342 research outputs found
Measuring cell adhesion forces with the atomic force microscope at the molecular level
In the past 25 years many techniques have been developed to characterize cell adhesion and to quantify adhesion forces. Atomic force microscopy (AFM) has been used to measure forces in the pico-newton range, an experimental technique known as force spectroscopy. We modified such an AFM to measure adhesion forces between live cells or between cells and surfaces. This strategy required functionalizing the surface of the sensors for immobilizing the cell. We used Dictyostelium discoideum cells which respond to starvation by surface expression of the adhesion molecule csA and consequent aggregation to measure the adhesion force of a single csA-csA bond. Relevant experimental parameters include the duration of contact between the interacting surfaces, the force against which this contact is maintained, the number and specificity of interacting adhesion molecules and the constituents of the medium in which the interaction occurs. This technology also permits the measurement of the viscoelastic properties of single cells or cell layers. Copyright (C) 2002 S, Karger AG, Basel
Velocity-Dependent Forces in Atomic Force Microscopy Imaging of Lipid Films
We have imaged adsorbed fluid lipid bilayers by atomic force microscopy. The patches were formed by
rupture of phospholipid vesicles onto magnesium fluoride. We show that the membrane patches are fluid
but can be stably imaged at scan rates higher than 6 p d s . At lower scan rates the tip penetrates through
the layer. The penetrating tip does not destroy the fluid patches, and the previous image can be restored
after increasing the scanning velocity. The dynamic forces that possibly explain the effect are discussed
Local motion and conformational changes in the cuticle of Clivia miniata Regel: A microfluorescence and spin-label study.
Atomic force microscopy-based mechanobiology
Mechanobiology emerges at the crossroads of medicine, biology, biophysics and engineering and describes how the responses of proteins, cells, tissues and organs to mechanical cues contribute to development, differentiation, physiology and disease. The grand challenge in mechanobiology is to quantify how biological systems sense, transduce, respond and apply mechanical signals. Over the past three decades, atomic force microscopy (AFM) has emerged as a key platform enabling the simultaneous morphological and mechanical characterization of living biological systems. In this Review, we survey the basic principles, advantages and limitations of the most common AFM modalities used to map the dynamic mechanical properties of complex biological samples to their morphology. We discuss how mechanical properties can be directly linked to function, which has remained a poorly addressed issue. We outline the potential of combining AFM with complementary techniques, including optical microscopy and spectroscopy of mechanosensitive fluorescent constructs, super-resolution microscopy, the patch clamp technique and the use of microstructured and fluidic devices to characterize the 3D distribution of mechanical responses within biological systems and to track their morphology and functional state.Peer ReviewedPostprint (published version
Imaging Single-Stranded DNA, Antigen-Antibody Reaction and Polymerized Langmuir-Blodgett Films with an Atomic Force Microscope
The combination of an (AFM) atomic force microscope together with microfabricated cantilevers that have integrated tips opens many possibilities for imaging systems of great importance in biology. We have imaged single-stranded 25mer DNA that was adsorbed on treated mica or that was covalently bound with a crosslinker to a polymerized Langmuir-Blodgett (LB) film, the top monolayer of a bilayer system. At low magnification the AFM shows cracks between solid domains, like in an image taken with a fluorescence microscope. At higher magnification, however, the AFM reveals much finer cracks and at still higher magnification it reveals rows of individual molecules in the polymerized LB film with a spacing of 0.45 nm. We have also imaged a LB film consisting of lipids in which 4% of the lipids had hapten molecules chemically bound to the lipid headgroups. Specific antibodies can then bind to these hapten molecules and be imaged with the AFM. This points to the possibility of using the AFM to monitor selective antibody binding
Angiotensin 1-7 Rescues Cognitive Decline and Neuronal Loss Following Traumatic Brain Injury in Mice
Purpose of study: Traumatic brain injury (TBI) is a leading cause of death and disability in the U.S., accounting for approximately 30% of all injury deaths and 3 million TBI-related emergency visits yearly. There is limited research in the area of mitigating the post-inflammatory effects of non-fatal traumatic brain injury. Angiotensin 1-7, an endogenous peptide that acts on the MAS receptor, has recently shown to be anti-inflammatory, anti-oxidative, and vasodilatory unlike its relative, angiotensin II. We asked the question of whether Ang 1-7 modulates neuroinflammation and improves cognitive function in mice following traumatic brain injury.
Methods: A controlled cortical impactor with a retractable piston was used to model a mild traumatic brain injury (mTBI). Mice either received Ang 1-7 (1 mg/kg, n=12) or normal saline (0.9%, n=12) 2 hours post-TBI and 30 minutes prior to novel objection recognition (NOR) testing on days 1, 3, 7, 14 post-TBI with tissue harvesting for hematoxylin and eosin (H&E) staining.
Results: Between-group studies showed that Ang 1-7 treated mice showed significantly higher NOR ratios compared to that of the control group. Additionally, statistically significant higher neuronal count in the ipsilateral hippocampal and cortical tissues days 1, 3, 7, and 14 post-TBI was observed in the Ang-1-7 group.
Conclusion: Together, these results demonstrate that Ang 1-7 significantly improves cognitive function and rescues further cell loss against secondary intrinsic injury following extrinsic mTBI and suggest that it may be a novel therapy to the effects of mild traumatic brain injury
Mechanisms of Nanonewton Mechanostability in a Protein Complex Revealed by Molecular Dynamics Simulations and Single-Molecule Force Spectroscopy
Can molecular dynamics simulations predict the mechanical behavior of protein complexes? Can simulations decipher the role of protein domains of unknown function in large macromolecular complexes? Here, we employ a wide-sampling computational approach to demonstrate that molecular dynamics simulations, when carefully performed and combined with single-molecule atomic force spectroscopy experiments, can predict and explain the behavior of highly mechanostable protein complexes. As a test case, we studied a previously unreported homologue from; Ruminococcus flavefaciens; called X-module-Dockerin (XDoc) bound to its partner Cohesin (Coh). By performing dozens of short simulation replicas near the rupture event, and analyzing dynamic network fluctuations, we were able to generate large simulation statistics and directly compare them with experiments to uncover the mechanisms involved in mechanical stabilization. Our single-molecule force spectroscopy experiments show that the XDoc-Coh homologue complex withstands forces up to 1 nN at loading rates of 10; 5; pN/s. Our simulation results reveal that this remarkable mechanical stability is achieved by a protein architecture that directs molecular deformation along paths that run perpendicular to the pulling axis. The X-module was found to play a crucial role in shielding the adjacent protein complex from mechanical rupture. These mechanisms of protein mechanical stabilization have potential applications in biotechnology for the development of systems exhibiting shear enhanced adhesion or tunable mechanics
Recognition of "mirror-image" DNA by small molecules
May the force be with you: “Mirror-image” hairpin polyamides distinguish the L enantiomer of DNA (L-DNA) in the presence of natural DNA (D-DNA). This specificity is investigated by a molecular force balance at a single-molecule level. The “DNA balance” allows the measurement of rupture forces of match/mismatch diastereomeric complexes in a single experiment
Recognition of "mirror-image" DNA by small molecules
May the force be with you: “Mirror-image” hairpin polyamides distinguish the L enantiomer of DNA (L-DNA) in the presence of natural DNA (D-DNA). This specificity is investigated by a molecular force balance at a single-molecule level. The “DNA balance” allows the measurement of rupture forces of match/mismatch diastereomeric complexes in a single experiment
Restoration of Visual Function by Expression of a Light-Gated Mammalian Ion Channel in Retinal Ganglion Cells or ON-Bipolar Cells
Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0460). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0460 was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0460 was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0460 in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0460 in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0460 was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation
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