Biophysics of high density nanometer regions extracted from super-resolution single particle trajectories: application to voltage-gated calcium channels and phospholipids.

The cellular membrane is very heterogenous and enriched with high-density regions forming microdomains, as revealed by single particle tracking experiments. However the organization of these regions remain unexplained. We determine here the biophysical properties of these regions, when described as a basin of attraction. We develop two methods to recover the dynamics and local potential wells (field of force and boundary). The first method is based on the local density of points distribution of trajectories, which differs inside and outside the wells. The second method focuses on recovering the drift field that is convergent inside wells and uses the transient field to determine the boundary. Finally, we apply these two methods to the distribution of trajectories recorded from voltage gated calcium channels and phospholipid anchored GFP in the cell membrane of hippocampal neurons and obtain the size and energy of high-density regions with a nanometer precision

Single particle trajectories reveal active endoplasmic reticulum luminal flow

The endoplasmic reticulum (ER), a network of membranous sheets and pipes, supports functions encompassing biogenesis of secretory proteins and delivery of functional solutes throughout the cell[1, 2]. Molecular mobility through the ER network enables these functionalities, but diffusion alone is not sufficient to explain luminal transport across supramicrometre distances. Understanding the ER structure–function relationship is critical in light of mutations in ER morphology-regulating proteins that give rise to neurodegenerative disorders[3, 4]. Here, super-resolution microscopy and analysis of single particle trajectories of ER luminal proteins revealed that the topological organization of the ER correlates with distinct trafficking modes of its luminal content: with a dominant diffusive component in tubular junctions and a fast flow component in tubules. Particle trajectory orientations resolved over time revealed an alternating current of the ER contents, while fast ER super-resolution identified energy-dependent tubule contraction events at specific points as a plausible mechanism for generating active ER luminal flow. The discovery of active flow in the ER has implications for timely ER content distribution throughout the cell, particularly important for cells with extensive ER-containing projections such as neurons.Wellcome Trust - 3-3249/Z/16/Z and 089703/Z/09/Z [Kaminski] UK Demential Research Institute [Avezov] Wellcome Trust - 200848/Z/16/Z, WT: UNS18966 [Ron] FRM Team Research Grant [Holcman] Engineering and Physical Sciences Research Council (EPSRC) - EP/L015889/1 and EP/H018301/1 [Kaminski] Medical Research Council (MRC) - MR/K015850/1 and MR/K02292X/1 [Kaminski

Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer–promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer–promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer–promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer–promoter relationships

Publisher Correction: Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD

Correction to: Nature Structural & Molecular Biology, published online 28 September 2023. In the version of the article initially published there were some errors in the affiliations. A. Ponjavic’s second and third affiliations have been corrected to Present address: School of Physics and Astronomy, University of Leeds, Leeds, UK and Present address: School of Food Science and Nutrition, University of Leeds, Leeds, UK; and L. Morey now has only two affiliations: Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain and Present address: Sylvester Comprehensive Cancer Center, Department of Human Genetics, University of Miami Miller School of Medicine, Biomedical Research Building, Miami, FL, USA. Additionally, the received date has been corrected to 14 April 2020 from 26 October 2021. These errors have been corrected in the HTML and PDF versions of the article

Synapsin 2a tetramerisation selectively controls the presynaptic nanoscale organisation of reserve synaptic vesicles

Abstract Neurotransmitter release relies on the regulated fusion of synaptic vesicles (SVs) that are tightly packed within the presynaptic bouton of neurons. The mechanism by which SVs are clustered at the presynapse, while preserving their ability to dynamically recycle to support neuronal communication, remains unknown. Synapsin 2a (Syn2a) tetramerization has been suggested as a potential clustering mechanism. Here, we used Dual-pulse sub-diffractional Tracking of Internalised Molecules (DsdTIM) to simultaneously track single SVs from the recycling and the reserve pools, in live hippocampal neurons. The reserve pool displays a lower presynaptic mobility compared to the recycling pool and is also present in the axons. Triple knockout of Synapsin 1-3 genes (SynTKO) increased the mobility of reserve pool SVs. Re-expression of wild-type Syn2a (Syn2aWT), but not the tetramerization-deficient mutant K337Q (Syn2aK337Q), fully rescued these effects. Single-particle tracking revealed that Syn2aK337QmEos3.1 exhibited altered activity-dependent presynaptic translocation and nanoclustering. Therefore, Syn2a tetramerization controls its own presynaptic nanoclustering and thereby contributes to the dynamic immobilisation of the SV reserve pool

Synapsin 2a tetramerisation selectively controls the presynaptic nanoscale organisation of reserve synaptic vesicles.

Acknowledgements: The super-resolution imaging was carried out at the Queensland Brain Institute’s (QBI’s) Advanced Microscopy Facility, led by Dr. Rumelo Amor and his team. We thank the QBI Information technology (IT) facility members and the entire QBI University of Queensland’s Biological Resources (UQBR) animal team for their ongoing technical assistance with our projects. We thank Dr. Alex McCann for critically appraising and editing the manuscript. We acknowledge Prof. Volker Haucke for providing the Synaptotagmin1-pHluorin plasmid, Prof. Vladislav Verkhusha for providing the pmTagBFP-N1 plasmid, Dr Sang-Ho Song for providing the Synapsin 2aWT-mEos3.1 and Synapsin 2aK337Q-mEos3.1 plasmids, Karen Chung for providing synapsin TKO mice and Melissa Yeow (Nanyang Technical University, Singapore) for dissecting the SynTKO hippocampi. This work was supported by an Australian Research Council Discovery Project grant (ARC) (DP230102278), a National Institute On Aging of the National Institutes of Health (NIH) grant R21AG080435, an ARC Linkage Infrastructure, Equipment, and Facilities grant (LE130100078), and a National Health and Medical Research Council (NHMRC) Fellowship (1155794) awarded to F.A.M., as well as grant OFIRG/MOH-000225-00 from the Singapore National Medical Research Council to G.J.A. R.M.M. was supported by a Clem Jones Foundation Fellowship, the University of Queensland (UQ) Research Stimulus Allocation 2 fellowship and the NHMRC Boosting Dementia Research Initiative. S.F.L. was supported by a Research Training Program (RTP) Scholarship. M.J. was supported by a UQ Amplify Fellowship, UQ Early Career Research Grant (2057309) and the Australian Research Council Discovery Early Career Researcher Award (DE190100565). D.H. was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 882673), and ANR AstroXcite.Funder: ARC Linkage Infrastructure, Equipment, and Facilities grant (LE130100078) National Health and Medical Research Council (NHMRC) Fellowship (1155794)Funder: European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 882673) and ANR AstroXcite.Neurotransmitter release relies on the regulated fusion of synaptic vesicles (SVs) that are tightly packed within the presynaptic bouton of neurons. The mechanism by which SVs are clustered at the presynapse, while preserving their ability to dynamically recycle to support neuronal communication, remains unknown. Synapsin 2a (Syn2a) tetramerization has been suggested as a potential clustering mechanism. Here, we used Dual-pulse sub-diffractional Tracking of Internalised Molecules (DsdTIM) to simultaneously track single SVs from the recycling and the reserve pools, in live hippocampal neurons. The reserve pool displays a lower presynaptic mobility compared to the recycling pool and is also present in the axons. Triple knockout of Synapsin 1-3 genes (SynTKO) increased the mobility of reserve pool SVs. Re-expression of wild-type Syn2a (Syn2aWT), but not the tetramerization-deficient mutant K337Q (Syn2aK337Q), fully rescued these effects. Single-particle tracking revealed that Syn2aK337QmEos3.1 exhibited altered activity-dependent presynaptic translocation and nanoclustering. Therefore, Syn2a tetramerization controls its own presynaptic nanoclustering and thereby contributes to the dynamic immobilisation of the SV reserve pool

Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

Acknowledgements: We thank T. Kretschmann for preparing the figures for publication, L. Lavis (Howard Hughes Medical Institute, Janelia Farm) for providing the JF549 dye, J. Wysocka (Stanford) for the Tbx3 constructs used for 2D enhancer tracking, A. Riddell for flow cytometry and the CSCI imaging (P. Humphreys and D. Clements) and DNA sequencing (M. Paramor and V. Murray) facilities. We thank K. Bowman, G. Brown and A. Crombie for preliminary computational analysis of NuRD-regulated genes and 2D enhancer tracking experiments, respectively. We thank the EU FP7 Integrated Project ‘4DCellFate’ (277899 E.D.L., B.D.H., I.B., C.S. and L.D.C.), the Medical Research Council (MR/P019471/1 E.D.L.) and the Wellcome Trust (206291/Z/17/Z E.D.L.) for program funding. We also thank the MRC (MR/R009759/1 B.D.H., and MR/M010082/1 E.D.L.), the Wellcome Trust (106115/Z/14/Z I.B. and 210701/Z/18/Z C.S.) and the Isaac Newton Trust (17.24(aa) B.D.H.) for project grant funding, and we thank the Wellcome Trust/MRC for core funding (203151/Z/16/Z) to the Cambridge Stem Cell Institute (including a starter grant to S.B.).To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships