Quantitative analysis of biochemical processes in living cells at a single-molecule level: a case of olaparib-PARP1 (DNA repair protein) interactions

The Analyst ◽  
2021 ◽  
Author(s):  
Aneta Karpinska ◽  
Marta Pilz ◽  
Joanna Buczkowska ◽  
Paweł Żuk ◽  
Karolina Kucharska ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Single Molecule ◽  
Protein Interactions ◽  
Quantitative Description ◽  
Living Cells ◽  
Single Molecule Level ◽  
Biochemical Processes ◽  
Repair Protein ◽  
Dna Repair Protein ◽  
Modern Instrumentation

Quantitative description of biochemical processes inside living cells and at single-molecule levels remains a challenge at the forefront of modern instrumentation and spectroscopy. This paper demonstrates such single-cell, single-molecule analyses...

scholarly journals Single-Molecule Analysis of Human Immunodeficiency Virus Type 1 gp120-Receptor Interactions in Living Cells

2005 ◽  
Vol 79 (23) ◽  
pp. 14748-14755 ◽  
Author(s):  
Melissa I. Chang ◽  
Porntula Panorchan ◽  
Terrence M. Dobrowsky ◽  
Yiider Tseng ◽  
Denis Wirtz
Keyword(s):  
Human Immunodeficiency Virus ◽  
Host Cell ◽  
Single Molecule ◽  
Quantitative Description ◽  
Living Cells ◽  
Viral Envelope ◽  
Binding Interactions ◽  
Immunodeficiency Virus ◽  
Single Molecule Analysis

ABSTRACT A quantitative description of the binding interactions between human immunodeficiency virus (HIV) type 1 envelope glycoproteins and their host cell surface receptors remains incomplete. Here, we introduce a single-molecule analysis that directly probes the binding interactions between an individual viral subunit gp120 and a single receptor CD4 and/or chemokine coreceptor CCR5 in living cells. This analysis differentiates single-molecule binding from multimolecule avidity and shows that, while the presence of CD4 is required for gp120 binding to CCR5, the force required to rupture a single gp120-coreceptor bond is significantly higher and its lifetime is much longer than those of a single gp120-receptor bond. The lifetimes of these bonds are themselves shorter than those of the P-selectin/PSGL-1 bond involved in leukocyte attachment to the endothelium bonds during an inflammation response. These results suggest an amended model of HIV entry in which, immediately after the association of gp120 to its receptor, gp120 seeks its coreceptor to rapidly form a new bond. This “bond transfer” occurs only if CCR5 is in close proximity to CD4 and CD4 is still attached to gp120. The analysis presented here may serve as a general framework to study mechanisms of receptor-mediated interactions between viral envelope proteins and host cell receptors at the single-molecule level in living cells.

scholarly journals An Optical Setup for the Study of Mechanotransduction in Living Cells at the Single Molecule Level

2017 ◽  
Vol 112 (3) ◽  
pp. 588a
Author(s):  
Marios Sergides ◽  
Tommaso Galgani ◽  
Claudia Arbore ◽  
Francesco S. Pavone ◽  
Marco Capitanio
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Single Molecule Level ◽  
Optical Setup

Micropatterning for quantitative analysis of protein-protein interactions in living cells

2008 ◽  
Vol 5 (12) ◽  
pp. 1053-1060 ◽  
Author(s):  
Michaela Schwarzenbacher ◽  
Martin Kaltenbrunner ◽  
Mario Brameshuber ◽  
Clemens Hesch ◽  
Wolfgang Paster ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Protein Interactions ◽  
Living Cells ◽  
Protein Protein Interactions

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 Complete Mapping of DNA‐Protein Interactions at the Single‐Molecule Level

2021 ◽  
pp. 2101383
Author(s):  
Wenzhe Liu ◽  
Jie Li ◽  
Yongping Xu ◽  
Dongbao Yin ◽  
Xin Zhu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Single Molecule Level ◽  
Complete Mapping ◽  
Dna Protein Interactions

scholarly journals Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA–Protein Interactions at the Single-Molecule Level

2017 ◽  
Vol 8 ◽  
Author(s):  
Huijun Xue ◽  
Yuanyuan Bei ◽  
Zhengyan Zhan ◽  
Xiuqiang Chen ◽  
Xin Xu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Single Molecule Level ◽  
Dna Protein Interactions
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