Precise Spatiotemporal Control Research Articles

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

Local tissue manipulation via a force- and pressure-controlled AFM micropipette for analysis of cellular processes

Local manipulation of complex tissues at the single-cell level is challenging and requires excellent sealing between the specimen and the micromanipulation device. Here, biological applications for a recently developed loading technique for a force- and pressure-controlled fluidic force microscope micropipette are described. This technique allows for the exact positioning and precise spatiotemporal control of liquid delivery. The feasibility of a local loading technique for tissue applications was investigated using two fluorescent dyes, with which local loading behaviour could be optically visualised. Thus, homogeneous intracellular distribution of CellTracker Red and accumulation of SYTO 9 Green within nuclei was realised in single cells of a tissue preparation. Subsequently, physiological micromanipulation experiments were performed. Salivary gland tissue was pre-incubated with the Ca2+-sensitive dye OGB-1. An intracellular Ca2+ rise was then initiated at the single-cell level by applying dopamine via micropipette. When pre-incubating tissue with the nitric oxide (NO)-sensitive dye DAF-FM, NO release and intercellular NO diffusion was observed after local application of the NO donor SNP. Finally, local micromanipulation of a well-defined area along irregularly shaped cell surfaces of complex biosystems was shown for the first time for the fluidic force microscope micropipette. Thus, this technique is a promising tool for the investigation of the spatiotemporal effects of locally applied substances in complex tissues.

Physicochemical and Biological Characterisation of Azobenzene-Containing Photoswitchable Surfactants

Abstract Surfactants are a versatile and widely used class of molecules, due to their valuable adsorption and self-assembly properties. In particular, surfactants that can respond to stimuli are of interest in modulating wetting, controlling delivery, and exploring mechanistic aspects of biological processes. Incorporating azobenzene into surfactants is a classic approach to rendering molecules that respond to light as an external stimulus; these molecules find wide utility in the precise spatiotemporal control of dispersed systems, from DNA to graphene. More recently, the creation of diverse libraries of such molecules has been achieved by coupling azobenzene-containing hydrophobic tail-groups to hydrophilic carbohydrate head-groups. Such a synthetic strategy offers fine control over adsorption and aggregation, as evidenced by physicochemical characterization of these molecules, uncovering rich phase behavior and diverse biological response. This article covers recent advances in the field of both ‘traditional’ and new azobenzene-containing photosurfactants, and offers directions for future study and use of this unique class of molecule.

A novel gene\u2019s role in an ancient mechanism: secreted Frizzled-related protein 1 is a critical component in the anterior\u2013posterior Wnt signaling network that governs the establishment of the anterior neuroectoderm in sea urchin embryos

The anterior neuroectoderm (ANE) in many deuterostome embryos (echinoderms, hemichordates, urochordates, cephalochordates, and vertebrates) is progressively restricted along the anterior–posterior axis to a domain around the anterior pole. In the sea urchin embryo, three integrated Wnt signaling branches (Wnt/β-catenin, Wnt/JNK, and Wnt/PKC) govern this progressive restriction process, which begins around the 32- to 60-cell stage and terminates by the early gastrula stage. We previously have established that several secreted Wnt modulators of the Dickkopf and secreted Frizzled-related protein families (Dkk1, Dkk3, and sFRP-1/5) are expressed within the ANE and play important roles in modulating the Wnt signaling network during this process. In this study, we use morpholino and dominant-negative interference approaches to characterize the function of a novel Frizzled-related protein, secreted Frizzled-related protein 1 (sFRP-1), during ANE restriction. sFRP-1 appears to be related to a secreted Wnt modulator, sFRP3/4, that is essential to block Wnt signaling and establish the ANE in vertebrates. Here, we show that the sea urchin sFRP3/4 orthologue is not expressed during ANE restriction in the sea urchin embryo. Instead, our results indicate that ubiquitously expressed maternal sFRP-1 and Fzl1/2/7 signaling act together as early as the 32- to 60-cell stage to antagonize the ANE restriction mechanism mediated by Wnt/β-catenin and Wnt/JNK signaling. Then, starting from the blastula stage, Fzl5/8 signaling activates zygotic sFRP-1 within the ANE territory, where it works with the secreted Wnt antagonist Dkk1 (also activated by Fzl5/8 signaling) to antagonize Wnt1/Wnt8–Fzl5/8–JNK signaling in a negative feedback mechanism that defines the outer ANE territory boundary. Together, these data indicate that maternal and zygotic sFRP-1 protects the ANE territory by antagonizing the Wnt1/Wnt8–Fzl5/8–JNK signaling pathway throughout ANE restriction, providing precise spatiotemporal control of the mechanism responsible for the establishment of the ANE territory around the anterior pole of the sea urchin embryo.

Ru(ii) polypyridyl complexes as photocages for bioactive compounds containing nitriles and aromatic heterocycles

Photocaging allows for precise spatiotemporal control over the release of biologically active compounds with light. Most photocaged molecules employ organic photolabile protecting groups; however, biologically active compounds often contain functionalities such as nitriles and aromatic heterocycles that cannot be caged with organic groups. Despite their prevalence, only a few studies have reported successful caging of nitriles and aromatic heterocycles. Recently, Ru(ii)-based photocaging has emerged as a powerful method for the release of bioactive molecules containing these functional groups, in many cases providing high levels of spatial and temporal control over biological activity. This Feature Article discusses recent developments in applying Ru(ii)-based photocaging towards biological problems. Our groups designed and synthesized Ru(ii)-based platforms for the photoinduced delivery of cysteine protease and cytochrome P450 inhibitors in order to achieve selective control over enzyme inhibition. We also reported Ru(ii) photocaging groups derived from higher-denticity ancillary ligands that possess photophysical and photochemical properties distinct from more traditional Ru(ii)-based caging groups. In addition, for the first time, we are able to rapidly synthesize and screen Ru(ii) polypyridyl complexes that elicit desired properties by solid-phase synthesis. Finally, our work also defined steric and orbital mixing effects that are important factors in controlling photoinduced ligand exchange.

Spatiotemporal Control of Doxorubicin Delivery from "Stealth-Like" Prodrug Micelles.

In the treatment of cancer, targeting of anticancer drugs to the tumor microenvironment is highly desirable. Not only does this imply accurate tumor targeting but also minimal drug release en route to the tumor and maximal drug release once there. Here we describe high-loading, “stealth-like” doxorubicin micelles as a pro-drug delivery system, which upon light activation, leads to burst-like doxorbicin release. Through this approach, we show precise spatiotemporal control of doxorubicin delivery to cells in vitro.

Optogenerapy: When bio-electronic implant enters the modern syringe era

Resort to medications dates back million years ago with the use of medicinal plants. In the nineteenth century, significant contributions in medicine appeared in different domains, among which the invention of a specific drug delivery device; the syringe. Nowadays, injection therapy of bio-manufactured drugs is routine practice for chronic diseases but remains constraining and painful. New emerging advanced therapies invest in genetic, electronics and cell-based therapy for addressing unmet needs for the caregivers and the patient. As digital process in health (eHealth) gains momentum, connected advanced bio-electronic devices now offer new strategies for personalized injection therapies. In this review, we take a journey along the genesis path of a new drug delivery system: the Optogenerapy, a synergy between optogenetic and gene therapy. Inside a bio-electronic implant, electronics and optogenetics are interfaced by light as a traceless inducer signal. By controlling a synthetic optogenetic pathway in the cell, therapeutics delivery can be fine-tuned with a precise spatiotemporal control. The technology holds promise of a new modern syringe era capable of producing a drug of interest at will directly inside the patient.

Precise spatio-temporal control of rapid optogenetic cell ablation with mem-KillerRed in Zebrafish

The ability to kill individual or groups of cells in vivo is important for studying cellular processes and their physiological function. Cell-specific genetically encoded photosensitizing proteins, such as KillerRed, permit spatiotemporal optogenetic ablation with low-power laser light. We report dramatically improved resolution and speed of cell targeting in the zebrafish kidney through the use of a selective plane illumination microscope (SPIM). Furthermore, through the novel incorporation of a Bessel beam into the SPIM imaging arm, we were able to improve on targeting speed and precision. The low diffraction of the Bessel beam coupled with the ability to tightly focus it through a high NA lens allowed precise, rapid targeting of subsets of cells at anatomical depth in live, developing zebrafish kidneys. We demonstrate that these specific targeting strategies significantly increase the speed of optoablation as well as fish survival.

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to "dial" the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

Optogenetics: Switching with red and blue.

Multiple optogenetic technologies are required to control biological activity simultaneously with different colors of light. Optimizing a near-infrared-induced heterodimerization system, which can be combined with blue-light-controlled domains, enables precise spatiotemporal control of target molecules in live mammalian cells.

Wavelength Dependent, Sequentially Activated, Dual Anticancer Drug Delivery System with Photoinduced Fluorescence off-on for Real Time Imaging

Development of new photoresponsive drug delivery systems for combination chemotherapy is sought-after due to their precise spatio-temporal control over delivering drugs. Further, design of wavelength dependent drug delivery systems with each individual drug release being “one on one” triggered by light of different activating wavelengths, is an emerging topic for its high selective control over the release of individual drug. Herein we are reporting design of a new wavelength dependent photoresponsive dual drug delivery system i. e. nitric oxide donor linked coumarin chlorambucil conjugate (NOD-Cou-Cbl) for sequentially delivering anticancer agents with orthogonal mode of actions. NOD-Cou-Cbl has shown a sequential release of nitric oxide (NO) followed by chlorambucil under step wise irradiation with visible light and UV light, respectively. We have also studied in vitro cytotoxicity activity and real time bioimaging property of dual DDS on MDA-MB-231 cell line.

AIDing-targeted protein degradation in Drosophila.

Conditional protein depletion is highly desirable for investigating protein functions in complex organisms. In this issue, Bence and colleagues combined auxin-inducible degradation with CRISPR, establishing an elegant tool to control protein levels. They achieve precise spatio-temporal control of protein degradation during Drosophila oogenesis and early embryogenesis by combining suitable GAL4 drivers (spatial control) with auxin feeding protocols (temporal control).

Simply constructed chitosan nanocarriers with precise spatiotemporal control for efficient intracellular drug delivery

A novel intelligent nanocarrier with pH and redox sensitivities was developed based on Carboxymethyl-chitosan (CMCS) and thioglycolic acid conjugated chitosan (TCS) to provide precise spatiotemporal control for efficient intracellular delivery. Doxorubicin (DOX) loaded nanocarriers (DOX/CMCS-TCS NPs) were simply prepared by ionic gelation and then oxidation crosslink. The nanocarriers exhibited decent stability at pH 7.4 for up to 3days and underwent aggregation under acidic pH (5.5) due to protonation of the carboxyl groups on CMCS. The TCS skeleton was stable at pH 5.5 (mimic endo/lysosomes) but disintegrated in the presence of 10mM glutathione (GSH) at pH 7.4 (mimic cytosol). In vitro DOX release from DOX/CMCS-TCS NPs was enhanced at pH 5.5 compared with physiological condition, with 64.2% and 31.6% DOX released in 2h, respectively. While 85.2% of DOX was released within 2h as treated with 10mM GSH, suggesting the release was closely associated with structural disintegration of nanocarriers. The maximum release of DOX was obtained at 10mM GSH and pH 5.5 with 92.3% of DOX released in 5h. Confocal laser scanning microscopy observation indicated that DOX/CMCS-TCS NPs efficiently escaped from endo/lysosomes within 1h incubation with MCF-7 cells and gave the best performance in delivering DOX into nucleus in 2h. Anticancer activity assay revealed that DOX/CMCS-TCS NPs had comparable or even better inhibition of cell viability at high drug concentrations than free DOX, with the IC50 of 0.6μg/mL following 48h incubation. In summary, the simply constructed DOX/CMCS-TCS NPs could not only respond to intracellular delivery temporally, they also achieve rapid release spatially in nucleus, which provide a precise spatiotemporal control of drug delivery for cancer therapy.

Optogenetic Control of Synaptic Composition and Function

The molecular composition of the postsynaptic membrane is sculpted by synaptic activity. During synaptic plasticity at excitatory synapses, numerous structural, signaling, and receptor molecules concentrate at the postsynaptic density (PSD) to regulate synaptic strength. We developed an approach that uses light to tune the abundance of specific molecules in the PSD. We used this approach to investigate the relationship between the number of AMPA-type glutamate receptors in the PSD and synaptic strength. Surprisingly, adding more AMPA receptors to excitatory contacts had little effect on synaptic strength. Instead, we observed increased excitatory input through the apparent addition of new functional sites. Our data support a model where adding AMPA receptors is sufficient to activate synapses that had few receptors to begin with, but that additional remodeling events are required to strengthen established synapses. More broadly, this approach introduces the precise spatiotemporal control of optogenetics to the molecular control of synaptic function.

Two Paralogous Tetraspanins TSP-12 and TSP-14 Function with the ADAM10 Metalloprotease SUP-17 to Promote BMP Signaling in Caenorhabditis elegans.

The highly conserved bone morphogenetic protein (BMP) signaling pathway regulates many developmental and homeostatic processes. While the core components of the BMP pathway have been well studied, much research is needed for understanding the mechanisms involved in the precise spatiotemporal control of BMP signaling in vivo. Here, we provide evidence that two paralogous and evolutionarily conserved tetraspanins, TSP-12 and TSP-14, function redundantly to promote BMP signaling in C. elegans. We further show that the ADAM10 (a disintegrin and metalloprotease 10) ortholog SUP-17 also functions to promote BMP signaling, and that TSP-12 can bind to and promote the cell surface localization of SUP-17. SUP-17/ADAM10 is known to be involved in the ligand-induced proteolytic processing of the Notch receptor. We have evidence that the function of SUP-17, and of TSP-12/TSP-14 in BMP signaling is independent of their roles in Notch signaling. Furthermore, presenilins, core components of the γ-secretase complex involved in processing Notch, do not appear to play a role in BMP signaling. These studies established a new role of the TSP-12/TSP-14/SUP-17 axis in regulating BMP signaling, in addition to their known function in the Notch signaling pathway. We also provide genetic evidence showing that a known BMP signaling modulator, UNC-40/neogenin/DCC, is one of the substrates of SUP-17/ADAM10 in the BMP signaling pathway.

The miR-124 family of microRNAs is crucial for regeneration of the brain and visual system in the planarian Schmidtea mediterranea.

Brain regeneration in planarians is mediated by precise spatiotemporal control of gene expression and is crucial for multiple aspects of neurogenesis. However, the mechanisms underpinning the gene regulation essential for brain regeneration are largely unknown. Here, we investigated the role of the miR-124 family of microRNAs in planarian brain regeneration. The miR-124 family (miR-124) is highly conserved in animals and regulates neurogenesis by facilitating neural differentiation, yet its role in neural wiring and brain organization is not known. We developed a novel method for delivering anti-miRs using liposomes for the functional knockdown of microRNAs. Smed-miR-124 knockdown revealed a key role for these microRNAs in neuronal organization during planarian brain regeneration. Our results also demonstrated an essential role for miR-124 in the generation of eye progenitors. Additionally, miR-124 regulates Smed-slit-1, which encodes an axon guidance protein, either by targeting slit-1 mRNA or, potentially, by modulating the canonical Notch pathway. Together, our results reveal a role for miR-124 in regulating the regeneration of a functional brain and visual system.

3D Chemical Patterning of Micromaterials for Encoded Functionality.

Programming local chemical properties of microscale soft materials with 3D complex shapes is indispensable for creating sophisticated functionalities, which has not yet been possible with existing methods. Precise spatiotemporal control of two-photon crosslinking is employed as an enabling tool for 3D patterning of microprinted structures for encoding versatile chemical moieties.

Control of cerebral ischemia with magnetic nanoparticles.

The precise manipulation of microcirculation in mice can facilitate mechanistic studies of brain injury and repair after ischemia, but this manipulation remains a technical challenge, particularly in conscious mice. We developed a technology that uses micromagnets to induce aggregation of magnetic nanoparticles to reversibly occlude blood flow in microvessels. This allowed induction of ischemia in a specific cortical region of conscious mice of any postnatal age, including perinatal and neonatal stages, with precise spatiotemporal control but without surgical intervention of the skull or artery. When combined with longitudinal live-imaging approaches, this technology facilitated the discovery of a feature of the ischemic cascade: selective loss of smooth muscle cells in juveniles but not adults shortly after onset of ischemia and during blood reperfusion.

Different Regulations of ROM2 and LRG1 Expression by Ccr4, Pop2, and Dhh1 in the Saccharomyces cerevisiae Cell Wall Integrity Pathway.

Ccr4, a component of the Ccr4-Not cytoplasmic deadenylase complex, is known to be required for the cell wall integrity (CWI) pathway in the budding yeast Saccharomyces cerevisiae. However, it is not fully understood how Ccr4 and other components of the Ccr4-Not complex regulate the CWI pathway. Previously, we showed that Ccr4 functions in the CWI pathway together with Khd1 RNA binding protein. Ccr4 and Khd1 modulate a signal from Rho1 small GTPase in the CWI pathway by regulating the expression of ROM2 mRNA and LRG1 mRNA, encoding a guanine nucleotide exchange factor (GEF) and a GTPase-activating protein (GAP) for Rho1, respectively. Here we examined the possible involvement of the POP2 gene encoding a subunit of the Ccr4-Not complex and the DHH1 gene encoding a DEAD box RNA helicase that associates with the Ccr4-Not complex in the regulation of ROM2 and LRG1 expression. Neither ROM2 mRNA level nor Rom2 function was impaired by pop2Δ or dhh1Δ mutation. The LRG1 mRNA level was increased in pop2Δ and dhh1Δ mutants, as well as the ccr4Δ mutant, and the growth defects caused by pop2Δ and dhh1Δ mutations were suppressed by lrg1Δ mutation. Our results suggest that LRG1 expression is regulated by Ccr4 together with Pop2 and Dhh1 and that ROM2 expression is regulated by Khd1 and Ccr4, but not by Pop2 and Dhh1. Thus, Rho1 activity in the CWI pathway is precisely controlled by modulation of the mRNA levels for Rho1-GEF Rom2 and Rho1-GAP Lrg1. IMPORTANCE We find here that Ccr4, Pop2, and Dhh1 modulate the levels of mRNAs for specific Rho1 regulators, Rom2 and Lrg1. In budding yeast, Rho1 activity is tightly regulated both temporally and spatially. It is anticipated that Ccr4, Pop2, and Dhh1 may contribute to the precise spatiotemporal control of Rho1 activity by regulating expression of its regulators temporally and spatially. Our finding on the roles of the components of the Ccr4-Not complex in yeast would give important information for understanding the roles of the evolutionary conserved Ccr4-Not complex.

Allosteric control of mammalian DNA methyltransferases - a new regulatory paradigm.

In mammals, DNA methylation is introduced by the DNMT1, DNMT3A and DNMT3B methyltransferases, which are all large multi-domain proteins containing a catalytic C-terminal domain and an N-terminal part with regulatory functions. Recently, two novel regulatory principles of DNMTs were uncovered. It was shown that their catalytic activity is under allosteric control of N-terminal domains with autoinhibitory function, the RFT and CXXC domains in DNMT1 and the ADD domain in DNMT3. Moreover, targeting and activity of DNMTs were found to be regulated in a concerted manner by interactors and posttranslational modifications (PTMs). In this review, we describe the structures and domain composition of the DNMT1 and DNMT3 enzymes, their DNA binding, catalytic mechanism, multimerization and the processes controlling their stability in cells with a focus on their regulation and chromatin targeting by PTMs, interactors and chromatin modifications. We propose that the allosteric regulation of DNMTs by autoinhibitory domains acts as a general switch for the modulation of the function of DNMTs, providing numerous possibilities for interacting proteins, nucleic acids or PTMs to regulate DNMT activity and targeting. The combined regulation of DNMT targeting and catalytic activity contributes to the precise spatiotemporal control of DNMT function and genome methylation in cells.