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Nanoscopic Imaging to Understand Synaptic Function
Published in Francesco S. Pavone, Shy Shoham, Handbook of Neurophotonics, 2020
Daniel Choquet, Anne-Sophie Hafner
Understanding synaptic function requires the determination of synaptic molecular components and their relative organization. As synapses are often considered as individual organelles, it is essential to their function that they sequester many of their key components. This is achieved in large part by scaffolding proteins. In fact, pre- and postsynapses express various types of scaffolding proteins providing binding sites for cytoplasmic and transmembrane proteins. On the presynaptic side, the main scaffolding proteins Bassoon, Munc13, and Piccolo have a pyramidal organization. They have been hypothesized to indirectly participate to drive synaptic vesicles to the presynaptic active zone. On the postsynaptic side, the anchoring of ionotropic glutamatergic receptors (i.e. AMPARs, NMDARs, Kainate receptors) in the postsynaptic density is essential to synaptic transmission. For instance, binding of the AMPAR auxiliary subunit stargazin to the main scaffolding protein of the postsynaptic density PSD-95 leads to AMPAR synaptic anchoring (Chen et al., 2000; Hafner et al., 2015; Opazo et al., 2010). Thus, on both sides of the synapse, scaffolding proteins play major roles that remain to be fully unraveled. With the aim of correlating synaptic structures with function, the laboratories of Xiaowei Zhuang and Catherine Dulac have developed a method and determined the relative position of ten key protein components of the glutamatergic synapse in the brain (Dani et al., 2010). By determining the position of each molecule with nanometer precision, STORM allows a high-resolution structural reconstruction of molecular assemblies. The authors systematically imaged a large number of synapses in various brain regions using Multicolor 3D STORM in order to map out the protein organization. Multicolor 3D STORM acquires its high resolution based on single-molecule imaging of photo-switchable fluorescent probes. As described above, the resolution of an optical system is based on its ability to differentiate the emission of two neighboring molecules. Hence, for molecules that give overlapping emission through a diffraction-limited system, STORM resolves these molecules by stochastically activating them at different times during image acquisition (Betzig et al., 2006; Hess et al., 2006; Rust et al., 2006). At any time, only a sparse optically resolvable subset of molecules is activated, allowing the images of these fluorophores to be readily separated from each other. As a result, the position of each individual fluorophore can be determined to a precision substantially beyond the diffraction-limited resolution. This procedure is repeated as many times as necessary to obtain the localizations of most fluorophores in the sample. A super-resolved image is subsequently reconstructed from these positions. For 3D STORM, a cylindrical lens is inserted in the detection path of the microscope to render the image of a single fluorophore elliptical. The lateral (x, y) and axial (z) coordinates of each fluorophore are determined from the centroid position and ellipticity of the image, respectively (Huang et al., 2008) (Figure 8.2).
Cambridge geneticists and the chromosome theory of inheritance: William Bateson, Leonard Doncaster and Reginald Punnett 1879–1940
Published in Annals of Science, 2022
Bateson did in fact begin research on cytology and genetics even before he appointed W. C. Frank Newton to the Innes staff later in 1922.131 Bateson and Caroline Pellew reported differences between parental sex in Pisum and the inheritance of rogue varieties in 1920. One of the Innes students Nesta Thomas counted the same haploid number of seven chromosomes in the type and rogue lines.132 In 1920 Bateson asked an Innes student Reginald Ruggles Gates to examine a variant lettuce cytologically. The haploid number was the same in stock and variant forms. But Gates observed synapsis of chromosomes in meiosis, first reported in animals, and interpreted his observation to coincide with Morgan’s model for synapsis, linkage and crossing over in Drosophila.133 One of Bateson’s early students R. P. Gregory had been breeding Primula sinesnsis for many seasons before he died in 1918. Bateson and the Innes staff reviewed the data cache on 18 inherited characters with the method of Bridges and defined two linkage groups. One had two characters; the other had four characters. One variant ‘moss curled’ was ‘cytologically distinct with an excess of chromosomes’.134 This report in 1923 certainly demonstrated Bateson’s recognition of the usefulness of the chromosome theory of heredity to analyze results of breeding plant species. Such observations also vitiated Bateson’s assertion that genetic segregation in plants was fundamentally different from that in animals.