Recent developments of imaging techniques have enabled fluorescence microscopy to investigate the localization and dynamics of intracellular substances of interest even at the single-molecule level. However, such sensitive detection is often hampered by autofluorescence arising from endogenous molecules. Those unwanted signals are generally reduced by utilizing differences in either wavelength or fluorescence lifetime; nevertheless, extraction of the signal of interest is often insufficient, particularly for in vivo imaging. Here, we describe a potential method for the selective imaging of nitrogen-vacancy centers (NVCs) in nanodiamonds. This method is based on the property of NVCs that the fluorescence intensity sensitively depends on the ground state spin configuration which can be regulated by electron spin magnetic resonance. Because the NVC fluorescence exhibits neither photobleaching nor photoblinking, this protocol allowed us to conduct long-term tracking of a single nanodiamond in both Caenorhabditis elegans and mice, with excellent imaging contrast even in the presence of strong background autofluorescence.
Animals cope with environmental changes by altering behavioral strategy. Environmental information is generally received by sensory neurons in the neural circuit that generates behavior. However, although environmental temperature inevitably influences an animal's entire body, the mechanism of systemic temperature perception remains largely unknown. We show here that systemic temperature signaling induces a change in a memory-based behavior in C. elegans. During behavioral conditioning, non-neuronal cells as well as neuronal cells respond to cultivation temperature through a heat-shock transcription factor that drives newly identified gene expression dynamics. This systemic temperature signaling regulates thermosensory neurons non-cell-autonomously through the estrogen signaling pathway, producing thermotactic behavior. We provide a link between systemic environmental recognition and behavioral plasticity in the nervous system.
The rotation of an object cannot be fully tracked without understanding a set of three angles, namely, roll, pitch, and yaw. Tracking these angles as a three-degrees-of-freedom (3-DoF) rotation is a fundamental measurement, facilitating, for example, attitude control of a ship, image stabilization to reduce camera shake, and self-driving cars. Until now, however, there has been no method to track 3-DoF rotation to measure nanometer-scale dynamics in biomolecules and live cells. Here we show that 3-DoF rotation of biomolecules can be visualized via nitrogen-vacancy centers in a fluorescent nanodiamond using a tomographic vector magnetometry technique. We demonstrate application of the method to three different types of biological systems. First, we tracked the rotation of a single molecule of the motor protein F1-ATPase by attaching a nanodiamond to the γ-subunit. We visualized the 3-step rotation of the motor in 3D space and, moreover, a delay of ATP binding or ADP release step in the catalytic reaction. Second, we attached a nanodiamond to a membrane protein in live cells to report on cellular membrane dynamics, showing that 3D rotational motion of the membrane protein correlates with intracellular cytoskeletal density. Last, we used the method to track nonrandom motions in the intestine of Caenorhabditis elegans. Collectively, our findings show that the method can record nanoscale 3-DoF rotation in vitro, in cells, and even in vivo. 3-DoF rotation tracking introduces a new perspective on microscopic biological samples, revealing in greater detail the functional mechanisms due to nanoscale dynamics in molecules and cells.
Identification of the AFD neuron as the site of action of the CREB protein in Caenorhabditis elegans thermotaxisThe CREB protein, a key molecule for learning and memory, is expressed in nearly all neurons. This study shows that Caenorhabditis elegans CREB activity is required exclusively in the AFD thermosensory neuron for thermotactic behavior.
Understanding physical rules underlying collective motions requires perturbation of controllable parameters in self-propelled particles. However, controlling parameters in animals is generally not easy, which makes collective behaviours of animals elusive. Here, we report an experimental system in which a conventional model animal, Caenorhabditis elegans, collectively forms dynamical networks of bundle-shaped aggregates. We investigate the dependence of our experimental system on various extrinsic parameters (material of substrate, ambient humidity and density of worms). Taking advantage of well-established C. elegans genetics, we also control intrinsic parameters (genetically determined motility) by mutations and by forced neural activation via optogenetics. Furthermore, we develop a minimal agent-based model that reproduces the dynamical network formation and its dependence on the parameters, suggesting that the key factors are alignment of worms after collision and smooth turning. Our findings imply that the concepts of active matter physics may help us to understand biological functions of animal groups.
Metabotropic glutamate receptors (mGluRs) function as neuronal G-protein-coupled receptors and this requires efficient membrane targeting through associations with cytoplasmic proteins. However, the molecular mechanism regulating mGluR cell-surface trafficking remains unknown. We report here that mGluR trafficking is controlled by the autoregulatory assembly of a scaffold protein Tamalin. In the absence of mGluR, Tamalin selfassembles into autoinhibited conformations, through its PDZ domain and C-terminal intrinsic ligand motif. X-ray crystallographic analyses visualized integral parts of the oligomeric self-assemblies of Tamalin, which require not only the novel hydrophobic dimerization interface but also canonical and noncanonical PDZ/ligand autoinhibitory interactions. The mGluR cytoplasmic region can competitively bind to Tamalin at a higher concentration, disrupting weak inhibitory interactions. The atomic view of mGluR association suggests that this rearrangement is dominated by electrostatic attraction and repulsion. We also observed in mammalian cells that the association liberates the intrinsic ligand toward a motor protein receptor, thereby facilitating mGluR cell-surface trafficking. Our study suggests a novel regulatory mechanism of the PDZ domain, by which Tamalin switches between the trafficking-inhibited and -active forms depending on mGluR association.
A major goal of neuroscience studies is to identify the neurons and molecules responsible for memory. Mechanosensory habituation in Caenorhabditis elegans is a simple form of learning and memory, in which a circuit of several sensory neurons and interneurons governs behavior. However, despite the usefulness of this paradigm, there are hardly any systems for rapid and accurate behavioral genetic analysis. Here, we developed a multiplexed optical system to genetically analyze C. elegans mechanosensory habituation, and identified two interneurons involved in memory formation. The system automatically trains large populations of animals and simultaneously quantifies the behaviors of various strains by optically discriminating between transgenic and nontransgenic animals. Biochemical and cell-specific behavioral analyses indicated that phosphorylation of cyclic AMP response element-binding protein (CREB), a factor known to regulate memory allocation, was facilitated during training and this phosphorylation in AVA and AVD interneurons was required for habituation. These interneurons are a potential target for cell-specific exploration of the molecular substrates of memory.Caenorhabditis elegans | mechanosensory habituation | memory | optical quantification | neural circuit I dentification of the physical substrates of memory in the brain, namely neurons and molecules encoding memory, is a major challenge in neuroscience. Recent studies have revealed that the transcription factor cAMP response element-binding protein (CREB) (1) is crucial for determining which neurons in the lateral amygdala participate in encoding an auditory fear memory, and that neurons with higher CREB activity are preferentially allocated into a unique memory trace (memory engram) (2-6). Thus, it has been generally thought through the accumulated weight of evidence that memory is represented by a specific population of neurons in the brain that forms memory engrams: only a portion of eligible neurons are recruited into a specific memory. Although this CREB-dependent memory allocation model is established (5), the identification of specific neurons supporting a given memory remains a long-standing challenge. Moreover, the extreme complexity of nervous systems in model animals still hampers cell-specific genetic analyses; therefore, even if neurons encoding memory could be identified, it would be difficult to address the subsequent question of how these neurons store memory at the molecular level in situ.The nematode Caenorhabditis elegans is a genetically tractable model organism with a neural circuit composed of only 302 neurons. Moreover, the wiring of this nervous system has been completely determined. These unique features of the C. elegans nervous system have proven to be advantageous for various behavioral experiments, such as genetic rescue experiments, ectopic expression analysis, overexpression analysis, and RNA interference-mediated gene disruption, because all of these transgenic studies can be conducted in situ in a cell-specific manner...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.