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Quantitative mRNA imaging throughout the entire Drosophila brain

Long, Xi; Colonell, Jennifer; Wong, Allan M; Singer, Robert H; Lionnet, Timothee
We describe a fluorescence in situ hybridization method that permits detection of the localization and abundance of single mRNAs (smFISH) in cleared whole-mount adult Drosophila brains. The approach is rapid and multiplexable and does not require molecular amplification; it allows facile quantification of mRNA expression with subcellular resolution on a standard confocal microscope. We further demonstrate single-mRNA detection across the entire brain using a custom Bessel beam structured illumination microscope (BB-SIM).
PMID: 28581495
ISSN: 1548-7105
CID: 2592002

mRNA quantification using single-molecule FISH in Drosophila embryos

Trcek, Tatjana; Lionnet, Timothee; Shroff, Hari; Lehmann, Ruth
Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. Here we describe an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e.g., short fluorescently labeled DNA oligonucleotides) in combination with wide-field epifluorescence, confocal or instant structured illumination microscopy (iSIM, a super-resolution imaging approach) and a spot-detection algorithm. Fixed Drosophila embryos are hybridized in solution with a mixture of smFISH probes, mounted onto coverslips and imaged in 3D. Individual fluorescently labeled mRNAs are then localized within tissues and counted using spot-detection software to generate quantitative, spatially resolved gene expression data sets. With minimum guidance, a graduate student can successfully implement this protocol. The smFISH procedure described here can be completed in 4-5 d.
PMID: 28594816
ISSN: 1750-2799
CID: 2590572

RNA Granule Organization [Meeting Abstract]

Lehmann, Ruth; Trcek, Tatjana; Grosch, Markus; Shroff, Hari; Lionnet, Timothee
ISI:000405461402579
ISSN: 1530-6860
CID: 2677112

Synthesis of Janelia Fluor HaloTag and SNAP-Tag Ligands and Their Use in Cellular Imaging Experiments

Grimm, Jonathan B; Brown, Timothy A; English, Brian P; Lionnet, Timothée; Lavis, Luke D
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.
PMID: 28924668
ISSN: 1940-6029
CID: 4309292

Bright photoactivatable fluorophores for single-molecule imaging

Grimm, Jonathan B; English, Brian P; Choi, Heejun; Muthusamy, Anand K; Mehl, Brian P; Dong, Peng; Brown, Timothy A; Lippincott-Schwartz, Jennifer; Liu, Zhe; Lionnet, Timothee; Lavis, Luke D
Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.
PMID: 27776112
ISSN: 1548-7105
CID: 2385112

Real-time quantification of single RNA translation dynamics in living cells

Morisaki, Tatsuya; Lyon, Kenneth; DeLuca, Keith F; DeLuca, Jennifer G; English, Brian P; Zhang, Zhengjian; Lavis, Luke D; Grimm, Jonathan B; Viswanathan, Sarada; Looger, Loren L; Lionnet, Timothee; Stasevich, Timothy J
Although messenger RNA (mRNA) translation is a fundamental biological process, it has never been imaged in real time in vivo with single-molecule precision. To achieve this, we developed nascent chain tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify protein synthesis dynamics at the single-mRNA level. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 seconds. Polysomes contain ~1 ribosome every 200 to 900 nucleotides and are globular rather than elongated in shape. By developing multicolor probes, we showed that 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.
PMID: 27313040
ISSN: 1095-9203
CID: 2385122

RNA Polymerase II cluster dynamics predict mRNA output in living cells

Cho, Won-Ki; Jayanth, Namrata; English, Brian P; Inoue, Takuma; Andrews, J Owen; Conway, William; Grimm, Jonathan B; Spille, Jan-Hendrik; Lavis, Luke D; Lionnet, Timothee; Cisse, Ibrahim I
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 beta-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.
PMCID:4929003
PMID: 27138339
ISSN: 2050-084x
CID: 2385152

Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging

Abrahamsson, Sara; Ilic, Rob; Wisniewski, Jan; Mehl, Brian; Yu, Liya; Chen, Lei; Davanco, Marcelo; Oudjedi, Laura; Fiche, Jean-Bernard; Hajj, Bassam; Jin, Xin; Pulupa, Joan; Cho, Christine; Mir, Mustafa; El Beheiry, Mohamed; Darzacq, Xavier; Nollmann, Marcelo; Dahan, Maxime; Wu, Carl; Lionnet, Timothee; Liddle, J Alexander; Bargmann, Cornelia I
Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a "precise color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is approximately 90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans.
PMCID:4866461
PMID: 27231594
ISSN: 2156-7085
CID: 2385142

Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes

Katz, Zachary B; English, Brian P; Lionnet, Timothee; Yoon, Young J; Monnier, Nilah; Ovryn, Ben; Bathe, Mark; Singer, Robert H
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 beta-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, beta-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.
PMCID:4764586
PMID: 26760529
ISSN: 2050-084x
CID: 2385172

TRICK: A Single-Molecule Method for Imaging the First Round of Translation in Living Cells and Animals

Halstead, J M; Wilbertz, J H; Wippich, F; Lionnet, T; Ephrussi, A; Chao, J A
The life of an mRNA is dynamic within a cell. The development of quantitative fluorescent microscopy techniques to image single molecules of RNA has allowed many aspects of the mRNA lifecycle to be directly observed in living cells. Recent advances in live-cell multicolor RNA imaging, however, have now made it possible to investigate RNA metabolism in greater detail. In this chapter, we present an overview of the design and implementation of the translating RNA imaging by coat protein knockoff RNA biosensor, which allows untranslated mRNAs to be distinguished from ones that have undergone a round of translation. The methods required for establishing this system in mammalian cell lines and Drosophila melanogaster oocytes are described here, but the principles may be applied to any experimental system.
PMID: 27241753
ISSN: 1557-7988
CID: 2385132