scholarly journals Super‐Resolution Microscopy and Single‐Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule

2017 ◽  
Vol 75 (1) ◽  
Author(s):  
Saumya Saurabh ◽  
Adam M. Perez ◽  
Colin J. Comerci ◽  
Lucy Shapiro ◽  
W. E. Moerner
Keyword(s):  
Super Resolution ◽  
Single Protein ◽  
Protein Tracking ◽  
Live Bacteria ◽  
Super Resolution Microscopy

scholarly journals A micromechanical cell stretching device compatible with super-resolution microscopy and single protein tracking

2020 ◽  
Author(s):  
Filipe Nunes Vicente ◽  
Sophie Massou ◽  
Franziska Wetzel ◽  
Amine Mehidi ◽  
Dan Strehle ◽  
...  
Keyword(s):  
Cell Biology ◽  
Molecular Mechanisms ◽  
Super Resolution ◽  
Pdms Layer ◽  
Cell Stretching ◽  
Glass Cover Slip ◽  
Single Protein ◽  
Protein Tracking ◽  
A Cell ◽  
Super Resolution Microscopy

Abstract Cell mechano-sensing is based on biomolecule deformations and reorganizations, yet the molecular mechanisms are still unclear. Super-resolution microscopy (SRM) and single protein tracking (SPT) techniques reveal the dynamic organization of proteins at the nanoscale. In parallel, stretchable substrates are used to investigate cellular responses to mechanical forces. However, simultaneous combination of SRM/SPT and cell stretching has never been achieved. Here, we present a cell stretching device compatible with SRM and SPT, composed of an ultra-thin Polydimethylsiloxane (PDMS) layer. The PDMS sheet is gliding on a glycerol-lubricated glass cover-slip to ensure flatness during uniaxial stretching, generated with a 3D-printed micromechanical device by a mobile arm connected to a piezoelectric translator. This method enables to obtain super-resolved images of protein reorganization after live stretching, and to monitor single protein deformation and recruitment inside mechanosensitive structures upon stretching. This protocol is related to the publication ‘Cell stretching is amplified by active actin remodeling to deform and recruit proteins in mechanosensitive structures’, in Nature Cell Biology.

scholarly journals An open microscopy framework suited for tracking dCas9 in live bacteria

2018 ◽  
Author(s):  
Koen J. A. Martens ◽  
Sam P. B. van Beljouw ◽  
Simon van der Els ◽  
Sander Baas ◽  
Jochem N. A. Vink ◽  
...  
Keyword(s):  
Target Genes ◽  
Life Sciences ◽  
Super Resolution ◽  
Single Particle Tracking ◽  
Spatiotemporal Resolution ◽  
Localization Microscopy ◽  
Target Sites ◽  
Heterogeneous Expression ◽  
Live Bacteria ◽  
Super Resolution Microscopy

Super-resolution microscopy is frequently employed in the life sciences, but the number of freely accessible and affordable microscopy frameworks, especially for single particle tracking photo-activation localization microscopy (sptPALM), remains limited. To this end, we designed the miCube: a versatile super-resolution capable fluorescence microscope, which combines high spatiotemporal resolution, good adaptability, low price, and easy installation. By providing all details, we hope to enable interested researchers to build an identical or derivative instrument. The capabilities of the miCube are assessed with a novel sptPALM assay relying on the heterogeneous expression of target genes. Here, we elucidate mechanistic details of catalytically inactive Cas9 (dead Cas9) in live Lactococcus lactis. We show that, lacking specific DNA target sites, the binding and unbinding of dCas9 to DNA can be described using simplified rate constants of kbound→free = 30−80 s−1 and kfree→bound = 15−40 s−1. Moreover, after providing specific DNA target sites via DNA plasmids, the plasmid-bound dCas9 population size decreases with increasing dCas9 copy number via a mono-exponential decay, indicative of simple disassociation kinetics.

scholarly journals Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

2020 ◽  
Author(s):  
Thomas Orré ◽  
Zeynep Karatas ◽  
Birgit Kastberger ◽  
Clément Cabriel ◽  
Ralph T. Böttcher ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Pleckstrin Homology Domain ◽  
Membrane Diffusion ◽  
Integrin Activation ◽  
Molecular Behavior ◽  
Protein Tracking ◽  
And Function ◽  
Super Resolution Microscopy

AbstractFocal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we linked the molecular behavior and tridimensional nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displayed free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation during cell adhesion and spreading. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.

Pushing the super-resolution limit: recent improvements in microscopy below the diffraction limit

2021 ◽  
Author(s):  
D. J. Nieves ◽  
M. A. B. Baker
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Information ◽  
Biological Systems ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Resolving Power ◽  
Resolution Limit ◽  
Single Protein ◽  
Super Resolution Microscopy ◽  
Near Future

Super-resolution microscopy has revolutionised the way we observe biological systems. These methods are now a staple of fluorescence microscopy. Researchers have used super-resolution methods in myriad systems to extract nanoscale spatial information on multiple interacting parts. These methods are continually being extended and reimagined to further push their resolving power and achieve truly single protein resolution. Here, we explore the most recent advances at the frontier of the ‘super-resolution’ limit and what opportunities remain for further improvements in the near future.

scholarly journals Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Thomas Orré ◽  
Adrien Joly ◽  
Zeynep Karatas ◽  
Birgit Kastberger ◽  
Clément Cabriel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Pleckstrin Homology Domain ◽  
Membrane Diffusion ◽  
Integrin Activation ◽  
Molecular Behavior ◽  
Protein Tracking ◽  
And Function ◽  
Super Resolution Microscopy

AbstractFocal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we link the molecular behavior and 3D nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displays free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation, cell spreading and FAs formation. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

Sign in / Sign up

Export Citation Format

Share Document