Kheya Sengupta

Quantitative Reflection Interference Contrast Microscopy (RICM) in Soft Matter and Cell Adhesion

Adhesion can be quantified by measuring the distance between the interacting surfaces. Reflection interference contrast microscopy (RICM), with its ability to measure inter-surface distances under water with nanometric precision and milliseconds time resolution, is ideally suited to studying the dynamics of adhesion in soft systems. Recent technical developments, which include innovative image analysis and the use of multi-coloured illumination, have led to renewed interest in this technique. Unambiguous quantitative measurements have been achieved for colloidal beads and model membranes, thus revealing new insights and applications. Quantification of data from cells shows exciting prospects. Herein, we review the basic principles and recent developments of RICM applied to studies of dynamical adhesion processes in soft matter and cell biology and provide practical hints to potential users.

Force-induced growth of adhesion domains is controlled by receptor mobility

In living cells, adhesion structures have the astonishing ability to grow and strengthen under force. Despite the rising evidence of the importance of this phenomenon, little is known about the underlying mechanism. Here, we show that force-induced adhesion-strengthening can occur purely because of the thermodynamic response to the elastic deformation of the membrane, even in the absence of the actively regulated cytoskeleton of the cell, which was hitherto deemed necessary. We impose pN-forces on two fluid membranes, locally pre-adhered by RGD-integrin binding. One of the binding partners is always mobile whereas the mobility of the other can be switched on or off. Immediate passive strengthening of adhesion structures occurs in both cases. When both binding partners are mobile, strengthening is aided by lateral movement of intact bonds as a transient response to force-induced membrane-deformation. By extending our microinterferometric technique to the suboptical regime, we show that the adhesion, as well as the resistance to force-induced de-adhesion, is greatly enhanced when both, rather than only one, of the binding partners are mobile. We formulate a theory that explains our observations by linking the macroscopic shape deformation with the microscopic formation of bonds, which further elucidates the importance of receptor mobility. We propose this fast passive response to be the first-recognition that triggers signaling events leading to mechanosensing in living cells.

Diffusion and intermembrane distance: case study of avidin and E-cadherin mediated adhesion.

We present a biomimetic model system for cell-cell adhesion consisting of a giant unilamellar vesicle (GUV) adhering via specific ligand-receptor interactions to a supported lipid bilayer (SLB). The modification of in-plane diffusion of tracer lipids and receptors in the SLB membrane due to adhesion to the GUV is reported. Adhesion was mediated by either biotin-neutravidin (an avidin analogue) or the extracellular domains of the cell adhesion molecule E-cadherin (Ecad). In the strong interaction (biotin-avidin) case, binding of soluble receptors to the SLB alone led to reduced diffusion of tracer lipids. From theoretical considerations, this could be attributed partially to introduction of obstacles and partially to viscous effects. Further specific binding of a GUV membrane caused additional slowing down of tracers (up to 15%) and immobilization of receptors, and led to accumulation of receptors in the adhesion zone until full coverage was achieved. The intermembrane distance was measured to be 7 nm from microinterferometry (RICM). We show that a crowding effect due to the accumulated receptors alone is not sufficient to account for the slowing downan additional friction from the membrane also plays a role. In the weak binding case (Ecad), the intermembrane distance was about 50 nm, corresponding to partial overlap of the Ecad domains. No significant change in diffusion of tracer lipids was observed upon either protein binding or subsequent vesicle binding. The former was probably due to very small effective size of the obstacles introduced into the bilayer by Ecad binding, whereas the latter was due to the fact that, with such high intermembrane distance, the resulting friction is negligible. We conclude that the effect of intermembrane adhesion on diffusion depends strongly on the choice of the receptors.

Probing Biomembrane Dynamics by Dual‐Wavelength Reflection Interference Contrast Microscopy

We present an improved analysis of reflection interference contrast microscopy (RICM) images, recorded to investigate model membrane systems that mimic cell adhesion. The model systems were giant unilamellar vesicles (GUV) adhering via specific ligand-receptor interactions to supported lipid bilayers (SLB) or to patterns of receptors. Conventional RICM and dual-wavelength RICM (DW-RICM) were applied to measure absolute optical distances between the biomembranes and planar substrates. We developed algorithms for a straightforward implementation of an automated, time-resolved reconstruction of the membrane conformations from RICM/DW-RICM images, taking into account all the interfaces in the system and blurring of the data due to camera noise. Finally, we demonstrate the validity and usefulness of this new approach by analyzing the topography and fluctuations of a bound membrane in the steady state and its dynamic adaptation to osmotic pressure changes. These measurements clearly show that macroscopic membrane flow through tightly adhered area is possible in our system.

Inter-membrane adhesion mediated by mobile linkers: Effect of receptor shortage

Giant unilamellar vesicles (GUVs) adhering to supported bilayers were used as a model system to mimic ligand–receptor mediated cell-cell adhesion. We present the effect of varying the concentration of receptors (neutravidin on the bilayer) and ligands (biotin on the vesicle) on GUV adhesion and the organization of receptors in the adhesion zone. At high concentrations of both ligands and receptors, the adhesion is strong, all the available membrane is adhered and receptors are accumulated under the adhered membrane up to the geometrical limit of close packing. At low concentrations of receptors (<0.5%), and an arbitrary concentration of ligands (≥0.1%), adhesion does not proceed to completion: the membrane is only partially bound and parts of it still fluctuate. The receptors tend to accumulate under the adhered membrane but the filling is partial. Receptors get jammed and form clusters with fractal like shapes along the rim of the adhered vesicle in such a way that the annular cluster prevents further filling of the adhesion disc. We characterize the filling in terms of a compaction factor and the final concentration. Interestingly, the closing of the ring of jammed clusters switches the interior of the adhesion disc from one thermodynamic ensemble to another. In the new ensemble the receptors sealed within the adhesion disc are mobile but their number is fixed. Under such conditions, the usually strong neutravidin/biotin bond is weak. The incomplete adhesion state can be attributed to a combination of the effects of diffusion, jamming and the competition of enthalpy and entropy on bond formation. The formation of jammed receptor clusters reported here represents a new mechanism that influences membrane adhesion.

Large-scale ordered plastic nanopillars for quantitative live-cell imaging.

Surfaces exhibiting ordered nanopillars have a wide range of potential biomedical applications based on the altered adhesivity of living cells on nanopatterned surfaces compared to planar ones. Examples include scaffolding for tissue engineering, designer bandages for wound dressing, and antifouling surfaces for implants. Although numerous experiments performed over the last decade have confirmed that cells respond to the chemistry (biochemical 2D imprint) and geometry (topographical 3D relief) of their surroundings at the nanoscale, the fundamental processes by which cells recognize nanostructures is a subject of on-going research. In this context, there is a need to ensure that the nanostructured surfaces have large-scale coverage and are compatible with quantitative optical microscopy (QOM), an important tool for studying living cells, especially the dynamics thereof. While biochemical patterning is not expected to pose a special challenge for QOM, topographical patterning may do so. Well-known techniques for topographical patterning are nanoimprint lithography (NIL, including thermal embossing and UV curing) and self-assembly based on colloidal beads or phase separation of polymers, all of which achieve large coverage. NIL is relatively resource intensive and usually depends on conventional techniques like electron-beam lithography for the initial stamp. Self-assembly, although increasingly refined, has limited flexibility for the choice of motif. Transparent substrates made using these

Measuring fast stochastic displacements of bio-membranes with dynamic optical displacement spectroscopy.

Stochastic displacements or fluctuations of biological membranes are increasingly recognized as an important aspect of many physiological processes, but hitherto their precise quantification in living cells was limited due to a lack of tools to accurately record them. Here we introduce a novel technique—dynamic optical displacement spectroscopy (DODS), to measure stochastic displacements of membranes with unprecedented combined spatiotemporal resolution of 20 nm and 10 μs. The technique was validated by measuring bending fluctuations of model membranes. DODS was then used to explore the fluctuations in human red blood cells, which showed an ATP-induced enhancement of non-Gaussian behaviour. Plasma membrane fluctuations of human macrophages were quantified to this accuracy for the first time. Stimulation with a cytokine enhanced non-Gaussian contributions to these fluctuations. Simplicity of implementation, and high accuracy make DODS a promising tool for comprehensive understanding of stochastic membrane processes.

Switching from Ultraweak to Strong Adhesion

Nature switches from weak to strong adhesion at the cellular level to spectacular effect – for example, for incredibly sensitive recognition and decisive action during immune response. [ 1 ] If realized in an artifi cial system, such a switching could one day be harnessed as a powerful tool to manipulate weakly interacting objects. The fi rst step towards realizing such a system involves understanding how to create and detect ultraweak adhesion and how to then switch-on a strong interaction. So far, in the context of model membranes, weak adhesion has been achieved only with a ligand-receptor of intrinsically low binding affi nity. [ 2 ] Whatever, the intrinsic strength of the bonds, so far they were usually found to be arranged in compact stable domains. [ 3 ] Here, we present experiments and simulations that indicate how to create and detect ultraweak adhesion in the context of fl uid two dimensional membranes interacting via specifi c ligand/receptor bonds. Thus, specifi c adhesion is mediated by transient domains consisting of sparsely distributed bonds. Amazingly, we demonstrate that the avidin/biotin pair – famous for forming the strongest receptor/ligand bond known in nature, mediates ultraweak adhesion under suitable circumstances. This choice of binders allows us to switch on strong binding once sensitive detection is achieved – without resorting to a second binding pair – something not possible with intrinsically weak binders. However, this goal necessitates an appropriate design strategy elaborated below. The in vitro system consists of two membranes: a solid supported lipid bilayer (SLB) and the freely fl uctuating membrane of a giant unillamelar vesicle (GUV). A GUV is a two

Inferring spatial organization of bonds within adhesion clusters by exploiting fluctuations of soft interfaces

Detecting the organization of bonds within adhesion domains connecting two interacting membranes is, at present, extremely challenging. Herein we present a technique, based on Reflection Interference Contrast Microscopy, which uses spontaneous thermal fluctuations of a soft interface as a tool to identify the organization of specific ligand-receptor bonds. The key is a time-resolved analysis of micro-interferometric data that systematically quantifies fluctuations and enables the detection of their suppression due to the formation of bonds, which, in turn, allows the identification of the bond organization without the use of fluorescent labelling. The identification of a new type of bond organization characterized by sparsely distributed bonds, as well as detection of pinning centres of nanometric size is presented.

Association rates of membrane-coupled cell adhesion molecules.

Thus far, understanding how the confined cellular environment affects the lifetime of bonds, as well as the extraction of complexation rates, has been a major challenge in studies of cell adhesion. Based on a theoretical description of the growth curves of adhesion domains, we present a new (to our knowledge) method to measure the association rate k(on) of ligand-receptor pairs incorporated into lipid membranes. As a proof of principle, we apply this method to several systems. We find that the k(on) for the interaction of biotin with neutravidin is larger than that for integrin binding to RGD or sialyl Lewis(x) to E-selectin. Furthermore, we find k(on) to be enhanced by membrane fluctuations that increase the probability for encounters between the binders. The opposite effect on k(on) could be attributed to the presence of repulsive polymers that mimic the glycocalyx, which points to two potential mechanisms for controlling the speed of protein complexation during the cell recognition process.