A mechanical approach to study the bending of the primary cilium in response to fluid flow

The primary cilium, a hair-like projection from the cellular membrane, is involved in fluid flow sensing. Bending the primary cilium is known to trigger several signaling pathways. In this study, the primary cilium is modeled as a thin beam undergoing large deflection due to fluid drag forces. For the first time, the bending response is analyzed with a model combining large angle rotations with the assumption of a linear drag force along the ciliary length. In addition, the initial curvature and the angle between the cell membrane and the cilium are integrated into the model. The model is applied on three dimensional confocal images of fluorescent cilia in living cells that are exposed to laminar shear flow. The coordinates of the cilia are computed by a simple and effective image processing algorithm. By fitting the equations of the model to the coordinates of the bent cilia, the flexural rigidity and the angle between the cilium and the cell membrane are estimated. The flexural rigidity of the primary cilium is approximately ${10}^{-23}$ Nm, but the values vary for different cilia, which suggests that the mechanical properties of the primary cilia are heterogeneous. Interestingly, the base of the cilium doesn't deflect under fluid flow which means that the primary cilium is firmly anchored. Using immunocytochemistry, the connections of the microtubule network to the base of the cilium could be resolved. The microtubules may provide the mechanical stability of the cilium. Incorporating a linear drag force and allowing a basal tilt is an important step towards a more realistic mechanical model of the primary cilium and towards more accurate values of its flexural rigidity. The model together with the imaging technique is a useful tool to study the mechanical behavior of the primary cilium and will eventually lead to new insight in the poorly understood mechanisms of mechanotransduction.

Cellular Responses to Bacterial Pore-Forming Toxins

Rodrigo Manuel Gonzalez

Pore-forming toxins (PFTs) represent the largest class of bacterial protein toxins and constitute major virulence factors produced by pathogenic bacteria during infection. Pore formation appears to be an ancient form of attack, which is also found in hydrozoans, plants and humans. Bacteria secrete PFTs in the medium as water-soluble monomeric proteins that diffuse and bind to target cells via specific receptors. After binding, monomers undergo multimerization and insertion into the plasma membrane, which leads to the formation of a hydrophilic transmembrane channel. Consequences of PFT pores are loss of membrane potential, disruption of ion gradients and changes in cytosolic ion composition. Cells have evolved different response to PFT injury through the activation of various signalling pathways. Two types of PFTs are used in this study – aerolysin produced by Aeromonas hydrophila and listeriolysin-O produced by Listeria monocytogenes – that form small and large pores, respectively. We show that changes in cellular ion composition are the initial event sensed by the cells. In particular, changes in potassium levels lead to the activation of different signalling pathways some of which promote cell survival. Depending on the extent and the duration of intoxication, these pathways can lead to the recovery of membrane integrity and contribute to restore ions homeostasis. Interestingly, this recovery process is independent on protein synthesis but requires the activation of the pre-existing mitogen-activated protein kinases (MAPK) p38 and ERK. In addition to these pathways, cells enter an energy saving state by inducing arrest in protein synthesis, activation of the autophagy recycling machinery and accumulation of cellular lipid droplets. Taken together, the results presented in this study raise the notion that upon membrane disruption, cells activate a large variety of signalling pathways. These pathways are similarly induced by small and large PFTs, whereas the specific involvement in the recovery process depends on the type of PFT. This suggests that the differences observed in recovery kinetics among the PFTs depend on the capacity of the cell the cope with different types of pores, more than on the type of pathway induced by intoxication.

EPFL2011

Activation and Spatial Organization of the Adenosine A2A Receptor in Supported Plasma Membrane Sheets

Sophie Roizard

G protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. The main mechanisms involving GPCR activation and signal transduction to the cell interior are understood but many of the processes which fine-tune GPCRs signalling need to be unraveled. The work performed in this thesis intended to develop new methods to study GPCRs in their native environment but in a simplified system compared to live cells. In this context, the tethering GPCR-containing membrane fragments on solid surfaces appeared to be interesting: it enabled studying the receptors in their real native environment with several high resolution imaging techniques and with the possibility to engineer and control the downstream intracellular signaling components. Two procedures have been established to produce plasma membrane supported on micrometer-sized porous beads or on microscope grids for fluorescence or electron microscopy studies. They have been applied in this thesis to study the Adenosine A2A receeptor (A2A) as a prototypical GPCR. The first procedure enabled the transfer of native membrane patches from live cells with an inside-out orientation onto lectin-coated beads. Using heterologously expressed A2ARs carrying a yellow fluorescent protein (A2AR-Citrine) we showed that the tethered membranes were simultaneously accessible from both sides and comprised fully functional receptors in terms of ligand and G protein binding. These beads were investigated by confocal (Chapter 2) and single molecule (Chapter 3) fluorescence microscopies to study agonist and antagonist binding to the A2AR and the assembly and disassembly of the ternary complexes agonist-A2AR-Gαβγ proteins. The interactions between the different signalling partners could be observed in real time using multicolor fluorescence microscopy. These beads were also used in collaboration with colleagues for screening ligand binding to other transmembrane receptors. The second procedure produced GPCRs-containing plasma membrane fragments ripped-off on electron microscope grids for investigation by transmission electron microscopy (TEM) (Chapter 4). This procedure was improved from an existing protocol in order to produce reproducibly and with a high yield large membrane sheets and to preserve the membrane-associated cytoskeleton during the ripping-off. A2AR-Citrine were specifically gold immuno-labelled on their extracellular terminus using the FLAG-tag or their intracellular side by targeting the protein Citrine. The high resolution of TEM showed that the gold immuno-labelled A2AR-Citrine clustered and compartmentalized on actin-based cytoskeletton. This approach provided the ability to sample multiple single cells on a single grid and presented excellent potential to probe and unravel the organization of protein signalling networks in direct association with the plasma membrane-associated cytoskeleton.

EPFL2013

Activation and Spatial Organization of the Adenosine A2A Receptor in Supported Plasma Membrane Sheets

Sophie Roizard

EPFL2013

Cellular Responses to Bacterial Pore-Forming Toxins

Rodrigo Manuel Gonzalez

EPFL2011