Nat commun: new technology can observe single protein in synapse

September 26, 2019 BIOON / -- our brain contains millions of synapses - connections that transmit information between neurons There are hundreds of different proteins in these synapses The dysfunction of these proteins will lead to schizophrenia and autism Recently, researchers at MIT, as well as at Harvard and MIT, have devised a new way to quickly image these synapses at high resolution Using fluorescent nucleic acid probes, they can label and observe an infinite number of different proteins In this study, they imaged 12 proteins in cell samples containing thousands of synapses "Multiple imaging is important because even in the same brain, there are big differences between synapses and cells," said Mark bate, associate professor of bioengineering at MIT (image source: www Pixabay Com) researchers plan to use this technology in the next step to study synapses and what happens when they block the expression of genes related to specific diseases, hoping to develop new therapies that can reverse these effects Relevant research is published in the recent journal Nature communications Synaptic proteins have many functions Many of them contribute to the formation of synaptic scaffolds, which are related to the secretion of neurotransmitters and the processing of incoming signals Although synapses contain hundreds of these proteins, traditional fluorescence microscopy techniques are limited to imaging up to four proteins at a time To increase the amount of protein, the MIT team developed a new technology based on an existing method called DNA paint Using this method, originally designed by Ralf Jungmann of the Max Planck Institute of Biochemistry, the researchers used DNA antibody probes to tag proteins or other molecules of interest They then made each protein visible by delivering fluorescent DNA "oligonucleotides" that bind to DNA antibody probes The intrinsic affinity between DNA strands is very low, so they can be combined and unbound regularly, and the flickering fluorescence can be imaged with ultra-high resolution microscope However, it takes about half an hour to image each protein, so many proteins in large samples cannot be imaged Bathe and his colleagues set out to create a faster way for them to analyze a large number of samples in a short time To achieve this, they optimized the DNA probe so that it could use the so-called lock-in nucleic acid to bind more closely to DNA antibodies This produces a brighter signal, so imaging can be done faster, but with a slightly lower resolution "When we do 12 or 15 color treatments on a single neuron pore, the whole experiment takes an hour, compared with the previous technology that took an entire night," says bate The researchers used the technique to label 12 different proteins found in synapses, including scaffold proteins, cytoskeleton related proteins, and proteins known to label excitatory or inhibitory synapses One of the proteins they studied was Shank3, a scaffold protein linked to autism and schizophrenia By analyzing the levels of proteins in thousands of neurons, researchers were able to identify proteomes that often interact more frequently than other proteins and understand the differences in different synapses in the proteins they contain Such information can be used to help classify synapses into subtypes that may help reveal their functions The researchers also showed that they could measure changes in synaptic protein levels after tetrodotoxin (TTX) treatment of neurons, a compound that had previously been shown to enhance synaptic connections "With conventional immunofluorescence, information can usually be extracted from three or four targets in the same sample, but through our technology, we can expand the number to 12 different targets in the same sample We applied this method to examine what happens after TTX treatment for synaptic remodeling Our findings confirm previous work and reveal the synergistic up regulation of synaptic proteins after TTX treatment " Researchers are now using the technique, called prism, to study how knockout genes related to various diseases affect the structure and composition of synapses After sequencing the genomes of patients with autism and schizophrenia, hundreds of disease-related genetic variations have been found, but for most of them, scientists do not know how they cause diseases "Understanding how genetic variation affects the development of neurons in the brain and their synaptic structure and function is a huge challenge in the field of neuroscience," says bate Source of information: technical can image individual proteins within synapses source: Syuan Ming Guo, Remi Veneziano, Simon Gordon, Li Li, Eric Danielson, Karen Perez de Arce, Demian Park, Anthony B kulesa, eike Christian wamhoff, Paul C Blainey, Edward S Boyden, Jeffrey R Cottrell, Mark Bathe Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes Nature Communications , 2019; 10: 4377 DOI: 10.1038/s41467-019-12372-6