Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling.

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic range of ∼10,000-fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by two orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially restricted subnuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

Correction to: A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.

A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

BACKGROUND: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. RESULTS: Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. CONCLUSION: K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.

Circadian regulation of locomotion, respiration, and arousability in adult blacklegged ticks (Ixodes scapularis).

The blacklegged tick, Ixodes scapularis, is an ectoparasitic arachnid and vector for infectious diseases, including Lyme borreliosis. Here, we investigate the diurnal activity and respiration of wild-caught and lab-reared adult ticks with long-term video recording, multi-animal tracking and high-resolution respirometry. We find male and female ticks are in a more active, more arousable state during circadian night. We find respiration is augmented by light, with dark onset triggering more frequent bouts of discontinuous gas exchange and a higher overall volume of CO2 respired. Observed inactivity during the day meets the criteria of sleep: homeostatic in nature, rapidly reversible, a characteristic pose, and reduced arousal threshold. Our findings indicate that blacklegged ticks are in a distinct, heightened state of activity and arousability during night and in dark, suggesting this period may carry higher risk for tick bites and subsequent contraction of tick-borne diseases.

Recovery from cold-induced reproductive dormancy is regulated by temperature-dependent AstC signaling.

Animals have evolved a variety of behaviors to cope with adverse environmental conditions. Similar to other insects, the fly, Drosophila melanogaster, responds to sustained cold by reducing its metabolic rate and arresting its reproduction. Here, we show that a subset of dorsal neurons (DN3s) that express the neuropeptide allatostatin C (AstC) facilitates recovery from cold-induced reproductive dormancy. The activity of AstC-expressing DN3s, as well as AstC peptide levels, are suppressed by cold. Cold temperature also impacts AstC levels in other Drosophila species and mosquitoes, Aedes aegypti, and Anopheles stephensi. The stimulatory effect of AstC on egg production is mediated by cholinergic AstC-R2 neurons. Our results demonstrate that DN3s coordinate female reproductive capacity with environmental temperature via AstC signaling. AstC/AstC-R2 is conserved across many insect species and their role in regulating female reproductive capacity makes them an ideal target for controlling the population of agricultural pests and human disease vectors.

Spatially resolved measurements of ballistic and total transmission in microscale tissue samples from 450†nm to 1624†nm.

We built a simple and versatile setup to measure tissue ballistic and total transmission with customizable wavelength range, spatial resolution, and sample sizes. We performed ballistic transmission and total transmission measurements of overlying structures from biological samples ex vivo. We obtained spatially resolved transmission maps to reveal transmission heterogeneity from five microscale tissue samples: Danionella skin, mouse skull bone, mosquito cuticle, wasp cuticle, and rat dura over a wide spectral range from 450 nm to 1624 nm at a spatial resolution of ∼25 µm for ballistic transmission measurements and ∼50 µm for total transmission measurements. We expect our method can be straightforwardly applied to measuring the transmission of other samples. The measurement results will be valuable for multiphoton microscopy. The total transmission of a sample is important for the collection of multiphoton excited fluorescence and the assessment of laser-induced sample heating. The ballistic transmission determines the excitation power at the focus and hence the fluorescence signal generation. Therefore, knowledge of ballistic transmission, total transmission, and transmission heterogeneity of overlying structures of animals and organs are essential to determine the optimal excitation wavelength and fluorophores for non-invasive multiphoton microscopy.

Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle.

We developed a multiphoton imaging method to capture neural structure and activity in behaving flies through the intact cuticle. Our measurements showed that the fly head cuticle has surprisingly high transmission at wavelengths >900nm, and the difficulty of through-cuticle imaging is due to the air sacs and/or fat tissue underneath the head cuticle. By compressing or removing the air sacs, we performed multiphoton imaging of the fly brain through the intact cuticle. Our anatomical and functional imaging results show that 2- and 3-photon imaging are comparable in superficial regions such as the mushroom body, but 3-photon imaging is superior in deeper regions such as the central complex and beyond. We further demonstrated 2-photon through-cuticle functional imaging of odor-evoked calcium responses from the mushroom body γ-lobes in behaving flies short term and long term. The through-cuticle imaging method developed here extends the time limits of in vivo imaging in flies and opens new ways to capture neural structure and activity from the fly brain.

In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.

Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1 µM to 10 mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed ∼3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms.

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.