Molecule Total Internal Reflection Fluorescence Research Articles

Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases.

Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases

Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence.

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit fromfunctional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (kcat). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

"Helicase" activity promoted through dynamic interactions between a ssDNA translocase and a diffusing SSB protein.

Replication protein A (RPA) is a eukaryotic single stranded (ss) DNA binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches we show that S. cerevisiae Pif1 can use its ATP-dependent 5’ to 3’ translocase activity to chemo-mechanically push human RPA (hRPA) directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity Pif1 can push RPA from an ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of RPA such that it can be readily reorganized even when bound tightly to ssDNA and demonstrate a new mechanism by which directional DNA unwinding can be achieved through the combined action of an ssDNA translocase that pushes an SSB protein. This work was supported by National Institutes of Health Grants 5R35GM136632 (to T.M.L.) and 1R35GM139508 (to R.G.) and American Cancer Society Grant PF-15-040-01-DMC (to J.E.S) and NIH Instrumentation Grant S10OD030315.

A single molecule TIRF-based platform to study DNA supercoiling effect on DNA processing enzymes.

DNA topology plays an important role in regulating DNA metabolic pathways. DNA supercoiling changes the DNA properties and proteins may differentially interact with DNA under various supercoiling states. A biophysical investigation of how proteins differentiate various topological states could further our understanding of protein-DNA interactions. We developed new single molecule total internal reflection fluorescence (smTIRF) -based platform to investigate how DNA topology affects protein-DNA interactions. We used this platform to study MutS, which detects a mismatched base pair to initiate mismatch repair, and CRISPR-Cas9. We generated plasmids that are site-specifically labeled with one or two fluorophores and a biotin. We prepared them in three different topological states: “Linear”, “relaxed circular”, and “negatively supercoiled”. For MutS study, we designed a Cy5 labeled plasmid with an adjacent mismatch. We observed FRET between Cy3-MutS and Cy5 when MutS bound the mismatch. In the presence of ADP, MutS bound the negative supercoiled DNA with a longer residence time compared to the relaxed circular DNA, suggesting that negative supercoiling helps mismatch recognition. For CRISPR-Cas9, we created a dual labeled plasmid to observe R-loop formation induced by dCas9. R-loop formation rate was similar for all three DNA topological states when the guide RNA sequence matched the target DNA sequence, while the rate was highest for the negatively supercoiled DNA and decreased progressively for the relaxed circular DNA and the linear DNA when we introduced one mismatch in either the PAM (protospacer adjacent motif)-proximal position or the PAM-distal position. Our observation confirms that DNA negative supercoiling assists DNA unwinding by Cas9, and this can exacerbate off-target effects in the cell. Collectively, our smTIRF-based platform allows high throughput investigation of supercoiling effects on DNA protein interactions as demonstrated using two different biological systems.

Single Molecule Analysis Reveals Dimer Formation of the Norepinephrine Transporter at the Plasma Membrane of Live Cells

BackgroundMonoamine transporters are the principal regulators of extracellular monoamine levels in the brain. At the plasma membrane, monoamine transporters form oligomers, which may play an important role in transporter trafficking, drug‐induced monoamine efflux, and drug tolerance development. Using single molecule microscopy, we previously reported that the serotonin transporter (SERT) co‐exists as monomers, dimers, and higher‐order oligomers, while the dopamine transporter (DAT) forms monomers and dimers only. Furthermore, whereas SERT oligomers are kinetically trapped at the plasma membrane by the phospholipid phosphatidylinositol (4,5)‐bisphosphate (PIP2), DAT dimerization is PIP2 independent. The subunit stoichiometry of the norepinephrine transporter (NET) is currently unknown. Based on a high degree of sequence homology between DAT and NET, we aimed to test the hypothesis that NET oligomerization resembles the one of DAT.MethodsWe fused monomeric green fluorescent protein (mGFP) to the N‐terminus of human NET and expressed the construct in Chinese hamster ovary cells. We assessed NET oligomerization using single molecule total internal reflection fluorescence (TIRF) microscopy in live cells. In order to determine the role of cell membrane lipids in NET oligomerization, we pharmacologically depleted the cell membrane of cholesterol and PIP2 before imaging.ResultsSingle molecule brightness analysis revealed that NET co‐exists as monomers (~60%) and dimers (~40%) at the plasma membrane. NET oligomerization displayed signs of transporter density dependence and subunit exchange. Cholesterol depletion did not affect NET dimerization, whereas PIP2 depletion resulted in a small but significant decrease in the average oligomeric state (Student's t‐test, P<0.05).ConclusionNET forms monomers and dimers but no higher‐order oligomers, thereby resembling the oligomerization of DAT. However, unlike DAT, NET dimers exchange subunits over time and dimerization is affected by the transporter density and PIP2 concentrations. This study therefore demonstrates that the oligomerization behavior of each monoamine transporters has unique features.

Novel Effect of Tau N-Terminal Mutation, R5L, on Microtubule Mediated Condensation

Understanding the physiological function of the intrinsically disordered microtubule associated protein (MAP) Tau has proven challenging. Tau, primarily expressed in neurons, is known to have a variety of axonal functions, including regulation of microtubule dynamics and modulation of kinesin motor motility. Interestingly, research on the microtubule binding behavior of Tau reveals that Tau binds to the microtubule surface in a dynamic equilibrium between static and diffusive states. Although it is understood that the binding state equilibrium plays a role in physiological Tau function, such as modulation of kinesin motility, it is less understood how disease associated mutations affect Tau binding behavior and function. The canonical theory states that mutations in Tau reduce Tau affinity for the microtubule. Here, we investigate the role of an N-terminal disease associated mutation in Tau, R5L, using an in vitro reconstituted system for single molecule Total Internal Reflection Fluorescence (smTIRF) Microscopy. Contrary to the canonical theory, we determined the R5L mutation does not reduce Tau affinity for the microtubule. Rather, the R5L mutation reduces the total amount of Tau bound to the microtubule at saturating conditions. Our data suggests the R5L mutation reduces Tau:Tau interactions, decreasing the ability of R5L-Tau to form larger order complexes, known as “Tau condensates.” Currently, we are determining the mechanism by which this occurs, potentially due to a structural change associated with the R5L mutation. Altogether, these results challenge the current paradigm of how mutations in Tau lead to disease.

Synergy between Wsp1 and Dip1 may initiate assembly of endocytic actin networks.

The actin filament nucleator Arp2/3 complex is activated at cortical sites in Schizosaccharomyces pombe to assemble branched actin networks that drive endocytosis. Arp2/3 complex activators Wsp1 and Dip1 are required for proper actin assembly at endocytic sites, but how they coordinately control Arp2/3-mediated actin assembly is unknown. Alone, Dip1 activates Arp2/3 complex without preexisting actin filaments to nucleate 'seed' filaments that activate Wsp1-bound Arp2/3 complex, thereby initiating branched actin network assembly. In contrast, because Wsp1 requires preexisting filaments to activate, it has been assumed to function exclusively in propagating actin networks by stimulating branching from preexisting filaments. Here we show that Wsp1 is important not only for propagation but also for initiation of endocytic actin networks. Using single molecule total internal reflection fluorescence microscopy we show that Wsp1 synergizes with Dip1 to co-activate Arp2/3 complex. Synergistic co-activation does not require preexisting actin filaments, explaining how Wsp1 contributes to actin network initiation in cells.

Study of Tau N-Terminal Mutation, R5L, on Tau Interaction with the Microtubule Lattice

Understanding the physiological function of the intrinsically disordered microtubule associated protein (MAP) Tau has proven challenging. Tau, primarily expressed in neurons, is known to have a variety of axonal functions, including regulation of microtubule dynamics, modulation of kinesin motor motility, and participation in signaling cascades. Interestingly, research on the microtubule binding behavior of Tau reveals that Tau binds to the microtubule surface in a dynamic equilibrium between static and diffusive states. Although it is understood that the binding state equilibrium plays a role in physiological Tau function, such as modulation of kinesin motility, it is less understood how disease associated mutations affect Tau binding behavior and function. The canonical theory states that mutations in Tau reduce Tau affinity for the microtubule. Here, we investigate the role of an N-terminal disease associated mutation, R5L, in both the 3RS and 4RS Tau isoforms using an in vitro reconstituted system for single molecule Total Internal Reflection Fluorescence (smTIRF) Microscopy. Contrary to the canonical theory, we determined the R5L mutation does not reduce Tau affinity to the microtubule. This was seen across microtubule lattices and Tau isoforms. Currently, we are investigating the single molecule properties of microtubule bound R5L-Tau to better comprehend the consequence of the R5L mutation. Altogether, these results show the canonical theory of disease mechanism does not encompass all Tau mutations and the theory needs to be expanded. This work will inform the mechanisms by which N-terminal disease associated mutations alter Tau function.

Direct Detection and Characterization of a Phosphoinositide Dependent Kinase-1 (PDK1) Homodimer on a Target Membrane Surface Via Single Molecule Fluorescence

Phosphoinositide Dependent Kinase-1 (PDK1, gene name PDPK1) is a Serine/Threonine kinase that acts as a master kinase, phosphorylating numerous targets from the AGC family of kinases, including protein kinase B (PKB/AKT1) and protein kinase C (PKC). PDK1 exhibits a monomer-dimer equilibrium in cells, with the regulation of this equilibrium and its effect on activity remaining poorly described. Presented here are in vitro studies describing propensity for full length, active PDK1 to form homodimers in a defined environment; namely, on a target membrane surface. Using single molecule total internal reflection fluorescence microscopy (smTIRFM) we measure two-dimensional diffusion of fluorescently labeled PDK1 molecules on a supported lipid bilayer. We observe a two-fold decrease in the diffusion coefficient of labeled PDK1 with increasing PDK1 concentration, consistent with homodimer formation on the supported lipid bilayer. Phosphorylation of threonine residue 513 on the PDK1 PH domain has been previously proposed to disrupt dimer formation. To test this hypothesis, we measured the apparent dimer KD for wild type PDK1, and point mutants T513A and T513E that are mimics for apo and phosphorylated states. The charge at residue 513 has little or no effect on the apparent KD of PDK1 dimerization in these experiments, inconsistent with regulation of dimerization by the charge of T513 previously observed in studies of the isolated PH domain. It follows that the isolated, bacterially expressed PDK1 PH domain possesses a different homodimer interface in the membrane-bound state than the full length PDK1 protein isolated from eukaryotic cells. Our most recent results will be presented.

Mg2+ -free ATP regulates the processivity of native cytoplasmic dynein.

Cytoplasmic dynein, a microtubule-based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 μm. We also identified the ratio of Mg2+ -free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping.

Understanding the Mechanisms of Human RNA Polymerase II Transcription using Single Molecule Fluorescence Colocalization

Transcription is the first step in cellular gene expression and a key regulatory point for determining how a cell responds to stimuli. In mammalian cells, transcription is initiated by the formation of a pre-initiation complex (PIC), an assembly of general transcription factors (GTFs) and RNA polymerase II (Pol II), on promoter DNA. Our goal is to use single molecule approaches to address mechanisms of PIC formation and activity that have previously been unattainable using traditional biochemical approaches. We have developed a novel single molecule total internal reflection fluorescence (TIRF) microscopy assay that allows us to visualize human Pol II transcriptional activity on individual DNA templates. With this single molecule setup, we have performed experiments to address the pathways by which active pre-initiation complexes can form. Ultimately we found that Pol II and three GTFs can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA.We are also developing a single molecule system to monitor dynamic protein-protein interactions that facilitate activation of transcription. It has long been known that transcriptional activators interact with subunits of transcription factor IID (TFIID) to potentiate high levels of transcription, however, the biophysical parameters defining these interactions are almost entirely uncharacterized. We have developed a novel method to label transcriptional activators and monitor their interactions with transcription factor TFIID in real-time using single molecule fluorescence colocalization. We observe that the activation domains from different activators exhibit different kinetic parameters for interactions with TFIID. We are positioned to address previously unanswered questions regarding how important protein-protein interactions between TFIID and transcriptional activators impact gene expression.

Superfamily 1 Helicase PIF1 Can Push Replication Protein a Along Single Stranded DNA and into Duplex DNA

Superfamily 1 helicase monomers are single stranded (ss) DNA translocases, but poor helicases. Replication protein A (RPA) is an ssDNA binding protein (SSB) that binds ssDNA with high affinity and can diffuse along ssDNA. On its own, RPA is capable of disrupting DNA secondary structure up to ∼7 base pairs (bp). We have previously shown using a novel single molecule total internal reflection fluorescence FRET assay that ssDNA translocases can push an E. coli SSB tetramer along ssDNA (Sokoloski et.al, PNAS.

Chemo-mechanical pushing of proteins along single-stranded DNA

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

Application of single molecule fluorescence microscopy to characterize the penetration of a large amphiphilic molecule in the stratum corneum of human skin.

We report here on the application of laser-based single molecule total internal reflection fluorescence microscopy (TIRFM) to study the penetration of molecules through the skin. Penetration of topically applied drug molecules is often observed to be limited by the size of the respective drug. However, the molecular mechanisms which govern the penetration of molecules through the outermost layer of the skin are still largely unknown. As a model compound we have chosen a larger amphiphilic molecule (fluorescent dye ATTO-Oxa12) with a molecular weight >700 Da that was applied to excised human skin. ATTO-Oxa12 penetrated through the stratum corneum (SC) into the viable epidermis as revealed by TIRFM of cryosections. Single particle tracking of ATTO-Oxa12 within SC sheets obtained by tape stripping allowed us to gain information on the localization as well as the lateral diffusion dynamics of these molecules. ATTO-Oxa12 appeared to be highly confined in the SC lipid region between (intercellular space) or close to the envelope of the corneocytes. Three main distinct confinement sizes of 52 ± 6, 118 ± 4, and 205 ± 5 nm were determined. We conclude that for this amphiphilic model compound several pathways through the skin exist.

Motor and Tail Homology 1 (TH1) Domains Antagonistically Control Myosin-1 Dynamics

Class 1 myosins are monomeric motor proteins that fulfill diverse functions at the membrane/cytoskeletal interface. All myosins-1 contain a motor domain, which binds actin, hydrolyzes ATP, and generates forces, and a TH1 domain, which interacts directly with membrane lipids. In most cases, TH1 is needed for proper subcellular localization and presumably function, although little is known about how this domain regulates the behavior of class 1 myosins in live cells. To address this, we used single molecule total internal reflection fluorescence microscopy to examine the dynamics of the well-characterized myosin-1a isoform during interactions with the cortex of living cells. Our studies revealed that full-length myosin-1a exhibits restricted mobility relative to TH1 alone. Motor domain mutations that disrupt actin binding increased the mobility of full-length myosin-1a, whereas mutations to the TH1 domain that are known to reduce steady-state targeting to the plasma membrane unexpectedly reduced mobility. Deletion of the calmodulin-binding lever arm in Myo1a mimicked the impact of actin-binding mutations. Finally, myosin-1b, which demonstrates exquisite sensitivity to mechanical load, exhibited dynamic behavior nearly identical to myosin-1a. These studies are the first, to our knowledge, to explore class 1 myosin dynamics at the single-molecule level in living cells; our results suggest a model where the motor domain restricts dynamics via a mechanism that requires the lever arm, whereas the TH1 domain allows persistent diffusion in close proximity to the plasma membrane.

Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation

Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The single molecule photobleaching (SMPB) technology for direct counting developed by our team (Shu et al., 2007 [18]; Zhang et al., 2007 [19]) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described.

Nucleosomes accelerate transcription factor dissociation

Transcription 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.

Breaking the Millisecond Barrier: Single Molecule Motors Wobble to Find their Next Binding Sites

Breaking the Millisecond Barrier: Single Molecule Motors Wobble to Find their Next Binding Sites

Single-Molecule Fluorescence Observations of Eukaryotic Translation

For the two last decades, structural and single-molecule investigations of the prokaryotic ribosome have provided a better understanding of the highly dynamic nature of translation. Eukaryotic ribosome has received far less attention, most of the conclusions being extrapolated from prokaryotic data. Although both ribosomes share functional and structural features, eukaryotic ribosome contains many additional domains of unknown function. Applying single-molecule techniques to eukaryote ribosomes would clarify the function of these additional domains.In order to study structural rearrangement of the ribosome during eukaryotic elongation, we bypass canonical initiation using an initiation factor free Internal Ribosome Entry Site (IRES) enabling elongation in the desired reading frame. Ribosomal subunits, purified from Rabbit Reticulocyte Lysate (RRL), are first bound to the IRES. Ribosome-mRNA complexes are then fixed on a microscope coverslip via the 5'end of the mRNA. All factors necessary for elongation are provided by injection of RRL. using single molecule Total Internal Reflection Fluorescence (TIRF) microscopy, we monitor elongation thanks to a fluorescently labelled oligonucleotide hybridized on the mRNA. The oligonucleotide is detached by the helicase activity of a translating ribosome. The evanescent wave generated by the TIRF apparatus allows solely the detection of molecules close to the surface. In this configuration, probe detachments induce the loss of fluorescence signals which are used to measure the distribution of translation speed. A thorough care is given to photobleaching during data analysis. Controls using antibiotics further show that the loss of fluorescence is a signature of a single ribosome activity.