scholarly journals Probing mechanotransduction in living cells by optical tweezers and FRET-based molecular force microscopy

2021 ◽  
Vol 136 (3) ◽  
Author(s):  
M. Sergides ◽  
L. Perego ◽  
T. Galgani ◽  
C. Arbore ◽  
F. S. Pavone ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Single Cells ◽  
Force Sensors ◽  
Force Microscopy ◽  
Mechanical Signals ◽  
Cell Plasma ◽  
Molecular Force ◽  
Wide Range ◽  
Biochemical Signals

AbstractCells sense mechanical signals and forces to probe the external environment and adapt to tissue morphogenesis, external mechanical stresses and a wide range of diverse mechanical cues. Here, we propose a combination of optical tools to manipulate single cells and measure the propagation of mechanical and biochemical signals inside them. Optical tweezers are used to trap microbeads that are used as handles to manipulate the cell plasma membrane; genetically encoded FRET-based force sensors inserted in F-actin and alpha-actinin are used to measure the propagation of mechanical signals to the cell cytoskeleton, while fluorescence microscopy with single-molecule sensitivity can be used with a huge array of biochemical and genetic sensors. We describe the details of the setup implementation, the calibration of the basic components and preliminary characterization of actin and alpha-actinin FRET-based force sensors.

scholarly journals Probing mechanotransduction in living cells by optical tweezers and FRET-based molecular force microscopy

2021 ◽  
Author(s):  
M. Sergides ◽  
L. Perego ◽  
T. Galgani ◽  
C. Arbore ◽  
F.S. Pavone ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Single Cells ◽  
Force Sensors ◽  
Force Microscopy ◽  
Mechanical Signals ◽  
Cell Plasma ◽  
Molecular Force ◽  
Wide Range ◽  
Biochemical Signals

AbstractCells sense mechanical signals and forces to probe the external environment and adapt to tissue morphogenesis, external mechanical stresses, and a wide range of diverse mechanical cues. Here, we propose a combination of optical tools to manipulate single cells and measure the propagation of mechanical and biochemical signals inside them. Optical tweezers are used to trap microbeads that are used as handles to manipulate the cell plasma membrane; genetically encoded FRET-based force sensors inserted in F-actin and alpha-actinin are used to measure the propagation of mechanical signals to the cell cytoskeleton; while fluorescence microscopy with single molecule sensitivity can be used with a huge array of biochemical and genetic sensors. We describe the details of the setup implementation, the calibration of the basic components and preliminary characterization of actin and alpha-actinin FRET-based force sensors.

scholarly journals Construction of 3D Cellular Composites with Stem Cells Derived from Adipose Tissue and Endothelial Cells by Use of Optical Tweezers in a Natural Polymer Solution

Materials ◽  
2019 ◽  
Vol 12 (11) ◽  
pp. 1759 ◽  
Author(s):  
Takehiro Yamazaki ◽  
Toshifumi Kishimoto ◽  
Paweł Leszczyński ◽  
Koichiro Sadakane ◽  
Takahiro Kenmotsu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Adipose Tissue ◽  
Optical Tweezers ◽  
Single Cells ◽  
Three Dimensional ◽  
Cell Types ◽  
Cellular Interactions ◽  
Research Fields ◽  
Wide Range

To better understand the regulation and function of cellular interactions, three-dimensional (3D) assemblies of single cells and subsequent functional analysis are gaining popularity in many research fields. While we have developed strategies to build stable cellular structures using optical tweezers in a minimally invasive state, methods for manipulating a wide range of cell types have yet to be established. To mimic organ-like structures, the construction of 3D cellular assemblies with variety of cell types is essential. Our recent studies have shown that the presence of nonspecific soluble polymers in aqueous solution is the key to creating stable 3D cellular assemblies efficiently. The present study further expands on the construction of 3D single cell assemblies using two different cell types. We have successfully generated 3D cellular assemblies, using GFP-labeled adipose tissue-derived stem cells and endothelial cells by using optical tweezers. Our findings will support the development of future applications to further characterize cellular interactions in tissue regeneration.

scholarly journals Broken force dispersal network in tip-links by the mutations at the Ca2+-binding residues induces hearing-loss

2019 ◽  
Vol 476 (16) ◽  
pp. 2411-2425 ◽  
Author(s):  
Jagadish P. Hazra ◽  
Amin Sagar ◽  
Nisha Arora ◽  
Debadutta Deb ◽  
Simerpreet Kaur ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Inner Ear ◽  
Single Molecule ◽  
Force Sensor ◽  
Force Sensors ◽  
Single Molecule Level ◽  
Conformational Variability ◽  
Atomic Force ◽  
Wide Range ◽  
And Function

Abstract Tip-link as force-sensor in hearing conveys the mechanical force originating from sound to ion-channels while maintaining the integrity of the entire sensory assembly in the inner ear. This delicate balance between structure and function of tip-links is regulated by Ca2+-ions present in endolymph. Mutations at the Ca2+-binding sites of tip-links often lead to congenital deafness, sometimes syndromic defects impairing vision along with hearing. Although such mutations are already identified, it is still not clear how the mutants alter the structure-function properties of the force-sensors associated with diseases. With an aim to decipher the differences in force-conveying properties of the force-sensors in molecular details, we identified the conformational variability of mutant and wild-type tip-links at the single-molecule level using FRET at the endolymphatic Ca2+ concentrations and subsequently measured the force-responsive behavior using single-molecule force spectroscopy with an Atomic Force Microscope (AFM). AFM allowed us to mimic the high and wide range of force ramps (103–106 pN s−1) as experienced in the inner ear. We performed in silico network analysis to learn that alterations in the conformations of the mutants interrupt the natural force-propagation paths through the sensors and make the mutant tip-links vulnerable to input forces from sound stimuli. We also demonstrated that a Ca2+ rich environment can restore the force-response of the mutant tip-links which may eventually facilitate the designing of better therapeutic strategies to the hearing loss.

scholarly journals Profound Nanoscale Structural and Biomechanical Changes in DNA Helix upon Treatment with Anthracycline Drugs

2020 ◽  
Vol 21 (11) ◽  
pp. 4142
Author(s):  
Aleksandra Kaczorowska ◽  
Weronika Lamperska ◽  
Kaja Frączkowska ◽  
Jan Masajada ◽  
Sławomir Drobczyński ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Regulatory Elements ◽  
Dna Molecules ◽  
Anthracycline Antibiotics ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Analysis ◽  
Dna Regulatory Elements

In our study, we describe the outcomes of the intercalation of different anthracycline antibiotics in double-stranded DNA at the nanoscale and single molecule level. Atomic force microscopy analysis revealed that intercalation results in significant elongation and thinning of dsDNA molecules. Additionally, using optical tweezers, we have shown that intercalation decreases the stiffness of DNA molecules, that results in greater susceptibility of dsDNA to break. Using DNA molecules with different GC/AT ratios, we checked whether anthracycline antibiotics show preference for GC-rich or AT-rich DNA fragments. We found that elongation, decrease in height and decrease in stiffness of dsDNA molecules was highest in GC-rich dsDNA, suggesting the preference of anthracycline antibiotics for GC pairs and GC-rich regions of DNA. This is important because such regions of genomes are enriched in DNA regulatory elements. By using three different anthracycline antibiotics, namely doxorubicin (DOX), epirubicin (EPI) and daunorubicin (DAU), we could compare their detrimental effects on DNA. Despite their analogical structure, anthracyclines differ in their effects on DNA molecules and GC-rich region preference. DOX had the strongest overall effect on the DNA topology, causing the largest elongation and decrease in height. On the other hand, EPI has the lowest preference for GC-rich dsDNA. Moreover, we demonstrated that the nanoscale perturbations in dsDNA topology are reflected by changes in the microscale properties of the cell, as even short exposition to doxorubicin resulted in an increase in nuclei stiffness, which can be due to aberration of the chromatin organization, upon intercalation of doxorubicin molecules.

Microfluidics Supporting an Optical Instrument for Multimodal Single Cell Biomechanics

2007 ◽  
Author(s):  
Nathalie Ne`ve ◽  
James K. Lingwood ◽  
Shelley R. Winn ◽  
Derek C. Tretheway ◽  
Sean S. Kohles
Keyword(s):  
Particle Image Velocimetry ◽  
Hydrostatic Pressure ◽  
Single Cell ◽  
Optical Tweezers ◽  
Particle Image ◽  
Single Cells ◽  
Optical Instrument ◽  
Image Velocimetry ◽  
Wide Range ◽  
Induced Stresses

Interfacing a novel micron-resolution particle image velocimetry and dual optical tweezers system (μPIVOT) with microfluidics facilitates the exposure of an individual biologic cell to a wide range of static and dynamic mechanical stress conditions. Single cells can be manipulated in a sequence of mechanical stresses (hydrostatic pressure variations, tension or compression, as well as shear and extensional fluid induced stresses) while measuring cellular deformation. The unique multimodal load states enable a new realm of single cell biomechanical studies.

scholarly journals Bioconjugation Strategies for Connecting Proteins to DNA-Linkers for Single-Molecule Force-Based Experiments

Nanomaterials ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2424
Author(s):  
Lyan M. van der Sleen ◽  
Katarzyna M. Tych
Keyword(s):  
Mechanical Properties ◽  
Atomic Force Microscopy ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Short Range ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Atomic Force

The mechanical properties of proteins can be studied with single molecule force spectroscopy (SMFS) using optical tweezers, atomic force microscopy and magnetic tweezers. It is common to utilize a flexible linker between the protein and trapped probe to exclude short-range interactions in SMFS experiments. One of the most prevalent linkers is DNA due to its well-defined properties, although attachment strategies between the DNA linker and protein or probe may vary. We will therefore provide a general overview of the currently existing non-covalent and covalent bioconjugation strategies to site-specifically conjugate DNA-linkers to the protein of interest. In the search for a standardized conjugation strategy, considerations include their mechanical properties in the context of SMFS, feasibility of site-directed labeling, labeling efficiency, and costs.

scholarly journals Generating kinetic environments to study dynamic cellular processes in single cells

2019 ◽  
Author(s):  
Alexander Thiemicke ◽  
Hossein Jashnsaz ◽  
Guoliang Li ◽  
Gregor Neuert
Keyword(s):  
Cell Culture ◽  
Single Molecule ◽  
Cellular Response ◽  
Single Cells ◽  
Mammalian Cell Culture ◽  
Time Lapse ◽  
Culture Conditions ◽  
Wide Range ◽  
Quantitative Manner ◽  
Fate Decision

AbstractCells of any organism are consistently exposed to changes over time in their environment. The kinetics by which these changes occur are critical for the cellular response and fate decision. It is therefore important to control the temporal changes of extracellular stimuli precisely to understand biological mechanisms in a quantitative manner. Most current cell culture and biochemical studies focus on instant changes in the environment and therefore neglect the importance of kinetic environments. To address these shortcomings, we developed two experimental methodologies to precisely control the environment of single cells. These methodologies are compatible with standard biochemistry, molecular, cell and quantitative biology assays. We demonstrate applicability by obtaining time series and time point measurements in both live and fixed cells. We demonstrate the feasibility of the methodology in yeast and mammalian cell culture in combination with widely used assays such as flow cytometry, time-lapse microscopy and single-molecule RNA Fluorescent in-situ Hybridization. Our experimental methodologies are easy to implement in most laboratory settings and allows the study of kinetic environments in a wide range of assays and different cell culture conditions.

Solid-State Nanopores: From Fabrication to Application

2012 ◽  
Vol 20 (5) ◽  
pp. 24-29 ◽  
Author(s):  
Adam R. Hall
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
High Speed ◽  
Super Resolution ◽  
Magnetic Tweezers ◽  
Fluorescent Imaging ◽  
Force Microscopy ◽  
Atomic Force ◽  
Labeling Requirements ◽  
Molecular Manipulation

There are relatively few technologies for measurement at the single-molecule scale. Fluorescent imaging, for example, can be used to directly visualize molecules and their interactions, but diffraction limitations and labeling requirements may push the system from its native state. Although recent advances in super-resolution imaging have been able to break this resolution barrier, important challenges remain. Atomic force microscopy (AFM) is capable of imaging molecules at high resolution and at high speed. However, AFM imaging is a surface technique, requiring sample preparation and some immobilization. Other technologies such as optical tweezers and magnetic tweezers are capable of molecular manipulation and spectroscopy to great effect but require a significant apparatus and have limited inherent analytical capabilities.

scholarly journals Characterization of G-Quadruplexes Folding/Unfolding Dynamics and Interactions with Proteins from Single-Molecule Force Spectroscopy

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1579
Author(s):  
Yuanlei Cheng ◽  
Yashuo Zhang ◽  
Huijuan You
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Multiple Structures ◽  
Regulation Functions ◽  
Nucleic Acid Structures

G-quadruplexes (G4s) are stable secondary nucleic acid structures that play crucial roles in many fundamental biological processes. The folding/unfolding dynamics of G4 structures are associated with the replication and transcription regulation functions of G4s. However, many DNA G4 sequences can adopt a variety of topologies and have complex folding/unfolding dynamics. Determining the dynamics of G4s and their regulation by proteins remains challenging due to the coexistence of multiple structures in a heterogeneous sample. Here, in this mini-review, we introduce the application of single-molecule force–spectroscopy methods, such as magnetic tweezers, optical tweezers, and atomic force microscopy, to characterize the polymorphism and folding/unfolding dynamics of G4s. We also briefly introduce recent studies using single-molecule force spectroscopy to study the molecular mechanisms of G4-interacting proteins.

scholarly journals On-Site Processing of Single Chromosomal DNA Molecules Using Optically Driven Microtools on A Microfluidic Workbench

2020 ◽  
Author(s):  
Akihito Masuda ◽  
Hidekuni Takao ◽  
Fusao Shimokawa ◽  
KYOHEI TERAO
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Single Cells ◽  
Optical Microscope ◽  
Optical Manipulation ◽  
Confined Space ◽  
Dna Molecules ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Immobilization Of Enzymes

Abstract We developed optically driven microtools for processing single biomolecules using a microfluidic workbench composed of a microfluidic platform that functions under an optical microscope. The optically driven microtools have enzymes immobilized on their surfaces, which catalyze chemical reactions for molecular processing in a confined space. Optical manipulation of the microtools enables them to be integrated with a microfluidic device for controlling the position, orientation, shape of the target sample. Here, we describe the immobilization of enzymes on the surface of microtools, the microfluidics workbench, including its microtool storage and sample positioning functions, and the use of this system for on-site cutting of single chromosomal DNA molecules. We fabricated microtools by UV lithography with SU-8 and selected ozone treatments for immobilizing enzymes. The microfluidic workbench has tool-stock chambers for tool storage and micropillars to trap and extend single chromosomal DNA molecules. The DNA cutting enzymes DNaseI and DNaseII were immobilized on microtools that were manipulated using optical tweezers. The DNaseI tool shows reliable cutting for on-site processing. This pinpoint processing provides an approach for analyzing chromosomal DNA at the single-molecule level. The flexibility of the microtool design allows for processing of various samples, including biomolecules and single cells.

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