scholarly journals Growth factor dependent changes in nanoscale architecture of focal adhesions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karin Legerstee ◽  
Tsion E. Abraham ◽  
Wiggert A. van Cappellen ◽  
Alex L. Nigg ◽  
Johan A. Slotman ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Intermediate Layer ◽  
Cell Movement ◽  
Focal Adhesions ◽  
Bottom Layer ◽  
Vertical Axis ◽  
Longitudinal Distribution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Mediate Cell

AbstractFocal adhesions (FAs) are flat elongated structures that mediate cell migration and link the cytoskeleton to the extracellular matrix. Along the vertical axis FAs were shown to be composed of three layers. We used structured illumination microscopy to examine the longitudinal distribution of four hallmark FA proteins, which we also used as markers for these layers. At the FA ends pointing towards the adherent membrane edge (heads), bottom layer protein paxillin protruded, while at the opposite ends (tails) intermediate layer protein vinculin and top layer proteins zyxin and VASP extended further. At the tail tips, only intermediate layer protein vinculin protruded. Importantly, head and tail compositions were altered during HGF-induced scattering with paxillin heads being shorter and zyxin tails longer. Additionally, FAs at protruding or retracting membrane edges had longer paxillin heads than FAs at static edges. These data suggest that redistribution of FA-proteins with respect to each other along FAs is involved in cell movement.

scholarly journals Optical Microscopy and the Extracellular Matrix Structure: A Review

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1760
Author(s):  
Joshua J. A. Poole ◽  
Leila B. Mostaço-Guidolin
Keyword(s):  
Extracellular Matrix ◽  
Confocal Microscopy ◽  
Laser Scanning ◽  
Biological Tissues ◽  
The Body ◽  
Stimulated Emission ◽  
Substantial Part ◽  
Fluorescence Lifetime Imaging ◽  
Structured Illumination ◽  
Structured Illumination Microscopy

Biological tissues are not uniquely composed of cells. A substantial part of their volume is extracellular space, which is primarily filled by an intricate network of macromolecules constituting the extracellular matrix (ECM). The ECM serves as the scaffolding for tissues and organs throughout the body, playing an essential role in their structural and functional integrity. Understanding the intimate interaction between the cells and their structural microenvironment is central to our understanding of the factors driving the formation of normal versus remodelled tissue, including the processes involved in chronic fibrotic diseases. The visualization of the ECM is a key factor to track such changes successfully. This review is focused on presenting several optical imaging microscopy modalities used to characterize different ECM components. In this review, we describe and provide examples of applications of a vast gamut of microscopy techniques, such as widefield fluorescence, total internal reflection fluorescence, laser scanning confocal microscopy, multipoint/slit confocal microscopy, two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG, THG), coherent anti-Stokes Raman scattering (CARS), fluorescence lifetime imaging microscopy (FLIM), structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), ground-state depletion microscopy (GSD), and photoactivated localization microscopy (PALM/fPALM), as well as their main advantages, limitations.

scholarly journals Total Internal Reflection Fluorescence Microscopy (TIRFM) – novel techniques and applications

2020 ◽  
Vol 8 (11) ◽  
Author(s):  
Verena Richter ◽  
Michael Wagner ◽  
Herbert Schneckenburger
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Plasma Membranes ◽  
Focal Adhesions ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Thin Layers ◽  
Structured Illumination ◽  
Structured Illumination Microscopy

Total Internal Reflection Fluorescence Microscopy (TIRFM) has been established almost 40 years ago for studies of plasma membranes or membrane proximal sites of living cells. The method is based on light incidence at an angle above the critical angle of total internal reflection and generation of an evanescent electromagnetic field penetrating about 100 nm into a sample and permitting selective excitation of membrane proximal fluorophores. Two techniques are presented here: prism-type TIRFM and objective-type TIRFM with high aperture microscope objective lenses. Furthermore, numerous applications are summarized, e.g. measurement of focal adhesions, cell-substrate topology, endocytosis or exocytosis of vesicles as well as single molecule detection within thin layers. Finally, highly innovative combinations of TIRFM with Förster Resonance Energy Transfer (FRET) measurements as well as with Structured Illumination Microscopy (SIM) and fluorescence reader technologies are presented.

scholarly journals Filopodome mapping identifies p130Cas as a mechanosensitive regulator of filopodia stability

2018 ◽  
Author(s):  
Guillaume Jacquemet ◽  
Rafael Saup ◽  
Hellyeh Hamidi ◽  
Mitro Miihkinen ◽  
Johanna Ivaska
Keyword(s):  
Binding Sites ◽  
Focal Adhesions ◽  
Structure And Function ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Cell Adhesion And Migration ◽  
And Migration ◽  
Cellular Behaviour ◽  
And Function ◽  
Fine Tune

AbstractFilopodia are adhesive cellular protrusions specialised in the detection of extracellular matrix (ECM)-derived cues. While ECM engagement at focal adhesions is known to trigger the recruitment of hundreds of proteins (“adhesome”) to fine-tune cellular behaviour, the components of the filopodia adhesions remain undefined. Here, we performed a structured illumination microscopy-based screen to map the localisation of 80 target proteins, linked to cell adhesion and migration, within filopodia. We demonstrate preferential enrichment of several adhesion proteins to either filopodia tips, filopodia shafts, or shaft subdomains suggesting divergent, spatially restricted functions for these proteins. Moreover, proteins with phospho-inositide (PI) binding sites are particularly enriched in filopodia. This, together with the strong localisation of PI(3,4)P2 in filopodia tips, predicts critical roles for PIs in regulating filopodia ultra-structure and function. Our mapping further reveals that filopodia adhesions consist of a unique set of proteins, the filopodome, that are distinct from classical nascent adhesions, focal adhesions and fibrillar adhesions. Using live imaging, we observe that filopodia adhesions can give rise to nascent adhesions, which, in turn, form focal adhesions. Finally, we demonstrate that p130Cas (BCAR1) is recruited to filopodia tips via its CCHD domain and acts as a mechanosensitive regulator of filopodia stability.

Turn-key super-resolution mapping of cell receptor force orientation and magnitude using a commercial structured illumination microscope

2020 ◽  
Author(s):  
Aaron Blanchard ◽  
J. Dale Combs ◽  
Joshua Brockman ◽  
Anna Kellner ◽  
Roxanne Glazier ◽  
...  
Keyword(s):  
Cell Biology ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Cell Receptor ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Surface Receptors ◽  
Cellular Processes ◽  
Resolution Mapping ◽  
Super Resolution Mapping

Abstract Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell surface receptors. Nucleic acid-based molecular tension probes allow one to quantify and visualize the piconewton (pN) forces applied by these receptors. Building on this technology, we recently imaged DNA tension probes using fluorescence polarization imaging to map the magnitude and 3D orientation of receptor forces with diffraction limited resolution (~ 250 nm). Further improvements in spatial resolution are desirable as many force-sensing receptors are organized at the nano-scale in supramolecular complexes such as focal adhesions. Here, we show that structured illumination microscopy (SIM), a super-resolution technique, can be used to perform super-resolution molecular force microscopy (MFM). Using SIM-MFM, we generate the highest resolution maps of both the magnitude and orientation of the pN traction forces applied by cells. We apply SIM-MFM to map platelet and fibroblast integrins forces, as well as T cell receptor forces. The method reveals that platelets dynamically re-arrange the orientation of their integrin forces during activation. Monte Carlo simulations validated the results and provided analysis of the sources of noise. Importantly, we envision that SIM-MFM will be broadly adopted by the cell biology and mechanobiology communities because it can be implemented on any standard SIM microscope without hardware modifications.

scholarly journals Phosphorylated paxillin and FAK constitute subregions within focal adhesions

2020 ◽  
Author(s):  
Michael Bachmann ◽  
Artiom Skripka ◽  
Bernhard Wehrle-Haller ◽  
Martin Bastmeyer
Keyword(s):  
Focal Adhesion ◽  
Structural Organization ◽  
Spatial Organization ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
Multiple Signaling

AbstractIntegrin-mediated adhesions are convergence points of multiple signaling pathways. Their inner structure and their diverse functions can be studied with super-resolution microscopy. We used structured illumination microscopy (SIM) to analyze spatial organization of paxillin phosphorylation (pPax) within adhesions. We found that pPax and focal adhesion kinase (FAK) form spot-like, spatially defined clusters within adhesions in several cell lines. In contrast, other adhesion proteins showed no consistent organization in such clusters. Live-cell super-resolution imaging revealed that pPax-FAK clusters persist over time but modify distance to each other dynamically. Moreover, we show that the distance between separate clusters of pPax is mechanosensitive. Thus, in this work we introduce a new structural organization within focal adhesions and demonstrate its regulation and dynamics.

Structured illumination microscopy reveals focal adhesions are composed of linear subunits

Cytoskeleton ◽  
2015 ◽  
Vol 72 (5) ◽  
pp. 235-245 ◽  
Author(s):  
Shiqiong Hu ◽  
Yee-Han Tee ◽  
Alexandre Kabla ◽  
Ronen Zaidel-Bar ◽  
Alexander Bershadsky ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
Structured Illumination ◽  
Structured Illumination Microscopy

Quantitative Freeze Fracture Studies of Gap Junctions in Somatic Cell Hybrids, Their Parents, and Revertants

1976 ◽  
Vol 34 ◽  
pp. 218-219
Author(s):  
W. J. Larsen ◽  
R. Azarnia ◽  
W. R. Loewenstein
Keyword(s):  
Gap Junctions ◽  
Striated Muscle ◽  
Freeze Fracture ◽  
Cell Movement ◽  
Dye Molecules ◽  
Somatic Cell Hybrids ◽  
Cell Coupling ◽  
Normal Cells ◽  
Mediate Cell ◽  
Almost All

Although the physiological significance of the gap junction remains unspecified, these membrane specializations are now recognized as common to almost all normal cells (excluding adult striated muscle and some nerve cells) and are found in organisms ranging from the coelenterates to man. Since it appears likely that these structures mediate the cell-to-cell movement of ions and small dye molecules in some electrical tissues, we undertook this study with the objective of determining whether gap junctions in inexcitable tissues also mediate cell-to-cell coupling.To test this hypothesis, a coupling, human Lesh-Nyhan (LN) cell was fused with a non-coupling, mouse cl-1D cell, and the hybrids, revertants, and parental cells were analysed for coupling with respect both to ions and fluorescein and for membrane junctions with the freeze fracture technique.

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