@article {69709, title = {In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors}, journal = {bioRxiv}, year = {2024}, month = {12/2024}, abstract = {

Neurochemical signals like dopamine (DA) play a crucial role in a variety of brain functions through intricate interactions with other neuromodulators and intracellular signaling pathways. However, studying these complex networks has been hindered by the challenge of detecting multiple neurochemicals in vivo simultaneously. To overcome this limitation, we developed a single-protein chemigenetic DA sensor, HaloDA1.0, which combines a cpHaloTag-chemical dye approach with the G protein-coupled receptor activation-based (GRAB) strategy, providing high sensitivity for DA, sub-second response kinetics, and an extensive spectral range from far-red to near-infrared. When used together with existing green and red fluorescent neuromodulator sensors, Ca2+ indicators, cAMP sensors, and optogenetic tools, HaloDA1.0 provides high versatility for multiplex imaging in cultured neurons, brain slices, and behaving animals, facilitating in-depth studies of dynamic neurochemical networks.Competing Interest StatementThe authors have declared no competing interest.

}, doi = {10.1101/2024.12.22.629999}, url = {https://www.biorxiv.org/content/early/2024/12/23/2024.12.22.629999}, author = {Zheng, Yu and Cai, Ruyi and Wang, Kui and Zhang, Junwei and Zhuo, Yizhou and Dong, Hui and Zhang, Yuqi and Wang, Yifan and Deng, Fei and Ji, En and Cui, Yiwen and Fang, Shilin and Zhang, Xinxin and Zhang, Kecheng and Wang, Jinxu and Li, Guochuan and Miao, Xiaolei and Wang, Zhenghua and Yang, Yuqing and Li, Shaochuang and Grimm, Jonathan and Johnsson, Kai and Schreiter, Eric and Lavis, Luke and Chen, Zhixing and Mu, Yu and Li, Yulong} } @article {68935, title = {Lysosomal release of amino acids at ER three-way junctions regulates transmembrane and secretory protein mRNA translation.}, journal = {bioRxiv}, year = {2023}, month = {2023 Aug 03}, abstract = {

One-third of the mammalian proteome is comprised of transmembrane and secretory proteins that are synthesized on endoplasmic reticulum (ER). Here, we investigate the spatial distribution and regulation of mRNAs encoding these membrane and secretory proteins (termed "secretome" mRNAs) through live cell, single molecule tracking to directly monitor the position and translation states of secretome mRNAs on ER and their relationship to other organelles. Notably, translation of secretome mRNAs occurred preferentially near lysosomes on ER marked by the ER junction-associated protein, Lunapark. Knockdown of Lunapark reduced the extent of secretome mRNA translation without affecting translation of other mRNAs. Less secretome mRNA translation also occurred when lysosome function was perturbed by raising lysosomal pH or inhibiting lysosomal proteases. Secretome mRNA translation near lysosomes was enhanced during amino acid deprivation. Addition of the integrated stress response inhibitor, ISRIB, reversed the translation inhibition seen in Lunapark knockdown cells, implying an eIF2 dependency. Altogether, these findings uncover a novel coordination between ER and lysosomes, in which local release of amino acids and other factors from ER-associated lysosomes patterns and regulates translation of mRNAs encoding secretory and membrane proteins.

}, doi = {10.1101/2023.08.01.551382}, author = {Choi, Heejun and Liao, Ya-Cheng and Yoon, Young J and Grimm, Jonathan and Lavis, Luke D and Singer, Robert H and Lippincott-Schwartz, Jennifer} } @article {68300, title = {Organization of translating secretome mRNAS on endoplasmic reticulum}, journal = {Biophysical Journal}, volume = {121}, year = {2022}, month = {2022 Feb 01}, pages = {33a}, abstract = {

The endoplasmic reticulum (ER) has a complex morphology comprised of stacked sheets, tubules, and three-way junctions, which together function as a platform for protein synthesis of membrane and secretory proteins. Specific ER subdomains are thought to be spatially organized to enable protein synthesis activity, but precisely where these domains are localized is unclear, especially relative to the plethora of organelle interactions taking place on the ER. Here, we use single-molecule tracking of ribosomes and mRNA in combination with simultaneous imaging of ER to assess the sites of membrane protein synthesis on the ER. We found that ribosomes were widely distributed throughout different ER morphologies, but the synthesis of membrane proteins (including Type I, II, and multi-spanning) and an ER luminal protein (Calreticulin) occurred primarily at three-way junctions. Lunapark played a key role in stabilizing transmembrane protein mRNA at three-way junctions. We additionally found that translating mRNAs coding for transmembrane proteins are in the vicinity of lysosomes and translate through a cap-independent but eIF2-dependent mechanism. These results support the idea that discrete ER subdomains co-exist with lysosomes to support specific types of protein synthesis activities, with ER-lysosome interactions playing an important role in the translation of secretome mRNAs.

}, issn = {00063495}, doi = {10.1016/j.bpj.2021.11.2550}, url = {https://linkinghub.elsevier.com/retrieve/pii/S0006349521035256}, author = {Choi, Heejun and Liao, Ya-Cheng and Yoon, Young J. and Grimm, Jonathan and Lavis, Luke D. and Singer, Robert H. and Lippincott-Schwartz, Jennifer} } @article {48859, title = {A dynamic interplay of enhancer elements regulates Klf4 expression in na{\"\i}ve pluripotency.}, journal = {Genes \& Development}, volume = {31}, year = {2017}, month = {2017 Sep 01}, pages = {1795-1808}, abstract = {

Transcription factor (TF)-directed enhanceosome assembly constitutes a fundamental regulatory mechanism driving spatiotemporal gene expression programs during animal development. Despite decades of study, we know little about the dynamics or order of events animating TF assembly at cis-regulatory elements in living cells and the long-range molecular "dialog" between enhancers and promoters. Here, combining genetic, genomic, and imaging approaches, we characterize a complex long-range enhancer cluster governing Kr{\"u}ppel-like factor 4 (Klf4) expression in na{\"\i}ve pluripotency. Genome editing by CRISPR/Cas9 revealed that OCT4 and SOX2 safeguard an accessible chromatin neighborhood to assist the binding of other TFs/cofactors to the enhancer. Single-molecule live-cell imaging uncovered that two na{\"\i}ve pluripotency TFs, STAT3 and ESRRB, interrogate chromatin in a highly dynamic manner, in which SOX2 promotes ESRRB target search and chromatin-binding dynamics through a direct protein-tethering mechanism. Together, our results support a highly dynamic yet intrinsically ordered enhanceosome assembly to maintain the finely balanced transcription program underlying na{\"\i}ve pluripotency.

}, issn = {1549-5477}, doi = {10.1101/gad.303321.117}, author = {Xie, Liangqi and Torigoe, Sharon E and Xiao, Jifang and Mai, Daniel H and Li, Li and Davis, Fred P and Dong, Peng and Marie-Nelly, Herve and Grimm, Jonathan and Lavis, Luke and Darzacq, Xavier and Cattoglio, Claudia and Liu, Zhe and Tjian, Robert} }