Pinching the cortex of live cells reveals thickness instabilities caused by Myosin II motors

AbstractThe cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration and division. Because the cortical thickness is below optical resolution, it has been generally considered as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension due to actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape changing capacity of highly contractile cells that use amoeboid-like migration.

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Pinching the cortex of live cells reveals thickness instabilities caused by myosin II motors

Science Advances ◽

10.1126/sciadv.abe3640 ◽

2021 ◽

Vol 7 (27) ◽

pp. eabe3640

Author(s):

Valentin Laplaud ◽

Nicolas Levernier ◽

Judith Pineau ◽

Mabel San Roman ◽

Lucie Barbier ◽

...

Keyword(s):

Magnetic Beads ◽

Optical Resolution ◽

Live Cells ◽

Cell Cortex ◽

Extracellular Medium ◽

Time Resolved ◽

Actomyosin Contractility ◽

Large Fluctuations ◽

Third Dimension ◽

Dynamic Layer

The cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration, and division. Because its thickness is below optical resolution, there is a tendency to consider the cortex as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time-resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension because of actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape-changing capacity of highly contractile cells that use amoeboid-like migration.

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KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

European Biophysics Journal ◽

10.1007/s00249-021-01560-2 ◽

2021 ◽

Author(s):

Diana Spiegelberg ◽

Jonas Stenberg ◽

Pascale Richalet ◽

Marc Vanhove

Keyword(s):

Flow Cytometry ◽

Live Cells ◽

Time Resolved ◽

Surface Receptors ◽

Full Characterization ◽

End Point ◽

Titration Curves ◽

Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

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3D Molecular Tracking in Live Cells with Simultaneous Time-Resolved Spectroscopy

Biophysical Journal ◽

10.1016/j.bpj.2010.12.2785 ◽

2011 ◽

Vol 100 (3) ◽

pp. 475a

Author(s):

James Werner ◽

Peter Goodwin ◽

Elizabeth Phipps ◽

Patrick Cutler ◽

Diane Lidke ◽

...

Keyword(s):

Live Cells ◽

Time Resolved ◽

Time Resolved Spectroscopy ◽

Molecular Tracking

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Imaging the Redox States of Live Cells with the Time-Resolved Fluorescence of Genetically Encoded Biosensors

Analytical Chemistry ◽

10.1021/acs.analchem.8b04292 ◽

2019 ◽

Vol 91 (6) ◽

pp. 3869-3876 ◽

Cited By ~ 3

Author(s):

Lei Li ◽

Changcheng Zhang ◽

Peng Wang ◽

Aoxue Wang ◽

Jiasheng Zhou ◽

...

Keyword(s):

Live Cells ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Redox States

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Mapping the Spatiotemporal Heterogeneity of Biomolecules Concentration, Mobility and Local Environment in Live Cells using Quantitative Time-Resolved Confocal Fluorescence Microscopy Imaging Without Scanning and Fluorescence Lifetime Imaging Microscopy

Biophysical Journal ◽

10.1016/j.bpj.2019.11.1735 ◽

2020 ◽

Vol 118 (3) ◽

pp. 307a

Author(s):

Sho Oasa ◽

Aleksandar Krmpot ◽

Stanko Nikolic ◽

Lars Terenius ◽

Rudolf Rigler ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Fluorescence Lifetime ◽

Local Environment ◽

Live Cells ◽

Fluorescence Lifetime Imaging Microscopy ◽

Fluorescence Lifetime Imaging ◽

Microscopy Imaging ◽

Time Resolved ◽

Lifetime Imaging ◽

Confocal Fluorescence

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Opto-magnetic capture of individual cells based on visual phenotypes

eLife ◽

10.7554/elife.45239 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 4

Author(s):

Loïc Binan ◽

François Bélanger ◽

Maxime Uriarte ◽

Jean François Lemay ◽

Jean Christophe Pelletier De Koninck ◽

...

Keyword(s):

Magnetic Beads ◽

Cost Effective ◽

Lipid Vesicles ◽

Population Based ◽

Live Cells ◽

Biologically Relevant ◽

Cost Effective Approach ◽

Magnetic Capture ◽

Radiation Induced ◽

Rare Cells

The ability to isolate rare live cells within a heterogeneous population based solely on visual criteria remains technically challenging, due largely to limitations imposed by existing sorting technologies. Here, we present a new method that permits labeling cells of interest by attaching streptavidin-coated magnetic beads to their membranes using the lasers of a confocal microscope. A simple magnet allows highly specific isolation of the labeled cells, which then remain viable and proliferate normally. As proof of principle, we tagged, isolated, and expanded individual cells based on three biologically relevant visual characteristics: i) presence of multiple nuclei, ii) accumulation of lipid vesicles, and iii) ability to resolve ionizing radiation-induced DNA damage foci. Our method constitutes a rapid, efficient, and cost-effective approach for isolation and subsequent characterization of rare cells based on observable traits such as movement, shape, or location, which in turn can generate novel mechanistic insights into important biological processes.

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Near-Field Second Harmonic Microscopy of Thin Ferroelectric Films

MRS Proceedings ◽

10.1557/proc-596-333 ◽

1999 ◽

Vol 596 ◽

Author(s):

I. I. Smolyaninov ◽

H. Y. Liang ◽

C. H. Lee ◽

C. C. Davis ◽

L. D. Rotter ◽

...

Keyword(s):

Second Harmonic ◽

Near Field ◽

Laser System ◽

Optical Resolution ◽

Optical Probe ◽

Ferroelectric Materials ◽

Scanning Probe ◽

Fast Time ◽

Regenerative Amplifier ◽

Time Resolved

AbstractNear-field second harmonic microscopy is ideally suited for studies of local nonlinearity and poling of ferroelectric materials at the microscopic level. Its main advantages in comparison with other scanning probe techniques are the possibility of fast time-resolved measurements, and substantially smaller perturbation of the sample under investigation caused by the optical probe. We report second harmonic imaging of the surface of thin BaTiO3 films obtained in a near-field microscopy setup using a Ti:sapphire laser system consisting of an oscillator and a regenerative amplifier operating at 810 nm. Optical resolution on the order of 80 nm has been achieved.

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