25 research outputs found
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Probing cellular protein complexes using single-molecule pull-down.
Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways
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Dissecting the sharp response of a canonical developmental enhancer reveals multiple sources of cooperativity.
Developmental enhancers integrate graded concentrations of transcription factors (TFs) to create sharp gene expression boundaries. Here we examine the hunchback P2 (HbP2) enhancer which drives a sharp expression pattern in the Drosophila blastoderm embryo in response to the transcriptional activator Bicoid (Bcd). We systematically interrogate cis and trans factors that influence the shape and position of expression driven by HbP2, and find that the prevailing model, based on pairwise cooperative binding of Bcd to HbP2 is not adequate. We demonstrate that other proteins, such as pioneer factors, Mediator and histone modifiers influence the shape and position of the HbP2 expression pattern. Comparing our results to theory reveals how higher-order cooperativity and energy expenditure impact boundary location and sharpness. Our results emphasize that the bacterial view of transcription regulation, where pairwise interactions between regulatory proteins dominate, must be reexamined in animals, where multiple molecular mechanisms collaborate to shape the gene regulatory function
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Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells
Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators
Protective Effect of Tat PTD-Hsp27 Fusion Protein on Tau Hyperphosphorylation Induced by Okadaic Acid in the Human Neuroblastoma Cell Line SH-SY5Y
Single-molecule study of the DNA helicase regulating genomic stability
A DNA helicase, PcrA is an essential protein in gram-positive bacteria. PcrA utilizes its helicase activity and translocation activity to regulate various cellular functions including plasmid replication and counteracting deleterious recombination.
Using single molecule FRET technique and site-directed mutation, we discovered enzymes new activity: PcrA act as a powerful motor and remove RecA protein effectively from single strand DNA (ssDNA) as it pulls the RecA-coated lagging strand from the DNA junction. Our observation provides plausible mechanistic explanation for how a helicase can protect stalled replication fork from uncontrolled recombination in vivo.
PcrA is a prototypical translocating motor of which structure and kinetics are heavily studied for its kinetic mechanism. Utilizing the persistent reeling-in activity of PcrA, we conducted heterogeneity-free analysis and identified that the fundamental unit of translocation kinetics is a ???one nucleotide uniform step???. Our result reconciles debates between the structural and biochemical studies on the motor mechanism, and moreover, clarifies a popular oversight regarding molecular heterogeneity.
Next, I developed new detection scheme to monitor translocation dynamics of unlabeled protein that carries metal containing domain. By utilizing protein???s ability to quench organic fluorophore in distance dependent manner, XPD helicase translocation on ssDNA was monitored and, the differential effect of single-strand binding protein, RPAs on the XPD translocation kinetics were evaluated.
Lastly, I aim to observe translocating motor on a physiological length of ssDNA. FRET has limited working distance and therefore about 80 nucleotides (nt) or further distance change is undetectable. Custom made DNA nano-structure was devised and it is capable of providing up to 2000 nt stretched ssDNA tracks immobilized on the surface. I use fluorescence colocalization and FIONA technique to monitor hundreds of nanometer scale motion of translocases and activity of other single strand interacting proteins
Dissecting the sharp response of a canonical developmental enhancer reveals multiple sources of cooperativity.
Selectively Impaired Endocannabinoid-Dependent Long-Term Depression in the Lateral Habenula in an Animal Model of Depression
Abnormal potentiation in the lateral habenula (LHb) has been suggested to mediate depression-like behaviors. However, the underlying mechanisms of the synaptic efficacy regulation of LHb synapses and the potential for their modulation are only poorly understood. Here, we report that long-term synaptic depression (LTD) occurs in the LHb upon both low-frequency stimulation (LFS) and moderate-frequency stimulation (MFS). LFS-induced LTD (LFS-LTD) is accompanied by a reduction in presynaptic release probability, which is endocannabinoid (eCB) signaling dependent. Surprisingly, exposure to an acute stressor completely masks the induction of LFS-LTD in the LHb while leaving the MFS-induced LTD intact. Pharmacological activation of cannabinoid receptor 1 (CB1R) or blockade of αCaMKII successfully restored LTD in the LHb in an animal model of depression. Thus, our findings reveal a form of synaptic strength regulation and a stress-induced shift of synaptic plasticity in the LHb
The ATP Hydrolysis Cycle and the Corresponding Motion of RecA Filament on Single-Stranded DNA
pH dependent stability change of RecA filament at 5′-disassembly end.
<p>(a) A model for RecA monomer binding and dissociation at the filament end. (b) Single molecule FRET histograms at different pH. The histogram was fit with three Gaussian peaks for M<sub>0</sub>, M<sub>1</sub>, and M<sub>2</sub> states, respectively, with additional peak at 0 for donor-only molecules. (c) The populations for each peak found in (b) at various pHs (symbols). Dashed lines are eye guides. (d) Average number of monomers bound between donor and acceptors (filled rectangles) calculated from (c): N<sub>average</sub> = 2*P(M<sub>2</sub>) + 1*P(M<sub>1</sub>) +0*P(M<sub>0</sub>), where P(M<sub>i</sub>) is the population in M<sub>i</sub> state. Red dashed line is a linear fit to the data with a slope −0.29.</p
PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps. Cell
Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA-bound proteins, and basic properties like the elementary step size remain controversial. Using single-molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a singlestranded loop. Static disorder limited previous ensemble studies of a PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 nt. This reeling-in activity requires the open conformation of PcrA and can rapidly dismantle a preformed RecA filament even at low PcrA concentrations, suggesting a mode of action for eliminating potentially deleterious recombination intermediates