Xiaowei Zhuang

Structural organization of the actin-spectrin–based membrane skeleton in dendrites and soma of neurons

Co-reporter:Chenglong Xia;Boran Han;Ruobo Zhou

PNAS 2017 Volume 114 (Issue 32 ) pp:E6678-E6685

Publication Date(Web):2017-08-08

DOI:10.1073/pnas.1705043114

Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. In the MPS, short actin filaments, capped by actin-capping proteins, form ring-like structures that wrap around the circumference of neurites, and these rings are periodically spaced along the neurite by spectrin tetramers, forming a quasi-1D lattice structure. This 1D MPS structure was initially observed in axons and exists extensively in axons, spanning nearly the entire axonal shaft of mature neurons. Such 1D MPS was also observed in dendrites, but the extent to which it exists and how it develops in dendrites remain unclear. It is also unclear whether other structural forms of the membrane skeleton are present in neurons. Here, we investigated the spatial organizations of spectrin, actin, and adducin, an actin-capping protein, in the dendrites and soma of cultured hippocampal neurons at different developmental stages, and compared results with those obtained in axons, using superresolution imaging. We observed that the 1D MPS exists in a substantial fraction of dendritic regions in relatively mature neurons, but this structure develops slower and forms with a lower propensity in dendrites than in axons. In addition, we observed that spectrin, actin, and adducin also form a 2D polygonal lattice structure, resembling the expanded erythrocyte membrane skeleton structure, in the somatodendritic compartment. This 2D lattice structure also develops substantially more slowly in the soma and dendrites than the development of the 1D MPS in axons. These results suggest membrane skeleton structures are differentially regulated across different subcompartments of neurons.

High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization

Co-reporter:Hazen P. Babcock;Guiping Wang;Jeffrey R. Moffitt;Kok Hao Chen;Junjie Hao

PNAS 2016 Volume 113 (Issue 39 ) pp:11046-11051

Publication Date(Web):2016-09-27

DOI:10.1073/pnas.1612826113

Image-based approaches to single-cell transcriptomics, in which RNA species are identified and counted in situ via imaging, have emerged as a powerful complement to single-cell methods based on RNA sequencing of dissociated cells. These image-based approaches naturally preserve the native spatial context of RNAs within a cell and the organization of cells within tissue, which are important for addressing many biological questions. However, the throughput of these image-based approaches is relatively low. Here we report advances that lead to a drastic increase in the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH), an image-based approach to single-cell transcriptomics. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule fluorescence in situ hybridization (smFISH) to read out these barcodes. Here we increase the throughput of MERFISH by two orders of magnitude through a combination of improvements, including using chemical cleavage instead of photobleaching to remove fluorescent signals between consecutive rounds of smFISH imaging, increasing the imaging field of view, and using multicolor imaging. With these improvements, we performed RNA profiling in more than 100,000 human cells, with as many as 40,000 cells measured in a single 18-h measurement. This throughput should substantially extend the range of biological questions that can be addressed by MERFISH.

Prevalent presence of periodic actin–spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species

Co-reporter:Jiang He;Monica A. Carrasco;Ruobo Zhou;Zhuhao Wu;Peri T. Kurshan;Jonathan E. Farley;David J. Simon;Guiping Wang;Boran Han;Junjie Hao;Evan Heller;Marc R. Freeman;Kang Shen;Tom Maniatis;Marc Tessier-Lavigne

PNAS 2016 Volume 113 (Issue 21 ) pp:6029-6034

Publication Date(Web):2016-05-24

DOI:10.1073/pnas.1605707113

Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens.

Spatial colocalization and functional link of purinosomes with mitochondria

Co-reporter:Tony Jun Huang;Doory Kim;Huayun Deng;Sara A. Jones;Jarrod B. French;Haibei Hu;Raymond J. Pugh;Ye Fang;Hong Zhao;Anthony M. Pedley;Chung Yu Chan;Youxin Zhang;Stephen J. Benkovic

Science 2016 Volume 351(Issue 6274) pp:733-737

Publication Date(Web):12 Feb 2016

DOI:10.1126/science.aac6054

Spatial control of cellular enzymes

Purine is a building block of DNA and also a component of ATP that is used as an energy source in the cell. Enzymes involved in purine biosynthesis organize into dynamic bodies called purinosomes. French et al. found that purinosomes colocalize with mitochondria, organelles that generate ATP (see the Perspective by Ma and Jones). Dysregulation of mitochondria caused an increase in the number of purinosomes. This suggests a synergy, with the purinosomes supplying the purine required for ATP production and in turn using ATP in the biosynthetic pathway. A master regulator of cellular metabolism, mTOR, appears to mediate the association of purinosomes and mitochondria. This could make purine and ATP synthesis responsive to changes in the metabolic needs of the cell.

Science, this issue p. 733; see also p. 670

Spatial organization of chromatin domains and compartments in single chromosomes

Co-reporter:Siyuan Wang;Bogdan Bintu;Brian J. Beliveau;Jeffrey R. Moffitt;Jun-Han Su;Chao-ting Wu

Science 2016 Volume 353(Issue 6299) pp:598-602

Publication Date(Web):05 Aug 2016

DOI:10.1126/science.aaf8084

Spatial organization inside the nucleus

In eukaryotic cells, DNA is packaged into a complex macromolecular structure called chromatin. Wang et al. have developed an imaging method to map the position of multiple regions on individual chromosomes, and the results confirm that chromatin is organized into large contact domains called TADS (topologically associating domains). Unexpectedly, though, folding deviates from the classical fractal-globule model at large length scales.

Science, this issue p. 598

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

Co-reporter:Jeffrey R. Moffitt;Dhananjay Bambah-Mukku;Catherine Dulac;Tian Lu;Junjie Hao

PNAS 2016 Volume 113 (Issue 50 ) pp:14456-14461

Publication Date(Web):2016-12-13

DOI:10.1073/pnas.1617699113

Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, background—from off-target binding of FISH probes and cellular autofluorescence—can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.

Spatially resolved, highly multiplexed RNA profiling in single cells

Co-reporter:Alistair N. Boettiger;Jeffrey R. Moffitt;Kok Hao Chen;Siyuan Wang

Science 2015 Volume 348(Issue 6233) pp:

Publication Date(Web):24 Apr 2015

DOI:10.1126/science.aaa6090

Multiplexed RNA imaging in single cells

The basis of cellular function is where and when proteins are expressed and in what quantities. Single-molecule fluorescence in situ hybridization (smFISH) experiments quantify the copy number and location of mRNA molecules; however, the numbers of RNA species that can be simultaneously measured by smFISH has been limited. Using combinatorial labeling with error-robust encoding schemes, Chen et al. simultaneously imaged 100 to 1000 RNA species in a single cell. Such large-scale detection allows regulatory interactions to be analyzed at the transcriptome scale.

Science, this issue p. 10.1126/science.aaa6090

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

Co-reporter:Siyuan Wang;Jeffrey R. Moffitt;Graham T. Dempsey;X. Sunney Xie

PNAS 2014 111 (23 ) pp:8452-8457

Publication Date(Web):2014-06-10

DOI:10.1073/pnas.1406593111

Photoactivatable fluorescent proteins (PAFPs) have been widely used for superresolution imaging based on the switching and localization of single molecules. Several properties of PAFPs strongly influence the quality of the superresolution images. These properties include (i) the number of photons emitted per switching cycle, which affects the localization precision of individual molecules; (ii) the ratio of the on- and off-switching rate constants, which limits the achievable localization density; (iii) the dimerization tendency, which could cause undesired aggregation of target proteins; and (iv) the signaling efficiency, which determines the fraction of target–PAFP fusion proteins that is detectable in a cell. Here, we evaluated these properties for 12 commonly used PAFPs fused to both bacterial target proteins, H-NS, HU, and Tar, and mammalian target proteins, Zyxin and Vimentin. Notably, none of the existing PAFPs provided optimal performance in all four criteria, particularly in the signaling efficiency and dimerization tendency. The PAFPs with low dimerization tendencies exhibited low signaling efficiencies, whereas mMaple showed the highest signaling efficiency but also a high dimerization tendency. To address this limitation, we engineered two new PAFPs based on mMaple, which we termed mMaple2 and mMaple3. These proteins exhibited substantially reduced or undetectable dimerization tendencies compared with mMaple but maintained the high signaling efficiency of mMaple. In the meantime, these proteins provided photon numbers and on–off switching rate ratios that are comparable to the best achieved values among PAFPs.

Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy

Co-reporter:Joshua C. Vaughan ; Graham T. Dempsey ; Eileen Sun

Journal of the American Chemical Society 2013 Volume 135(Issue 4) pp:1197-1200

Publication Date(Web):January 11, 2013

DOI:10.1021/ja3105279

We report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine tris(2-carboxyethyl)phosphine (TCEP). Using Cy5 as a model, we show that the quenching reaction occurs by 1,4-addition of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination with UV light dissociates the adduct and returns the dye to the fluorescent state. We demonstrate that TCEP quenching can be used for super-resolution imaging as well as for other applications, such as differentiating between molecules inside and outside the cell.

Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons

Co-reporter:Ke Xu;Guisheng Zhong

Science 2013 Volume 339(Issue 6118) pp:452-456

Publication Date(Web):25 Jan 2013

DOI:10.1126/science.1232251

Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection

Co-reporter:Dr. Mark Bates;Graham T. Dempsey;Kok Hao Chen; Xiaowei Zhuang

ChemPhysChem 2012 Volume 13( Issue 1) pp:99-107

Publication Date(Web):

DOI:10.1002/cphc.201100735

Abstract

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution.

A high-density 3D localization algorithm for stochastic optical reconstruction microscopy

Co-reporter:Hazen Babcock;Yaron M Sigal

Optical Nanoscopy 2012 Volume 1( Issue 1) pp:

Publication Date(Web):2012 December

DOI:10.1186/2192-2853-1-6

Stochastic optical reconstruction microscopy (STORM) and related methods achieves sub-diffraction-limit image resolution through sequential activation and localization of individual fluorophores. The analysis of image data from these methods has typically been confined to the sparse activation regime where the density of activated fluorophores is sufficiently low such that there is minimal overlap between the images of adjacent emitters. Recently several methods have been reported for analyzing higher density data, allowing partial overlap between adjacent emitters. However, these methods have so far been limited to two-dimensional imaging, in which the point spread function (PSF) of each emitter is assumed to be identical.In this work, we present a method to analyze high-density super-resolution data in three dimensions, where the images of individual fluorophores not only overlap, but also have varying PSFs that depend on the z positions of the fluorophores.This approach accurately analyzed data sets with an emitter density five times higher than previously possible with sparse emitter analysis algorithms. We applied this algorithm to the analysis of data sets taken from membrane-labeled retina and brain tissues which contain a high-density of labels, and obtained substantially improved super-resolution image quality.

Coupled, Circumferential Motions of the Cell Wall Synthesis Machinery and MreB Filaments in B. subtilis

Co-reporter:Ethan C. Garner;Remi Bernard;Wenqin Wang;David Z. Rudner;Tim Mitchison

Science 2011 Volume 333(Issue 6039) pp:222-225

Publication Date(Web):08 Jul 2011

DOI:10.1126/science.1203285

Bacteria elongation involves moving synthetic complexes around the cell wall.

Chromosome Organization by a Nucleoid-Associated Protein in Live Bacteria

Co-reporter:Gene-Wei Li;Wenqin Wang;X. Sunney Xie;Chongyi Chen

Science 2011 Volume 333(Issue 6048) pp:1445-1449

Publication Date(Web):09 Sep 2011

DOI:10.1126/science.1204697

Super-resolution imaging in live Escherichia coli reveals protein clusters that sequester DNA loci and organize the chromosome.

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Co-reporter:Mariana Mihalusova;John Y. Wu

PNAS 2011 108 (51 ) pp:

Publication Date(Web):2011-12-20

DOI:10.1073/pnas.1017686108

Telomerase ribonucleoprotein (RNP) employs an RNA subunit to template the addition of telomeric repeats onto chromosome ends. Previous studies have suggested that a region of the RNA downstream of the template may be important for telomerase activity and that the region could fold into a pseudoknot. Whether the pseudoknot motif is formed in the active telomerase RNP and what its functional role is have not yet been conclusively established. Using single-molecule FRET, we show that the isolated pseudoknot sequence stably folds into a pseudoknot. However, in the context of the full-length telomerase RNA, interference by other parts of the RNA prevents the formation of the pseudoknot. The protein subunits of the telomerase holoenzyme counteract RNA-induced misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of the pseudoknot but also reveal the critical role played by telomerase proteins in pseudoknot folding.

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Co-reporter:Mariana Mihalusova;John Y. Wu

PNAS 2011 108 (51 ) pp:

Publication Date(Web):2011-12-20

DOI:10.1073/pnas.1017686108

Telomerase ribonucleoprotein (RNP) employs an RNA subunit to template the addition of telomeric repeats onto chromosome ends. Previous studies have suggested that a region of the RNA downstream of the template may be important for telomerase activity and that the region could fold into a pseudoknot. Whether the pseudoknot motif is formed in the active telomerase RNP and what its functional role is have not yet been conclusively established. Using single-molecule FRET, we show that the isolated pseudoknot sequence stably folds into a pseudoknot. However, in the context of the full-length telomerase RNA, interference by other parts of the RNA prevents the formation of the pseudoknot. The protein subunits of the telomerase holoenzyme counteract RNA-induced misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of the pseudoknot but also reveal the critical role played by telomerase proteins in pseudoknot folding.

Photoswitching Mechanism of Cyanine Dyes

Co-reporter:Graham T. Dempsey ; Mark Bates ; Walter E. Kowtoniuk ; David R. Liu ; Roger Y. Tsien

Journal of the American Chemical Society 2009 Volume 131(Issue 51) pp:18192-18193

Publication Date(Web):December 4, 2009

DOI:10.1021/ja904588g

Cyanine dyes have been shown to undergo reversible photoswitching, where the fluorophore can be switched between a fluorescent state and a dark state upon illumination at different wavelengths. The photochemical mechanism by which switching occurs has yet to be elucidated. In this study, we have determined the mechanism of photoswitching by characterizing the kinetics of dark state formation and the spectral and structural properties of the dark state. The rate of switching to the dark state depends on the concentration of the primary thiol in the solution and the solution pH in a manner quantitatively consistent with the formation of an encounter complex between the cyanine dye and ionized thiol prior to their conjugation. Mass spectrometry suggests that the photoconversion product is a thiol−cyanine adduct in which covalent attachment of the thiol to the polymethine bridge disrupts the original conjugated π-electron system of the dye.

Dynamics of nucleosome remodelling by individual ACF complexes

Co-reporter:Timothy R. Blosser, Janet G. Yang, Michael D. Stone, Geeta J. Narlikar & Xiaowei Zhuang

Nature 2009 462(7276) pp:1022

Publication Date(Web):2009-12-24

DOI:10.1038/nature08627

The ATP-dependent chromatin assembly factor (ACF) generates regularly spaced nucleosomes, but the mechanism by which ACF mobilizes nucleosomes remains poorly understood. Here, single-molecule FRET is used to monitor the remodelling of individual nucleosomes by ACF in real time; the study reveals previously unknown remodelling intermediates and dynamics, and indicates that ACF is a highly processive and bidirectional nucleosome translocase.

Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy

Co-reporter:Bo Huang;Wenqin Wang;Mark Bates

Science 2008 Vol 319(5864) pp:810-813

Publication Date(Web):08 Feb 2008

DOI:10.1126/science.1153529

Abstract

Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.

Super-resolution microscopy by nanoscale localization of photo-switchable fluorescent probes

Co-reporter:Mark Bates, Bo Huang, Xiaowei Zhuang

Current Opinion in Chemical Biology 2008 Volume 12(Issue 5) pp:505-514

Publication Date(Web):October 2008

DOI:10.1016/j.cbpa.2008.08.008

A new form of super-resolution fluorescence microscopy has emerged in recent years, based on the high accuracy localization of individual photo-switchable fluorescent labels. Image resolution as high as 20 nm in the lateral dimensions and 50 nm in the axial direction has been attained with this concept, representing an order of magnitude improvement over the diffraction limit. The demonstration of multicolor imaging with molecular specificity, three-dimensional (3D) imaging of cellular structures, and time-resolved imaging of living cells further illustrates the exciting potential of this method for biological imaging at the nanoscopic scale.

Slide into Action: Dynamic Shuttling of HIV Reverse Transcriptase on Nucleic Acid Substrates

Co-reporter:Jason W. Rausch;Elio A. Abbondanzieri;Shixin Liu;Stuart F. J. Le Grice

Science 2008 Volume 322(Issue 5904) pp:1092-1097

Publication Date(Web):14 Nov 2008

DOI:10.1126/science.1163108

Abstract

The reverse transcriptase (RT) of human immunodeficiency virus (HIV) catalyzes a series of reactions to convert single-stranded viral RNA into double-stranded DNA for host cell integration. This process requires a variety of enzymatic activities, including DNA polymerization, RNA cleavage, strand transfer, and strand displacement synthesis. We used single-molecule fluorescence resonance energy transfer to probe the interactions between RT and nucleic acid substrates in real time. RT was observed to slide on nucleic acid duplexes, rapidly shuttling between opposite termini of the duplex. Upon reaching the DNA 3′ terminus, RT can spontaneously flip into a polymerization orientation. Sliding kinetics were regulated by cognate nucleotides and anti-HIV drugs, which stabilized and destabilized the polymerization mode, respectively. These long-range translocation activities facilitate multiple stages of the reverse transcription pathway, including normal DNA polymerization and strand displacement synthesis.

Dynamic binding orientations direct activity of HIV reverse transcriptase

Co-reporter:Elio A. Abbondanzieri, Gregory Bokinsky, Jason W. Rausch, Jennifer X. Zhang, Stuart F. J. Le Grice & Xiaowei Zhuang

Nature 2008 453(7192) pp:184

Publication Date(Web):2008-05-08

DOI:10.1038/nature06941

The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.

Virus trafficking – learning from single-virus tracking

Co-reporter:Boerries Brandenburg and Xiaowei Zhuang

Nature Reviews Microbiology 2007 5(3) pp:197

Publication Date(Web):2007-03-01

DOI:10.1038/nrmicro1615

What could be a better way to study virus trafficking than 'miniaturizing oneself' and 'taking a ride with the virus particle' on its journey into the cell? Single-virus tracking in living cells potentially provides us with the means to visualize the virus journey. This approach allows us to follow the fate of individual virus particles and monitor dynamic interactions between viruses and cellular structures, revealing previously unobservable infection steps. The entry, trafficking and egress mechanisms of various animal viruses have been elucidated using this method. The combination of single-virus trafficking with systems approaches and state-of-the-art imaging technologies should prove exciting in the future.

Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes

Co-reporter:Mark Bates;Bo Huang;Graham T. Dempsey

Science 2007 Volume 317(Issue 5845) pp:1749-1753

Publication Date(Web):21 Sep 2007

DOI:10.1126/science.1146598

Abstract

Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with a spatial resolution of 20 to 50 nanometers. Multicolor super-resolution imaging, however, remains a challenging task. Here, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable “reporter” fluorophore that can be cycled between fluorescent and dark states, and an “activator” that facilitates photo-activation of the reporter. Combinatorial pairing of reporters and activators allows the creation of probes with many distinct colors. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a super-resolution STORM image. Using this approach, we demonstrate multicolor imaging of DNA model samples and mammalian cells with 20- to 30-nanometer resolution. This technique will facilitate direct visualization of molecular interactions at the nanometer scale.

Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein

Co-reporter:Michael D. Stone, Mariana Mihalusova, Catherine M. O'Connor, Ramadevi Prathapam, Kathleen Collins and Xiaowei Zhuang

Nature 2007 446(7134) pp:458

Publication Date(Web):2007-02-25

DOI:10.1038/nature05600

Telomerase is an essential cellular ribonucleoprotein (RNP) that solves the end replication problem and maintains chromosome stability by adding telomeric DNA to the termini of linear chromosomes1, 2, 3. Genetic mutations that abrogate the normal assembly of telomerase RNP cause human disease4. It is therefore of fundamental and medical importance to decipher cellular strategies for telomerase biogenesis, which will require new insights into how specific interactions occur in a precise order along the RNP assembly pathway. Here we use a single-molecule approach to dissect the individual assembly steps of telomerase. Direct observation of complex formation in real time revealed two sequential steps of protein-induced RNA folding, establishing a hierarchical RNP assembly mechanism: interaction with the telomerase holoenzyme protein p65 induces structural rearrangement of telomerase RNA, which in turn directs the binding of the telomerase reverse transcriptase to form the functional ternary complex. This hierarchical assembly process is facilitated by an evolutionarily conserved structural motif within the RNA. These results identify the RNA folding pathway during telomerase biogenesis and define the mechanism of action for an essential telomerase holoenzyme protein.

Dissecting the multistep reaction pathway of an RNA enzyme by single-molecule kinetic “fingerprinting”

Co-reporter:Shixin Liu;Gregory Bokinsky;Nils G. Walter

PNAS 2007 Volume 104 (Issue 31 ) pp:12634-12639

Publication Date(Web):2007-07-31

DOI:10.1073/pnas.0610597104

Single-molecule FRET is a powerful tool for probing the kinetic mechanism of a complex enzymatic reaction. However, not every reaction intermediate can be identified via a distinct FRET value, making it difficult to fully dissect a multistep reaction pathway. Here, we demonstrate a method using sequential kinetic experiments to differentiate each reaction intermediate by a distinct time sequence of FRET signal (a kinetic “fingerprint”). Our model system, the two-way junction hairpin ribozyme, catalyzes a multistep reversible RNA cleavage reaction, which comprises two structural transition steps (docking/undocking) and one chemical reaction step (cleavage/ligation). Whereas the docked and undocked forms of the enzyme display distinct FRET values, the cleaved and ligated forms do not. To overcome this difficulty, we used Mg2+ pulse–chase experiments to differentiate each reaction intermediate by a distinct kinetic fingerprint at the single-molecule level. This method allowed us to unambiguously determine the rate constant of each reaction step and fully characterize the reaction pathway by using the chemically competent enzyme–substrate complex. We found that the ligated form of the enzyme highly favors the docked state, whereas undocking becomes accelerated upon cleavage by two orders of magnitude, a result different from that obtained with chemically blocked substrate and product analogs. The overall cleavage reaction is rate-limited by the docking/undocking kinetics and the internal cleavage/ligation equilibrium, contrasting the rate-limiting mechanism of the four-way junction ribozyme. These results underscore the kinetic interdependence of reversible steps on an enzymatic reaction pathway and demonstrate a potentially general route to dissect them.

Electrical detection of single viruses

Co-reporter:Fernando Patolsky;Gengfeng Zheng;Charles M. Lieber;Oliver Hayden;Melike Lakadamyali

PNAS 2004 Volume 101 (Issue 39 ) pp:14017-14022

Publication Date(Web):2004-09-28

DOI:10.1073/pnas.0406159101

We report direct, real-time electrical detection of single virus particles with high selectivity by using nanowire field effect transistors. Measurements made with nanowire arrays modified with antibodies for influenza A showed discrete conductance changes characteristic of binding and unbinding in the presence of influenza A but not paramyxovirus or adenovirus. Simultaneous electrical and optical measurements using fluorescently labeled influenza A were used to demonstrate conclusively that the conductance changes correspond to binding/unbinding of single viruses at the surface of nanowire devices. pH-dependent studies further show that the detection mechanism is caused by a field effect, and that the nanowire devices can be used to determine rapidly isoelectric points and variations in receptor-virus binding kinetics for different conditions. Lastly, studies of nanowire devices modified with antibodies specific for either influenza or adenovirus show that multiple viruses can be selectively detected in parallel. The possibility of large-scale integration of these nanowire devices suggests potential for simultaneous detection of a large number of distinct viral threats at the single virus level.

Assembly of endocytic machinery around individual influenza viruses during viral entry

Co-reporter:

Nature Structural and Molecular Biology 2004 11(6) pp:567-573

Publication Date(Web):02 May 2004

DOI:10.1038/nsmb769

Most viruses enter cells via receptor-mediated endocytosis. However, the entry mechanisms used by many of them remain unclear. Also largely unknown is the way in which viruses are targeted to cellular endocytic machinery. We have studied the entry mechanisms of influenza viruses by tracking the interaction of single viruses with cellular endocytic structures in real time using fluorescence microscopy. Our results show that influenza can exploit clathrin-mediated and clathrin- and caveolin-independent endocytic pathways in parallel, both pathways leading to viral fusion with similar efficiency. Remarkably, viruses taking the clathrin-mediated pathway enter cells via the de novo formation of clathrin-coated pits (CCPs) at viral-binding sites. CCP formation at these sites is much faster than elsewhere on the cell surface, suggesting a virus-induced CCP formation mechanism that may be commonly exploited by many other types of viruses.

Single-molecule enzymology of RNA: Essential functional groups impact catalysis from a distance

Co-reporter:David Rueda;Gregory Bokinsky;Maria M. Rhodes;Michael J. Rust;Nils G. Walter;

Proceedings of the National Academy of Sciences 2004 101(27) pp:10066-10071

Publication Date(Web):June 24, 2004

DOI:10.1073/pnas.0403575101

The hairpin ribozyme is a minimalist paradigm for studying RNA folding and function. In this enzyme, two domains dock by induced fit to form a catalytic core that mediates a specific backbone cleavage reaction. Here, we have fully dissected its reversible reaction pathway, which comprises two structural transitions (docking/undocking) and a chemistry step (cleavage/ligation), by applying a combination of single-molecule fluorescence resonance energy transfer (FRET) assays, ensemble cleavage assays, and kinetic simulations. This has allowed us to quantify the effects that modifications of essential functional groups remote from the site of catalysis have on the individual rate constants. We find that all ribozyme variants show similar fractionations into effectively noninterchanging molecule subpopulations of distinct undocking rate constants. This leads to heterogeneous cleavage activity as commonly observed for RNA enzymes. A modification at the domain junction additionally leads to heterogeneous docking. Surprisingly, most modifications not only affect docking/undocking but also significantly impact the internal chemistry rate constants over a substantial distance from the site of catalysis. We propose that a network of coupled molecular motions connects distant parts of the RNA with its reaction site, which suggests a previously undescribed analogy between RNA and protein enzymes. Our findings also have broad implications for applications such as the action of drugs and ligands distal to the active site or the engineering of allostery into RNA.

Single-molecule transition-state analysis of RNA folding

Co-reporter:Gregory Bokinsky;David Rueda;Vinod K. Misra;Maria M. Rhodes;Andrew Gordus;Hazen P. Babcock;Nils G. Walter;

Proceedings of the National Academy of Sciences 2003 100(16) pp:9302-9307

Publication Date(Web):July 17, 2003

DOI:10.1073/pnas.1133280100

How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozyme's two helical domains dock to make several tertiary contacts. Our single-molecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions.

Visualizing infection of individual influenza viruses

Co-reporter:Michael J. Rust;Melike Lakadamyali;Hazen P. Babcock

PNAS 2003 Volume 100 (Issue 16 ) pp:9280-9285

Publication Date(Web):2003-08-05

DOI:10.1073/pnas.0832269100

Influenza is a paradigm for understanding viral infections. As an opportunistic pathogen exploiting the cellular endocytic machinery for infection, influenza is also a valuable model system for exploring the cell's constitutive endocytic pathway. We have studied the transport, acidification, and fusion of single influenza viruses in living cells by using real-time fluorescence microscopy and have dissected individual stages of the viral entry pathway. The movement of individual viruses revealed a striking three-stage active transport process that preceded viral fusion with endosomes starting with an actin-dependent movement in the cell periphery, followed by a rapid, dynein-directed translocation to the perinuclear region, and finally an intermittent movement involving both plus- and minus-end-directed microtubule-based motilities in the perinuclear region. Surprisingly, the majority of viruses experience their initial acidification in the perinuclear region immediately following the dynein-directed rapid translocation step. This finding suggests a previously undescribed scenario of the endocytic pathway toward late endosomes: endosome maturation, including initial acidification, largely occurs in the perinuclear region.

Superresolution Imaging of Chemical Synapses in the Brain

Co-reporter:Adish Dani, Bo Huang, Joseph Bergan, Catherine Dulac, Xiaowei Zhuang

Neuron (9 December 2010) Volume 68(Issue 5) pp:843-856

Publication Date(Web):9 December 2010

DOI:10.1016/j.neuron.2010.11.021

Determination of the molecular architecture of synapses requires nanoscopic image resolution and specific molecular recognition, a task that has so far defied many conventional imaging approaches. Here, we present a superresolution fluorescence imaging method to visualize the molecular architecture of synapses in the brain. Using multicolor, three-dimensional stochastic optical reconstruction microscopy, the distributions of synaptic proteins can be measured with nanometer precision. Furthermore, the wide-field, volumetric imaging method enables high-throughput, quantitative analysis of a large number of synapses from different brain regions. To demonstrate the capabilities of this approach, we have determined the organization of ten protein components of the presynaptic active zone and the postsynaptic density. Variations in synapse morphology, neurotransmitter receptor composition, and receptor distribution were observed both among synapses and across different brain regions. Combination with optogenetics further allowed molecular events associated with synaptic plasticity to be resolved at the single-synapse level.Highlights► Superresolution fluorescence imaging of brain tissue ► High-throughput ultrastructural imaging of synapses ► High-precision position and orientation analyses of synaptic proteins ► Quantitative analyses of composition and plasticity of individual synapses

Rapid Actin-Dependent Viral Motility in Live Cells

Co-reporter:Joshua C. Vaughan, Boerries Brandenburg, James M. Hogle, Xiaowei Zhuang

Biophysical Journal (16 September 2009) Volume 97(Issue 6) pp:

Publication Date(Web):16 September 2009

DOI:10.1016/j.bpj.2009.07.011

During the course of an infection, viruses take advantage of a variety of mechanisms to travel in cells, ranging from diffusion within the cytosol to active transport along cytoskeletal filaments. To study viral motility within the intrinsically heterogeneous environment of the cell, we have developed a motility assay that allows for the global and unbiased analysis of tens of thousands of virus trajectories in live cells. Using this assay, we discovered that poliovirus exhibits anomalously rapid intracellular movement that was independent of microtubules, a common track for fast and directed cargo transport. Such rapid motion, with speeds of up to 5 μm/s, allows the virus particles to quickly explore all regions of the cell with the exception of the nucleus. The rapid, microtubule-independent movement of poliovirus was observed in multiple human-derived cell lines, but appeared to be cargo-specific. Other cargo, including a closely related picornavirus, did not exhibit similar motility. Furthermore, the motility is energy-dependent and requires an intact actin cytoskeleton, suggesting an active transport mechanism. The speed of this microtubule-independent but actin-dependent movement is nearly an order of magnitude faster than the fastest speeds reported for actin-dependent transport in animal cells, either by actin polymerization or by myosin motor proteins.

Statistical Deconvolution for Superresolution Fluorescence Microscopy

Co-reporter:Eran A. Mukamel, Hazen Babcock, Xiaowei Zhuang

Biophysical Journal (16 May 2012) Volume 102(Issue 10) pp:

Publication Date(Web):16 May 2012

DOI:10.1016/j.bpj.2012.03.070

Superresolution microscopy techniques based on the sequential activation of fluorophores can achieve image resolution of ∼10 nm but require a sparse distribution of simultaneously activated fluorophores in the field of view. Image analysis procedures for this approach typically discard data from crowded molecules with overlapping images, wasting valuable image information that is only partly degraded by overlap. A data analysis method that exploits all available fluorescence data, regardless of overlap, could increase the number of molecules processed per frame and thereby accelerate superresolution imaging speed, enabling the study of fast, dynamic biological processes. Here, we present a computational method, referred to as deconvolution-STORM (deconSTORM), which uses iterative image deconvolution in place of single- or multiemitter localization to estimate the sample. DeconSTORM approximates the maximum likelihood sample estimate under a realistic statistical model of fluorescence microscopy movies comprising numerous frames. The model incorporates Poisson-distributed photon-detection noise, the sparse spatial distribution of activated fluorophores, and temporal correlations between consecutive movie frames arising from intermittent fluorophore activation. We first quantitatively validated this approach with simulated fluorescence data and showed that deconSTORM accurately estimates superresolution images even at high densities of activated fluorophores where analysis by single- or multiemitter localization methods fails. We then applied the method to experimental data of cellular structures and demonstrated that deconSTORM enables an approximately fivefold or greater increase in imaging speed by allowing a higher density of activated fluorophores/frame.

TOPICS

Spatial control of cellular enzymes

Spatial organization inside the nucleus

Multiplexed RNA imaging in single cells

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