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112 Publications

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    08/03/23 | Lysosomal release of amino acids at ER three-way junctions regulates transmembrane and secretory protein mRNA translation.
    Choi H, Liao Y, Yoon YJ, Grimm J, Lavis LD, Singer RH, Lippincott-Schwartz J
    bioRxiv. 2023 Aug 03:. doi: 10.1101/2023.08.01.551382

    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.

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    06/13/23 | Dynamic 1D Search and Processive Nucleosome Translocations by RSC and ISW2 Chromatin Remodelers
    Jee Min Kim , Claudia C. Carcamo , Sina Jazani , Zepei Xie , Xinyu A. Feng , Matthew Poyton , Katie L. Holland , Jonathan B. Grimm , Luke D. Lavis , Taekjip Ha , Carl Wu
    bioRxiv. 2023 Jun 13:. doi: 10.1101/2023.06.13.544671

    Eukaryotic gene expression is linked to chromatin structure and nucleosome positioning by ATP-dependent chromatin remodelers that establish and maintain nucleosome-depleted regions (NDRs) near transcription start-sites. Conserved yeast RSC and ISW2 remodelers exert antagonistic effects on nucleosomes flanking NDRs, but the temporal dynamics of remodeler search, nucleosome engagement and mobilization for promoter accessibility are unknown. Using optical tweezers and 2-color single-particle imaging, we investigated the Brownian diffusion of RSC and ISW2 on free DNA and sparse nucleosome arrays. RSC and ISW2 rapidly scan DNA by one-dimensional hopping and sliding respectively, with dynamic collisions between remodelers followed by recoil or apparent co-diffusion. Static nucleosomes block remodeler diffusion resulting in remodeler recoil or sequestration. Remarkably, both RSC and ISW2 use ATP hydrolysis to translocate mono-nucleosomes processively at ∼30 bp/sec for surprising distances on extended linear DNA. Processivity and opposing push-pull directionalities of nucleosome translocation shown by RSC and ISW2 shape the distinctive landscape of promoter chromatin.

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    06/01/23 | Rejuvenating old fluorophores with new chemistry.
    Schnermann MJ, Lavis LD
    Current Opinions in Chemical Biology. 2023 Jun 01;75:102335. doi: 10.1016/j.cbpa.2023.102335

    The field of organic chemistry began with 19th century scientists identifying and then expanding upon synthetic dye molecules for textiles. In the 20th century, dye chemistry continued with the aim of developing photographic sensitizers and laser dyes. Now, in the 21st century, the rapid evolution of biological imaging techniques provides a new driving force for dye chemistry. Of the extant collection of synthetic fluorescent dyes for biological imaging, two classes reign supreme: rhodamines and cyanines. Here, we provide an overview of recent examples where modern chemistry is used to build these old-but-venerable classes of optically responsive molecules. These new synthetic methods access new fluorophores, which then enable sophisticated imaging experiments leading to new biological insights.

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    05/29/23 | Mapping memories: pulse-chase labeling reveals AMPA receptor dynamics during memory formation.
    Doyeon Kim , Pojeong Park , Xiuyuan Li , J. David Wong Campos , He Tian , Eric M. Moult , Jonathan B. Grimm , Luke Lavis , Adam E. Cohen
    bioRxiv. 2023 May 29:. doi: 10.1101/2023.05.26.541296

    A tool to map changes in synaptic strength during a defined time window could provide powerful insights into the mechanisms governing learning and memory. We developed a technique, Extracellular Protein Surface Labeling in Neurons (EPSILON), to map α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) insertion in vivo by pulse-chase labeling of surface AMPARs with membrane-impermeable dyes. This approach allows for single-synapse resolution maps of plasticity in genetically targeted neurons during memory formation. We investigated the relationship between synapse-level and cell-level memory encodings by mapping synaptic plasticity and cFos expression in hippocampal CA1 pyramidal cells upon contextual fear conditioning (CFC). We observed a strong correlation between synaptic plasticity and cFos expression, suggesting a synaptic mechanism for the association of cFos expression with memory engrams. The EPSILON technique is a useful tool for mapping synaptic plasticity and may be extended to investigate trafficking of other transmembrane proteins.

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    05/26/23 | Voltage dynamics of dendritic integration and back-propagation in vivo
    J. David Wong-Campos , Pojeong Park , Hunter Davis , Yitong Qi , He Tian , Daniel G. Itkis , Doyeon Kim , Sarah E. Plutkis , Luke Lavis , Adam E. Cohen
    bioRxiv. 2023 May 26:. doi: 10.1101/2023.05.25.542363

    Neurons integrate synaptic inputs within their dendrites and produce spiking outputs, which then propagate down the axon and back into the dendrites where they contribute to plasticity. Mapping the voltage dynamics in dendritic arbors of live animals is crucial for understanding neuronal computation and plasticity rules. Here we combine patterned channelrhodopsin activation with dual-plane structured illumination voltage imaging, for simultaneous perturbation and monitoring of dendritic and somatic voltage in Layer 2/3 pyramidal neurons in anesthetized and awake mice. We examined the integration of synaptic inputs and compared the dynamics of optogenetically evoked, spontaneous, and sensory-evoked back-propagating action potentials (bAPs). Our measurements revealed a broadly shared membrane voltage throughout the dendritic arbor, and few signatures of electrical compartmentalization among synaptic inputs. However, we observed spike rate acceleration-dependent propagation of bAPs into distal dendrites. We propose that this dendritic filtering of bAPs may play a critical role in activity-dependent plasticity.

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    05/17/23 | Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator
    Abdelfattah AS, Zheng J, Singh A, Huang Y, Reep D, Tsegaye G, Tsang A, Arthur BJ, Rehorova M, Olson CV, Shuai Y, Zhang L, Fu T, Milkie DE, Moya MV, Weber TD, Lemire AL, Baker CA, Falco N, Zheng Q, Grimm JB, Yip MC, Walpita D, Chase M, Campagnola L, Murphy GJ, Wong AM, Forest CR, Mertz J, Economo MN, Turner GC, Koyama M, Lin B, Betzig E, Novak O, Lavis LD, Svoboda K, Korff W, Chen T, Schreiter ER, Hasseman JP, Kolb I
    Neuron. 2023 May 17;111(10):1547-1563. doi: 10.1016/j.neuron.2023.03.009

    The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

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    05/01/23 | Time-tagged ticker tapes for intracellular recordings.
    Lin D, Li X, Moult E, Park P, Tang B, Shen H, Grimm JB, Falco N, Jia BZ, Baker D, Lavis LD, Cohen AE
    Nature Biotechnology. 2023 May 01;41(5):631-9. doi: 10.1038/s41587-022-01524-7

    Recording transcriptional histories of a cell would enable deeper understanding of cellular developmental trajectories and responses to external perturbations. Here we describe an engineered protein fiber that incorporates diverse fluorescent marks during its growth to store a ticker tape-like history. An embedded HaloTag reporter incorporates user-supplied dyes, leading to colored stripes that map the growth of each individual fiber to wall clock time. A co-expressed eGFP tag driven by a promoter of interest records a history of transcriptional activation. High-resolution multi-spectral imaging on fixed samples reads the cellular histories, and interpolation of eGFP marks relative to HaloTag timestamps provides accurate absolute timing. We demonstrate recordings of doxycycline-induced transcription in HEK cells and cFos promoter activation in cultured neurons, with a single-cell absolute accuracy of 30-40 minutes over a 12-hour recording. The protein-based ticker tape design we present here could be generalized to achieve massively parallel single-cell recordings of diverse physiological modalities.

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    03/30/23 | Sequence and Structural Motifs Controlling the Broad Substrate Specificity of the Mycobacterial Hormone-Sensitive Lipase LipN
    Schemenauer DE, Pool EH, Raynor SN, Ruiz GP, Goehring LM, Koelper AJ, Wilson MA, Durand AJ, Kourtoglou EC, Larsen EM, Lavis LD, Esteb JJ, Hoops GC, Johnson RJ
    ACS Omega. 2023 Mar 30;8(14):13252 - 13264. doi: 10.1021/acsomega.3c0053410.1021/acsomega.3c00534.s001

    Mycobacterium tuberculosis has a complex life cycle transitioning between active and dormant growth states depending on environmental conditions. LipN (Rv2970c) is a conserved mycobacterial serine hydrolase with regulated catalytic activity at the interface between active and dormant growth conditions. LipN also catalyzes the xenobiotic degradation of a tertiary ester substrate and contains multiple conserved motifs connected with the ability to catalyze the hydrolysis of difficult tertiary ester substrates. Herein, we expanded a library of fluorogenic ester substrates to include more tertiary and constrained esters and screened 33 fluorogenic substrates for activation by LipN, identifying its unique substrate signature. LipN preferred short, unbranched ester substrates, but had its second highest activity against a heteroaromatic five-membered oxazole ester. Oxazole esters are present in multiple mycobacterial serine hydrolase inhibitors but have not been tested widely as ester substrates. Combined structural modeling, kinetic measurements, and substitutional analysis of LipN showcased a fairly rigid binding pocket preorganized for catalysis of short ester substrates. Substitution of diverse amino acids across the binding pocket significantly impacted the folded stability and catalytic activity of LipN with two conserved motifs (HGGGW and GDSAG) playing interconnected, multidimensional roles in regulating its substrate specificity. Together this detailed substrate specificity profile of LipN illustrates the complex interplay between structure and function in mycobacterial hormone-sensitive lipase homologues and indicates oxazole esters as promising inhibitor and substrate scaffolds for mycobacterial hydrolases.

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    11/13/22 | Brain-wide measurement of protein turnover with high spatial and temporal resolution
    Boaz Mohar , Jonathan B. Grimm , Ronak Patel , Timothy A. Brown , Paul Tillberg , Luke D. Lavis , Nelson Spruston , Karel Svoboda
    bioRxiv. 2022 Nov 13:. doi: 10.1101/2022.11.12.516226

    Cells regulate function by synthesizing and degrading proteins. This turnover ranges from minutes to weeks, as it varies across proteins, cellular compartments, cell types, and tissues. Current methods for tracking protein turnover lack the spatial and temporal resolution needed to investigate these processes, especially in the intact brain, which presents unique challenges. We describe a pulse-chase method (DELTA) for measuring protein turnover with high spatial and temporal resolution throughout the body, including the brain. DELTA relies on rapid covalent capture by HaloTag of fluorophores that were optimized for bioavailability in vivo. The nuclear protein MeCP2 showed brain region- and cell type-specific turnover. The synaptic protein PSD95 was destabilized in specific brain regions by behavioral enrichment. A novel variant of expansion microscopy further facilitated turnover measurements at individual synapses. DELTA enables studies of adaptive and maladaptive plasticity in brain-wide neural circuits.

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    09/01/22 | A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility
    Shu-Hsien Sheu , Srigokul Upadhyayula , Vincent Dupuy , Song Pang , Andrew L. Lemire , Deepika Walpita , H. Amalia Pasolli , Fei Deng , Jinxia Wan , Lihua Wang , Justin Houser , Silvia Sanchez-Martinez , Sebastian E. Brauchi , Sambashiva Banala , Melanie Freeman , C. Shan Xu , Tom Kirchhausen , Harald F. Hess , Luke Lavis , Yu-Long Li , Séverine Chaumont-Dubel , David E. Clapham
    Cell. 2022 Sep 01;185(18):3390-3407. doi: 10.1016/j.cell.2022.07.026

    Chemical synapses between axons and dendrites mediate much of the brain’s intercellular communication. Here we describe a new kind of synapse – the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam – scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

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