The histogram of vertical localization coordinates was calculated for each focal adhesion region. The local z=0 nm level was defined by nonspecific fluorescence from the media that adsorbed to the coverglass, and was used to account for sample tilt. The centre positions (zcentre) and width parameter (σvert) were calculated from Gaussian fits or from the first and second moment of the distributions for non-Gaussian cases such as actin and α-actinin. For more detailed information see Methods and Supplementary Information .
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature .
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Anatomy
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Cell Component
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Focal Adhesions
Focal Adhesions
Focal adhesiions are specialized regions of the cell surface where the cytoskeleton connects to the extracellular matrix.
These structures are critical for cell adhesion, migration, and signaling.
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These structures are critical for cell adhesion, migration, and signaling.
PubCompare.ai's AI-driven platform helps optimize research protocols related to focal adhesions, enhancing reproducibility and accuracy.
Easily locate relevant protocols from literature, preprints, and patents, while utilizing AI-driven comparisons to identify the best protocols and products.
Improve your focal adhesion research efficiency with PubCompare.ai's innovative solution.
Most cited protocols related to «Focal Adhesions»
Actinin
Actins
Fluorescence
Focal Adhesions
Seizures
Cells
Fibronectins
Focal Adhesions
Green Fluorescent Proteins
Proteins
red fluorescent protein
Anabolism
Breast
Cancer Stem Cells
Carcinogenesis
CD44 protein, human
Cells
Focal Adhesions
Glycogen
Homo sapiens
Malignant Neoplasm of Breast
Malignant Neoplasms
Mammary Carcinoma, Human
MicroRNAs
Multiplex Polymerase Chain Reaction
Oligonucleotide Primers
Population Group
Real-Time Polymerase Chain Reaction
Reverse Transcription
Small Nuclear RNA
Stem, Plant
Stem Cells
trizol
All biological images were recorded on a custom-built setup (Supplementary Fig. 6 ) based on a commercial microscope stand (Axio Observer D1, Carl Zeiss MicroImaging) with a 100×/1.46 NA oil immersion objective (alpha Plan-Apochromat 100×/1.46 Oil, Zeiss). The setup is equipped with lasers emitting at 405 nm (CrystaLaser, 50 mW), 568 nm (Coherent Innova 300, ~400 mW) and 642 nm (MPB Communications, 500 mW). Fluorescence was recorded by our sCMOS camera through the side port of the stand. All data was recorded at room temperature.
Fixed microtubule structures were imaged in a 128 × 128 pixel ROI for 40,000 frames at 1,600 fps (Fig. 1g–k ), a 512 × 512 pixel ROI for 16,000 frames at 400 fps (Fig. 2a,b ) and a 64 × 64 pixel ROI for 30,000 frames at 3,200 fps (Fig. 2e–h ). The 642 nm laser was used at intensities of 18.4 kW/cm2, 5.5 kW/cm2, and 7.4 kW/cm2, respectively. Images were acquired using HCimage software (Hamamatsu). Focal adhesions were recorded in a 256 × 256 pixel ROI for 2,400 frames at 800 fps with the 642 nm laser intensity set to 9.2 kW/cm2. Clathrin data was recorded in a 256 × 256 pixel ROI for 50,000 frames at 600 fps with the 568 nm laser intensity set to 5.3 kW/cm2. Mitochondria were recorded in a 256 × 256 pixel ROI for 80,000 frames at 400 fps with the 568 nm laser intensity set to 5.3 kW/cm2. EB3 data was recorded in a 256 × 256 pixel ROI for 30,000 frames at 600 fps with the 568 nm laser intensity set to 5.3 kW/cm2. Peroxisomes were recorded in a 256 × 256 pixel ROI for 50,000 frames at 600 fps with the 568 nm laser intensity set to 5.3 kW/cm2. Transferrin data was recorded in a 128 × 128 pixel ROI at 1,600 fps with the 642 nm laser intensity set to 7.4 kW/cm2. During imaging, the intensity of the 405 nm activation laser was manually increased from 0 to 0.3 W/cm2 (for 512 × 512 pixel ROI) and 0 to 1.8 W/cm2 (for 256 × 256, 128 × 128 and 64 × 64 pixel ROI) to ensure optimal particle densities for either single emitter fitting or multi-emitter fitting31 (link).
Fixed microtubule structures were imaged in a 128 × 128 pixel ROI for 40,000 frames at 1,600 fps (
Biopharmaceuticals
Clathrin
Fluorescence
Focal Adhesions
Immersion
Microscopy
Microtubules
Mitochondria
Peroxisome
Reading Frames
Transferrin
Biopharmaceuticals
Clathrin
Fluorescence
Focal Adhesions
Immersion
Microscopy
Microtubules
Mitochondria
Peroxisome
Reading Frames
Transferrin
Most recents protocols related to «Focal Adhesions»
Thoracolumbar spinal MRI examinations were performed at the Department of Radiology, Carlanderska Hospital using a 1.5 T scanner (Signa, GE Healthcare, Chicago, IL, USA). The MRI protocol included sagittal T1-and T2-weighted sequences (Th1-S1). In the thoracic spine, a field of view of 360 × 360mm2 and slice thickness of 3 mm was used. In the lumbar spine a field of view of 320 × 320mm2 and slice thickness of 3.5 mm was utilized.
The MRI images were classified by a senior radiologist (> 15 years of experience) according to a predetermined standardized protocol. Disc degeneration was classified according to the Pfirrmann classification [23 (link)]. In the thoracic spine, no distinction between Pfirrmann grade 1 and grade 2 was made since the resolution of the images was not considered adequate for reliable differentiation between these grades. Vertebral and endplate changes were classified according to the Modic classification [24 (link)] and a modified Endplate defect score, adapted to our MRI protocol. The Endplate defect score [25 (link)] was modified where Type I-III (representing no degeneration) were pooled (Table1 ). Schmorl’s nodes were classified as present or not present and defined as a vertebral endplate irregularity associated with intraspongious disc herniation, irrespective of the size, at either the cranial or caudal endplate, or at both endplates relative to the lumbar disc level. Spondylolisthesis was assessed as either present or not [26 , 27 (link)]. Similarly, vertebral apophyseal injury, defined as any irregularity or signal changes in the apophyseal region, was categorized as either present or not.
The MRI images were classified by a senior radiologist (> 15 years of experience) according to a predetermined standardized protocol. Disc degeneration was classified according to the Pfirrmann classification [23 (link)]. In the thoracic spine, no distinction between Pfirrmann grade 1 and grade 2 was made since the resolution of the images was not considered adequate for reliable differentiation between these grades. Vertebral and endplate changes were classified according to the Modic classification [24 (link)] and a modified Endplate defect score, adapted to our MRI protocol. The Endplate defect score [25 (link)] was modified where Type I-III (representing no degeneration) were pooled (Table
Modified endplate score, based on the original endplate defect score [25 (link)]
| Modified endplate defect score | Original endplate defect score |
|---|---|
| 1 | Type I—Normal endplate with no interruption |
| Type II—Thinning of the endplate, no obvious break | |
| Type III—Focal endplate defect with established disc marrow contact but with maintained endplate contour | |
| 2 | Type IV—Endplate defects < 25% of the endplate area |
| 3 | Type V—Endplate defects up to 50% of the endplate area |
| 4 | Type VI—Extensive damaged endplates up to total destruction |
Full text: Click here
Back Pain
Cranium
Focal Adhesions
Intervertebral Disc Degeneration
Intervertebral Disk Displacement
Lumbar Region
Marrow
Patients
Physical Examination
Radiologist
Spinal Injuries
Spondylolisthesis
Vertebra
Vertebrae, Lumbar
Vertebral Column
X-Rays, Diagnostic
This cross-sectional study was conducted between August 15 and 26, 2018, at the time of Hajj (1439 Hijri), and included all healthcare workers in the ICU departments of King Abdullah Medical City and Al Noor Specialist Hospital, Makkah, Saudi Arabia.
The participants included physicians, nurses, pharmacists, and respiratory therapists of medical, surgical, coronary care, and neuro-critical care units.
The study was conducted after obtaining the ethical approval from the Institutional Review Board of King Abdullah Medical City.
The participants included physicians, nurses, pharmacists, and respiratory therapists of medical, surgical, coronary care, and neuro-critical care units.
The study was conducted after obtaining the ethical approval from the Institutional Review Board of King Abdullah Medical City.
Ethics Committees, Research
Focal Adhesions
Health Personnel
Heart
Nurses
Physicians
Respiratory Rate
MEFs expressing full-length WT talin1 and full-length talin1-17b variant expressing cells were seeded on glass-bottom dishes for 8 h. Hoechst (1 µg/ml) was added for 1 h before imaging. Live-cell imaging was performed at 20X on Leica SP8 live-imaging system. Images were acquired every 5 min for 4 h. The movement of the cell was tracked in each frame in ImageJ using the Track-mate plugin and the velocity of each cell was calculated.
FRAP was performed to assess the dynamics of focal adhesions containing WT talin1 or 17b splice variant. Live-cell imaging was performed at 63X on Leica SP8 live-imaging system. Before photobleaching, three pre-bleach images of GFP-talin were acquired, using a 488 nm laser set at 10% of the maximum power. Photobleaching of GFP was conducted for 10 s at 100% laser power. Fluorescence recovery images were acquired every 30 s for 9 min using a 488 nm laser set at 10% of the maximum power. The mean fluorescence intensity pre-bleach was set to 100%. Photobleaching due to continuous illumination during recording was corrected by normalizing the fluorescence intensity at the FA with total cell fluorescence intensity. Corrected recovery fluorescence intensities were normalized to the pre-bleach intensity. The intensity was considered 100 and 0% for pre-bleach and bleach points. The fractional recovery post-bleach was calculated by normalizing the corrected recovery fluorescence intensities at each time point to pre-bleach intensity.
FRAP was performed to assess the dynamics of focal adhesions containing WT talin1 or 17b splice variant. Live-cell imaging was performed at 63X on Leica SP8 live-imaging system. Before photobleaching, three pre-bleach images of GFP-talin were acquired, using a 488 nm laser set at 10% of the maximum power. Photobleaching of GFP was conducted for 10 s at 100% laser power. Fluorescence recovery images were acquired every 30 s for 9 min using a 488 nm laser set at 10% of the maximum power. The mean fluorescence intensity pre-bleach was set to 100%. Photobleaching due to continuous illumination during recording was corrected by normalizing the fluorescence intensity at the FA with total cell fluorescence intensity. Corrected recovery fluorescence intensities were normalized to the pre-bleach intensity. The intensity was considered 100 and 0% for pre-bleach and bleach points. The fractional recovery post-bleach was calculated by normalizing the corrected recovery fluorescence intensities at each time point to pre-bleach intensity.
Full text: Click here
Cells
Fluorescence
Focal Adhesions
Hyperostosis, Diffuse Idiopathic Skeletal
Light
Motility, Cell
Reading Frames
Talin
Full-length WT talin-1 plasmid containing internal GFP and RFP (Kumar et al., 2016 (link)) was used in this study. Nucleotides corresponding to the 17-residue region of the 17b splice variant were cloned into WT talin-1 by Gibson assembly. Briefly, the WT talin-1 plasmid was digested with NotI and XhoI. Primers containing overhangs of nucleotides corresponding to the 17-residue region of the splice variant were used to amplify R2. Two fragments containing nucleotides between the NotI site to R1 domain and beyond R2 domain to XhoI site were also amplified by PCR. All fragments were incubated with Gibson assembly mix (New England Biolabs) as per manufacturer’s instructions.
Talin-2 was knocked down in Tln1−/− cells (Priddle et al., 1998 (link)) by transient transfection of Tln2 siRNA (ONTARGET-plus Smartpool siRNA, Catalog ID:L-065877-00-0005, Horizon Discovery) and scrambled siRNA (AM4636; Ambion), using Lipofectamine RNAimax, as described previously (Kumar et al., 2016 (link)). Knockdown was confirmed by immunoblotting for talin-2 using mouse anti-talin-2 antibody (AC14-0126; Abcore). These cells were transfected with full-length WT talin-1 and full-length talin-1-17b variant, using Lipofectamine 2000 (Thermo Fisher Scientific) for 24 h and trypsinized for all experiments at this time point. For early adhesion analysis, Tln1−/−transfected cells were held in suspension for 30 min and plated on fibronectin-coated glass bottom dishes for 15 min, 30 min, 1 h, and 4 h. Cells were fixed in 4% paraformaldehyde and counterstained with Alexa 405 phalloidin (Thermo Fisher Scientific) and anti-vinculin antibody (V9131; Sigma-Aldrich). Cells were imaged using a 63× objective on a Leica SP8 confocal microscope. ImageJ was used to assess cell area, focal adhesion area, focal adhesion number and mean fluorescence intensities of vinculin and talin within each adhesion. To assess substrate stiffness–dependent cell spreading, cells were trypsinized and plated on either fibronectin-coated (10 µg/ml) glass-bottom dishes or polyacrylamide gels of varying stiffness. At 6 h, cells were fixed in 4% paraformaldehyde and counterstained with Alexa 647 phalloidin (Thermo Fisher Scientific). The cell area was calculated using ImageJ.
For tensin-1 localization, cells were plated for 48 h and counter-stained with tensin-1 (SAB4200283; Sigma-Aldrich). Talin adhesions that were within 7 µm from centroid of the cell were considered central adhesions and tensin intensity was quantified within these adhesions. Mean of tensin intensity within central adhesions was divided by mean of tensin intensity of peripheral adhesions in each cell and the ratio was plotted. β-catenin staining was also performed on densely seeded cells using anti-β-catenin antibody (9562; CST).
Talin-2 was knocked down in Tln1−/− cells (Priddle et al., 1998 (link)) by transient transfection of Tln2 siRNA (ONTARGET-plus Smartpool siRNA, Catalog ID:L-065877-00-0005, Horizon Discovery) and scrambled siRNA (AM4636; Ambion), using Lipofectamine RNAimax, as described previously (Kumar et al., 2016 (link)). Knockdown was confirmed by immunoblotting for talin-2 using mouse anti-talin-2 antibody (AC14-0126; Abcore). These cells were transfected with full-length WT talin-1 and full-length talin-1-17b variant, using Lipofectamine 2000 (Thermo Fisher Scientific) for 24 h and trypsinized for all experiments at this time point. For early adhesion analysis, Tln1−/−transfected cells were held in suspension for 30 min and plated on fibronectin-coated glass bottom dishes for 15 min, 30 min, 1 h, and 4 h. Cells were fixed in 4% paraformaldehyde and counterstained with Alexa 405 phalloidin (Thermo Fisher Scientific) and anti-vinculin antibody (V9131; Sigma-Aldrich). Cells were imaged using a 63× objective on a Leica SP8 confocal microscope. ImageJ was used to assess cell area, focal adhesion area, focal adhesion number and mean fluorescence intensities of vinculin and talin within each adhesion. To assess substrate stiffness–dependent cell spreading, cells were trypsinized and plated on either fibronectin-coated (10 µg/ml) glass-bottom dishes or polyacrylamide gels of varying stiffness. At 6 h, cells were fixed in 4% paraformaldehyde and counterstained with Alexa 647 phalloidin (Thermo Fisher Scientific). The cell area was calculated using ImageJ.
For tensin-1 localization, cells were plated for 48 h and counter-stained with tensin-1 (SAB4200283; Sigma-Aldrich). Talin adhesions that were within 7 µm from centroid of the cell were considered central adhesions and tensin intensity was quantified within these adhesions. Mean of tensin intensity within central adhesions was divided by mean of tensin intensity of peripheral adhesions in each cell and the ratio was plotted. β-catenin staining was also performed on densely seeded cells using anti-β-catenin antibody (9562; CST).
Full text: Click here
Antibodies, Anti-Idiotypic
ARID1A protein, human
Cells
CTNNB1 protein, human
Fluorescence
FN1 protein, human
Focal Adhesions
Hyperostosis, Diffuse Idiopathic Skeletal
Lipofectamine
lipofectamine 2000
Microscopy, Confocal
Mus
Nucleotides
Oligonucleotide Primers
paraform
Phalloidine
Plasmids
polyacrylamide gels
RNA, Small Interfering
Talin
Tensin
Tissue Adhesions
Transfection
Transients
Vinculin
Protocol full text hidden due to copyright restrictions
Open the protocol to access the free full text link
Cell Adhesion
Cells
ethidium homodimer
Fibroblasts
Fibrosis
fluorexon
Focal Adhesions
Hydrogels
Malignant Neoplasms
Phenotype
Transforming Growth Factor beta
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DAPI is a fluorescent dye that binds strongly to adenine-thymine (A-T) rich regions in DNA. It is commonly used as a nuclear counterstain in fluorescence microscopy to visualize and locate cell nuclei.
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Rhodamine phalloidin is a fluorescent dye used for staining and visualizing actin filaments in cells. It binds specifically to actin and can be used to label the cytoskeleton in fixed cells for microscopy analysis.
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The Olympus Confocal Microscope is an advanced imaging system that uses a focused laser beam to scan and capture high-resolution, three-dimensional images of microscopic samples. It provides optical sectioning capabilities, allowing for the visualization of specific layers within a specimen.
More about "Focal Adhesions"
Focal adhesions are specialized regions of the cell surface where the cytoskeleton connects to the extracellular matrix.
These structures, also known as cell-matrix junctions or cell-substratum adhesions, play a crucial role in cell adhesion, migration, and signaling.
Focal adhesions are comprised of a complex of proteins, including integrins, talin, vinculin, paxillin, and focal adhesion kinase (FAK).
Integrins act as transmembrane receptors, linking the extracellular matrix to the cytoskeleton.
Talin, vinculin, and paxillin are adapter proteins that facilitate this connection, while FAK is an important signaling molecule involved in regulating cell behavior.
Researchers often use various techniques to study focal adhesions, such as immunofluorescence staining with Rhodamine phalloidin to visualize the actin cytoskeleton, and DAPI to stain cell nuclei.
Detergents like Triton X-100 may be used to permeabilize cells, and bovine serum albumin (BSA) is commonly used as a blocking agent.
Antibodies like Ab32084 can be employed to detect specific focal adhesion proteins.
To analyze focal adhesion dynamics and quantify their properties, researchers may utilize software like MATLAB and imaging techniques such as confocal microscopy.
The PubCompare.ai platform can help optimize research protocols related to focal adhesions, enhancing reproducibility and accuracy by providing AI-driven comparisons of protocols from literature, preprints, and patents.
This innovative solution can improve the efficiency of focal adhesion research.
These structures, also known as cell-matrix junctions or cell-substratum adhesions, play a crucial role in cell adhesion, migration, and signaling.
Focal adhesions are comprised of a complex of proteins, including integrins, talin, vinculin, paxillin, and focal adhesion kinase (FAK).
Integrins act as transmembrane receptors, linking the extracellular matrix to the cytoskeleton.
Talin, vinculin, and paxillin are adapter proteins that facilitate this connection, while FAK is an important signaling molecule involved in regulating cell behavior.
Researchers often use various techniques to study focal adhesions, such as immunofluorescence staining with Rhodamine phalloidin to visualize the actin cytoskeleton, and DAPI to stain cell nuclei.
Detergents like Triton X-100 may be used to permeabilize cells, and bovine serum albumin (BSA) is commonly used as a blocking agent.
Antibodies like Ab32084 can be employed to detect specific focal adhesion proteins.
To analyze focal adhesion dynamics and quantify their properties, researchers may utilize software like MATLAB and imaging techniques such as confocal microscopy.
The PubCompare.ai platform can help optimize research protocols related to focal adhesions, enhancing reproducibility and accuracy by providing AI-driven comparisons of protocols from literature, preprints, and patents.
This innovative solution can improve the efficiency of focal adhesion research.