Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell's ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.
How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.
The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity.
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.