Neurotransmission in complex animals depends on a choir of functionally distinct synapses releasing neurotransmitters in a highly coordinated manner. During synaptic signaling, vesicles fuse with the ...plasma membrane to release their contents. The rate of vesicle fusion is high and can exceed the rate at which synaptic vesicles can be re-supplied by distant sources. Thus, local compensatory endocytosis is needed to replenish the synaptic vesicle pools. Over the last four decades, various experimental methods and model systems have been used to study the cellular and molecular mechanisms underlying synaptic vesicle cycle. Clathrin-mediated endocytosis is thought to be the predominant mechanism for synaptic vesicle recycling. However, recent studies suggest significant contribution from other modes of endocytosis, including fast compensatory endocytosis, activity-dependent bulk endocytosis, ultrafast endocytosis, as well as kiss-and-run. Currently, it is not clear whether a universal model of vesicle recycling exist for all types of synapses. It is possible that each synapse type employs a particular mode of endocytosis. Alternatively, multiple modes of endocytosis operate at the same synapse, and the synapse toggles between different modes depending on its activity level. Here we compile review and research articles based on well-characterized model systems: frog neuromuscular junctions,
neuromuscular junctions,
neuromuscular junctions, lamprey reticulospinal giant axons, goldfish retinal ribbon synapses, the calyx of Held, and rodent hippocampal synapses. We will compare these systems in terms of their known modes and kinetics of synaptic vesicle endocytosis, as well as the underlying molecular machineries. We will also provide the future development of this field.
Electron microscopy depicts subcellular structures at synapses exquisitely but only captures static images. To visualize membrane dynamics, we have developed a novel technique, called ...flash-and-freeze, which induces neuronal activity with a flash of light and captures the membrane dynamics by rapid freezing. For characterizing membrane movements during synaptic transmission, a light-sensitive cation channel, channelrhodopsin, is heterologously expressed in mouse hippocampal neurons or in Caenorhabditis elegans motor neurons. A brief pulse of blue light activates channelrhodopsin and induces an action potential, leading to synaptic transmission. Following the light stimulation, neurons are frozen at different time intervals ranging from 10 ms to 20 s. Electron micrographs are then acquired from each time point to visualize the morphological changes. Using this approach, we have characterized a novel form of endocytosis, ultrafast endocytosis, which rapidly removes excess membrane added to the surface during neurotransmission. The flash-and-freeze approach can be adapted to study other cellular phenomena that can be induced by light-sensitive genetic or pharmacological tools.
Abstract
Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their ...physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we design a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid defunctionalization of organelles via remodeling of the membrane phospholipids. In particular, we identify catalytically active PLAAT truncates with minimal unfavorable characteristics. Chemically-induced translocation of the optimized PLAAT to the mitochondria surface results in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapt the molecular tool in peroxisomes, and observe leakage of matrix-resident functional proteins. The technique is compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should prove useful in studying organelle biology of diverse contexts.
To address the open and diverse situation of future optical networks, it is necessary to find a methodology to accurately estimate the value of a target quantity in an optical performance monitor ...(OPM) depending on the high-level monitoring objectives declared by the network operator. Using machine learning techniques partially enables a trainable OPM; however, it still requires the feature selection before the learning process. Here, we show the OPM that uses a convolutional neural network (CNN) with a digital coherent receiver to deal with the abundance of training data required for convergence and pre-processing of input data by human engineers needed for feature (representation) extraction. To proof a concept of the OPM based on CNN, we experimentally demonstrate that a CNN can learn an accurate optical signal-to-noise-ratio (OSNR) estimation functionality from asynchronously sampled data right after intradyne coherent detection. We evaluate bias errors and standard deviations of a CNN-based OSNR estimator for six combinations of modulation formats and symbol rates and confirm that the proposed OSNR estimator can provide accurate estimation results (<0.4 dB bias errors and standard deviations). Additionally, we investigate filters in the trained CNN to reveal what the CNN learned in the training phase. This is a valuable step toward designing autonomous "self-driving" optical networks.
The high in vivo stability of 2,2-dihydroxymethyl-3-18Ffluoropropyl-2-nitroimidazole (18FDiFA) prompted us to evaluate neopentyl as a scaffold to prepare a radiotheranostic system with radioiodine ...and astatine. Three DiFA analogues with one, two, or without a hydroxyl group were synthesized. While all 125I-labeled compounds remained stable against nucleophilic substitution, only a 125I-labeled neopentyl glycol was stable against cytochrome P450 (CYP)-mediated metabolism and showed high stability against in vivo deiodination. 211At-labeled neopentyl glycol also remained stable against both nucleophilic substitution and CYP-mediated metabolism. 211At-labeled neopentyl glycol showed the biodistribution profiles similar to those of its radioiodinated counterpart in contrast to the 125I/211At-labeled benzoate pair. The urine analyses confirmed that 211At-labeled neopentyl glycol was excreted in the urine as a glucuronide conjugate with the absence of free 211AtAt–. These findings indicate that neopentyl glycol would constitute a promising scaffold to prepare a radiotheranostic system with radioiodine and 211At.
Some protein components of intracellular non-membrane-bound entities, such as RNA granules, are known to form hydrogels in vitro. The physico-chemical properties and functional role of these ...intracellular hydrogels are difficult to study, primarily due to technical challenges in probing these materials in situ. Here, we present iPOLYMER, a strategy for a rapid induction of protein-based hydrogels inside living cells that explores the chemically inducible dimerization paradigm. Biochemical and biophysical characterizations aided by computational modelling show that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that acts as a size-dependent molecular sieve. We functionalize these polymers with RNA-binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. These results show that iPOLYMER can be used to synthetically reconstitute the nucleation of biologically functional entities, including RNA granules in intact cells.
Many long-lived organisms, including humans, can regenerate some adult tissues lost to physical injury or disease. Much of the previous research on mechanisms of regeneration has focused on adult ...stem cells, which give rise to new tissue necessary for the replacement of missing body parts. Here we report that apoptosis of differentiated cells complements stem cell division during regeneration in the planarian
Schmidtea mediterranea. Specifically, we developed a whole-mount TUNEL assay that allowed us to document two dramatic increases in the rate of apoptosis following amputation—an initial localized response near the wound site and a subsequent systemic response that varies in magnitude depending on the type of fragment examined. The latter cell death response can be induced in uninjured organs, occurs in the absence of planarian stem cells, and can also be triggered by prolonged starvation. Taken together, our results implicate apoptosis in the restoration of proper anatomical scale and proportion through remodeling of existing tissues. We also report results from initial mechanistic studies of apoptosis in planarians, which revealed that a
S. mediterranea homolog of the antiapoptotic gene
BCL2 is required for cell survival in adult animals. We propose that apoptosis is a central mechanism working in concert with stem cell division to restore anatomical form and function during metazoan regeneration.
Accurate optical monitors are critical for automating operations of fiber-optic networks. Deep neural network (DNN) based optical monitors have been investigated as accurate optical monitors to ...leverage a large amount of data obtained from fiber-optic networks. Although DNN-based optical monitors have been trained and tested to ensure the given accuracy criteria, this does not ensure sufficient accuracy under unexpected conditions, that is, out of test conditions, e.g., a newly developed modulation format that is not included in the test dataset. Thus, it is necessary to prepare a monitor to assess the current accuracy of a DNN-based optical monitor's output for robust automation of networks. We present a DNN-based optical monitor that simultaneously outputs an optical signal-to-noise ratio and its uncertainty information using a dropout method at the inference phase. This monitor was evaluated in cases in which the DNNs were trained with either a limited number of records or partially missing records in a training dataset. The proposed monitor successfully informed that own output has large uncertainties due to a limited amount of training data or a missing part in training dataset. Additionally, to improve an accuracy of estimated uncertainty, the number of partial neural networks by dropout at the inference phase was optimized. This is a valuable step toward designing robust "self-driving" optical networks.
A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal ...locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection protein in electron micrographs.
Neurotransmitter is released synchronously and asynchronously following an action potential. Our recent study indicates that the release sites of these two phases are segregated within an active ...zone, with asynchronous release sites enriched near the center in mouse hippocampal synapses. Here we demonstrate that synchronous and asynchronous release sites are aligned with AMPA receptor and NMDA receptor clusters, respectively. Computational simulations indicate that this spatial and temporal arrangement of release can lead to maximal membrane depolarization through AMPA receptors, alleviating the pore-blocking magnesium leading to greater activation of NMDA receptors. Together, these results suggest that release sites are likely organized to activate NMDA receptors efficiently.
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