Central dogma at the single-molecule level in living cells

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.

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Using bespoke fluorescence microscopy to study the soft condensed matter of living cells at the single molecule level

Journal of Physics Conference Series ◽

10.1088/1742-6596/286/1/012001 ◽

2011 ◽

Vol 286 ◽

pp. 012001

Author(s):

Q Xue ◽

O Harriman ◽

M C Leake

Keyword(s):

Fluorescence Microscopy ◽

Single Molecule ◽

Condensed Matter ◽

Living Cells ◽

Soft Condensed Matter ◽

Single Molecule Level

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Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

10.1101/2020.06.26.174102 ◽

2020 ◽

Author(s):

Sven A. Szilagyi ◽

Moritz Burmeister ◽

Q. Tyrell Davis ◽

Gero L. Hermsdorf ◽

Suman De ◽

...

Keyword(s):

Single Molecule ◽

Signal To Noise Ratio ◽

Numerical Aperture ◽

Light Sheet ◽

Living Cells ◽

High Signal ◽

Active Optics ◽

Single Molecule Level ◽

Light Sheet Microscopy ◽

Reflected Light

AbstractObservation of highly dynamic processes inside living cells at the single molecule level is key for a quantitative understanding of biological systems. However, imaging of single molecules in living cells usually is limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination have recently been developed. For example, a reflected light-sheet design combines the illumination by a thin light-sheet with a high numerical aperture objective for single-molecule detection. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-color acquisition of z-stacks. We demonstrate fast volume scanning by imaging a two-color giant unilamellar vesicle (GUV) hemisphere. In addition, the high signal-to-noise ratio enabled the imaging and tracking of single lipids in the cap of a GUV. In the long term, the enhanced reflected scanning light sheet microscope enables fast 3D scanning of artificial membrane systems and cells with single-molecule sensitivity and thereby will provide quantitative and molecular insight into the operation of cells.

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Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level

International Journal of Molecular Sciences ◽

10.3390/ijms14023961 ◽

2013 ◽

Vol 14 (2) ◽

pp. 3961-3992 ◽

Cited By ~ 31

Author(s):

Carina Monico ◽

Marco Capitanio ◽

Gionata Belcastro ◽

Francesco Vanzi ◽

Francesco Pavone

Keyword(s):

Single Molecule ◽

Living Cells ◽

Optical Methods ◽

Dna Interactions ◽

Single Molecule Level ◽

Protein Dna Interactions

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Detection and quantification of single mRNA dynamics with the Riboglow fluorescent RNA tag

10.1101/701649 ◽

2019 ◽

Author(s):

Esther Braselmann ◽

Timothy J. Stasevich ◽

Kenneth Lyon ◽

Robert T. Batey ◽

Amy E. Palmer

Keyword(s):

Single Molecule ◽

Fluorescent Probes ◽

Mammalian Cells ◽

Fluorescent Antibody ◽

Living Cells ◽

Rna Molecules ◽

Single Molecule Level ◽

Rna Biology ◽

Tagging System ◽

Detection And Quantification

AbstractLabeling and tracking biomolecules with fluorescent probes on the single molecule level enables quantitative insights into their dynamics in living cells. We previously developed Riboglow, a platform to label RNAs in live mammalian cells, consisting of a short RNA tag and a small organic probe that increases fluorescence upon binding RNA. Here, we demonstrate that Riboglow is capable of detecting and tracking single RNA molecules. We benchmark RNA tracking by comparing results with the established MS2 RNA tagging system. To demonstrate versatility of Riboglow, we assay translation on the single molecule level, where the translated mRNA is tagged with Riboglow and the nascent polypeptide is labeled with a fluorescent antibody. The growing effort to investigate RNA biology on the single molecule level requires sophisticated and diverse fluorescent probes for multiplexed, multi-color labeling of biomolecules of interest, and we present Riboglow as a new member in this toolbox.

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Monitoring HIV-1 Assembly in Living Cells: Insight from Dynamic and Single Molecule Microscopy

10.20944/preprints201811.0596.v1 ◽

2018 ◽

Author(s):

Kaushik Inamdar ◽

Charlotte Floderer ◽

Cyril Favard ◽

Delphine Muriaux

Keyword(s):

Host Cell ◽

Single Molecule ◽

High Speed ◽

Time Course ◽

Super Resolution ◽

Living Cells ◽

Single Molecule Level ◽

Host Cell Factors ◽

Immature Virus ◽

Hiv 1

HIV-1 assembly is a complex mechanism taking place at the plasma membrane of the host cell. It requires nice spatial and temporal coordination to end up with a full immature virus. Researchers have extensively studied HIV-1 assembly molecular mechanism during the past decades, in order to dissect the respective roles of viral proteins, viral genome and host cell factors. Nevertheless, the time course of the process has been observed in living cells only a decade ago. The very recent revolution of optical microscopy, combining high speed and high spatial resolution now permit to study assemblies and their consequences at the single molecule level within (living) cells. In this review, after a short description of these new approaches, we will show how HIV-1 assembly in cells has been revisited using these advanced super resolution microscopy techniques and how much it could make a bridge in studying assembly from the single molecule to the host cell.

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Quantifying the spatiotemporal dynamics of IRES versus Cap translation with single-molecule resolution in living cells

10.1101/2020.01.09.900829 ◽

2020 ◽

Cited By ~ 2

Author(s):

Amanda Koch ◽

Luis Aguilera ◽

Tatsuya Morisaki ◽

Brian Munsky ◽

Timothy J. Stasevich

Keyword(s):

Single Molecule ◽

Living Cells ◽

Host Cells ◽

Spatial Distributions ◽

Dependent Manner ◽

Single Molecule Tracking ◽

Single Molecule Level ◽

The One ◽

Kinetics Of ◽

Relative Kinetics

ABSTRACTViruses use IRES sequences within their RNA to hijack translation machinery and thereby rapidly replicate in host cells. While this process has been extensively studied in bulk assays, the dynamics of hijacking at the single-molecule level remain unexplored in living cells. To achieve this, we developed a bicistronic biosensor encoding complementary repeat epitopes in two ORFs, one translated in a Cap-dependent manner and the other translated in an IRES-mediated manner. Using a pair of complementary probes that bind the epitopes co-translationally, our biosensor lights up in different colors depending on which ORF is being translated. In combination with single-molecule tracking and computational modeling, we measured the relative kinetics of Cap versus IRES translation and show: (1) Two non-overlapping ORFs can be simultaneously translated within a single mRNA; (2) EMCV IRES-mediated translation sites recruit ribosomes less efficiently than Cap-dependent translation sites but are otherwise nearly indistinguishable, having similar mobilities, sizes, spatial distributions, and ribosomal initiation and elongation rates; (3) Both Cap-dependent and IRES-mediated ribosomes tend to stretch out translation sites; (4) Although the IRES recruits two to three times fewer ribosomes than the Cap in normal conditions, the balance shifts dramatically in favor of the IRES during oxidative and ER stresses that mimic viral infection; and (5) Translation of the IRES is enhanced by translation of the Cap, demonstrating upstream translation can positively impact the downstream translation of a non-overlapping ORF. With the ability to simultaneously quantify two distinct translation mechanisms in physiologically relevant live-cell environments, we anticipate bicistronic biosensors like the one we developed here will become powerful new tools to dissect both canonical and non-canonical translation dynamics with single-molecule precision.Graphical Abstract

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

The Analyst ◽

10.1039/d1an01769a ◽

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...

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