scholarly journals Computational Investigation of Dynamic Properties of Actin Networks With Crosslinking Proteins

2010 ◽  
Author(s):  
Taeyoon Kim ◽  
Wonmuk Hwang ◽  
Roger D. Kamm
Keyword(s):  
Single Molecule ◽  
Viscoelastic Properties ◽  
Dynamic Properties ◽  
Living Cells ◽  
Biological Processes ◽  
Computational Investigation ◽  
Actin Networks ◽  
Physical Understanding ◽  
Single Molecule Studies ◽  
Similar Range

Due to the increasing recognition of the role that force plays in biological processes, a new field, mechanobiology, has recently emerged. One aspect of this is the need to gain a physical understanding of the viscoelastic properties of the cytoskeleton. Numerous studies, both in living cells and in reconstituted actin gels, have been conducted, but important questions still remain. Of these an important issue revolves around the role played by actin crosslinking proteins (ACPs), and whether they undergo unfolding or unbinding under stress. This issue is complicated by the fact that single molecule studies show that both events occur within a similar range of forces, on the order of 20–100 pN.

Nanomechanochemical Delivery of Nanoparticles for Nanomechanics Inside Living Cells

2010 ◽  
Author(s):  
Kyungsuk Yum ◽  
Sungsoo Na ◽  
Yang Xiang ◽  
Ning Wang ◽  
Min-Feng Yu
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Endocytic Pathway ◽  
Great Promise ◽  
Biological Processes ◽  
Temporal Precision ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Biological Studies

Studying biological processes and mechanics in living cells is challenging but highly rewarding. Recent advances in experimental techniques have provided numerous ways to investigate cellular processes and mechanics of living cells. However, most of existing techniques for biomechanics are limited to experiments outside or on the membrane of cells, due to the difficulties in physically accessing the interior of living cells. On the other hand, nanomaterials, such as fluorescent quantum dots (QDs) and magnetic nanoparticles, have shown great promise to overcome such limitations due to their small sizes and excellent functionalities, including bright and stable fluorescence and remote manipulability. However, except a few systems, the use of nanoparticles has been limited to the study of biological studies on cell membranes or related to endocytosis, because of the difficulty of delivering dispersed and single nanoparticles into living cells. Various strategies have been explored, but delivered nanoparticles are often trapped in the endocytic pathway or form aggregates in the cytoplasm, limiting their further use. Here we show a nanoscale direct delivery method, named nanomechanochemical delivery, where we manipulate a nanotube-based nanoneedle, carrying “cargo” (QDs in this study), to mechanically penetrate the cell membrane, access specific areas inside cells, and release the cargo [1]. We selectively delivered well-dispersed QDs into either the cytoplasm or the nucleus of living cells. We quantified the dynamics of the delivered QDs by single-molecule tracking and demonstrated the applicability of the QDs as a nanoscale probe for studying nanomechanics inside living cells (by using the biomicrorhology method), revealing the biomechanical heterogeneity of the cellular environment. This method may allow new strategies for studying biological processes and mechanics in living cells with spatial and temporal precision, potentially at the single-molecule level.

scholarly journals Watching biological nanomotors at work : insights from single-molecule studies

2017 ◽  
Author(s):  
◽  
Nagaraju Chada
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Halobacterium Salinarum ◽  
Biological Processes ◽  
Enzyme Dynamics ◽  
Oligomeric State ◽  
Force Microscopy ◽  
Atomic Force ◽  
Single Molecule Studies

Recent decades have seen several complimentary biophysics tools emerge to study single protein macromolecules. Most of these techniques use glass as a specimen support. The atomic force microscope, a vital tool in biophysics suited to study proteins in their near native environments, uses mica as a specimen support, as it is known for its extreme flatness and ease of use. Here we optimized glass as a specimen support for atomic force microscopy. This enables the combination of other single molecule techniques with atomic force microscopy to study the same protein macromolecular system in unison. Using bacteriorhodopsin from Halobacterium salinarum and the Sec-translocase (SecA/SecYEG) from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on common microscope cover glass following a straight-forward cleaning procedure. Repeated association and dissociation of SecA with SecYEG indicated that the proteins remain competent for biological processes on glass supports for long periods of time. This work opens the door for combining high resolution biological AFM with other powerful complementary single molecule techniques that require glass as a specimen support. In the second part of this work we studied SecA-ATP hydrolysis and catalase enzyme dynamics. Both of these protein macromolecules were observed to be highly dynamic during catalytic turnover. Single molecule studies of catalase indicated that the enzyme undergoes significant dynamics including oligomeric state changes when exposed to H2O2. Conformational dynamics of the SecA-ATPase was visualized at the single molecule level and the protein macromolecule was observed to flicker between a compact and expanded state in the presence of ATP, indicating reversible conformational changes. Future studies in the lab will shed more light onto these important biological processes.

Single-Molecule Studies of Nucleic Acids and Their Proteins

2018 ◽  
Author(s):  
David Bensimon ◽  
Vincent Croquette ◽  
Jean-François Allemand ◽  
Xavier Michalet ◽  
Terence Strick
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Radiative Decay ◽  
Dynamic Properties ◽  
Rna Polymerases ◽  
Chiral Discrimination ◽  
Triplet States ◽  
Comprehensive Overview ◽  
Dna And Rna ◽  
Single Molecule Studies

This book presents a comprehensive overview of the foundations of single-molecule studies, based on manipulation of the molecules and observation of these with fluorescent probes. It first discusses the forces present at the single-molecule scale, the methods to manipulate them, and their pros and cons. It goes on to present an introduction to single-molecule fluorescent studies based on a quantum description of absorption and emission of radiation due to Einstein. Various considerations in the study of single molecules are introduced (including signal to noise, non-radiative decay, triplet states, etc.) and some novel super-resolution methods are sketched. The elastic and dynamic properties of polymers, their relation to experiments on DNA and RNA, and the structural transitions observed in those molecules upon stretching, twisting, and unzipping are presented. The use of these single-molecule approaches for the investigation of DNA–protein interactions is highlighted via the study of DNA and RNA polymerases, helicases, and topoisomerases. Beyond the confirmation of expected mechanisms (e.g., the relaxation of DNA torsion by topoisomerases in quantized steps) and the discovery of unexpected ones (e.g., strand-switching by helicases, DNA scrunching by RNA polymerases, and chiral discrimination by bacterial topoII), these approaches have also fostered novel (third generation) sequencing technologies.

scholarly journals Experimental approaches for addressing fundamental biological questions in living, functioning cells with single molecule precision

Open Biology ◽  
2012 ◽  
Vol 2 (6) ◽  
pp. 120090 ◽  
Author(s):  
Tchern Lenn ◽  
Mark C. Leake
Keyword(s):  
Single Molecule ◽  
Molecular Basis ◽  
Life Sciences ◽  
Short Review ◽  
Living Cells ◽  
Cellular Systems ◽  
Biological Processes ◽  
Sensitivity Level ◽  
Experimental Approaches ◽  
The Impact

In recent years, single molecule experimentation has allowed researchers to observe biological processes at the sensitivity level of single molecules in actual functioning, living cells, thereby allowing us to observe the molecular basis of the key mechanistic processes in question in a very direct way, rather than inferring these from ensemble average data gained from traditional molecular and biochemical techniques. In this short review, we demonstrate the impact that the application of single molecule bioscience experimentation has had on our understanding of various cellular systems and processes, and the potential that this approach has for the future to really address very challenging and fundamental questions in the life sciences.

Approaches to quantitative single-molecule studies in living cells

2007 ◽  
Author(s):  
Christian Roth ◽  
Pia Heinlein ◽  
Dirk-Peter Herten
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Single Molecule Studies

scholarly journals Dynamic actin crosslinking governs the cytoplasm’s transition to fluid-like behavior

2019 ◽  
Author(s):  
Loïc Chaubet ◽  
Abdullah R. Chaudhary ◽  
Hossein K. Heris ◽  
Allen J. Ehrlicher ◽  
Adam G. Hendricks
Keyword(s):  
Viscoelastic Properties ◽  
Myosin Ii ◽  
Optical Trap ◽  
Living Cells ◽  
Actin Network ◽  
Wild Type ◽  
Actin Networks ◽  
Long Timescales

AbstractCells precisely control their mechanical properties to organize and differentiate into tissues. The architecture and connectivity of cytoskeletal filaments changes in response to mechanical and biochemical cues, allowing the cell to rapidly tune its mechanics from highly-crosslinked, elastic networks to weakly-crosslinked viscous networks. While the role of actin crosslinking in controlling actin network mechanics is well-characterized in purified actin networks, its mechanical role in the cytoplasm of living cells remains unknown. Here, we probe the frequency-dependent intracellular viscoelastic properties of living cells using multifrequency excitation and in situ optical trap calibration. At long timescales in the intracellular environment, we observe that the cytoskeleton becomes fluid-like. The mechanics are well-captured by a model in which actin filaments are dynamically connected by a single dominant crosslinker. A disease-causing point mutation (K255E) of the actin crosslinker α-actinin 4 (ACTN4) causes its binding kinetics to be insensitive to tension. Under normal conditions, the viscoelastic properties of wild type (WT) and K255E+/- cells are similar. However, when tension is reduced through myosin II inhibition, WT cells relax 3x faster to the fluid-like regime while K255E+/- cells are not affected. These results indicate that dynamic actin crosslinking enables the cytoplasm to flow at long timescales.

scholarly journals DNA hybridisation kinetics using single-molecule fluorescence imaging

2021 ◽  
Author(s):  
Rebecca Andrews
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Biological Processes ◽  
Single Molecule Fluorescence ◽  
Extrinsic Factors ◽  
Factors Affecting ◽  
Dna Hybridisation ◽  
Single Molecule Studies ◽  
Kinetics Of ◽  
Insight Into

Abstract Deoxyribonucleic acid (DNA) hybridisation plays a key role in many biological processes and nucleic acid biotechnologies, yet surprisingly there are many aspects about the process which are still unknown. Prior to the invention of single-molecule microscopy, DNA hybridisation experiments were conducted at the ensemble level, and thus it was impossible to directly observe individual hybridisation events and understand fully the kinetics of DNA hybridisation. In this mini-review, recent single-molecule fluorescence-based studies of DNA hybridisation are discussed, particularly for short nucleic acids, to gain more insight into the kinetics of DNA hybridisation. As well as looking at single-molecule studies of intrinsic and extrinsic factors affecting DNA hybridisation kinetics, the influence of the methods used to detect hybridisation of single DNAs is considered. Understanding the kinetics of DNA hybridisation not only gives insight into an important biological process but also allows for further advancements in the growing field of nucleic acid biotechnology.

scholarly journals Single-molecule imaging and tracking of molecular dynamics in living cells

2017 ◽  
Vol 4 (5) ◽  
pp. 739-760 ◽  
Author(s):  
Nan Li ◽  
Rong Zhao ◽  
Yahong Sun ◽  
Zi Ye ◽  
Kangmin He ◽  
...  
Keyword(s):  
Single Molecule ◽  
Quantitative Information ◽  
Living Cells ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Biomolecular Interaction ◽  
Ensemble Averaging ◽  
Spatiotemporal Resolution ◽  
Detection And Tracking ◽  
Single Molecule Studies

Abstract Unlike the ensemble-averaging measurements, the single-molecule imaging and tracking (SMIT) in living cells provides the real-time quantitative information about the locations, kinetics, dynamics and interactions of individual molecules in their native environments with high spatiotemporal resolution and minimal perturbation. The past decade has witnessed a transforming development in the methods of SMIT with living cells, including fluorescent probes, labeling strategies, fluorescence microscopy, and detection and tracking algorithms. In this review, we will discuss these aspects with a particular focus on their recent advancements. We will then describe representative single-molecule studies to illustrate how the single-molecule approaches can be applied to monitor biomolecular interaction/reaction dynamics, and extract the molecular mechanistic information for different cellular systems.

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