Visualizing the ‘backbone’ of focal adhesions

2018 ◽  
Vol 2 (5) ◽  
pp. 677-680 ◽  
Author(s):  
Samuel F. H. Barnett ◽  
Pakorn Kanchanawong
Keyword(s):  
Protein Engineering ◽  
Structural Organization ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Biological Processes ◽  
Recent Application ◽  
Super Resolution Microscopy

To understand how complex machines perform their functions, it is essential to map out the ‘blueprints’ of how their internal components are organized. Focal adhesions (FAs) are complex mechanobiological structures involved in a plethora of cell biological processes. The application of super-resolution microscopy in concert with protein engineering offers one approach to unravel the complexity of how individual proteins are organized within FAs. In our recent application, the FA protein talin was found to form a direct structural and physical link between integrin and actin. Interestingly, engineered talin constructs with alternate lengths rescaled the FA nanostructure accordingly. This helped establish that talin could be analogous to the backbone of FAs, serving as the mechanosensitive master coordinator of FA structural organization.

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.

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 Nanoscale organization of rotavirus replication machineries

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yasel Garcés Suárez ◽  
Jose L Martínez ◽  
David Torres Hernández ◽  
Haydee Olinca Hernández ◽  
Arianna Pérez-Delgado ◽  
...  
Keyword(s):  
Optical Microscopy ◽  
Spatial Resolution ◽  
Structural Organization ◽  
Super Resolution ◽  
Viral Proteins ◽  
Genome Replication ◽  
Molecular Scale ◽  
Viral Components ◽  
Super Resolution Microscopy ◽  
Outermost Zone

Rotavirus genome replication and assembly take place in cytoplasmic electron dense inclusions termed viroplasms (VPs). Previous conventional optical microscopy studies observing the intracellular distribution of rotavirus proteins and their organization in VPs have lacked molecular-scale spatial resolution, due to inherent spatial resolution constraints. In this work we employed super-resolution microscopy to reveal the nanometric-scale organization of VPs formed during rotavirus infection, and quantitatively describe the structural organization of seven viral proteins within and around the VPs. The observed viral components are spatially organized as five concentric layers, in which NSP5 localizes at the center of the VPs, surrounded by a layer of NSP2 and NSP4 proteins, followed by an intermediate zone comprised of the VP1, VP2, VP6. In the outermost zone, we observed a ring of VP4 and finally a layer of VP7. These findings show that rotavirus VPs are highly organized organelles.

scholarly journals Nanoscale organization of rotavirus replication machineries

2018 ◽  
Author(s):  
Yasel Garcés ◽  
José L. Martínez ◽  
David T. Hernández ◽  
Haydee O. Hernández ◽  
Mayra Méndez ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Structural Organization ◽  
Super Resolution ◽  
Viral Proteins ◽  
Genome Replication ◽  
Viral Dsrna ◽  
Molecular Scale ◽  
Viral Components ◽  
Super Resolution Microscopy ◽  
Outermost Zone

AbstractRotavirus genome replication and assembly take place in cytoplasmic electron dense inclusions termed viro-plasms (VPs). Previous conventional optical microscopy studies observing the intracellular distribution of rotavirus proteins and their organization in VPs have lacked molecular-scale spatial resolution, due to inherent spatial resolution constraints. In this work we employed super-resolution microscopy to reveal the nanometric-scale organization of VPs formed during rotavirus infection, and quantitatively describe the structural organization of seven viral proteins and viral dsRNA within and around the VPs. The observed viral components are spatially organized as 6 concentric layers, in which NSP5 localizes at the center of the VPs, surrounded by a layer of NSP2 and NSP4 proteins, followed by an intermediate zone comprised of the VP1, VP2, VP6 proteins and the dsRNA. In the outermost zone, we observed a ring of VP4 and finally a layer of VP7. These findings show that rotavirus VPs are highly organized organelles.

scholarly journals Subcellular drug targeting illuminates local kinase action

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Paula J Bucko ◽  
Chloe K Lombard ◽  
Lindsay Rathbun ◽  
Irvin Garcia ◽  
Akansha Bhat ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Super Resolution ◽  
Biological Processes ◽  
Anchoring Proteins ◽  
Intracellular Compartments ◽  
Mitotic Spindle Assembly ◽  
Targeted Inhibition ◽  
Super Resolution Microscopy

Deciphering how signaling enzymes operate within discrete microenvironments is fundamental to understanding biological processes. A-kinase anchoring proteins (AKAPs) restrict the range of action of protein kinases within intracellular compartments. We exploited the AKAP targeting concept to create genetically encoded platforms that restrain kinase inhibitor drugs at distinct subcellular locations. Local Kinase Inhibition (LoKI) allows us to ascribe organelle-specific functions to broad specificity kinases. Using chemical genetics, super resolution microscopy, and live-cell imaging we discover that centrosomal delivery of Polo-like kinase 1 (Plk1) and Aurora A (AurA) inhibitors attenuates kinase activity, produces spindle defects, and prolongs mitosis. Targeted inhibition of Plk1 in zebrafish embryos illustrates how centrosomal Plk1 underlies mitotic spindle assembly. Inhibition of kinetochore-associated pools of AurA blocks phosphorylation of microtubule-kinetochore components. This versatile precision pharmacology tool enhances investigation of local kinase biology.

scholarly journals Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

2020 ◽  
Author(s):  
Ryu J. Iwatate ◽  
Akira Yoshinari ◽  
Noriyoshi Yagi ◽  
Marek Grzybowski ◽  
Hiroaki Ogasawara ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Super Resolution ◽  
Cell Plate ◽  
Protein Labeling ◽  
Synthetic Dyes ◽  
Biological Processes ◽  
Major Drawback ◽  
Specific Proteins ◽  
Super Resolution Microscopy

AbstractSynthetic chemical fluorescent dyes are promising tools for many applications in biology. SNAP tagging provides a unique opportunity for labeling of specific proteins in vivo with synthetic dyes for studying for example endocytosis, or super-resolution microscopy. However, despite the potential, chemical dye tagging has not been used effectively in plants. A major drawback was the limited knowledge regarding cell wall and membrane permeability of synthetic dyes. Twenty-six out of 31 synthetic dyes were taken up into BY-2 cells, eight were not taken up and can thus serve for measuring endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine (TMR) and silicon-rhodamine (SiR) 647 were used to SNAP tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubules arrays in interphase and during mitosis. Fluorescence activation-coupled protein labeling (FAPL) with DRBG-488 was used to observe PIN2 endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the newly forming cell plate during mitosis. Together the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety to cell biological processes.

scholarly journals Size-dependent secondary nucleation and amplification of α-synuclein amyloid fibrils

2021 ◽  
Author(s):  
Arunima Sakunthala ◽  
Debalina Datta ◽  
Ambuja Navalkar ◽  
Laxmikant Gadhe ◽  
Pradeep Kadu ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Super Resolution ◽  
Cellular Internalization ◽  
Biological Processes ◽  
Secondary Nucleation ◽  
Size Dependent ◽  
Distinct Mechanism ◽  
In Vitro Reconstitution ◽  
Super Resolution Microscopy

The size of the amyloid seeds is known to modulate their autocatalytic amplification and cellular toxicity. However, the seed size-dependent secondary nucleation mechanism, toxicity, and disease-associated biological processes mediated by α-synuclein (α-Syn) fibrils are largely unknown. Using the cellular model and in vitro reconstitution, we showed that the size of α-Syn fibril seeds not only dictates its cellular internalization and associated cell death; but also the distinct mechanisms of fibril amplification pathways involved in the pathological conformational change of α-Syn. Specifically, small-sized fibril seeds showed elongation possibly through monomer addition at the fibril termini; whereas longer fibrils template the fibril amplification by surface-mediated nucleation as demonstrated by super-resolution microscopy. The distinct mechanism of fibril amplification, and cellular uptake along with toxicity suggest that breakage of fibrils into different sizes of seeds determine the underlying pathological outcome of synucleinopathies.

Towards structural biology with super-resolution microscopy

Nanoscale ◽  
2018 ◽  
Vol 10 (35) ◽  
pp. 16416-16424 ◽  
Author(s):  
Julia Molle ◽  
Leonhard Jakob ◽  
Johann Bohlen ◽  
Mario Raab ◽  
Philip Tinnefeld ◽  
...  
Keyword(s):  
Structural Biology ◽  
Single Molecule ◽  
Structural Organization ◽  
Dna Nanotechnology ◽  
Super Resolution ◽  
Super Resolution Microscopy

The combination of DNA nanotechnology and single-molecule biochemistry allows the first step towards the investigation of the structural organization of a protein via SR microscopy.

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