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.

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.

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

scholarly journals Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy

2017 ◽  
Vol 56 (22) ◽  
pp. 6131-6135 ◽  
Author(s):  
Anne Burgert ◽  
Jan Schlegel ◽  
Jérôme Bécam ◽  
Sören Doose ◽  
Erhard Bieberich ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals Ligand Geometry Controls Adhesion formation via Integrin Clustering

2018 ◽  
Author(s):  
Rishita Changede ◽  
Haogang Cai ◽  
Shalom Wind ◽  
Michael P. Sheetz
Keyword(s):  
Adhesion Formation ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Total Width ◽  
Cellular Processes ◽  
Cell Matrix ◽  
Integrin Clustering ◽  
Super Resolution Microscopy ◽  
Fiber Mesh

AbstractIntegrin-mediated cell matrix adhesions are key to sensing the geometry and rigidity of the extracellular environment to regulate vital cellular processes. In vivo, the extracellular matrix (ECM) is composed of a fibrous mesh. To understand the geometry that supports adhesion formation on fibrous substrates, we patterned 10 nm gold-palladium single lines or pairs of lines (total width within 100 nm), mimicking thin single ECM fibers or a minimal mesh geometry, respectively and functionalized it with integrin binding ligand Arg-Gly-Asp (RGD). Single lines showed reduced focal adhesion kinase (FAK) recruitment and did not support cell spreading or formation of focal adhesions, despite the presence of a high density of integrin-binding ligands. Using super resolution microscopy, we observed transient integrin clusters on single lines, whereas stable 110 nm integrin clusters formed on pairs of lines similar to those on continuous substrates. This indicated that two-dimensional ligand geometry is required for adhesion formation on rigid substrates. A mechanism to form modular 100nm integrin clusters bridging the minimal fiber mesh would require unliganded integrins. We observed that integrin mutants unable to bind ligand co-clustered with ligand-bound integrins when present in an active extended conformation. Thus, these results indicate that functional integrin clusters are required to form focal adhesions and unliganded integrins can co-cluster to bridge between thin matrix fibers and can form stable integrin adhesions on dense fibrous networks.

scholarly journals Nanotopography controls single-molecule mobility to determine overall cell fate

2020 ◽  
Author(s):  
Marie FA Cutiongco ◽  
Paul M Reynolds ◽  
Christopher D Syme ◽  
Nikolaj Gadegaard
Keyword(s):  
Cell Fate ◽  
Single Molecule ◽  
Molecular Diffusion ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Local Area ◽  
Surface Areas ◽  
Modular Units ◽  
Nanoscale Sensing ◽  
Super Resolution Microscopy

AbstractThe addition of nanoscale distortion to ordered nanotopographies consistently determines an osteogenic fate in stem cells. Although disordered and ordered nanopit arrays have identical surface areas, array symmetry has opposite effects on cell fate. We aimed to understand how cells sense disorder at the nanoscale. We observed effects in the early formation of cell and focal adhesions that controlled long-term cell fate. Disordered nanopits consistently yielded larger focal adhesions at a faster rate, prompting us to investigate this at the molecular scale. Super-resolution microscopy revealed that the nanopits did not act as nucleation points, as previously thought. Rather, nanopit arrays altered the plasma membrane and acted as barriers that changed molecular diffusion. The local areas corralled by four nanopits were the smallest structures that exerted diverging effects between ordered and disordered arrays. Heterogeneity in the local area on disordered arrays increased the proportion of fastest and slowest diffusing molecules. This resulted in higher quantity, more frequent formation and clustered arrangement of nascent adhesions, i.e., the modular units on which focal adhesions are built. This work presents a new pathway to exploit nanoscale sensing to dictate cell fate.

scholarly journals Microscope-AOtools: A generalised adaptive optics implementation

2020 ◽  
Author(s):  
Nicholas Hall ◽  
Josh Titlow ◽  
Martin J. Booth ◽  
Ian M. Dobbie
Keyword(s):  
Image Quality ◽  
Adaptive Optics ◽  
Super Resolution ◽  
Biological Imaging ◽  
New Techniques ◽  
Reduce Image Quality ◽  
Set Up ◽  
And Function ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

AbstractMicroscope-AOtools is a software package which allows for a simple, robust and generalised implementation of adaptive optics (AO) elements. It contains all the necessary methods for set-up, calibration, and aberration correction which are simple to use and function in a robust manner. Aberrations arising from sources such as sample hetero-geneity and refractive index mismatches are constant problems in biological imaging. These aberrations reduce image quality and the achievable depth of imaging, particularly in super-resolution microscopy techniques. AO technology has been proven to be effective in correcting for these aberrations and thereby improving the image quality. However, it has not been widely adopted by the biological imaging community due, in part, to difficulty in set-up and operation of AO, particularly by non-specialist users. Microscope-AOtools offers a robust, easy-to-use implementation of the essential methods for set-up and use of AO techniques. These methods are constructed in a generalised manner that can utilise a range of adaptive optics elements, wavefront sensing techniques and sensorless AO correction methods. Furthermore, the methods are designed to be easily extensible as new techniques arise, leading to a streamlined pipeline for new AO technology and techniques to be adopted by the wider microscopy community.

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