@article {69441, title = {Compartmentalized Cytoplasmic Flows Direct Protein Transport to the Cell{\textquoteright}s Leading Edge}, journal = {bioRxiv}, year = {2024}, month = {2024 May 14}, abstract = {

Inside the cell, proteins essential for signaling, morphogenesis, and migration navigate complex pathways, typically via vesicular trafficking or microtubule-driven mechanisms 1-3. However, the process by which soluble cytoskeletal monomers maneuver through the cytoplasm{\textquoteright}s ever-changing environment to reach their destinations without using these pathways remains unknown. 4-6 Here, we show that actin cytoskeletal treadmilling leads to the formation of a semi-permeable actin-myosin barrier, creating a specialized compartment separated from the rest of the cell body that directs proteins toward the cell edge by advection, diffusion facilitated by fluid flow. Contraction at this barrier generates a molecularly non-specific fluid flow that transports actin, actin-binding proteins, adhesion proteins, and even inert proteins forward. The local curvature of the barrier specifically targets these proteins toward protruding edges of the leading edge, sites of new filament growth, effectively coordinating protein distribution with cellular dynamics. Outside this compartment, diffusion remains the primary mode of protein transport, contrasting sharply with the directed advection within. This discovery reveals a novel protein transport mechanism that redefines the front of the cell as a pseudo-organelle, actively orchestrating protein mobilization for cellular front activities such as protrusion and adhesion. By elucidating a new model of protein dynamics at the cellular front, this work contributes a critical piece to the puzzle of how cells adapt their internal structures for targeted and rapid response to extracellular cues. The findings challenge the current understanding of intracellular transport, suggesting that cells possess highly specialized and previously unrecognized organizational strategies for managing protein distribution efficiently, providing a new framework for understanding the cellular architecture{\textquoteright}s role in rapid response and adaptation to environmental changes.

}, doi = {10.1101/2024.05.12.593794}, url = {https://www.biorxiv.org/content/early/2024/05/14/2024.05.12.593794}, author = {Galbraith, Catherine G and English, Brian P and Boehm, Ulrike and Galbraith, James A} } @article {68011, title = {BRD2 compartmentalizes the accessible genome.}, journal = {Nature Genetics}, volume = {54}, year = {2022}, month = {2022 Apr 11}, pages = {481-491}, abstract = {

Mammalian chromosomes are organized into megabase-sized compartments that are further subdivided into topologically associating domains (TADs). While the formation of TADs is dependent on cohesin, the mechanism behind compartmentalization remains enigmatic. Here, we show that the bromodomain and extraterminal (BET) family scaffold protein BRD2 promotes spatial mixing and compartmentalization of active chromatin after cohesin loss. This activity is independent of transcription but requires BRD2 to recognize acetylated targets through its double bromodomain and interact with binding partners with its low-complexity domain. Notably, genome compartmentalization mediated by BRD2 is antagonized on the one hand by cohesin and on the other hand by the BET homolog protein BRD4, both of which inhibit BRD2 binding to chromatin. Polymer simulation of our data supports a BRD2-cohesin interplay model of nuclear topology, in which genome compartmentalization results from a competition between loop extrusion and chromatin-state-specific affinity interactions.

}, keywords = {Animals, Cell Cycle Proteins, Chromatin, Chromosomes, Mammals, Nuclear Proteins, Protein Binding, Protein Domains, Transcription Factors}, issn = {1546-1718}, doi = {10.1038/s41588-022-01044-9}, author = {Xie, Liangqi and Dong, Peng and Qi, Yifeng and Hsieh, Tsung-Han S and English, Brian P and Jung, Seolkyoung and Chen, Xingqi and De Marzio, Margherita and Casellas, Rafael and Chang, Howard Y and Zhang, Bin and Tjian, Robert and Liu, Zhe} } @article {68462, title = {Cap-dependent translation initiation monitored in living cells.}, journal = {Nature Communications}, volume = {13}, year = {2022}, month = {2022 Nov 02}, pages = {6558}, abstract = {

mRNA translation is tightly regulated to preserve cellular homeostasis. Despite extensive biochemical, genetic, and structural studies, a detailed understanding of mRNA translation regulation is lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-mRNA resolution were necessary to fully elucidate regulation of this paramount process. Here live-cell spectroscopy and single-particle tracking were combined to interrogate the binding dynamics of endogenous initiation factors to the 5{\textquoteright}cap. The diffusion of initiation factors (IFs) changed markedly upon their association with mRNA. Quantifying their diffusion characteristics revealed the sequence of IFs assembly and disassembly in cell lines and the clustering of translation in neurons. This approach revealed translation regulation at high spatial and temporal resolution that can be applied to the formation of any endogenous complex that results in a measurable shift in diffusion.

}, keywords = {Peptide Chain Initiation, Translational, Peptide Initiation Factors, Protein Biosynthesis, RNA Caps, RNA, Messenger}, issn = {2041-1723}, doi = {10.1038/s41467-022-34052-8}, author = {Gandin, Valentina and English, Brian P and Freeman, Melanie and Leroux, Louis-Philippe and Preibisch, Stephan and Walpita, Deepika and Jaramillo, Maritza and Singer, Robert H} } @article {68556, title = {Inhibition of coronavirus HCoV-OC43 by targeting the eIF4F complex.}, journal = {Frontiers in Pharmacology}, volume = {13}, year = {2022}, month = {2022 Dec 01}, pages = {1029093}, abstract = {

The translation initiation complex 4F (eIF4F) is a rate-limiting factor in protein synthesis. Alterations in eIF4F activity are linked to several diseases, including cancer and infectious diseases. To this end, coronaviruses require eIF4F complex activity to produce proteins essential for their life cycle. Efforts to target coronaviruses by abrogating translation have been largely limited to repurposing existing eIF4F complex inhibitors. Here, we report the results of a high throughput screen to identify small molecules that disrupt eIF4F complex formation and inhibit coronavirus RNA and protein levels. Of 338,000 small molecules screened for inhibition of the eIF4F-driven, CAP-dependent translation, we identified SBI-1232 and two structurally related analogs, SBI-5844 and SBI-0498, that inhibit human coronavirus OC43 (HCoV-OC43; OC43) with minimal cell toxicity. Notably, gene expression changes after OC43 infection of Vero E6 or A549 cells were effectively reverted upon treatment with SBI-5844 or SBI-0498. Moreover, SBI-5844 or SBI-0498 treatment effectively impeded the eIF4F complex assembly, with concomitant inhibition of newly synthesized OC43 nucleocapsid protein and OC43 RNA and protein levels. Overall, we identify SBI-5844 and SBI-0498 as small molecules targeting the eIF4F complex that may limit coronavirus transcripts and proteins, thereby representing a basis for developing novel therapeutic modalities against coronaviruses.

}, issn = {1663-9812}, doi = {10.3389/fphar.2022.1029093}, author = {Feng, Yongmei and Grotegut, Stefan and Jovanovic, Predrag and Gandin, Valentina and Olson, Steven H and Murad, Rabi and Beall, Anne and Colayco, Sharon and De-Jesus, Paul and Chanda, Sumit and English, Brian P and Singer, Robert H and Jackson, Michael and Topisirovic, Ivan and Ronai, Ze{\textquoteright}ev A} } @article {65581, title = {Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction.}, journal = {eLife}, volume = {9}, year = {2020}, month = {2020 Apr 27}, abstract = {

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

}, issn = {2050-084X}, doi = {10.7554/eLife.55667}, author = {Ranjan, Anand and Nguyen, Vu Q and Liu, Sheng and Wisniewski, Jan and Kim, Jee Min and Tang, Xiaona and Mizuguchi, Gaku and Elalaoui, Ejlal and Nickels, Timothy J and Jou, Vivian and English, Brian P and Zheng, Qinsi and Luk, Ed and Lavis, Luke D and Lionnet, Timoth{\'e}e and Wu, Carl} } @article {48804, title = {Synthesis of Janelia Fluor HaloTag and SNAP-Tag Ligands and Their Use in Cellular Imaging Experiments.}, journal = {Methods in Molecular Biology (Clifton, N.J.)}, volume = {1663}, year = {2017}, month = {2017}, pages = {179-188}, abstract = {

The development of genetically encoded self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in microscopy. Intracellular labeling using these systems requires small, cell-permeable dyes with high brightness and photostability. We recently discovered a general method to improve the properties of classic fluorophores by replacing N,N-dimethylamino groups with four-membered azetidine rings to create the "Janelia Fluor" dyes. Here, we describe the synthesis of the HaloTag and SNAP-tag ligands of Janelia Fluor 549 and Janelia Fluor 646 as well as standard labeling protocols for use in ensemble and single-molecule cellular imaging.

}, issn = {1940-6029}, doi = {10.1007/978-1-4939-7265-4_15}, author = {Grimm, Jonathan B and Brown, Timothy A and English, Brian P and Lionnet, Timoth{\'e}e and Lavis, Luke D} } @article {47729, title = {Bright photoactivatable fluorophores for single-molecule imaging.}, journal = {Nature Methods}, volume = {13}, year = {2016}, month = {2016 Oct 24}, pages = {985-8}, abstract = {

Small molecule fluorophores are important tools for advanced imaging experiments. The development of self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in live-cell microscopy. We recently described a general method for improving the brightness and photostability of small, cell-permeable fluorophores, resulting in the novel azetidine-containing "Janelia Fluor" (JF) dyes. Here, we refine and extend the utility of the JF dyes by synthesizing photoactivatable derivatives that are compatible with live cell labeling strategies. These compounds retain the superior brightness of the JF dyes once activated, but their facile photoactivation also enables improved single-particle tracking and localization microscopy experiments.

}, doi = {10.1038/nmeth.4034}, author = {Lavis, Luke D and Grimm, Jonathan B and English, Brian P and Choi, Heejin and Muthusamy, Anand K and Mehl, Brian P and Dong, Peng and Brown, Timothy A and Lippincott-Schwartz, Jennifer and Liu, Zhe and Lionnet, Timoth{\'e}e} } @article {47967, title = {Glutamate-induced RNA localization and translation in neurons.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, year = {2016}, month = {2016 Sep 13}, abstract = {

Localization of mRNA is required for protein synthesis to occur within discrete intracellular compartments. Neurons represent an ideal system for studying the precision of mRNA trafficking because of their polarized structure and the need for synapse-specific targeting. To investigate this targeting, we derived a quantitative and analytical approach. Dendritic spines were stimulated by glutamate uncaging at a diffraction-limited spot, and the localization of single β-actin mRNAs was measured in space and time. Localization required NMDA receptor activity, a dynamic actin cytoskeleton, and the transacting RNA-binding protein, Zipcode-binding protein 1 (ZBP1). The ability of the mRNA to direct newly synthesized proteins to the site of localization was evaluated using a Halo-actin reporter so that RNA and protein were detected simultaneously. Newly synthesized Halo-actin was enriched at the site of stimulation, required NMDA receptor activity, and localized preferentially at the periphery of spines. This work demonstrates that synaptic activity can induce mRNA localization and local translation of β-actin where the new actin participates in stabilizing the expanding synapse in dendritic spines.

}, doi = {10.1073/pnas.1614267113}, author = {Yoon, Young J and Wu, Bin and Buxbaum, Adina R and Das, Sulagna and Tsai, Albert and English, Brian P and Grimm, Jonathan B and Lavis, Luke D and Singer, Robert H} } @article {47398, title = {Mapping translation {\textquoteright}hot-spots{\textquoteright} in live cells by tracking single molecules of mRNA and ribosomes.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016}, abstract = {

Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.

}, issn = {2050-084X}, doi = {10.7554/eLife.10415}, author = {Katz, Zachary B and English, Brian P and Lionnet, Timoth{\'e}e and Yoon, Young J and Monnier, Nilah and Ovryn, Ben and Bathe, Mark and Singer, Robert H} } @article {47602, title = {Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies.}, journal = {Nature Biotechnology}, volume = {34}, year = {2016}, month = {2016 Jul 4}, pages = {987-92}, abstract = {

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (\~{}70 nm) imaging of cells and mammalian tissues on conventional microscopes.

}, issn = {1546-1696}, doi = {10.1038/nbt.3625}, author = {Tillberg, Paul and Chen, Fei and Piatkevich, Kiryl D and Zhao, Yongxin and Yu, Chih-Chieh Jay and English, Brian P and Gao, Linyi and Martorell, Anthony and Suk, Ho-Jun and Yoshida, Fumiaki and DeGennaro, Ellen M and Roossien, Douglas H and Gong, Guanyu and Seneviratne, Uthpala and Tannenbaum, Steven R and Desimone, Robert and Cai, Dawen and Boyden, Edward S} } @article {47938, title = {Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.}, journal = {Genes and Development}, volume = {30}, year = {2016}, month = {2016 Sep 15}, pages = {2106-18}, abstract = {

Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A {\textquotedblleft}step-wise{\textquotedblright} preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB{\textendash}promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II{\textendash}TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

}, doi = {10.1101/gad.285395.116}, author = {Zhang, Zhengjian and English, Brian P and Grimm, Jonathan B and Kazane, Stephanie A and Hu, Wenxin and Tsai, Albert and Inouye, Carla and You, Changjiang and Piehler, Jacob and Schultz, Peter G and Lavis, Luke D and Revyakin, Andrey and Tjian, Robert} } @article {47512, title = {Real-time quantification of single RNA translation dynamics in living cells.}, journal = {Science}, volume = {352}, year = {2016}, month = {2016 May 05}, pages = {1425-9}, abstract = {

Although mRNA translation is a fundamental biological process, it has never been imaged in real-time with single molecule precision in vivo. To achieve this, we developed Nascent Chain Tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify single mRNA protein synthesis dynamics. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 s. Polysomes contain ~1 ribosome every 200-900 nucleotides and are globular rather than elongated in shape. By developing multi-color probes, we show most polysomes act independently; however, a small fraction (~5\%) form complexes in which two distinct mRNAs can be translated simultaneously. The sensitivity and versatility of NCT make it a powerful new tool for quantifying mRNA translation kinetics.

}, doi = {10.1126/science.aaf0899}, author = {Morisaki, Tatsuya and Lyon, Kenneth and DeLuca, Keith F and DeLuca, Jennifer G and English, Brian P and Zhang, Zhengjian and Lavis, Luke D and Grimm, Jonathan B and Viswanathan, Sarada and Looger, Loren L} } @article {47753, title = {RNA Polymerase II cluster dynamics predict mRNA output in living cells.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 May 03}, abstract = {

Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output.

}, issn = {2050-084X}, doi = {10.7554/eLife.13617}, author = {Cho, Won-Ki and Jayanth, Namrata and English, Brian P and Inoue, Takuma and Andrews, J Owen and Conway, William and Grimm, Jonathan B and Spille, Jan-Hendrik and Lavis, Luke D and Lionnet, Timoth{\'e}e and Cisse, Ibrahim I} } @article {46267, title = {A general method to improve fluorophores for live-cell and single-molecule microscopy.}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Jan 19}, pages = {244-50}, abstract = {

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

}, issn = {1548-7105}, doi = {10.1038/nmeth.3256}, author = {Grimm, Jonathan B and English, Brian P and Chen, Jiji and Slaughter, Joel P and Zhang, Zhengjian and Revyakin, Andrey and Patel, Ronak and Macklin, John J and Normanno, Davide and Singer, Robert H and Lionnet, Timoth{\'e}e and Lavis, Luke D} } @article {46929, title = {Inferring transient particle transport dynamics in live cells.}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Jul 20}, pages = {838-40}, abstract = {

Live-cell imaging and particle tracking provide rich information on mechanisms of intracellular transport. However, trajectory analysis procedures to infer complex transport dynamics involving stochastic switching between active transport and diffusive motion are lacking. We applied Bayesian model selection to hidden Markov modeling to infer transient transport states from trajectories of mRNA-protein complexes in live mouse hippocampal neurons and metaphase kinetochores in dividing human cells. The software is available at http://hmm-bayes.org/.

}, issn = {1548-7105}, doi = {10.1038/nmeth.3483}, author = {Monnier, Nilah and Barry, Zachary and Park, Hye Yoon and Su, Kuan-Chung and Katz, Zachary and English, Brian P and Dey, Arkajit and Pan, Keyao and Cheeseman, Iain M and Singer, Robert H and Bathe, Mark} } @article {47266, title = {A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells.}, journal = {Proceedings of SPIE}, volume = {9550}, year = {2015}, month = {2015 Aug 21}, pages = {955008 }, abstract = {

Our aim is to develop quantitative single-molecule assays to study when and where molecules are interacting inside living cells and where enzymes are active. To this end we present a three-camera imaging microscope for fast tracking of multiple interacting molecules simultaneously, with high spatiotemporal resolution. The system was designed around an ASI RAMM frame using three separate tube lenses and custom multi-band dichroics to allow for enhanced detection efficiency. The frame times of the three Andor iXon Ultra EMCCD cameras are hardware synchronized to the laser excitation pulses of the three excitation lasers, such that the fluorophores are effectively immobilized during frame acquisitions and do not yield detections that are motion-blurred. Stroboscopic illumination allows robust detection from even rapidly moving molecules while minimizing bleaching, and since snapshots can be spaced out with varying time intervals, stroboscopic illumination enables a direct comparison to be made between fast and slow molecules under identical light dosage. We have developed algorithms that accurately track and co-localize multiple interacting biomolecules. The three-color microscope combined with our co-movement algorithms have made it possible for instance to simultaneously image and track how the chromosome environment affects diffusion kinetics or determine how mRNAs diffuse during translation. Such multiplexed single-molecule measurements at a high spatiotemporal resolution inside living cells will provide a major tool for testing models relating molecular architecture and biological dynamics.

}, doi = {10.1117/12.2190246}, author = {English, Brian P and Singer, Robert H} } @article {46369, title = {Tracking surface glycans on live cancer cells with single-molecule sensitivity.}, journal = {Angewandte Chemie International Edition English}, volume = {54}, year = {2015}, month = {2015 Feb 2}, pages = {1765-9}, abstract = {

Using a combination of metabolically labeled glycans, a bioorthogonal copper(I)-catalyzed azide-alkyne cycloaddition, and the controlled bleaching of fluorescent probes conjugated to azide- or alkyne-tagged glycans, a sufficiently low spatial density of dye-labeled glycans was achieved, enabling dynamic single-molecule tracking and super-resolution imaging of N-linked sialic acids and O-linked N-acetyl galactosamine (GalNAc) on the membrane of live cells. Analysis of the trajectories of these dye-labeled glycans in mammary cancer cells revealed constrained diffusion of both N- and O-linked glycans, which was interpreted as reflecting the mobility of the glycan rather than to be caused by transient immobilization owing to spatial inhomogeneities on the plasma membrane. Stochastic optical reconstruction microscopy (STORM) imaging revealed the structure of dynamic membrane nanotubes.

}, issn = {1521-3773}, doi = {10.1002/anie.201407976}, author = {Jiang, Hao and English, Brian P and Hazan, Rachel B and Wu, Peng and Ovryn, Ben} } @article {45617, title = {Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.}, journal = {Science}, volume = {346}, year = {2014}, month = {2014 Oct 24}, pages = {1257998}, abstract = {

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

}, issn = {1095-9203}, doi = {10.1126/science.1257998}, author = {Chen, Bi-Chang and Legant, Wesley R and Wang, Kai and Shao, Lin and Milkie, Daniel E and Davidson, Michael W and Janetopoulos, Chris and Wu, Xufeng S and Hammer, John A and Liu, Zhe and English, Brian P and Mimori-Kiyosue, Yuko and Romero, Daniel P and Ritter, Alex T and Lippincott-Schwartz, Jennifer and Fritz-Laylin, Lillian and Mullins, R Dyche and Mitchell, Diana M and Bembenek, Joshua N and Reymann, Anne-Cecile and B{\"o}hme, Ralph and Grill, Stephan W and Wang, Jennifer T and Seydoux, Geraldine and Tulu, U Serdar and Kiehart, Daniel P and Betzig, Eric} } @article {45699, title = {Photoswitchable red fluorescent protein with a large stokes shift.}, journal = {Chemistry \& Biology}, volume = {21}, year = {2014}, month = {2014 Oct 23}, pages = {1402-14}, abstract = {

A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622\ nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621\ nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in\ vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us\ to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

}, issn = {1879-1301}, doi = {10.1016/j.chembiol.2014.08.010}, author = {Piatkevich, Kiryl D and English, Brian P and Malashkevich, Vladimir N and Xiao, Hui and Almo, Steven C and Singer, Robert H and Verkhusha, Vladislav V} }