chains made of self-assembling subunits, human-designed filament-forming proteins bearing different epitope tags that each correspond to a different cellular state or function (for example, gene expression downstream of neural activity or pharmacological exposure), allowing the physiological history to be read out along the ordered subunits of protein chains with conventional optical microscopy. We use expression recording islands to record gene expression timecourse downstream of specific pharmacological and physiological stimuli in cultured neurons and in living mouse brain, with a time resolution of a fraction of a day, over periods of days to weeks.

Experimental Validation of Diffraction Lithography for Fabrication of Solid Microneedles.

Microneedles are highly sought after for medicinal and cosmetic applications. However, the current manufacturing process for microneedles remains complicated, hindering its applicability to a broader variety of applications. As diffraction lithography has been recently reported as a simple method for fabricating solid microneedles, this paper presents the experimental validation of the use of ultraviolet light diffraction to control the liquid-to-solid transition of photosensitive resin to define the microneedle shape. The shapes of the resultant microneedles were investigated utilizing the primary experimental parameters including the photopattern size, ultraviolet light intensity, and the exposure time. Our fabrication results indicated that the fabricated microneedles became taller and larger in general when the experimental parameters were increased. Additionally, our investigation revealed four unique crosslinked resin morphologies during the first growth of the microneedle: microlens, first harmonic, first bell-tip, and second harmonic shapes. Additionally, by tilting the light exposure direction, a novel inclined microneedle array was fabricated for the first time. The fabricated microneedles were characterized with skin insertion and force-displacement tests. This experimental study enables the shapes and mechanical properties of the microneedles to be predicted in advance for mass production and wide practical use for biomedical or cosmetic applications.

Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics.

In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus.

Precision Calcium Imaging of Dense Neural Populations via a Cell-Body-Targeted Calcium Indicator.

Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and artifactual correlations of neural activity. We address this problem by engineering cell-body-targeted variants of the fluorescent calcium indicators GCaMP6f and GCaMP7f. We screened fusions of GCaMP to natural, as well as artificial, peptides and identified fusions that localized GCaMP to within 50 µm of the cell body of neurons in mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP in dense neural circuits reported fewer artifactual spikes from neuropil, an increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators facilitates usage of simple, powerful, one-photon methods for imaging neural calcium dynamics.

Coiled-Coils: the Molecular Zippers that Self-Assemble Protein Nanostructures.

Coiled-coils, the bundles of intertwined helical protein motifs, have drawn much attention as versatile molecular toolkits. Because of programmable interaction specificity and affinity as well as well-established sequence-to-structure relationships, coiled-coils have been used as subunits that self-assemble various molecular complexes in a range of fields. In this review, I describe recent advances in the field of protein nanotechnology, with a focus on programming assembly of protein nanostructures using coiled-coil modules. Modular design approaches to converting the helical motifs into self-assembling building blocks are described, followed by a discussion on the molecular basis and principles underlying the modular designs. This review also provides a summary of recently developed nanostructures with a variety of structural features, which are in categories of unbounded nanostructures, discrete nanoparticles, and well-defined origami nanostructures. Challenges existing in current design strategies, as well as desired improvements for controls over material properties and functionalities for applications, are also provided.

Modular assembly of a protein nanotriangle using orthogonally interacting coiled coils.

Synthetic protein assemblies that adopt programmed shapes would support many applications in nanotechnology. We used a rational design approach that exploits the modularity of orthogonally interacting coiled coils to create a self-assembled protein nanotriangle. Coiled coils have frequently been used to construct nanoassemblies and materials, but rarely with successful prior specification of the resulting structure. We designed a heterotrimer from three pairs of heterodimeric coiled coils that mediate specific interactions while avoiding undesired crosstalk. Non-associating pairs of coiled-coil units were strategically fused to generate three chains that were predicted to preferentially form the heterotrimer, and a rational annealing process led to the desired oligomer. Extensive biophysical characterization and modeling support the formation of a molecular triangle, which is a shape distinct from naturally occurring supramolecular nanostructures. Our approach can be extended to design more complex nanostructures using additional coiled-coil modules, other protein parts, or templated surfaces.

Colloidal Assembly of Hierarchically Structured Porous Supraparticles from Flower-Shaped Protein-Inorganic Hybrid Nanoparticles.

Mimicry of biomineralization is an attractive strategy to fabricate nanostructured hybrid materials. While biomineralization involves processes that organize hybrid clusters into complex structures with hierarchy, arrangement of artificial components in biomimetic approaches has been challenging. Here, we demonstrate self-assembly of hierarchically structured porous supraparticles from protein-inorganic hybrid flower-shaped (FS) nanoparticle building blocks. In our strategy, the FS nanoparticles self-assemble via high valency interactions in combination with interfacial adsorption and compression. The flower-like shape directed robust assembly of the FS nanoparticles into chain-like clusters in solution, which were further assembled into spherical supraparticles during rotation of FS nanoparticle solution. Continuously expanding and contracting the air-water interface during rotation catalyzed assembly of FS nanoparticle clusters, indicating that adsorption and compression of the building blocks at the interface were critical. The resulting supraparticles contain hierarchical pores which are translated from the structural characteristics of individual FS nanoparticle building blocks. The protein-inorganic supraparticles are protein-compatible, have large surface area, and provide specific affinity recognition for robust protein immobilization. A variety of functional proteins could be immobilized to the porous supraparticles, making it a general platform that could provide benefits for many applications.

Self-assembled hybrid supraparticles that proteolytically degrade tumor necrosis factor-α.

The strategies of pathogens to evade the human immune system are highly sophisticated and modulate a variety of inflammatory pathways. The similarities in the demands for modulation of inflammatory responses during disease treatment and during pathogenic infection provide opportunities to use pathogenic virulence factors to develop a new class of therapeutic materials that control inflammation. In this work, we harness a strategy from Porphyromonas gingivalis by transforming its major virulence factor, an arginine-specific cysteine protease, into self-assembled protease-inorganic hybrid supraparticles. The cysteine protease degrades the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α). It is an irreversible inhibition of TNF-α, which avoids some of the adverse effects of current TNF-α antagonists. We fabricated self-assembled porous supraparticles that specifically incorporate the pathogen-derived protease and showed improved inactivation of TNF-α over soluble enzyme, creating a potential therapeutic for various autoimmune diseases or other sources of inflammation.

Thermally triggered self-assembly of folded proteins into vesicles.

We report thermally triggered self-assembly of folded proteins into vesicles that incorporates globular proteins as building blocks. Leucine zipper coiled coils were combined with either globular proteins or elastin-like polypeptides as recombinant fusion proteins, which form "rod-coil" and "globule-rod-coil" protein complex amphiphiles. In aqueous solution, they self-assembled into hollow vesicles via temperature-responsive inverse phase transition. The characteristic of the protein vesicle membranes enables preferential encapsulation of simultaneously formed protein coacervate. Furthermore, the type of encapsulated cargo extends to small molecules and nanoparticles. Our approach offers a versatile strategy to create protein vesicles as vehicles with biological functionality.