Organ-Specific Gene Expression Control Using DNA Origami-Based Nanodevices.

Nanodevices that function in specific organs or cells are one of the ultimate goals of synthetic biology. The recent progress in DNA nanotechnology such as DNA origami has allowed us to construct nanodevices to deliver a payload (e.g., drug) to the tumor. However, delivery to specific organs remains difficult due to the fragility of the DNA nanostructure and the low targeting capability of the DNA nanostructure. Here, we constructed tough DNA origami that allowed us to encapsulate the DNA origami into lipid-based nanoparticles (LNPs) under harsh conditions (low pH), harnessing organ-specific delivery of the gene of interest (GOI). We found that DNA origami-encapsulated LNPs can increase the functionality of payload GOIs (mRNA and siRNA) inside mouse organs through the contribution from different LNP structures revealed by cryogenic electron microscope (Cryo-EM). These data should be the basis for future organ-specific gene expression control using DNA origami nanodevices.

Implication of thermal signaling in neuronal differentiation revealed by manipulation and measurement of intracellular temperature.

Neuronal differentiation-the development of neurons from neural stem cells-involves neurite outgrowth and is a key process during the development and regeneration of neural functions. In addition to various chemical signaling mechanisms, it has been suggested that thermal stimuli induce neuronal differentiation. However, the function of physiological subcellular thermogenesis during neuronal differentiation remains unknown. Here we create methods to manipulate and observe local intracellular temperature, and investigate the effects of noninvasive temperature changes on neuronal differentiation using neuron-like PC12 cells. Using quantitative heating with an infrared laser, we find an increase in local temperature (especially in the nucleus) facilitates neurite outgrowth. Intracellular thermometry reveals that neuronal differentiation is accompanied by intracellular thermogenesis associated with transcription and translation. Suppression of intracellular temperature increase during neuronal differentiation inhibits neurite outgrowth. Furthermore, spontaneous intracellular temperature elevation is involved in neurite outgrowth of primary mouse cortical neurons. These results offer a model for understanding neuronal differentiation induced by intracellular thermal signaling.

Heat-hypersensitive mutants of ryanodine receptor type 1 revealed by microscopic heating.

Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca2+ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca2+ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca2+ imaging using the endoplasmic reticulum-targeted fluorescent probes demonstrates that the Ca2+ burst originates from heat-induced Ca2+ release (HICR) through RyR1-mutant channels because of the channels' heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.

Cell-autonomous control of intracellular temperature by unsaturation of phospholipid acyl chains.

Intracellular temperature affects a wide range of cellular functions in living organisms. However, it remains unclear whether temperature in individual animal cells is controlled autonomously as a response to fluctuations in environmental temperature. Using two distinct intracellular thermometers, we find that the intracellular temperature of steady-state Drosophila S2 cells is maintained in a manner dependent on Δ9-fatty acid desaturase DESAT1, which introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA. The DESAT1-mediated increase of intracellular temperature is caused by the enhancement of F1Fo-ATPase-dependent mitochondrial respiration, which is coupled with thermogenesis. We also reveal that F1Fo-ATPase-dependent mitochondrial respiration is potentiated by cold exposure through the remodeling of mitochondrial cristae structures via DESAT1-dependent unsaturation of mitochondrial phospholipid acyl chains. Based on these findings, we propose a cell-autonomous mechanism for intracellular temperature control during environmental temperature changes.

In situ measurements of intracellular thermal conductivity using heater-thermometer hybrid diamond nanosensors.

Understanding heat dissipation processes at nanoscale during cellular thermogenesis is essential to clarify the relationships between the heat and biological processes in cells and organisms. A key parameter determining the heat flux inside a cell is the local thermal conductivity, a factor poorly investigated both experimentally and theoretically. Here, using a nanoheater/nanothermometer hybrid made of a polydopamine encapsulating a fluorescent nanodiamond, we measured the intracellular thermal conductivities of HeLa and MCF-7 cells with a spatial resolution of about 200 nm. The mean values determined in these two cell lines are both 0.11 ± 0.04 W m-1 K-1, which is significantly smaller than that of water. Bayesian analysis of the data suggests there is a variation of the thermal conductivity within a cell. These results make the biological impact of transient temperature spikes in a cell much more feasible, and suggest that cells may use heat flux for short-distance thermal signaling.

Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes.

Cytoplasmic RIG-I-like receptor (RLR) proteins in mammalian cells recognize viral RNA and initiate an antiviral response that results in IFN-ß induction. Melanoma differentiation-associated protein 5 (MDA5) forms fibers along viral dsRNA and propagates an antiviral response via a signaling domain, the tandem CARD. The most enigmatic RLR, laboratory of genetics and physiology (LGP2), lacks the signaling domain but functions in viral sensing through cooperation with MDA5. However, it remains unclear how LGP2 coordinates fiber formation and subsequent MDA5 activation. We utilized biochemical and biophysical approaches to observe fiber formation and the conformation of MDA5. LGP2 facilitated MDA5 fiber assembly. LGP2 was incorporated into the fibers with an average inter-molecular distance of 32 nm, suggesting the formation of hetero-oligomers with MDA5. Furthermore, limited protease digestion revealed that LGP2 induces significant conformational changes on MDA5, promoting exposure of its CARDs. Although the fibers were efficiently dissociated by ATP hydrolysis, MDA5 maintained its active conformation to participate in downstream signaling. Our study demonstrated the coordinated actions of LGP2 and MDA5, where LGP2 acts as an MDA5 nucleator and requisite partner in the conversion of MDA5 to an active conformation. We revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses.

Multifunctional temozolomide-loaded lipid superparamagnetic nanovectors: dual targeting and disintegration of glioblastoma spheroids by synergic chemotherapy and hyperthermia treatment.

Aiming at finding new solutions for fighting glioblastoma multiforme, one of the most aggressive and lethal human cancer, here an in vitro validation of multifunctional nanovectors for drug delivery and hyperthermia therapy is proposed. Hybrid magnetic lipid nanoparticles have been fully characterized and tested on a multi-cellular complex model resembling the tumor microenvironment. Investigations of cancer therapy based on a physical approach (namely hyperthermia) and on a pharmaceutical approach (by exploiting the chemotherapeutic drug temozolomide) have been extensively carried out, by evaluating its antiproliferative and pro-apoptotic effects on 3D models of glioblastoma multiforme. A systematic study of transcytosis and endocytosis mechanisms has been moreover performed with multiple complimentary investigations, besides a detailed description of local temperature increments following hyperthermia application. Finally, an in-depth proteomic analysis corroborated the obtained findings, which can be summarized in the preparation of a versatile, multifunctional, and effective nanoplatform able to overcome the blood-brain barrier and to induce powerful anti-cancer effects on in vitro complex models.

Live Cell Imaging Using Photoswitchable Diarylethene-Doped Fluorescent Polymer Dots.

Fluorescence photoswitching using nanomaterials has recently emerged as a promising approach for the imaging of biological targets. However, despite intensive research efforts during the last decade, practical microscopy of biological targets using photoswitchable nanoparticles in real time remains challenging. To address this problem, we have developed live macrophage cell imaging and single particle imaging methods, using photoswitchable fluorescent diarylethene-doped polymer nanoparticles (P-dots) under Xe lamp irradiation. We established a 34-times prolonged "off-state", using P-dots doped with a diarylethene-containing methoxy substituent, upon visible-light irradiation using a Xe lamp and a green fluorescent protein filter cube. To demonstrate the practicality of doped P-dots imaging, we imaged lysosomes in macrophage cells, and observed 11-times slower recovery of the fluorescence from the "off-state" to the "on-state", indicating their potential for cellular imaging.

Synergistic effect of ATP for RuvA-RuvB-Holliday junction DNA complex formation.

The Escherichia coli RuvB hexameric ring motor proteins, together with RuvAs, promote branch migration of Holliday junction DNA. Zero mode waveguides (ZMWs) constitute of nanosized holes and enable the visualization of a single fluorescent molecule under micromolar order of the molecules, which is applicable to characterize the formation of RuvA-RuvB-Holliday junction DNA complex. In this study, we used ZMWs and counted the number of RuvBs binding to RuvA-Holliday junction DNA complex. Our data demonstrated that different nucleotide analogs increased the amount of Cy5-RuvBs binding to RuvA-Holliday junction DNA complex in the following order: no nucleotide, ADP, ATPγS, and mixture of ADP and ATPγS. These results suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA-RuvB-Holliday junction DNA complex formation.

ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues.

Elucidating the dynamic organization of nuclear RNA foci is important for understanding and manipulating these functional sites of gene expression in both physiological and pathological states. However, such studies have been difficult to establish in vivo as a result of the absence of suitable RNA imaging methods. Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks. Upon in vivo electroporation, exciton-controlled sequence-specific oligonucleotide probes revealed focally concentrated endogenous 28S rRNA and U3 snoRNA at nucleoli and poly(A) RNA at nuclear speckles. Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue. Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior.

Quantitative in vivo fluorescence cross-correlation analyses highlight the importance of competitive effects in the regulation of protein-protein interactions.

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction.

Real-time single-cell imaging of protein secretion.

Protein secretion, a key intercellular event for transducing cellular signals, is thought to be strictly regulated. However, secretion dynamics at the single-cell level have not yet been clarified because intercellular heterogeneity results in an averaging response from the bulk cell population. To address this issue, we developed a novel assay platform for real-time imaging of protein secretion at single-cell resolution by a sandwich immunoassay monitored by total internal reflection microscopy in sub-nanolitre-sized microwell arrays. Real-time secretion imaging on the platform at 1-min time intervals allowed successful detection of the heterogeneous onset time of nonclassical IL-1ß secretion from monocytes after external stimulation. The platform also helped in elucidating the chronological relationship between loss of membrane integrity and IL-1ß secretion. The study results indicate that this unique monitoring platform will serve as a new and powerful tool for analysing protein secretion dynamics with simultaneous monitoring of intracellular events by live-cell imaging.

In vivo fluorescent adenosine 5'-triphosphate (ATP) imaging of Drosophila melanogaster and Caenorhabditis elegans by using a genetically encoded fluorescent ATP biosensor optimized for low temperatures.

Adenosine 5'-triphosphate (ATP) is the major energy currency of all living organisms. Despite its important functions, the spatiotemporal dynamics of ATP levels inside living multicellular organisms is unclear. In this study, we modified the genetically encoded Förster resonance energy transfer (FRET)-based ATP biosensor ATeam to optimize its affinity at low temperatures. This new biosensor, AT1.03NL, detected ATP changes inside Drosophila S2 cells more sensitively than the original biosensor did, at 25 °C. By expressing AT1.03NL in Drosophila melanogaster and Caenorhabditis elegans, we succeeded in imaging the in vivo ATP dynamics of these model animals at single-cell resolution.

Single-molecule imaging of the oligomer formation of the nonhexameric Escherichia coli UvrD helicase.

Superfamily I helicases are nonhexameric helicases responsible for the unwinding of nucleic acids. However, whether they unwind DNA in the form of monomers or oligomers remains a controversy. In this study, we addressed this question using direct single-molecule fluorescence visualization of Escherichia coli UvrD, a superfamily I DNA helicase. We performed a photobleaching-step analysis of dye-labeled helicases and determined that the helicase is bound to 18-basepair (bp) double-stranded DNA (dsDNA) with a 3' single-stranded DNA (ssDNA) tail (12, 20, or 40 nt) in a dimeric or trimeric form in the absence of ATP. We also discovered through simultaneous visualization of association/dissociation of the helicase with/from DNA and the DNA unwinding dynamics of the helicase in the presence of ATP that these dimeric and trimeric forms are responsible for the unwinding of DNA. We can therefore propose a new kinetic scheme for the helicase-DNA interaction in which not only a dimeric helicase but also a trimeric helicase can unwind DNA. This is, to our knowledge, the first direct single-molecule nonhexameric helicase quantification study, and it strongly supports a model in which an oligomer is the active form of the helicase, which carries important implications for the DNA unwinding mechanism of all superfamily I helicases.

ParA-mediated plasmid partition driven by protein pattern self-organization.

DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low-copy plasmids, such as the plasmids P1 and F, employ a three-component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker-type ATPase, typically called ParA, which also binds non-specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP-driven patterning is involved in partition is unknown. We reconstituted and visualized ParA-mediated plasmid partition inside a DNA-carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB-stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion-ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid.

Direct observation of strand passage by DNA-topoisomerase and its limited processivity.

Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIα, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.51 ± 0.33 µm (11 ± 6 turns) of a braid was unlinked in a burst of reactions taking 8 ± 4 s, the unlinked length being essentially independent of the enzyme concentration between 0.25-37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being ~100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of ~10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break ~10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments.

Facilitated intracellular transport of TrkA by an interaction with nerve growth factor.

Intracellular transport of neurotrophin receptors together with neurotrophins is one of the key events of neurotrophin signaling for the growth and the survival of neurons. However, the involvement of neurotrophin signaling in the regulation of intracellular transport of neurotrophin receptors has been remained unclear. We visualized the behavior of TrkA, a receptor of nerve growth factor (NGF), by labeling with GFP in PC12 cells. We found remarkable changes of the behavior of TrkA-GFP upon the application of NGF. Before the application, only ~37% of the fluorescent dots of TrkA showed translocations along neurites of PC12 cells. After the application, number of the dots showing the directional movement increased to ~65%. The averaged velocities of the directional movement of TrkA-GFP dots became higher after the application of NGF. We tested the idea whether NGF binding accelerated the translocations of TrkA by simultaneously observing TrkA-GFP and fluorescently labeled NGF, Cy3.5-NGF. The velocity of TrkA-GFP dots associated with Cy3.5-NGF was remarkably higher than that of TrkA-GFP dots without Cy3.5-NGF. On the basis of these observations, we hypothesize that there is a signaling mechanism within a single vesicle that facilitates the intracellular transport of each vesicle containing the activated TrkA.

Real time monitoring of endogenous cytoplasmic mRNA using linear antisense 2'-O-methyl RNA probes in living cells.

Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

Strain through the neck linker ensures processive runs: a DNA-kinesin hybrid nanomachine study.

The motor protein kinesin has two heads and walks along microtubules processively using energy derived from ATP. However, how kinesin heads are coordinated to generate processive movement remains elusive. Here we created a hybrid nanomachine (DNA-kinesin) using DNA as the skeletal structure and kinesin as the functional module. Single molecule imaging of DNA-kinesin hybrid allowed us to evaluate the effects of both connect position of the heads (N, C-terminal or Mid position) and sub-nanometer changes in the distance between the two heads on motility. Our results show that although the native structure of kinesin is not essential for processive movement, it is the most efficient. Furthermore, forward bias by the power stroke of the neck linker, a 13-amino-acid chain positioned at the C-terminus of the head, and internal strain applied to the rear of the head through the neck linker are crucial for the processive movement. Results also show that the internal strain coordinates both heads to prevent simultaneous detachment from the microtubules. Thus, the inter-head coordination through the neck linker facilitates long-distance walking.

Visualization of RecA filaments and DNA by fluorescence microscopy.

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.