Project description:This review reports on the use of the atomic force microscopy (AFM) in the investigation of cell scaffolds in recent years. It is shown how the technique is able to deliver information about the scaffold surface properties (e.g., topography), as well as about its mechanical behavior (Young's modulus, viscosity, and adhesion). In addition, this short review also points out the utilization of the atomic force microscope technique beyond its usual employment in order to investigate another type of basic questions related to materials physics, chemistry, and biology. The final section discusses in detail the novel uses that those alternative measuring modes can bring to this field in the future.
Project description:In atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS), it is assumed that the pulling angle is negligible and that the force applied to the molecule is equivalent to the force measured by the instrument. Recent studies, however, have indicated that the pulling geometry errors can drastically alter the measured force-extension relationship of molecules. Here we describe a software-based alignment method that repositions the cantilever such that it is located directly above the molecule's substrate attachment site. By aligning the applied force with the measurement axis, the molecule is no longer undergoing combined loading, and the full force can be measured by the cantilever. Simulations and experimental results verify the ability of the alignment program to minimize pulling geometry errors in AFM-SMFS studies.
Project description:G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors. Despite considerable insights into their pharmacology, the GPCR architecture at the cell surface still remains largely unexplored. Herein, we present the specific unfolding of different GPCRs at the surface of living mammalian cells by atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS). Mathematical analysis of the GPCR unfolding distances at resting state revealed the presence of different receptor populations relying on distinct oligomeric states which are receptor-specific and receptor expression-dependent. Moreover, we show that the oligomer size dictates the receptor spatial organization with nanoclusters of high-order oligomers while lower-order complexes spread over the whole cell surface. Finally, the receptor activity reshapes both the oligomeric populations and their spatial arrangement. These results add an additional level of complexity to the GPCR pharmacology until now considered to arise from a single receptor population at the cell surface.
Project description:Misfolding and aggregation of amyloid β-40 (Aβ-40) peptide play key roles in the development of Alzheimer's disease (AD). However, very little is known about the molecular mechanisms underlying these molecular processes. We developed a novel experimental approach that can directly probe aggregation-prone states of proteins and their interactions. In this approach, the proteins are anchored to the surface of the atomic force microscopy substrate (mica) and the probe, and the interaction between anchored molecules is measured in the approach-retraction cycles. We used dynamic force spectroscopy (DFS) to measure the stability of transiently formed dimers. One of the major findings from DFS analysis of α-synuclein (α-Syn) is that dimeric complexes formed by misfolded α-Syn protein are very stable and dissociate over a range of seconds. This differs markedly from the dynamics of monomers, which occurs on a microsecond to nanosecond time scale. Here we applied the same approach to quantitatively characterize interactions of Aβ-40 peptides over a broad range of pH values. These studies showed that misfolded dimers are characterized by lifetimes in the range of seconds. This value depends on pH and varies between 2.7 s for pH 2.7 and 0.1 s for pH 7, indicating that the aggregation properties of Aβ-40 are modulated by the environmental conditions. The analysis of the contour lengths revealed the existence of various pathways for dimer dissociation, suggesting that dimers with different conformations are formed. These structural variations result in different aggregation pathways, leading to different types of oligomers and higher-order aggregates, including fibrils.
Project description:Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. Here we describe these techniques and illustrate them with examples highlighting current capabilities and limitations.
Project description:Cell membranes are typically very complex, consisting of a multitude of different lipids and proteins. Supported lipid bilayers are widely used as model systems to study biological membranes. Atomic force microscopy and force spectroscopy techniques are nanoscale methods that are successfully used to study supported lipid bilayers. These methods, especially force spectroscopy, require the reliable preparation of supported lipid bilayers with extended coverage. The unreliability and a lack of a complete understanding of the vesicle fusion process though have held back progress in this promising field. We document here robust protocols for the formation of fluid phase DOPC and gel phase DPPC bilayers on mica. Insights into the most crucial experimental parameters and a comparison between DOPC and DPPC preparation are presented. Finally, we demonstrate force spectroscopy measurements on DOPC surfaces and measure rupture forces and bilayer depths that agree well with X-ray diffraction data. We also believe our approach to decomposing the force-distance curves into depth sub-components provides a more reliable method for characterising the depth of fluid phase lipid bilayers, particularly in comparison with typical image analysis approaches.
Project description:A new experimental platform for probing nanoscale molecular changes in living bacteria using atomic force microscopy-infrared (AFM-IR) spectroscopy is demonstrated. This near-field technique is eminently suited to the study of single bacterial cells. Here, we report its application to monitor dynamical changes occurring in the cell wall during cell division in Staphylococcus aureus using AFM to demonstrate the division of the cell and AFM-IR to record spectra showing the thickening of the septum. This work was followed by an investigation into single cells, with particular emphasis on cell-wall signatures, in several bacterial species. Specifically, mainly cell wall components from S. aureus and Escherichia coli containing complex carbohydrate and phosphodiester groups, including peptidoglycans and teichoic acid, could be identified and mapped at nanometre spatial resolution. Principal component analysis of AFM-IR spectra of six living bacterial species enabled the discrimination of Gram-positive from Gram-negative bacteria based on spectral bands originating mainly from the cell wall components. The ability to monitor in vivo molecular changes during cellular processes in bacteria at the nanoscale opens a new platform to study environmental influences and other factors that affect bacterial chemistry.
Project description:Atomic force microscopy (AFM) techniques have provided and continue to provide increasingly important insights into surface morphology, mechanics, and other critical material characteristics at the nanoscale. One attractive implementation involves extracting meaningful material properties, which demands physically accurate models specifically designed for AFM experimentation and simulation. The AFM community has pursued the precise quantification and extraction of rate-dependent material properties, in particular, for a significant period of time, attempting to describe the standard viscoelastic response of materials. AFM static force spectroscopy (SFS) is one approach commonly used in pursuit of this goal. It is capable of acquiring rich temporal insight into the behavior of a sample. During AFM-SFS experiments the cantilever base approaches samples with a nearly constant velocity, which is manipulated to investigate different timescales of the mechanical response. This manuscript seeks to build upon our previous work and presents an approach to extracting useful linear viscoelastic information from AFM-SFS experiments. In addition, the basis for selecting and restricting the model parameters for fitting is discussed from the perspective of applying this technique on a practical level. This work begins with a guided discussion that develops a fit function from fundamental laws, continues with conditioning a raw SFS experimental dataset, and concludes with the fit and prediction of viscoelastic response parameters such as storage modulus, loss modulus, loss angle, and compliance. These steps constitute a complete guide to leveraging AFM-SFS data to estimate key material parameters, with a series of detailed insights into both the methodology and supporting analytical choices.
Project description:The adhesive and mechanical properties of a modular fusion protein consisting of two different types of binding units linked together via a flexible resilin-like-polypeptide domain are quantified. The adhesive domains have been constructed from fungal cellulose-binding modules (CBMs) and an amphiphilic hydrophobin HFBI. This study is carried out by single-molecule force spectroscopy, which enables stretching of single molecules. The fusion proteins are designed to self-assemble on the cellulose surface, leading into the submonolayer of proteins having the HFBI pointing away from the surface. A hydrophobic atomic force microscopy (AFM) tip can be employed for contacting and lifting the single fusion protein from the HFBI-functionalized terminus by the hydrophobic interaction between the tip surface and the hydrophobic patch of the HFBI. The work of rupture, contour length at rupture and the adhesion forces of the amphiphilic end domains are evaluated under aqueous environment at different pHs.
Project description:Postovulatory aging is a process occurring in the mature (MII) oocyte leading the unfertilized ones to apoptosis. The optimal time window of fertility for different mammalian species after oocytes maturation depends on its timeliness: the higher the time elapsed from the accomplishment of the MII stage, the lower are the chances of fertilization and of development of a viable embryo. In the in vitro fertilization, the selection of competent oocytes for intracytoplasmic sperm injection (ICSI) is mostly made by the visual inspection of the MII oocyte morphology, which does not allow to determine the oocyte postovulatory age. On the other hand, more specific tests usually involve some kind of staining, thus compromising the viability of the oocyte for reproductive purposes. Hence, the need of a noninvasive analysis of oocyte aging to improve the success rate of in vitro fertilization procedures. Here, we exploit atomic force microscopy to examine the evolution of the mechanical properties of mouse oocytes during in vitro postovulatory aging. Three hours before the occurrence of any visual morphological feature related to degradation, we observe a sudden change of the mechanical parameters: the elastic modulus doubles its initial value, while the viscosity decreases significantly. These mechanical variations are temporally correlated with the release of the cortical granules, investigated by fluorescence microscopy. Interestingly, the oocyte mechanics correlates as well with the yield of embryo formation, evaluated up to the blastocyst formation stage. These results demonstrate that minimally invasive mechanical measurements are very sensitive to the aging of the oocyte and can be used as a label-free method to detect the age of the postovulatory oocytes.