9 research outputs found
Asynchronous release sites align with NMDA receptors in mouse hippocampal synapses
© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Li, S., Raychaudhuri, S., Lee, S. A., Brockmann, M. M., Wang, J., Kusick, G., Prater, C., Syed, S., Falahati, H., Ramos, R., Bartol, T. M., Hosy, E., & Watanabe, S. Asynchronous release sites align with NMDA receptors in mouse hippocampal synapses. Nature Communications, 12(1), (2021): 677, https://doi.org/10.1038/s41467-021-21004-x.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.e also thank the Marine Biological Laboratory and their Neurobiology course for supporting the initial set of experiments (course supported by National Institutes of Health grant R25NS063307). S.W. and this work were supported by start-up funds from the Johns Hopkins University School of Medicine, Johns Hopkins Discovery funds, and the National Science Foundation (1727260), the National Institutes of Health (1DP2 NS111133-01 and 1R01 NS105810-01A1) awarded to S.W. S.W. is an Alfred P. Sloan fellow, McKnight Foundation Scholar, and Klingenstein and Simons Foundation scholar. G.K. was supported by a grant from the National Institutes of Health to the Biochemistry, Cellular and Molecular Biology Program of the Johns Hopkins University School of Medicine (T32 GM007445) and is a National Science Foundation Graduate Research Fellow (2016217537). E.H. and T.M.B. are supported by CRCNS-NIH-ANR grant AMPAR-T. The EM ICE high-pressure freezer was purchased partly with funds from an equipment grant from the National Institutes of Health (S10RR026445) awarded to Scot C Kuo
A Mechanistic Study of Complex Biological Phenomena: From Membrane-less Organelle Assembly to Cell Cycle Regulation
This work is an effort to disentangle different mechanisms that drive complex biological phenomena, from the assembly of membrane-less organelles and their spatiotemporal regulation to the regulation of cell cycle events. To do so, we have taken advantage of the differential sensitivity of distinct mechanisms to temperature by utilizing a microfluidics-based temperature assay, in combination with quantitative live imaging and genetic approaches, in early Drosophila embryos. Our results indicate that the quintessential membrane-less organelle, the nucleolus, forms through two independent mechanisms, namely active assembly and thermodynamically-driven phase separation. These two independent mechanisms are coordinated by rDNA to ensure that the organelle forms at the right time and the right place. Transcription of rRNA spatiotemporally regulates the phase separation by overcoming the initial nucleation step in the formation of these assemblies. rDNA is also necessary for the formation of active assemblies, and therefore can be considered as a common coordinator of the active and thermodynamic mechanisms of the nucleolus assembly. Finally, by using a similar temperature-base assay we are able uncouple various cell cycle events and show that while entry into prophase can occur independent of CDK1 activity, initiation of prometaphase always coincides with the activation of CDK1
De novo nucleolus formation in fly embryos
First time nucleolus formation is depicted wild type early fly embryos. Nucleolus is absent in early embryos, and forms for the first time at nuclear cycle 13 (bright foci in the nuclei). tagRFP-Fibrillarin is used as the marker of the nucleolus. Nuclei at nuclear cycles 10 to 14 are shown. Images are obtained using Leica SP5, and are maximum projected, 82 x 41 microns
Nuclear divisions in early fly embryos
Nuclear division is depicted in wild type fly embryos at nuclear cycles 10-14. H2Av-RFP marks the chromosomes and is shown in magenta, and Jupiter-GFP depicts the microtubules and is shown in yellow. Images were obtained every 10s with Leica SP5 confocal microscope. Images are maximum projected, and 82 x 41 microns in size
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Nucleation by rRNA Dictates the Precision of Nucleolus Assembly.
Membrane-less organelles are intracellular compartments specialized to carry out specific cellular
functions. There is growing evidence supporting the possibility that such organelles form as a new
phase, separating from cytoplasm or nucleoplasm. However, a main challenge to such phase
separation models is that the initial assembly, or nucleation, of the new phase is typically a highly
stochastic process, and does not allow for the spatiotemporal precision observed in biological
systems. Here we investigate the initial assembly of the nucleolus, a membrane-less organelle
involved in different cellular functions including ribosomal biogenesis. We demonstrate that the
nucleolus formation is precisely timed in D. melanogaster embryos and follows the transcription
of rRNA. We provide evidence that transcription of rRNA is necessary for overcoming the highly
stochastic nucleation step in the formation of the nucleolus, through a seeding mechanism. In the
absence of rDNA, the nucleolar proteins studied are able to form high concentration assemblies.
However, unlike the nucleolus, these assemblies are highly variable in number, location and time
at which they form. In addition, quantitative study of the changes in the nucleoplasmic
concentration and distribution of these nucleolar proteins in the wild-type embryos is consistent
with the role of rRNA in seeding the nucleolus formation
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Mitosis-associated repression in development
Transcriptional repression is a pervasive feature of animal development. Here, we employ live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. We propose that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail
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Mitosis-associated repression in development
Transcriptional repression is a pervasive feature of animal development. Here, we employ live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. We propose that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail
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Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells.
Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28°C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes