Elastic membrane heterogeneity of living cells revealed by stiff nanoscale membrane domains

Many approaches have been developed to characterize the heterogeneity of membranes in living cells. In this study, the elastic properties of specific membrane domains in living cells are characterized by atomic force microscopy. Our data reveal the existence of heterogeneous nanometric scale domains with specific biophysical properties. We focused on glycosylphosphatidylinositol (GPI)-anchored proteins, which play an important role in membrane trafficking and cell signaling under both physiological and pathological conditions and which are known to partition preferentially into cholesterol-rich microdomains. We demonstrate that these GPI-anchored proteins reside within domains that are stiffer than the surrounding membrane. In contrast, membrane domains containing the transferrin receptor, which does not associate with cholesterol-rich regions, manifest no such feature. The heightened stiffness of GPI domains is consistent with existing data relating to the specific condensation of lipids and the slow diffusion rates of lipids and proteins therein. Our quantitative data may forge the way to unveiling the links that exist between membrane stiffness, molecular diffusion, and signaling activation.

Structural basis and biophysical properties of synaptic plasticity studied by atomic force microscopy

Alexandre Yersin

In this thesis, we studied two systems important for synaptic plasticity, one presynaptic and another postsynaptic. The protein complex composed of VAMP 2, SNAP-25 and syntaxin 1 (SNARE complex) is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. In order to better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope (AFM) and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces, dissociation kinetics of the SNAREs and led us to propose a sequence of formation for the complex. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein nSec1 injected into the AFM chamber prevented the SNARE complex formation. Finally we measured the effect of tetanus toxin protease (TeTx) on the SNARE complex and its activity by on-line registration during TeTx injection. These experiments reveal that AFM is a valuable tool to investigate complicate interactions among a protein complex containing up to four components. Trafficking of glutamate receptors AMPA is closely related to postsynaptic induction of long-term potentiation. In a second part of this thesis, we used AFM to explore the distribution of AMPA receptors on-line at the surface of living cells in correlation with cellular viscoelastic properties. First, we showed that AFM permits to detect specifically AMPA receptors at the surface of living neurons and we proved that the specificity of detection is stable during time. Moreover, we were able to visualize variations in AMPA receptor density on surface in response to different stimulations. Finally, AFM also allowed us to study local viscoelastic properties of the cell and their modifications occurring during AMPA receptor trafficking. Our measurements reveal that AMPA receptors are situated at the cell surface on local stiffness maxima. We have noticed that an excitatory stimulation removes these stiffness maxima before producing internalization of receptors. Our results suggest also that AMPA receptors may be constituted of two subpopulations depending on the cell stiffness. A "hard" subpopulation may be preferentially internalized after an excitatory stimulation whereas a "soft" one seems to remain at the cell surface.

EPFL2004

Investigating molecular interactions of G protein coupled receptors by fluorescence techniques

Jean-Baptiste Perez

G Protein coupled receptors (GPCRs) constitute an abundant family of membrane proteins which play a central role in cellular signaling and are important targets for modern medicine. Despite intensive research, central questions of the molecular mechanism of GPCR mediated signal transduction remain unresolved. This thesis concerns α1b-adrenergic receptor (α1b-AR) as a prototypic GPCR. The question whether ARs form homo- or hetero-oligomers was investigated using fluorescence resonance energy transfer (FRET). An important finding was that both α1a and α1b-AR subtypes form homo-oligomers. Also hetero-oligomers have been observed between the α1b- and the α1a-AR subtypes, but not with less related receptors. Another interesting finding concerns α1-AR co-internalization which correlates with the ability of the receptor to hetero-oligomerize. Investigating particular processes in living cells is often complicated by the complex cellular environment. Here we have developed a rather simple approach to study transmembrane signaling by using supported cell membrane sheets. They were prepared by pressing a glass coverslip on the apical part of cultured cells, ripping off large regions of the native plasma membrane. For the α1b-AR we still could observe ligand binding to such sheets, indicating that GPCR functionality was at least partly conserved. Due to the absence of cytosolic autofluorescence of the cells, supported membrane sheets were also found to be suited for single-molecule microscopy. Because both leaflets of the supported membranes remained fluid, it was possible to follow the diffusion of certain membrane proteins. For example using fluorescence recovery after photobleaching (FRAP) and single-molecule microscopy, we investigated how G proteins diffused along the membrane and compartmentalized. The predominant role of lipid anchors in G protein localization was highlighted by measuring the diffusion of two Gαq proteins fused with citrine at two different positions and citrine based constructs designed to mimic the monomeric and the trimeric form of a G protein. Finally, first results were obtained showing the feasibility to form supported cell membrane sheets on microstructured devices consisting of a planar silicon support perforated with arrays of holes. This geometry provides a full accessibility to both intra- and extracellular sides of the bilayer. This concept could potentially be applied for the development of chip-based screening assays.

EPFL2005

Oxidative stress on biomaterials

Reactive Oxygen Species (ROS) is a family of oxygen-based active molecules, which are deeply involved in numerous crucial processes of cellular metabolism, where the redox equilibrium is very important. An imbalance between ROS and the cell ability to neutralize them with antioxidants results in an Oxidative Stress (OS). This leads to cell alterations and impaired physiological functions. OS is involved in a growing list of human pathologies, from brain disorder, like Alzheimer and Parkinson's disease, to various forms of cancer (skin melanoma) and eye pathologies (cataract, macular disease), as well as in the aging process. Sometimes, inducing OS selectively in diseased cells can also act as a powerful therapy. This is the basis of PhotoDynamic Therapy (PDT), a newly developed medical application to cure cancer. It is based on ROS generation through photo-sensitive molecules (also referred to as PhotoSensitizers, or PS) under specific illumination. The goal of this work was to develop an efficient way to tune and characterize OS and related damage to biological systems. For this purpose, we synthesized a water-soluble derivative of fullerene-based PS, since the pristine molecule is known to generate ROS under visible illumination at very high yield. The efficiency of our custom-made PS in generating ROS was characterized by Electron Spin Resonance (ESR). Parallel studies were performed on a porphyrin-based PS and on TiO2 nanoparticles. The effects of oxidative stress provoked via PS were investigated for selected proteins and for a large variety of cells. The extent of OS was finely tuned by adjusting the experimental conditions such as PS concentration in the buffer. The changes upon OS were studied by three modern experimental techniques: ESR, Synchrotron Infrared MicroSpectroscopy (SIRMS) and Atomic Force Microscopy (AFM). We have obtained a promising set of data on the consequences of OS detected by these local techniques. During the OS certain chemical bonds are interrupted and others are created which manifest in the change of the lattice vibrations detected for entire cells using SIRMS. These stresses affect, first of all, the constituents of the cells. For example, OS on a protein induces conformational changes as measured by ESR using spin labeled proteins. We have shown that larger organizations of proteins like cytoskeletal filaments must also change under OS, since the cell stiffness decreases considerably. AFM measurements on various cell types (neurons, fibroblast, bladder cell, glioblastoma…) and on healthy and cancerous cells have clearly shown the progressive decrease of the Young's modulus as OS was switched on.

EPFL2006