@article {69579, title = {Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking.}, journal = {Elife}, volume = {13}, year = {2024}, month = {2024 Aug 15}, abstract = {

AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.

}, keywords = {Actins, Animals, Cells, Cultured, Dendrites, Exocytosis, Hippocampus, Neuronal Plasticity, Neurons, Polymerization, Protein Transport, Rats, Receptors, AMPA}, issn = {2050-084X}, doi = {10.7554/eLife.80622}, url = {https://doi.org/10.7554/eLife.80622}, author = {Wong, Victor C and Houlihan, Patrick R and Liu, Hui and Walpita, Deepika and DeSantis, Michael C and Liu, Zhe and O{\textquoteright}Shea, Erin K} } @article {68146, title = {Direct Observation of Compartment-Specific Localization and Dynamics of Voltage-Gated Sodium Channels.}, journal = {Journal of Neuroscience}, year = {2022}, month = {2022 Jun 06}, abstract = {

Brain enriched voltage-gated sodium channel (VGSC) Na1.2 and Na1.6 are critical for electrical signaling in the central nervous system. Previous studies have extensively characterized cell-type specific expression and electrophysiological properties of these two VGSCs and how their differences contribute to fine-tuning of neuronal excitability. However, due to lack of reliable labeling and imaging methods, the sub-cellular localization and dynamics of these homologous Na1.2 and Na1.6 channels remain understudied. To overcome this challenge, we combined genome editing, super-resolution and live-cell single molecule imaging to probe subcellular composition, relative abundances and trafficking dynamics of Na1.2 and Na1.6 in cultured mouse and rat neurons and in male and female mouse brain. We discovered a previously uncharacterized trafficking pathway that targets Na1.2 to the distal axon of unmyelinated neurons. This pathway utilizes distinct signals residing in the intracellular loop 1 (ICL1) between transmembrane domain I and II to suppress the retention of Na1.2 in the axon initial segment (AIS) and facilitate its membrane loading at the distal axon. As mouse pyramidal neurons undergo myelination, Na1.2 is gradually excluded from the distal axon as Na1.6 becomes the dominant VGSC in the axon initial segment and nodes of Ranvier. In addition, we revealed exquisite developmental regulation of Na1.2 and Na1.6 localizations in the axon initial segment and dendrites, clarifying the molecular identity of sodium channels in these subcellular compartments. Together, these results unveiled compartment-specific localizations and trafficking mechanisms for VGSCs, which could be regulated separately to modulate membrane excitability in the brain.Direct observation of endogenous voltage-gated sodium channels reveals a previously uncharacterized distal axon targeting mechanism and the molecular identity of sodium channels in distinct subcellular compartments.

}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.0086-22.2022}, author = {Liu, Hui and Wang, Hong-Gang and Pitt, Geoffrey S and Liu, Zhe J} } @article {67735, title = {A dominant-negative SOX18 mutant disrupts multiple regulatory layers essential to transcription factor activity.}, journal = {Nucleic Acids Research}, year = {2021}, month = {2021 Sep 27}, abstract = {

Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function.

}, issn = {1362-4962}, doi = {10.1093/nar/gkab820}, author = {McCann, Alex J and Lou, Jieqiong and Moustaqil, Mehdi and Graus, Matthew S and Blum, Ailisa and Fontaine, Frank and Liu, Hui and Luu, Winnie and Rudolffi-Soto, Paulina and Koopman, Peter and Sierecki, Emma and Gambin, Yann and Meunier, Fr{\'e}d{\'e}ric A and Liu, Zhe and Hinde, Elizabeth and Francois, Mathias} } @article {67746, title = {A dominant-negative SOX18 mutant disrupts multiple regulatory layers essential to transcription factor activity.}, journal = {Nucleic Acids Research}, year = {2021}, month = {2021 Sep 27}, abstract = {

Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function.

}, issn = {1362-4962}, doi = {10.1093/nar/gkab820}, author = {McCann, Alex J and Lou, Jieqiong and Moustaqil, Mehdi and Graus, Matthew S and Blum, Ailisa and Fontaine, Frank and Liu, Hui and Luu, Winnie and Rudolffi-Soto, Paulina and Koopman, Peter and Sierecki, Emma and Gambin, Yann and Meunier, Fr{\'e}d{\'e}ric A and Liu, Zhe and Hinde, Elizabeth and Francois, Mathias} } @article {65658, title = {Microdomains form on the luminal face of neuronal extracellular vesicle membranes.}, journal = {Scientific Reports}, volume = {10}, year = {2020}, month = {2020 Jul 20}, pages = {11953}, abstract = {

Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology.

}, issn = {2045-2322}, doi = {10.1038/s41598-020-68436-x}, author = {Matthies, Doreen and Lee, Nathanael Y J and Gatera, Ian and Pasolli, H Amalia and Zhao, Xiaowei and Liu, Hui and Walpita, Deepika and Liu, Zhe and Yu, Zhiheng and Ioannou, Maria S} } @article {65081, title = {Bright and photostable chemigenetic indicators for extended in vivo voltage imaging.}, journal = {Science}, volume = {365}, year = {2019}, month = {2019 Aug 13}, pages = {699-704}, abstract = {

Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, "Voltron", that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 min of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

}, issn = {1095-9203}, doi = {10.1126/science.aav6416}, author = {Abdelfattah, Ahmed S and Kawashima, Takashi and Singh, Amrita and Novak, Ondrej and Liu, Hui and Shuai, Yichun and Huang, Yi-Chieh and Campagnola, Luke and Seeman, Stephanie C and Yu, Jianing and Zheng, Jihong and Grimm, Jonathan B and Patel, Ronak and Friedrich, Johannes and Mensh, Brett D and Paninski, Liam and Macklin, John J and Murphy, Gabe J and Podgorski, Kaspar and Lin, Bei-Jung and Chen, Tsai-Wen and Turner, Glenn C and Liu, Zhe and Koyama, Minoru and Svoboda, Karel and Ahrens, Misha B and Lavis, Luke D and Schreiter, Eric R} } @article {65067, title = {Neuron-astrocyte metabolic coupling protects against activity-induced fatty acid toxicity.}, journal = {Cell}, volume = {177}, year = {2019}, month = {2019 May 30}, pages = {1522-1535.e14}, abstract = {

Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial β-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2019.04.001}, author = {Ioannou, Maria S and Jackson, Jesse and Sheu, Shu-Hsien and Chang, Chi-Lun and Weigel, Aubrey V and Liu, Hui and Pasolli, H Amalia and Xu, C Shan and Pang, Song and Matthies, Doreen and Hess, Harald F and Lippincott-Schwartz, Jennifer and Liu, Zhe} } @article {48346, title = {Stochastic protein labeling enables long-term single molecule observation in vivo.}, journal = {bioRxiv}, year = {2017}, month = {2017 Mar 13}, abstract = {Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control protein copy number in a cell. This system has a dynamic titration range of more than 10,000 fold, enabling sparse labeling of proteins expressed at widely different levels. Combined with fluorescence signal amplification tags, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in live zebrafish. We found that axon initial segment utilizes a waterfall mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor Sox2 samples clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements for a quantitative understanding of complex control of molecular dynamics in vivo.}, doi = {10.1101/116186}, author = {Liu, Hui and Dong, Peng and Ioannou, Maria S and Li, Li and Shea, Jamien and Pasolli, H Amalia and Grimm, Jonathan B and Rivlin, Patricia K and Lavis, Luke D and Koyama, Minoru and Liu, Zhe} } @article {48752, title = {Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {115}, year = {2017}, month = {2017 Jan 09}, pages = {343-8}, abstract = {

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of \>10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact\ zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics\ in vivo.

}, doi = {10.1073/pnas.1713895115}, author = {Liu, Hui and Dong, Peng and Ioannou, Maria S and Li, Li and Shea, Jamien and Pasolli, H Amalia and Grimm, Jonathan B and Rivlin, Patricia K and Lavis, Luke D and Koyama, Minoru and Liu, Zhe} } @article {47741, title = {Real-time imaging of Huntingtin aggregates diverting target search and gene transcription.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 Aug 03}, abstract = {

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells - 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form {\textquoteright}sticky{\textquoteright} decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells.

}, issn = {2050-084X}, doi = {10.7554/eLife.17056}, author = {Li, Li and Liu, Hui and Dong, Peng and Li, Dong and Legant, Wesley R and Grimm, Jonathan B and Lavis, Luke D and Betzig, Eric and Tjian, Robert and Liu, Zhe} }