Nanomechanochemical Delivery of Nanoparticles for Nanomechanics Inside 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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An Optical Setup for the Study of Mechanotransduction in Living Cells at the Single Molecule Level

Biophysical Journal ◽

10.1016/j.bpj.2016.11.3163 ◽

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

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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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A Fluorogenic Array Tag for Temporally Unlimited Single Molecule Tracking

10.1101/159004 ◽

2017 ◽

Cited By ~ 1

Author(s):

Rajarshi P Ghosh ◽

J Matthew Franklin ◽

Will E. Draper ◽

Quanming Shi ◽

Jan T. Liphardt

Keyword(s):

Single Molecule ◽

Precision Measurement ◽

Single Molecules ◽

Living Cells ◽

Background Suppression ◽

Molecular Heterogeneity ◽

Single Molecule Tracking ◽

High Brightness ◽

Cellular Processes ◽

State Switching

AbstractCellular processes take place over many timescales, prompting the development of precision measurement technologies that cover milliseconds to hours. Here we describe ArrayG, a bipartite fluorogenic system composed of a GFP-nanobody array and monomeric wtGFP binders. The free binders are initially dim but brighten 15 fold upon binding the array, suppressing background fluorescence. By balancing rates of intracellular binder production, photo-bleaching, and stochastic binder exchange on the array, we achieved temporally unlimited tracking of single molecules. Fast (20-180Hz) tracking of ArrayG tagged kinesins and integrins, for thousands of frames, revealed repeated state-switching and molecular heterogeneity. Slow (0.5 Hz) tracking of single histones for as long as 1 hour showed fractal dynamics of chromatin. We also report ArrayD, a DHFR-nanobody-array tag for dual color imaging. The arrays are aggregation resistant and combine high brightness, background suppression, fluorescence replenishment, and extended choice of fluorophores, opening new avenues for seeing and tracking single molecules in living cells.

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New approaches to manipulating the epigenome

Dialogues in Clinical Neuroscience ◽

10.31887/dcns.2014.16.3/jday ◽

2014 ◽

Vol 16 (3) ◽

pp. 345-357 ◽

Cited By ~ 2

Keyword(s):

Epigenetic Mechanisms ◽

Temporal Precision ◽

Cellular Processes ◽

Disease States ◽

Cellular Environment ◽

Health And Disease ◽

Potential Impact ◽

Epigenetic Editing ◽

Insight Into ◽

Epigenetic Processes

Cellular processes that control transcription of genetic information are critical for cellular function, and are often implicated in psychiatric and neurological disease states. Among the most critical of these processes are epigenetic mechanisms, which serve to link the cellular environment with genomic material. Until recently our understanding of epigenetic mechanisms has been limited by the lack of tools that can selectively manipulate the epigenome with genetic, cellular, and temporal precision, which in turn diminishes the potential impact of epigenetic processes as therapeutic targets. This review highlights an emerging suite of tools that enable robust yet selective interrogation of the epigenome. In addition to allowing site-specific epigenetic editing, these tools can be paired with optogenetic approaches to provide temporal control over epigenetic processes, allowing unparalleled insight into the function of these mechanisms. This improved control promises to revolutionize our understanding of epigenetic modifications in human health and disease states.

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Dendrimers as Modulators of Brain Cells

Molecules ◽

10.3390/molecules25194489 ◽

2020 ◽

Vol 25 (19) ◽

pp. 4489

Author(s):

Dusica Maysinger ◽

Qiaochu Zhang ◽

Ashok Kakkar

Keyword(s):

Translational Medicine ◽

Early Stage ◽

Living Cells ◽

Mixed Population ◽

Neural Cells ◽

Biological Processes ◽

Clinical Investigations ◽

Biological Studies ◽

Essential Properties ◽

Functional Complexity

Nanostructured hyperbranched macromolecules have been extensively studied at the chemical, physical and morphological levels. The cellular structural and functional complexity of neural cells and their cross-talk have made it rather difficult to evaluate dendrimer effects in a mixed population of glial cells and neurons. Thus, we are at a relatively early stage of bench-to-bedside translation, and this is due mainly to the lack of data valuable for clinical investigations. It is only recently that techniques have become available that allow for analyses of biological processes inside the living cells, at the nanoscale, in real time. This review summarizes the essential properties of neural cells and dendrimers, and provides a cross-section of biological, pre-clinical and early clinical studies, where dendrimers were used as nanocarriers. It also highlights some examples of biological studies employing dendritic polyglycerol sulfates and their effects on glia and neurons. It is the aim of this review to encourage young scientists to advance mechanistic and technological approaches in dendrimer research so that these extremely versatile and attractive nanostructures gain even greater recognition in translational medicine.

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

Open Biology ◽

10.1098/rsob.120090 ◽

2012 ◽

Vol 2 (6) ◽

pp. 120090 ◽

Cited By ~ 31

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.

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Actin kinetics shapes cortical network structure and mechanics

Science Advances ◽

10.1126/sciadv.1501337 ◽

2016 ◽

Vol 2 (4) ◽

pp. e1501337 ◽

Cited By ~ 70

Author(s):

Marco Fritzsche ◽

Christoph Erlenkämper ◽

Emad Moeendarbary ◽

Guillaume Charras ◽

Karsten Kruse

Keyword(s):

Single Molecule ◽

Actin Filaments ◽

Length Distribution ◽

Living Cells ◽

Stochastic Simulations ◽

Cortical Actin ◽

Animal Cells ◽

Cellular Mechanics ◽

Cellular Processes ◽

Actin Cortex

The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs.

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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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