Nanosecond-scale kinetics of nematocyst discharge.
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Animals↗
Biomedical subjects
Martin Benoit
Publications and source records attributed to Martin Benoit.
Combined atomic force microscopy and optical microscopy measurements as a method to investigate particle uptake by cells.
We propose a combination of atomic force microscopy (AFM) and optical microscopy for the investigation of particle uptake by cells. Positively and negatively charged polymer microcapsules were chosen as model particles, because their interaction with cells had already been investigated in detail. AFM measurements allowed the recording of adhesion forces on a single-molecule level. Due to the micrometer size of the capsules, the number of ingested capsules could be counted by optical microscopy. The combination of both methods allowed combined measurement of the adhesion forces and the uptake rate for the same model particle. As a demonstration of this system, the correlation between the adhesion of positively or negatively charged polymer microcapsules onto cell surfaces and the uptake of these microcapsules by cells has been investigated for several cell lines. As is to be expected, we find a correlation between both processes, which is in agreement with adsorption-dependent uptake of the polymer microcapsules by cells.
Adhesiveness↗
Alpha4beta1-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the alpha4-cytoplasmic domain.
The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.
Cell Adhesion↗
Phase contrast and DIC illumination for AFM hybrids.
High-resolution optical microscopy is an essential pre-requisite for life science force microscopy, particularly for applications in cell biology and medicine. Identification and validation of cells is typically established with techniques like phase contrast microscopy or differential interference contrast microscopy. The option to select or monitor individual cells online with such light microscopy techniques while performing atomic force microscopy (AFM) measurements is therefore extremely beneficial. Here, we report two conceptually different strategies to implement these light microscopy techniques in a fully functional AFM head at the ultimate resolution of the Abbe diffraction limit.
Epithelial Cells↗
Double-chip protein arrays: force-based multiplex sandwich immunoassays with increased specificity.
Protein assays provide direct access to biologically and pharmacologically relevant information. To obtain a maximum of information from the very smallest amounts of complex biological samples, highly multiplexed protein assays are needed. However, at present, cross-reactions of binding reagents restrict the use of such assays to selected cases and severely limit the potential for up-scaling the technology. Here we describe a double-chip format, which can effectively overcome this specificity problem for sandwich immunoassays. This format consists of a capture array and a reference array with fluorescent labeled detection antibodies coupled to the reference array via DNA duplexes. This format allows for the local application of the labeled detection antibodies onto their corresponding specific spots on the capture array. Here we show that this double-chip format allows for the use of cross-reactive antibodies without generating false positive signals, and an assay for the parallel detection of seven different cytokines was set up. Even without further optimization, the dynamic range and the limit of detection for interleukin 8 were found to be comparable to those obtained with other types of multiplexed sandwich immunoassays.
Animals↗
Cell adhesion measured by force spectroscopy on living cells.
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Animals↗
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.
Animals↗