scholarly journals Build Your Own Microscope: Step-By-Step Guide for Building a Prism-Based TIRF Microscope

2018 ◽  
Vol 1 (4) ◽  
pp. 40 ◽  
Author(s):  
Dalton Gibbs ◽  
Anisa Kaur ◽  
Anoja Megalathan ◽  
Kumar Sapkota ◽  
Soma Dhakal
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Life Sciences ◽  
Inverted Microscope ◽  
Sophisticated Instrument ◽  
High Level ◽  
Single Molecule Analysis ◽  
Molecule Dynamics ◽  
Vast Range

Prism-based total internal reflection fluorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum dots via evanescent field of a confined region of space, which is beneficial not only for single molecule detection but also for analysis of single molecule dynamics and for acquiring kinetics data. However, there is neither a commercial microscope available for purchase nor a detailed guide dedicated for building this microscope. Thus far, pTIRF microscopes are custom-built with the use of a commercially available inverted microscope, which requires high level of expertise in selecting and handling sophisticated instrument-parts. To directly address this technology gap, here we describe a step-by-step guide on how to build and characterize a pTIRF microscope for in vitro single-molecule imaging, nanostructure analysis and other life sciences research.

scholarly journals Large domain movements upon UvrD dimerization and helicase activation

2017 ◽  
Vol 114 (46) ◽  
pp. 12178-12183 ◽  
Author(s):  
Binh Nguyen ◽  
Yerdos Ordabayev ◽  
Joshua E. Sokoloski ◽  
Elizabeth Weiland ◽  
Timothy M. Lohman
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Self Assembly ◽  
Total Internal Reflection ◽  
Dna Helicase ◽  
Conformational State ◽  
Helicase Activity ◽  
Multiple Conformations ◽  
Dna Substrate

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.

scholarly journals Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yann Gambin ◽  
Nicholas Ariotti ◽  
Kerrie-Ann McMahon ◽  
Michele Bastiani ◽  
Emma Sierecki ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Building Blocks ◽  
Scaffolding Protein ◽  
Cell Extracts ◽  
Single Molecule Analysis ◽  
Exquisite Specificity

In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.

scholarly journals Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation

2016 ◽  
Vol 214 (6) ◽  
pp. 705-718 ◽  
Author(s):  
Ye Jin Chai ◽  
Emma Sierecki ◽  
Vanesa M. Tomatis ◽  
Rachel S. Gormal ◽  
Nichole Giles ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Chaperone ◽  
Neurotransmitter Release ◽  
Lewy Body ◽  
Epileptic Encephalopathy ◽  
Chaperone Activity ◽  
Wild Type ◽  
Developmental Defects ◽  
Single Molecule Analysis

Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body–like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-SynWT and that of the Parkinson’s disease–causing α-SynA30P mutant, an effect rescued by Munc18-1WT expression, indicative of chaperone activity. Coexpression of the α-SynA30P mutant with Munc18-1 reduced the number of α-SynA30P aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration.

scholarly journals BLM helicase facilitates telomere replication during leading strand synthesis of telomeres

2015 ◽  
Vol 210 (2) ◽  
pp. 191-208 ◽  
Author(s):  
William C. Drosopoulos ◽  
Settapong T. Kosiyatrakul ◽  
Carl L. Schildkraut
Keyword(s):  
Single Molecule ◽  
Werner Syndrome ◽  
Telomeric Dna ◽  
G4 Dna ◽  
Genome Wide ◽  
Blm Helicase ◽  
Telomere Replication ◽  
Leading Strand ◽  
Single Molecule Analysis

Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome–associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, we found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, our findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis.

scholarly journals Mapping DNA replication with nanopore sequencing

2018 ◽  
Author(s):  
Magali Hennion ◽  
Jean-Michel Arbona ◽  
Corinne Cruaud ◽  
Florence Proux ◽  
Benoît Le Tallec ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Electrical Signal ◽  
Yeast Cells ◽  
Nanopore Sequencing ◽  
Single Molecule Level ◽  
Experimental Systems ◽  
Genome Wide ◽  
Single Molecule Analysis

ABSTRACTWe have harnessed nanopore sequencing to study DNA replication genome-wide at the single-molecule level. Using in vitro prepared DNA substrates, we characterized the effect of bromodeoxyuridine (BrdU) substitution for thymidine on the MinION nanopore electrical signal. Using a neural-network basecaller trained on yeast DNA containing either BrdU or thymidine, we identified BrdU-labelled tracts in yeast cells synchronously entering S phase in the presence of hydroxyurea and BrdU. As expected, the BrdU-labelled tracts coincided with previously identified early-firing, but not late-firing, replication origins. These results open the way to high-throughput, high-resolution, single-molecule analysis of DNA replication in many experimental systems.

scholarly journals Distinct roles and actions of PDI family enzymes in catalysis of nascent-chain disulfide formation

2021 ◽  
Author(s):  
Chihiro Hirayama ◽  
Kodai Machida ◽  
Kentaro Noi ◽  
Tadayoshi Murakawa ◽  
Masaki Okumura ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Single Molecule ◽  
Disulfide Bonds ◽  
High Speed ◽  
Binding Pocket ◽  
Face To Face ◽  
Disulfide Formation ◽  
Nascent Chain ◽  
Single Molecule Analysis

AbstractThe mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed an in vitro system for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains (RNC) of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with short segments exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Similarly to ERp46, a PDI mutant with an occluded substrate-binding pocket displayed shorter-time RNC binding and higher efficiency in disulfide introduction than wild-type PDI. Altogether, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation in RNC when longer nascent chains emerge out from ribosome.

scholarly journals Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Chantal L Márquez ◽  
Derrick Lau ◽  
James Walsh ◽  
Vaibhav Shah ◽  
Conall McGuinness ◽  
...  
Keyword(s):  
Single Molecule ◽  
Nuclear Import ◽  
Rate Limiting Step ◽  
Viral Cdna ◽  
Microscopy Method ◽  
Single Molecule Analysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hiv 1

Uncoating of the metastable HIV-1 capsid is a tightly regulated disassembly process required for release of the viral cDNA prior to nuclear import. To understand the intrinsic capsid disassembly pathway and how it can be modulated, we have developed a single-particle fluorescence microscopy method to follow the real-time uncoating kinetics of authentic HIV capsids in vitro immediately after permeabilizing the viral membrane. Opening of the first defect in the lattice is the rate-limiting step of uncoating, which is followed by rapid, catastrophic collapse. The capsid-binding inhibitor PF74 accelerates capsid opening but stabilizes the remaining lattice. In contrast, binding of a polyanion to a conserved arginine cluster in the lattice strongly delays initiation of uncoating but does not prevent subsequent lattice disassembly. Our observations suggest that different stages of uncoating can be controlled independently with the interplay between different capsid-binding regulators likely to determine the overall uncoating kinetics.

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