Combined Optical Tweezers Research Articles

Single-molecule analysis of purified proteins and nuclear extracts: Insights from 8-oxoguanine glycosylase 1

By observing one molecule at a time, single-molecule studies can offer detailed insights about biomolecular processes including on rates, off rates, and diffusivity of molecules on strands of DNA. A recent technological advance (Single-molecule Analysis of DNA-binding proteins from Nuclear Extracts, SMADNE) has lowered the barrier to entry for single-molecule studies, and single-molecule dynamics can now be determined directly out of nuclear extracts, providing information in an intermediate environment between purified proteins in isolation and the heterogeneity of a nucleus. To compare and contrast the single-molecule DNA binding dynamics in nuclear extracts versus purified proteins, combined optical tweezers and fluorescence microscopy experiments were performed with purified GFP-tagged 8-oxoguanine glycosylase 1 (OGG1), purified GFP-OGG1 spiked into nuclear extracts, and nuclear extracts from human cells overexpressing GFP-OGG1. We observed differences in undamaged DNA binding during DNA damage search in each of the three conditions. Purified GFP-OGG1 engaged undamaged DNA for a weighted average lifetime of 5.7 s and 21% of these events underwent DNA diffusion after binding. However, unlike other glycosylases studied by SMADNE, OGG1 does not bind non-damaged DNA efficiently in nuclear extracts. In contrast, GFP-OGG1 binding dynamics on DNA substrates containing oxidative damage were relatively similar in all three conditions, with the weighted average binding lifetimes varying from 2.2 s in nuclear extracts to 7.8 s with purified GFP-OGG1 in isolation. Finally, we compared the purified protein and nuclear extract approaches for a catalytically dead OGG1 variant (GFP-OGG1-K249Q). This variant greatly increased the binding lifetime for oxidative DNA damage, with the weighted average lifetime for GFP-OGG1–249Q in nuclear extracts at 15.4 s vs 10.7 s for the purified protein. SMADNE will provide a new window of observation into the behavior of nucleic acid binding proteins only accessible by biophysicists trained in protein purification and protein labeling.

High-Resolution, High-Contrast Optical Interface for Defect Qubits

Point defects in crystals provide important building blocks for quantum applications. To initialize, control, and read-out their quantum states, an efficient optical interface for addressing defects with photons is required. However, conventional confocal fluorescence microscopy with high refractive index crystals has limited photon collection efficiency and spatial resolution. Here, we demonstrate high resolution, high contrast imaging for defects qubits using microsphere-assisted confocal microscopy. A microsphere provides an excellent optical interface for point defects with a magnified virtual image that improves spatial resolution up to ~${\lambda}$/5 as well as an optical signal-to-noise ratio by four times. These features enable individual optical addressing of single photons and single spins of spatially-unresolved defects in conventional confocal microscopy with improved signal contrast. The combined optical tweezers show the possibility of positioning or scanning the microspheres for deterministic coupling and wide-field imaging of defects. The approach does not require any complicated fabrication and additional optical system but uses simple micro-optics off-the-shelf. From these distinctive advantages of the microspheres, our approach can provide an efficient way for imaging and addressing closely-spaced defects with higher resolution and sensitivity.

CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS.

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single-molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced by the presence of CTP or the non-hydrolysable analogue CTPγS. However, ParB proteins are also detected at a lower density in distal non-specific DNA. This requires the presence of a parS loading site and is prevented by protein roadblocks, consistent with one-dimensional diffusion by a sliding clamp. ParB diffusion on non-specific DNA is corroborated by direct visualization and quantification of movement of individual quantum dot labelled ParB. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations.

The Mitotic Crosslinking Protein PRC1 Acts as a Mechanical Dashpot to Resist Microtubule Sliding

Cell division in eukaryotes requires the regulated assembly of the spindle apparatus. The proper organization of microtubules within the spindle is driven by motor proteins that exert forces to push and slide filaments, while non-motor proteins can crosslink filaments into higher order motifs such as overlapping bundles. It has not been clear how active and passive forces are integrated to produce regulated mechanical outputs within spindles. Here we employ a combined optical tweezers and TIRF microscopy instrument to directly measure the resistive forces produced by the mitotic crosslinking protein PRC1. We observe that PRC1 generates frictional forces that resist microtubule sliding. These forces scale with microtubule sliding velocity and the number of PRC1 crosslinks, but do not depend on overlap length or PRC1 density within overlaps. Our results suggest that PRC1 ensembles act like a mechanical dashpot, producing significant resistance against fast motions, but minimal resistance against slow motions, allowing for the integration of diverse motor activities into a single mechanical outcome.

Unravelling the structural plasticity of stretched DNA under torsional constraint.

Regions of the genome are often held under torsional constraint. Nevertheless, the influence of such constraint on DNA–protein interactions during genome metabolism is still poorly understood. Here using a combined optical tweezers and fluorescence microscope, we quantify and explain how torsional constraint influences the structural stability of DNA under applied tension. We provide direct evidence that concomitant basepair melting and helical unwinding can occur in torsionally constrained DNA at forces >∼50 pN. This striking result indicates that local changes in linking number can be absorbed by the rest of the DNA duplex. We also present compelling new evidence that an overwound DNA structure (likely P-DNA) is created (alongside underwound structures) at forces >∼110 pN. These findings substantiate previous theoretical predictions and highlight a remarkable structural plasticity of torsionally constrained DNA. Such plasticity may be required in vivo to absorb local changes in linking number in DNA held under torsional constraint.

Cell manipulation tool with combined microwell array and optical tweezers for cell isolation and deposition

Isolation from rare cells and deposition of sorted cells with high accuracy for further study are critical to a wide range of biomedical applications. In the current paper, we report an automated cell manipulation tool with combined optical tweezers and a uniquely designed microwell array, which functions for recognition, isolation, assembly, transportation and deposition of the interesting cells. The microwell array allows the passive hydrodynamic docking of cells, while offering the opportunity to inspect the interesting cell phenotypes with high spatio-temporal resolution based on the flexible image processing technique. In addition, dynamic and parallel cell manipulation in three dimensions can realize the target cell levitation from microwell and pattern assembly with multiple optical traps. Integrated with the programmed motorized stage, the optically levitated and assembled cells can be transported and deposited to the predefined microenvironment, so the tool can facilitate the integration of other on-chip functionalities for further study without removing these isolated cells from the chip. Experiments on human embryonic stem cells and yeast cells are performed to demonstrate the effectiveness of the proposed cell manipulation tool. Besides the application to cell isolation and deposition, three other biological applications with this tool are also presented.

Effects of Plasma Membrane Cholesterol Level and Cytoskeleton F-Actin on Cell Protrusion Mechanics

Protrusions are deformations that form at the surface of living cells during biological activities such as cell migration. Using combined optical tweezers and fluorescent microscopy, we quantified the mechanical properties of protrusions in adherent human embryonic kidney cells in response to application of an external force at the cell surface. The mechanical properties of protrusions were analyzed by obtaining the associated force-length plots during protrusion formation, and force relaxation at constant length. Protrusion mechanics were interpretable by a standard linear solid (Kelvin) model, consisting of two stiffness parameters, k 0 and k 1 (with k 0>k 1), and a viscous coefficient. While both stiffness parameters contribute to the time-dependant mechanical behavior of the protrusions, k 0 and k 1 in particular dominated the early and late stages of the protrusion formation and elongation process, respectively. Lowering the membrane cholesterol content by 25% increased the k 0 stiffness by 74%, and shortened the protrusion length by almost half. Enhancement of membrane cholesterol content by nearly two-fold increased the protrusion length by 30%, and decreased the k 0 stiffness by nearly two-and-half-fold as compared with control cells. Cytoskeleton integrity was found to make a major contribution to protrusion mechanics as evidenced by the effects of F-actin disruption on the resulting mechanical parameters. Viscoelastic behavior of protrusions was further characterized by hysteresis and force relaxation after formation. The results of this study elucidate the coordination of plasma membrane composition and cytoskeleton during protrusion formation.

A microengineered cell fusion approach with combined optical tweezers and microwell array technologies

Cell fusion in vitro can be artificially achieved through microengineering technology. This paper presents a laser-induced cell fusion methodology on the microwell array-based microfluidic chip, with high selectivity and controllability at the single cell level. Optical tweezers and optical scissors are employed to achieve cell pairing and fusion, respectively. The specific cells are first characterized with high spatio-temporal resolution and preselected from the mixture through an on-chip isolation method prior to pairing. The paired cells are then transported, deposited, fused, and released at the desired location with high controllability. Biophysical analysis on the fused cells shows that the fusion efficiency of the homotypical pairs is higher than that of the heterogenic pairs obtained based on different combinations of sample cells, such as human embryonic stem cells and Jurkat cells. This laser-induced cell fusion technique offers a new opportunity to explore specific targeted therapy in stem cell research for the treatment of human diseases.