Precise Spatiotemporal Control Research Articles

A single-cell transcriptomic atlas characterizes the silk-producing organ in the silkworm

The silk gland of the domesticated silkworm Bombyx mori, is a remarkable organ that produces vast amounts of silk with exceptional properties. Little is known about which silk gland cells execute silk protein synthesis and its precise spatiotemporal control. Here, we use single-cell RNA sequencing to build a comprehensive cell atlas of the silkworm silk gland, consisting of 14,972 high-quality cells representing 10 distinct cell types, in three early developmental stages. We annotate all 10 cell types and determine their distributions in each region of the silk gland. Additionally, we decode the developmental trajectory and gene expression status of silk gland cells. Finally, we discover marker genes involved in the regulation of silk gland development and silk protein synthesis. Altogether, this work reveals the heterogeneity of silkworm silk gland cells and their gene expression dynamics, affording a deeper understanding of silk-producing organs at the single-cell level.

Activatable Cancer Sono-Immunotherapy using Semiconducting Polymer Nanobodies.

Despite the great promises of sonodynamic therapy (SDT) in combination cancer therapy, its clinical applications are hindered by the "always-on" pharmacological activities of therapeutic agents and the lack of efficient sonosensitizers. Herein, the development of semiconducting polymers as efficient sonosensitizers and further development of sono-immunotherapeutic nanobodies (SPNAb ) for activatable cancer sono-immunotherapy are reported. Conjugation of anti-CTLA-4 antibodies onto the polymer nanoparticles through a 1 O2 -cleavable linker affords SPNAb with relatively low CTLA-4 binding affinity. Upon sono-irradiation, SPNAb generates 1 O2 not only to elicit a sonodynamic effect to induce immunogenic cell death, but also to release anti-CTLA-4 antibodies and trigger in situ checkpoint blockade. Such a synergistic therapeutic action mediated by SPNAb modulates the tumoricidal function of T-cell immunity by promoting the proliferation of cytotoxic T lymphocytes and depleting immunosuppressive regulatory T cells, resulting in effective tumor regression, metastasis inhibition, durable immunological memory, and prevention of relapse. Therefore, this study represents a proof-of-concept sonodynamic strategy using semiconducting polymers for precise spatiotemporal control over immunotherapy.

Spatiotemporal Control of Neuronal Remodeling by Cell Adhesion Molecules: Insights From Drosophila.

Developmental neuronal remodeling is required for shaping the precise connectivity of the mature nervous system. Remodeling involves pruning of exuberant neural connections, often followed by regrowth of adult-specific ones, as a strategy to refine neural circuits. Errors in remodeling are associated with neurodevelopmental disorders such as schizophrenia and autism. Despite its fundamental nature, our understanding of the mechanisms governing neuronal remodeling is far from complete. Specifically, how precise spatiotemporal control of remodeling and rewiring is achieved is largely unknown. In recent years, cell adhesion molecules (CAMs), and other cell surface and secreted proteins of various families, have been implicated in processes of neurite pruning and wiring specificity during circuit reassembly. Here, we review some of the known as well as speculated roles of CAMs in these processes, highlighting recent advances in uncovering spatiotemporal aspects of regulation. Our focus is on the fruit fly Drosophila, which is emerging as a powerful model in the field, due to the extensive, well-characterized and stereotypic remodeling events occurring throughout its nervous system during metamorphosis, combined with the wide and constantly growing toolkit to identify CAM binding and resulting cellular interactions in vivo. We believe that its many advantages pose Drosophila as a leading candidate for future breakthroughs in the field of neuronal remodeling in general, and spatiotemporal control by CAMs specifically.

Photo-Controlled [4+4] Cycloaddition of Anthryl-Polymer Systems: A Versatile Approach to Fabricate Functional Materials.

The reversible photo-induced [4+4] cycloaddition reaction of anthracene enables multiple cycles of dimerization and scission, allowing phototunable linkage of molecular fragments for the synthesis of polymer scaffolds. New functional materials ranging from hydrogels to shape-memory polymers were designed from anthryl-polymer systems because of their diverse photochemical reactivity and responsiveness. Light as an external stimulus allows for the remote and precise spatiotemporal control of materials without the need for additional reagents. Depending on how the photoreactive anthracene moieties were introduced, the interaction of anthryl-polymer systems with light results in various processes such as polymerization, cyclization, and cross-linking. Structural modifications of anthracene derivatives could shift their absorption from the ultraviolet to the visible light region, widening their range of applications including biologically relevant studies. These applications are further diversified and enhanced by the reversibility of the dimerization reaction using light and heat as stimuli. In this review, current developments in the synthesis and photodimerization of anthracene-containing polymers and their emerging applications in the fabrication of new materials are discussed.

Cells feel the beat – temporal effect of cyclic mechanical actuation on muscle cells

Understanding how muscle cells (myoblasts) react to mechanical actuation with a precise spatio-temporal control is challenging and requires new material systems to achieve a better insights to mechanotransduction pathways involved in myogenic differentiation. Here, we employ a recently developed light-responsive hydrogel as an actuating substrate with native tissue-like modulus to stimulate myoblast cells in the absence of any additional differentiation factors. A near-infrared micro-projection system allows to locally define the actuation frequency and duration and investigate cellular processes of proliferation and differentiation. We observe that actuation enhances cell proliferation and migration, associated with nuclear translocation of myogenic proteins, such as MyoD and Yes-associated protein (YAP). Furthermore, we record a significant reduction of collagen type I and fibronectin deposition, suggesting an antifibrotic effect of the actuation. In conclusion, our findings emphasize the need of controlling the actuation time (training schedule) and amplitude for inducing myoblasts proliferation and mechanotransduction, underling the importance of using a synthetic material system that can be adjusted to support myogenic differentiation and other cell processes in vitro.

Electrically Fueled Active Supramolecular Materials.

Fuel-driven dissipative self-assemblies play essential roles in living systems, contributing both to their complex, dynamic structures and emergent functions. Several dissipative supramolecular materials have been created using chemicals or light as fuel. However, electrical energy, one of the most common energy sources, has remained unexplored for such purposes. Here, we demonstrate a new platform for creating active supramolecular materials using electrically fueled dissipative self-assembly. Through an electrochemical redox reaction network, a transient and highly active supramolecular assembly is achieved with rapid kinetics, directionality, and precise spatiotemporal control. As electronic signals are the default information carriers in modern technology, the described approach offers a potential opportunity to integrate active materials into electronic devices for bioelectronic applications.

Controlling CRISPR-Cas9 by guide RNA engineering.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a product of million years of evolution by microbes to fight against invading genetic materials. Around 10 years ago, scientists started to repurpose the CRISPR as genetic tools by molecular engineering approaches. The guide RNA provides a versatile and unique platform for the innovation to improve and expand the application of CRISPR-Cas9 system. In this review, we will first introduce the basic sequence and structure of guide RNA and its role during the function of CRISPR-Cas9. We will then summarize recent progress on the development of various guide RNA engineering strategies. These strategies have been dedicated to improve the performance of CRISPR-Cas9, to achieve precise spatiotemporal control of CRISPR-Cas9, and to broaden the application of CRISPR-Cas9. Finally, we will briefly discuss the uniqueness and advantage of guide RNA-engineering based systems versus those with engineered Cas9 proteins and speculate potential future directions in guide RNA engineering. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico RNA Methods > RNA Nanotechnology Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Chirality transfer from a 3D macro shape to the molecular level by controlling asymmetric secondary flows

Homochirality is a fundamental feature of living systems, and its origin is still an unsolved mystery. Previous investigations showed that external physical forces can bias a spontaneous symmetry breaking process towards deterministic enantioselection. But can the macroscopic shape of a reactor play a role in chiral symmetry breaking processes? Here we show an example of chirality transfer from the chiral shape of a 3D helical channel to the chirality of supramolecular aggregates, with the handedness of the helical channel dictating the direction of enantioselection in the assembly of an achiral molecule. By combining numerical simulations of fluid flow and mass transport with experimental data, we demonstrated that the chiral information is transferred top-down thanks to the interplay between the hydrodynamics of asymmetric secondary flows and the precise spatiotemporal control of reagent concentration fronts. This result shows the possibility of controlling enantioselectively molecular processes at the nanometer scale by modulating the geometry and the operating conditions of fluidic reactors.

A Review on Microfluidic Platforms Applied to Nerve Regeneration

In recent decades, microfluidics have significantly advanced nerve regeneration research. Microfluidic devices can provide an accurate simulation of in vivo microenvironment for different research purposes such as analyzing myelin growth inhibitory factors, screening drugs, assessing nerve growth factors, and exploring mechanisms of neural injury and regeneration. The microfluidic platform offers technical supports for nerve regeneration that enable precise spatio-temporal control of cells, such as neuron isolation, single-cell manipulation, neural patterning, and axon guidance. In this paper, we review the development and recent advances of microfluidic platforms for nerve regeneration research.

Optogenetics Illuminates Applications in Microbial Engineering.

Optogenetics has been used in a variety of microbial engineering applications, such as chemical and protein production, studies of cell physiology, and engineered microbe-host interactions. These diverse applications benefit from the precise spatiotemporal control that light affords, as well as its tunability, reversibility, and orthogonality. This combination of unique capabilities has enabled a surge of studies in recent years investigating complex biological systems with completely new approaches. We briefly describe the optogenetic tools that have been developed for microbial engineering, emphasizing the scientific advancements that they have enabled. In particular, we focus on the unique benefits and applications of implementing optogenetic control, from bacterial therapeutics to cybergenetics. Finally, we discuss future research directions, with special attention given to the development of orthogonal multichromatic controls. With an abundance of advantages offered by optogenetics, the future is bright in microbial engineering.

A Temperature-Controlled Cell-Free Expression System by Dynamic Repressor.

The cell-free protein synthesis (CFPS) system is a typical protein production platform in the field of synthetic biology. However, there are limitations in controlling protein synthesis in the CFPS system. Compared with the traditional method of adding chemicals, temperature is an ideal control switch to achieve precise spatiotemporal control with few side effects. Hence, the design of a temperature-controlled cell-free protein synthesis (tcCFPS) system based on E.coli was carried out with the repressor cI protein in this study. The corresponding tcCFPS achieved a 143-fold dynamic protein expression level at 37 °C than that at 30 °C. Besides, the artificial cell controlled by temperature was constructed to expand the applications of tcCFPS. This study provides a new effective method for active protein synthesis in a cell-free system and a potential means of drug synthesis and delivery.

Microfluidics-enabled functional 3D printing.

Microfluidic technology has established itself as a powerful tool to enable highly precise spatiotemporal control over fluid streams for mixing, separations, biochemical reactions, and material synthesis. 3D printing technologies such as extrusion-based printing, inkjet, and stereolithography share similar length scales and fundamentals of fluid handling with microfluidics. The advanced fluidic manipulation capabilities afforded by microfluidics can thus be potentially leveraged to enhance the performance of existing 3D printing technologies or even develop new approaches to additive manufacturing. This review discusses recent developments in integrating microfluidic elements with several well-established 3D printing technologies, highlighting the trend of using microfluidic approaches to achieve functional and multimaterial 3D printing as well as to identify potential future research directions in this emergent area.

Photopolymerized Features via Beam Pen Lithography as a Novel Tool for the Generation of Large Area Protein Micropatterns.

A cantilever-free scanning probe lithography (CF-SPL)-based method for the rapid polymerization of nanoscale features onasurfacevia crosslinking and thiol-acrylate photoreactions is described, whereinthenanoscale position, height, and diameter of each feature can be finely and independently tuned. With precise spatiotemporal control over the illumination pattern, beam pen lithography (BPL) allows for the photo-crosslinking of polymers into ultrahigh resolution features over centimeter-scale areas using massively parallel >160 000 pen arrays of individually addressable pens that guide and focus light onto the surface with sub-diffraction resolution. The photoinduced crosslinking reaction of the ink material, which is composed of photoinitiator, diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide, poly(ethylene glycol) diacrylate, and thiol-modified functional binding molecules (i.e., thiol-PEG-biotin or 16-mercaptohexanoic acid), proceeds to ≈80% conversion with UV exposure (72mWcm-2 ) for short time periods (0.5s). Such polymer patterns are further reacted with proteins (streptavidin and fibronectin) to yield protein arrays with feature arrangements at high resolution and densities controlled by local UV exposure. This platform, which combines polymer photochemistry and massive arrays of scanning probes, constitutes a new approach to making biomolecular microarrays in a high-throughput and high-yielding manner, opening new routes for biochip synthesis, bioscreening, and cell biology research.

Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation

Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate-binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family.

Ubiquitin ligases: guardians of mammalian development.

Mammalian development demands precision. Millions of molecules must be properly located in temporal order, and their function regulated, to orchestrate important steps in cell cycle progression, apoptosis, migration and differentiation, to shape developing embryos. Ubiquitin and its associated enzymes act as cellular guardians to ensure precise spatio-temporal control of key molecules during each of these important cellular processes. Loss of precision results in numerous examples of embryological disorders or even cancer. This Review discusses the crucial roles of E3 ubiquitin ligases during key steps of early mammalian development and their roles in human disease, and considers how new methods to manipulate and exploit the ubiquitin regulatory machinery - for example, the development of molecular glues and PROTACs - might facilitate clinical therapy.

In vivo spatiotemporal control of voltage-gated ion channels by using photoactivatable peptidic toxins

Photoactivatable drugs targeting ligand-gated ion channels open up new opportunities for light-guided therapeutic interventions. Photoactivable toxins targeting ion channels have the potential to control excitable cell activities with low invasiveness and high spatiotemporal precision. As proof-of-concept, we develop HwTxIV-Nvoc, a UV light-cleavable and photoactivatable peptide that targets voltage-gated sodium (NaV) channels and validate its activity in vitro in HEK293 cells, ex vivo in brain slices and in vivo on mice neuromuscular junctions. We find that HwTxIV-Nvoc enables precise spatiotemporal control of neuronal NaV channel function under all conditions tested. By creating multiple photoactivatable toxins, we demonstrate the broad applicability of this toxin-photoactivation technology.

A dental implant-on-a-chip for 3D modeling of host-material-pathogen interactions and therapeutic testing platforms.

The precise spatiotemporal control and manipulation of fluid dynamics on a small scale granted by lab-on-a-chip devices provide a new biomedical research realm as a substitute for in vivo studies of host-pathogen interactions. While there has been a rise in the use of various medical devices/implants for human use, the applicability of microfluidic models that integrate such functional biomaterials is currently limited. Here, we introduced a novel dental implant-on-a-chip model to better understand host-material-pathogen interactions in the context of peri-implant diseases. The implant-on-a-chip integrates gingival cells with relevant biomaterials - keratinocytes with dental resin and fibroblasts with titanium while maintaining a spatially separated co-culture. To enable this co-culture, the implant-on-a-chip's core structure necessitates closely spaced, tall microtrenches. Thus, an SU-8 master mold with a high aspect-ratio pillar array was created by employing a unique backside UV exposure with a selective optical filter. With this model, we successfully replicated the morphology of keratinocytes and fibroblasts in the vicinity of dental implant biomaterials. Furthermore, we demonstrated how photobiomodulation therapy might be used to protect the epithelial layer from recurrent bacterial challenges (∼3.5-fold reduction in cellular damage vs. control). Overall, our dental implant-on-a-chip approach proposes a new microfluidic model for multiplexed host-material-pathogen investigations and the evaluation of novel treatment strategies for infectious diseases.

Seeing the Light: The Use of Zebrafish for Optogenetic Studies of the Heart.

Optogenetics, involving the optical measurement and manipulation of cellular activity with genetically encoded light-sensitive proteins (“reporters” and “actuators”), is a powerful experimental technique for probing (patho-)physiological function. Originally developed as a tool for neuroscience, it has now been utilized in cardiac research for over a decade, providing novel insight into the electrophysiology of the healthy and diseased heart. Among the pioneering cardiac applications of optogenetic actuators were studies in zebrafish, which first demonstrated their use for precise spatiotemporal control of cardiac activity. Zebrafish were also adopted early as an experimental model for the use of optogenetic reporters, including genetically encoded voltage- and calcium-sensitive indicators. Beyond optogenetic studies, zebrafish are becoming an increasingly important tool for cardiac research, as they combine many of the advantages of integrative and reduced experimental models. The zebrafish has striking genetic and functional cardiac similarities to that of mammals, its genome is fully sequenced and can be modified using standard techniques, it has been used to recapitulate a variety of cardiac diseases, and it allows for high-throughput investigations. For optogenetic studies, zebrafish provide additional advantages, as the whole zebrafish heart can be visualized and interrogated in vivo in the transparent, externally developing embryo, and the relatively small adult heart allows for in situ cell-specific observation and control not possible in mammals. With the advent of increasingly sophisticated fluorescence imaging approaches and methods for spatially-resolved light stimulation in the heart, the zebrafish represents an experimental model with unrealized potential for cardiac optogenetic studies. In this review we summarize the use of zebrafish for optogenetic investigations in the heart, highlighting their specific advantages and limitations, and their potential for future cardiac research.

Photo controlled release of nitric oxide (NO) from amphiphilic and nanoscale vesicles based ruthenium nitrosyl complex: NO release and cytotoxicity studies

Nitric oxide (NO) is an important gaseous signaling molecule. Precise spatiotemporal control release of NO for therapeutic applications is still challenging. We report herein a new amphiphilic and nano scale vesicles based NO-delivery platform that is capable of light-controlled NO release. 1.Ru-NO was incorporated into the lipid bilayers to form nanoscale vesicles (Lip-1.Ru-NO) to make it bio-compatible and 100% water soluble. The liposome Lip-1.Ru-NO gradually released NO in blue light over a period of 120 min, with relatively high efficiency as compared to 1.Ru-NO in CH3CN-water mixture. Amount of NO release from liposomes could be easily controlled and modulated by changing the concentration of 1.Ru-NO in nano scale vesicles. Light triggered controlled NO release, water solubility, and non-toxic nature of the complex before and after NO release are fulfilled by incorporation of amphiphilic ruthenium complex in to the vesicles surface.

Phase separation in synthetic biology

BackgroundThe concept of phase separation has been used to describe and interpret physicochemical phenomena in biological systems for decades. Many intracellular macromolecules undergo phase separation, where it plays important roles in gene regulation, cellular signaling, metabolic reactions and so on, due to its unique dynamic properties and biological effects. As the noticeable importance of phase separation, pioneer researchers have explored the possibility to introduce the synthetically engineered phase separation for applicable cell function.ResultsIn this article, we illustrated the application value of phase separation in synthetic biology. We described main states of phase separation in detail, summarized some ways to implement synthetic condensates and several methods to regulate phase separation, and provided a substantial amount of identical examples to illuminate the applications and perspectives of phase separation in synthetic biology.ConclusionsMultivalent interactions implement phase separation in synthetic biology. Small molecules, light control and spontaneous interactions induce and regulate phase separation. The sy nthetic condensates are widely used in signal amplifications, designer orthogonally non‐membrane‐bound organelles, metabolic pathways, gene regulations, signaling transductions and controllable platforms. Studies on quantitative analysis, more standardized modules and precise spatiotemporal control of synthetic phase separation may promote the further development of this field.