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132 Publications

Showing 1-10 of 132 results
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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12/05/23 | Imaging neuronal voltage beyond the scattering limit
Tsai-Wen Chen , Xian-Bin Huang , Sarah E. Plutkis , Katie L. Holland , Luke D. Lavis , Bei-Jung Lin
bioRxiv. 2023 Dec 05:. doi: 10.1101/2023.12.03.568403

Voltage imaging is a promising technique for high-speed recording of neuronal population activity. However, tissue scattering severely limits its application in dense neuronal populations. Here, we adopted the principle of localization microscopy, a technique that enables super-resolution imaging of single-molecules, to resolve dense neuronal activities in vivo. Leveraging the sparse activation of neurons during action potentials (APs), we precisely localize the fluorescence change associated with each AP, creating a super-resolution image of neuronal activities. This approach, termed Activity Localization Imaging (ALI), identifies overlapping neurons and separates their activities with over 10-fold greater precision than what tissue scattering permits. Using ALI, we simultaneously recorded over a hundred densely-labeled CA1 neurons, creating a map of hippocampal theta oscillation at single-cell and single-cycle resolution.

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11/13/23 | 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
bioRxiv. 2023 Nov 13:. doi: 10.1101/2023.11.09.566470

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 developed 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 demonstrated 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 impacted nucleosome diffusive properties in a manner that was dependent on local chromatin density and supportive of a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Our results reveal that nuclear heterogeneity arises from both active and passive process and highlights the need to account for different organizational principals when modeling different chromatin environments.

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10/27/23 | Nanoscale imaging reveals the mechanisms of ER-to-Golgi transport via a dynamic tubular-vesicular network
Luis Wong-Dilworth , Gresy Bregu , Steffen Restel , Carmen Rodilla-Ramirez , Svenja Ebeling , Shelly Harel , Paula Leupold , Jonathan Grimm , Luke D. Lavis , Jessica Angulo-Capel , Felix Campelo , Francesca Bottanelli
bioRxiv. 2023 Oct 27:. doi: 10.1101/2023.10.27.563951

The endoplasmic reticulum (ER) and the Golgi apparatus are the first sorting stations along the secretory pathway of mammalian cells and have a crucial role in protein quality control and cellular homeostasis. While machinery components mediating ER-to-Golgi transport have been mapped, it is unclear how exchange between the two closely juxtaposed organelles is coordinated in living cells. Here, using gene editing to tag machinery components, live-cell confocal and stimulated emission depletion (STED) super-resolution microscopy, we show that ER-to-Golgi transport occurs via a dynamic network of tubules positive for the small GTPase ARF4. swCOPI machinery is tightly associated to this network and moves with tubular-vesicular structures. Strikingly, the ARF4 network appears to be continuous with the ER and ARF4 tubules remodel around static ER exit sites (ERES) defined by COPII machinery. We were further able to dissect the steps of ER-to-Golgi transport with functional trafficking assays. A wave of cargo released from the ER percolates through peripheral and Golgi-tethered ARF4 structures before filling the cis-Golgi. Perturbation via acute degradation of ARF4 shows an active regulatory role for the GTPase and COPI in anterograde transport. Our data supports a model in which anterograde ER-to-Golgi transport occurs via an ARF4 tubular-vesicular network directly connecting the ER and Golgi-associated pre-cisternae.

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10/16/23 | Optimized Red-Absorbing Dyes for Imaging and Sensing
Grimm JB, Tkachuk AN, Patel R, Hennigan ST, Gutu A, Dong P, Gandin V, Osowski AM, Holland KL, Liu ZJ, Brown TA, Lavis LD
Journal of the American Chemical Society. 2023 Oct 16:. doi: 10.1021/jacs.3c0527310.1021/jacs.3c05273

Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property of rhodamines is their equilibrium between a colorless lactone and fluorescent zwitterion. Tuning the lactone–zwitterion equilibrium constant (KL–Z) can optimize dye properties for specific biological applications. Here, we use known and novel organic chemistry to prepare a comprehensive collection of rhodamine dyes to elucidate the structure–activity relationships that govern KL–Z. We discovered that the auxochrome substituent strongly affects the lactone–zwitterion equilibrium, providing a roadmap for the rational design of improved rhodamine dyes. Electron-donating auxochromes, such as julolidine, work in tandem with fluorinated pendant phenyl rings to yield bright, red-shifted fluorophores for live-cell single-particle tracking (SPT) and multicolor imaging. The N-aryl auxochrome combined with fluorination yields red-shifted Förster resonance energy transfer (FRET) quencher dyes useful for creating a new semisynthetic indicator to sense cAMP using fluorescence lifetime imaging microscopy (FLIM). Together, this work expands the synthetic methods available for rhodamine synthesis, generates new reagents for advanced fluorescence imaging experiments, and describes structure–activity relationships that will guide the design of future probes.

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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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