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124 Publications

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    06/02/24 | Dynamic assemblies of parvalbumin interneurons in brain oscillations.
    Huang Y, Chen H, Lin Y, Lin S, Zheng Q, Abdelfattah AS, Lavis LD, Schreiter ER, Lin B, Chen T
    Neuron. 2024 Jun 02:. doi: 10.1016/j.neuron.2024.05.015

    Brain oscillations are crucial for perception, memory, and behavior. Parvalbumin-expressing (PV) interneurons are critical for these oscillations, but their population dynamics remain unclear. Using voltage imaging, we simultaneously recorded membrane potentials in up to 26 PV interneurons in vivo during hippocampal ripple oscillations in mice. We found that PV cells generate ripple-frequency rhythms by forming highly dynamic cell assemblies. These assemblies exhibit rapid and significant changes from cycle to cycle, varying greatly in both size and membership. Importantly, this variability is not just random spiking failures of individual neurons. Rather, the activities of other PV cells contain significant information about whether a PV cell spikes or not in a given cycle. This coordination persists without network oscillations, and it exists in subthreshold potentials even when the cells are not spiking. Dynamic assemblies of interneurons may provide a new mechanism to modulate postsynaptic dynamics and impact cognitive functions flexibly and rapidly.

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    05/18/24 | Dendritic excitations govern back-propagation via a spike-rate accelerometer
    Park P, Wong-Campos D, Itkis DG, Lee BH, Qi Y, Davis H, Antin B, Pasarkar A, Grimm JB, Plutkis SE, Holland KL, Paninski L, Lavis LD, Cohen AE
    bioRxiv. 2024 May 18:. doi: 10.1101/2023.06.02.543490

    Dendrites on neurons support nonlinear electrical excitations, but the computational significance of these events is not well understood. We developed molecular, optical, and analytical tools to map sub-millisecond voltage dynamics throughout the dendritic trees of CA1 pyramidal neurons under diverse optogenetic and synaptic stimulus patterns, in acute brain slices. We observed history-dependent spike back-propagation in distal dendrites, driven by locally generated Na+ spikes (dSpikes). Dendritic depolarization created a transient window for dSpike propagation, opened by A-type KV channel inactivation, and closed by slow NaV inactivation. Collisions of dSpikes with synaptic inputs triggered calcium channel and N-methyl-D-aspartate receptor (NMDAR)-dependent plateau potentials, with accompanying complex spikes at the soma. This hierarchical ion channel network acts as a spike-rate accelerometer, providing an intuitive picture of how dendritic excitations shape associative plasticity rules.

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    05/16/24 | Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment.
    Daugird TA, Shi Y, Holland KL, Rostamian H, Liu Z, Lavis LD, Rodriguez J, Strahl BD, Legant WR
    Nat. Commun.. 2024 May 16:. doi: 10.1038/s41467-024-48562-0

    In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we present an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrate that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacts nucleosome diffusive properties in a manner that is dependent both on local chromatin density and on relative location within the nucleus. Our results support a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Additionally, they reveal that nuclear heterogeneity arises from both active and passive processes and highlight the need to account for different organizational principles when modeling different chromatin environments.

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    05/12/24 | Coumarin as a general switching auxiliary to prepare photochromic and spontaneously blinking fluorophores
    Jradi FM, English BP, Brown TA, Aaron J, Khuon S, Galbraith JA, Galbraith CG, Lavis LD
    bioRxiv. 2024 May 12:. doi: 10.1101/2024.05.12.593749

    Single-molecule localization microscopy (SMLM) uses activatable or switchable fluorophores to create non-diffraction limited maps of molecular location in biological samples. Despite the utility of this imaging technique, the portfolio of appropriate labels for SMLM remains limited. Here, we describe a general strategy for the construction of “glitter bomb” labels by simply combining rhodamine and coumarin dyes though an amide bond. Condensation of the ortho-carboxyl group on the pendant phenyl ring of rhodamine dyes with a 7-aminocoumarin yields photochromic or spontaneously blinking fluorophores depending on the parent rhodamine structure. We apply this strategy to prepare labels useful super-resolution experiments in fixed cells using different attachment techniques. This general glitter bomb strategy should lead to improved labels for SMLM, ultimately enabling the creation of detailed molecular maps in biological samples.

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    05/10/24 | Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore
    Fiore A, Yu G, Northey JJ, Patel R, Ravenscroft TA, Ikegami R, Kolkman W, Kumar P, Grimm JB, Dilan TL, Ruetten VM, Ahrens MB, Shroff H, Lavis LD, Wang S, Weaver VM, Pedram K
    bioRxiv. 2024 May 10:. doi: 10.1101/2024.05.09.593460

    All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

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    03/25/24 | Evaluation of the Cytosolic Uptake of HaloTag Using a pH-Sensitive Dye
    Giancola JB, Grimm JB, Jun JV, Petri YD, Lavis LD, Raines RT
    ACS Chemical Biology. 2024 Mar 25:. doi: 10.1021/acschembio.3c0071310.1021/acschembio.3c00713.s001

    The efficient cytosolic delivery of proteins is critical for advancing novel therapeutic strategies. Current delivery methods are severely limited by endosomal entrapment, and detection methods lack sophistication in tracking the fate of delivered protein cargo. HaloTag, a commonly used protein in chemical biology and a challenging delivery target, is an exceptional model system for understanding and exploiting cellular delivery. Here, we employed a combinatorial strategy to direct HaloTag to the cytosol. We established the use of Virginia Orange, a pH-sensitive fluorophore, and Janelia Fluor 585, a similar but pH-agnostic fluorophore, in a fluorogenic assay to ascertain protein localization within human cells. Using this assay, we investigated HaloTag delivery upon modification with cell-penetrating peptides, carboxyl group esterification, and cotreatment with an endosomolytic agent. We found efficacious cytosolic entry with two distinct delivery methods. This study expands the toolkit for detecting the cytosolic access of proteins and highlights that multiple intracellular delivery strategies can be used synergistically to effect cytosolic access. Moreover, HaloTag is poised to serve as a platform for the delivery of varied cargo into human cells.

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    03/18/24 | 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
    eLife. 2024 Mar 18:. doi: 10.7554/eLife.91433

    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, engagement, and directional nucleosome mobilization for promoter accessibility are unknown. Using optical tweezers and two-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/s on extended linear DNA under tension. 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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    03/07/24 | Dendritic voltage imaging maps the biophysical basis of plateau potentials in the hippocampus
    Pojeong Park , J. David Wong-Campos , Daniel Itkis , Byung Hun Lee , Yitong Qi , Hunter C. Davis , Jonathan B. Grimm , Sarah E. Plutkis , Luke Lavis , Adam Ezra Cohen
    bioRxiv. 2024 Mar 7:. doi: 10.1101/2023.06.02.543490

    Dendrites on neurons integrate synaptic inputs to determine spike timing. Dendrites also convey back-propagating action potentials (bAPs) which interact with synaptic inputs to produce plateau potentials and to mediate synaptic plasticity. The biophysical rules which govern the timing, spatial structures, and ionic character of dendritic excitations are not well understood. We developed molecular, optical, and computational tools to map sub-millisecond voltage dynamics throughout the dendritic trees of CA1 pyramidal neurons under diverse optogenetic and synaptic stimulus patterns, in acute brain slices. We observed history-dependent bAP propagation in distal dendrites, driven by locally generated Na+ spikes (dSpikes). Dendritic depolarization creates a transient window for dSpike propagation, opened by A-type KV channel inactivation, and closed by slow NaV inactivation. Collisions of dSpikes with synaptic inputs triggered calcium channel and N-methyl-D-aspartate receptor (NMDAR)-dependent plateau potentials, with accompanying complex spikes at the soma. This hierarchical ion channel network acts as a spike-rate accelerometer, providing an intuitive picture of how dendritic excitations shape associative plasticity rules.Competing Interest StatementThe authors have declared no competing interest.

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    02/24/24 | A series of spontaneously blinking dyes for super-resolution microscopy
    Katie L. Holland , Sarah E. Plutkis , Timothy A. Daugird , Abhishek Sau , Jonathan B. Grimm , Brian P. English , Qinsi Zheng , Sandeep Dave , Fariha Rahman , Liangqi Xie , Peng Dong , Ariana N. Tkachuk , Timothy A. Brown , Robert H. Singer , Zhe Liu , Catherine G. Galbraith , Siegfried M. Musser , Wesley R. Legant , Luke D. Lavis
    bioRxiv. 2024 Feb 24:. doi: 10.1101/2024.02.23.581625

    Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.

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    01/01/24 | Transforming chemigenetic bimolecular fluorescence complementation systems into chemical dimerizers using chemistry.
    Pratik Kumar , Alina Gutu , Amelia Waring , Timothy A. Brown , Luke D. Lavis , Alison G. Tebo
    bioRxiv. 2024 Jan 01:. doi: 10.1101/2023.12.30.573644

    Chemigenetic tags are versatile labels for fluorescence microscopy that combine some of the advantages of genetically encoded tags with small molecule fluorophores. The Fluorescence Activating and absorbance Shifting Tags (FASTs) bind a series of highly fluorogenic and cell-permeable chromophores. Furthermore, FASTs can be used in complementation-based systems for detecting or inducing protein-protein interactions, depending on the exact FAST protein variant chosen. In this study, we systematically explore substitution patterns on FAST fluorogens and generate a series of fluorogens that bind to FAST variants, thereby activating their fluorescence. This effort led to the discovery of a novel fluorogen with superior properties, as well as a fluorogen that transforms splitFAST systems into a fluorogenic dimerizer, eliminating the need for additional protein engineering.

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