Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer

DNA-interacting proteins have roles in multiple processes, many operating as molecular machines which undergo dynamic meta-stable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner, fast enough to sample the molecular transitions across the free-energy landscape. Progress has been made over the past decade in utilizing cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection signal-to-noise of molecular signatures and transition events. In the present paper, we discuss a range of techniques we have developed to monitor DNA–protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our laboratory made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new super-resolution fluorescence microscope.

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Assay Systems for Profiling Deubiquitinating Activity

International Journal of Molecular Sciences ◽

10.3390/ijms21165638 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5638

Author(s):

Jinhong Cho ◽

Jinyoung Park ◽

Eunice EunKyeong Kim ◽

Eun Joo Song

Keyword(s):

Protein Degradation ◽

Protein Interactions ◽

Live Cells ◽

Deubiquitinating Enzyme ◽

Protein Protein Interactions ◽

Deubiquitinating Enzymes ◽

Cell Lysates ◽

Cellular Processes

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

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High throughput mechanobiology: Force modulation of ensemble biochemical and cell-based assays

10.1101/2020.05.05.065912 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ália dos Santos ◽

Natalia Fili ◽

David S. Pearson ◽

Yukti Hari-Gupta ◽

Christopher P. Toseland

Keyword(s):

High Throughput ◽

Single Molecule ◽

Receptor Activation ◽

Magnetic Tweezers ◽

Live Cells ◽

Mechanical Stimuli ◽

Cellular Assays ◽

Novel Approach ◽

Microplate Reader

ABSTRACTMechanobiology is focused on how the physical forces and the mechanical properties of proteins, cells and tissues contribute to physiology and disease. While the response of proteins and cells to mechanical stimuli is critical for function, the tools to probe these activities are typically restricted to single molecule manipulations. Here, we have developed a novel microplate reader assay to encompass mechanical measurements with ensemble biochemical and cellular assays, using a microplate lid modified with magnets. This configuration enables multiple static magnetic tweezers to function simultaneously across the microplate, thereby greatly increasing throughput. The broad applicability and versatility of our approach has been demonstrated through in vitro force-induced enzymatic activity and conformation changes, along with force-induced receptor activation and their downstream signalling pathways in live cells. Overall, our methodology allows for the first-time ensemble biochemical and cell-based assays to be performed under force, in high throughput format. This novel approach would substantially add to the mechano-biological toolbox and increase the availability of mechanobiology measurements.

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Stratification relieves constraints from steric hindrance in the generation of compact actomyosin asters at the membrane cortex

Science Advances ◽

10.1126/sciadv.aay6093 ◽

2020 ◽

Vol 6 (11) ◽

pp. eaay6093

Author(s):

Amit Das ◽

Abrar Bhat ◽

Rastko Sknepnek ◽

Darius Köster ◽

Satyajit Mayor ◽

...

Keyword(s):

Myosin Ii ◽

Super Resolution ◽

Two Dimensions ◽

Coarse Grained ◽

In Vivo Studies ◽

Agent Based ◽

Molecular Stratification ◽

Rectangular Slab

Recent in vivo studies reveal that several membrane proteins are driven to form nanoclusters by active contractile flows arising from localized dynamic patterning of F-actin and myosin at the cortex. Since myosin-II assemble as minifilaments with tens of myosin heads, one might worry that steric considerations would obstruct the emergence of nanoclustering. Using coarse-grained, agent-based simulations that account for steric constraints, we find that the patterns exhibited by actomyosin in two dimensions, do not resemble the steady-state patterns in our in vitro reconstitution of actomyosin on a supported bilayer. We perform simulations in a thin rectangular slab, separating the layer of actin filaments from myosin-II minifilaments. This recapitulates the observed features of in vitro patterning. Using super resolution microscopy, we find evidence for such stratification in our in vitro system. Our study suggests that molecular stratification may be an important organizing feature of the cortical cytoskeleton in vivo.

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Lipid-Protein Interactions: are in Vitro and in Vivo Studies Contradictory Or Complementary?

Biophysical Journal ◽

10.1016/j.bpj.2010.12.2085 ◽

2011 ◽

Vol 100 (3) ◽

pp. 345a

Author(s):

Jean-Marie Ruysschaert ◽

Cedric Govaerts

Keyword(s):

Protein Interactions ◽

In Vivo Studies ◽

Lipid Protein Interactions

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The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1504822112 ◽

2015 ◽

Vol 112 (23) ◽

pp. 7189-7194 ◽

Cited By ~ 484

Author(s):

Shana Elbaum-Garfinkle ◽

Younghoon Kim ◽

Krzysztof Szczepaniak ◽

Carlos Chih-Hsiung Chen ◽

Christian R. Eckmann ◽

...

Keyword(s):

Phase Separation ◽

Phase Boundary ◽

Single Molecule ◽

Protein Interactions ◽

Driving Forces ◽

Early Embryo ◽

P Granules ◽

Intrinsically Disordered

P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA–protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA–protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.

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Nucleosomes accelerate transcription factor dissociation

Nucleic Acids Research ◽

10.1093/nar/gkt1319 ◽

2013 ◽

Vol 42 (5) ◽

pp. 3017-3027 ◽

Cited By ~ 82

Author(s):

Yi Luo ◽

Justin A. North ◽

Sean D. Rose ◽

Michael G. Poirier

Keyword(s):

Single Molecule ◽

Protein Interactions ◽

Protein Complexes ◽

High Specificity ◽

Dissociation Rate ◽

Duplex Dna ◽

Recognition Sequence ◽

Nucleosome Arrays

AbstractTranscription factors (TF) bind DNA-target sites within promoters to activate gene expression. TFs target their DNA-recognition sequences with high specificity by binding with resident times of up to hours in vitro. However, in vivo TFs can exchange on the order of seconds. The factors that regulate TF dynamics in vivo and increase dissociation rates by orders of magnitude are not known. We investigated TF binding and dissociation dynamics at their recognition sequence within duplex DNA, single nucleosomes and short nucleosome arrays with single molecule total internal reflection fluorescence (smTIRF) microscopy. We find that the rate of TF dissociation from its site within either nucleosomes or nucleosome arrays is increased by 1000-fold relative to duplex DNA. Our results suggest that TF binding within chromatin could be responsible for the dramatic increase in TF exchange in vivo. Furthermore, these studies demonstrate that nucleosomes regulate DNA–protein interactions not only by preventing DNA–protein binding but by dramatically increasing the dissociation rate of protein complexes from their DNA-binding sites.

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Chromatin fiber breaks into clutches under tension and crowding

10.1101/2021.11.16.468645 ◽

2021 ◽

Author(s):

Shuming Liu ◽

Xingcheng Lin ◽

Bin Zhang

Keyword(s):

Single Molecule ◽

Sol Gel ◽

In Vivo Studies ◽

Binding Modes ◽

Cell Nuclei ◽

Force Extension ◽

Fiber Breaks ◽

Experimental Findings

The arrangement of nucleosomes inside chromatin is of extensive interest. While in vitro experiments have revealed the formation of 30 nm fibers, most in vivo studies have failed to confirm their presence in cell nuclei. To reconcile the diverging experimental findings, we characterized chromatin organization using a near atomistic model. The computed force-extension curve matches well with measurements from single-molecule experiments. Notably, we found that a dodeca-nucleosome in the two-helix zigzag conformation breaks into structures with nucleosome clutches and a mix of trimers and tetramers under tension. Such unfolded configurations can also be stabilized through trans interactions with other chromatin chains. Our study supports a hypothesis that disordered, in vivo chromatin configurations arise as folding intermediates from regular fibril structures. We further revealed that chromatin segments with fibril or clutch structures engaged in distinct binding modes and discussed the implications of these inter-chain interactions for a potential sol-gel phase transition.

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In Vitro and In Vivo Studies Identify Important Features of Dengue Virus pr-E Protein Interactions

PLoS Pathogens ◽

10.1371/journal.ppat.1001157 ◽

2010 ◽

Vol 6 (10) ◽

pp. e1001157 ◽

Cited By ~ 43

Author(s):

Aihua Zheng ◽

Mahadevaiah Umashankar ◽

Margaret Kielian

Keyword(s):

Dengue Virus ◽

Protein Interactions ◽

In Vivo Studies ◽

E Protein

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Of numbers and movement – understanding transcription factor pathogenesis by advanced microscopy

Disease Models & Mechanisms ◽

10.1242/dmm.046516 ◽

2020 ◽

Vol 13 (12) ◽

pp. dmm046516

Author(s):

Julia M. T. Auer ◽

Jack J. Stoddart ◽

Ioannis Christodoulou ◽

Ana Lima ◽

Kassiani Skouloudaki ◽

...

Keyword(s):

Gene Expression ◽

Single Molecule ◽

Molecular Mechanisms ◽

Gene Dosage ◽

Super Resolution ◽

Loss Of Function ◽

Developmental Defects ◽

Tf Gene

ABSTRACTTranscription factors (TFs) are life-sustaining and, therefore, the subject of intensive research. By regulating gene expression, TFs control a plethora of developmental and physiological processes, and their abnormal function commonly leads to various developmental defects and diseases in humans. Normal TF function often depends on gene dosage, which can be altered by copy-number variation or loss-of-function mutations. This explains why TF haploinsufficiency (HI) can lead to disease. Since aberrant TF numbers frequently result in pathogenic abnormalities of gene expression, quantitative analyses of TFs are a priority in the field. In vitro single-molecule methodologies have significantly aided the identification of links between TF gene dosage and transcriptional outcomes. Additionally, advances in quantitative microscopy have contributed mechanistic insights into normal and aberrant TF function. However, to understand TF biology, TF-chromatin interactions must be characterised in vivo, in a tissue-specific manner and in the context of both normal and altered TF numbers. Here, we summarise the advanced microscopy methodologies most frequently used to link TF abundance to function and dissect the molecular mechanisms underlying TF HIs. Increased application of advanced single-molecule and super-resolution microscopy modalities will improve our understanding of how TF HIs drive disease.

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