FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments

Fluorescence recovery after photobleaching (FRAP) is a standard method used to study the dynamics of lipids and proteins in artificial and cellular membrane systems. The advent of confocal microscopy two decades ago has made quantitative FRAP easily available to most laboratories. Usually, a single bleaching pattern/area is used and the corresponding recovery time is assumed to directly provide a diffusion coefficient, although this is only true in the case of unrestricted Brownian motion. Here, we propose some general guidelines to perform FRAP experiments under a confocal microscope with different bleaching patterns and area, allowing the experimentalist to establish whether the molecules undergo Brownian motion (free diffusion) or whether they have restricted or directed movements. Using in silico simulations of FRAP measurements, we further indicate the data acquisition criteria that have to be verified in order to obtain accurate values for the diffusion coefficient and to be able to distinguish between different diffusive species. Using this approach, we compare the behavior of lipids in three different membrane platforms (supported lipid bilayers, giant liposomes and sponge phases), and we demonstrate that FRAP measurements are consistent with results obtained using other techniques such as Fluorescence Correlation Spectroscopy (FCS) or Single Particle Tracking (SPT). Finally, we apply this method to show that the presence of the synaptic protein Munc18-1 inhibits the interaction between the synaptic vesicle SNARE protein, VAMP2, and its partner from the plasma membrane, Syn1A

Membrane Recruitment of Scaffold Proteins Drives Specific Signaling

Cells must give the right response to each stimulus they receive. Scaffolding, a signaling process mediated by scaffold proteins, participates in the decoding of the cues by specifically directing signal transduction. The aim of this paper is to describe the molecular mechanisms of scaffolding, i.e. the principles by which scaffold proteins drive a specific response of the cell. Since similar scaffold proteins are found in many species, they evolved according to the purpose of each organism. This means they require adaptability. In the usual description of the mechanisms of scaffolding, scaffold proteins are considered as reactors where molecules involved in a cascade of reactions are simultaneously bound with the right orientation to meet and interact. This description is not realistic: (i) it is not verified by experiments and (ii) timing and orientation constraints make it complex which seems to contradict the required adaptability. A scaffold protein, Ste5, is used in the MAPK pathway of Saccharomyces Cerevisiae for the cell to provide a specific response to stimuli. The massive amount of data available for this pathway makes it ideal to investigate the actual mechanisms of scaffolding. Here, a complete treatment of the chemical reactions allows the computation of the distributions of all the proteins involved in the MAPK pathway when the cell receives various cues. These distributions are compared to several experimental results. It turns out that the molecular mechanisms of scaffolding are much simpler and more adaptable than previously thought in the reactor model. Scaffold proteins bind only one molecule at a time. Then, their membrane recruitment automatically drives specific, amplified and localized signal transductions. The mechanisms presented here, which explain how the membrane recruitment of a protein can produce a drastic change in the activity of cells, are generic and may be commonly used in many biological processes

The elongated cell.

<p>When a cell receives a signal on a small fraction of its surface, we will consider it as an object that is stimulated on a flat area, S, (where the signal is received). The cell is self similar along the x axis. The volume of the cell is equal to S.l.The shape of S is not important, the only requirement to obtain a cell where the system can be solved in one dimension is that the shape of the cell is self similar by translation along an axis normal to S (the simplest shapes are a cylinder or a parallelepiped shape).</p

Signal localization.

<p>The figure represents the ratio of the concentration of active kinase normalized by the concentration close to the surface that receives the signal as a function of the distance from the membrane. When Ste5 is recruited to the membrane, the polarization of the cell is significantly increased. It is assumed that the effective affinities between the kinases and Ste5 are 1 µM for the three kinases Ste11, Ste7 and Fus3. The localization is not as relevant in the case of a spherical cell since the only polarization that can be achieved is between the vicinity of the membrane and the center of the cell.</p

MAPK pathway in <i>Saccharomyces Cerevisiae.</i>

<p>The MAPK pathway in <i>Saccharomyces cerevisiae</i> consists in a cascade of four successive kinases: Ste20, Ste11, Ste7 and a MAPK. In the results presented in the text, two MAPK are studied, Fus3 and Kss1. Activation of Kss1 has been recognized to be predominant during filamentation while Fus3 is relevant for the mating response. During mating, a scaffold protein, Ste5 is recruited to the membrane and bind to Ste11, Ste7 and Fus3. When activated, the Rho family protein cdc42, a small GTPase localized to the plasma membrane, binds and activates Ste20 through a CRIB domain. Ste20 remains at the plasma membrane. This point is the start of the cascade of reactions where a kinase activates the next kinase by phosphorylation. Ste20 activates the MAPK kinase kinase Ste11, which in turn activates a MAPK kinase, Ste7, which activates two different MAPKs, Kss1 and Fus3.</p

Spectroscopie dynamique de force (méthodes d'analyse, application au couple streptavidine-biotine et à l'ancrage d'une protéine dans une membrane cellulaire)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

How a scaffold protein works.

<p>1a: Reactor model In the reactor model, the scaffold protein is able to simultaneously bind several kinases (three in this figure) that will activate each other in series very quickly. The scaffold protein is a reactor where the kinases are correctly oriented in order to facilitate the reactions. 1b: Constraints in the reactor model and in the simple membrane recruitment. A cascade of 4 kinases is represented: K0, K1, K2 and K3. K0 is assumed to be membrane bound (like Ste20 in the case of the MAPK pathway studied in the text). The arrows represent the activation of a kinase by another one (* indicates that the kinase is active). In the reactor model, the successive kinases, K1, K2 and K3, bind, meet and interact onto a scaffold protein (noted “Scaffold”). This model requires many geometrical and energetic constraints. It also implies that the binding sites for the various kinases on the scaffold protein must be blocked in the cytosol to prevent the scaffolding process to happen in volume. In the simple membrane recruitment, the scaffold protein binds independently the three kinases: when in the cytosol, it does not affect the signal transduction; when recruited to the membrane, it creates a source of active molecules at the membrane. The only constraint in this description is that the binding site between two successive kinases has to remain accessible when the kinase to be activated is bound to the scaffold protein.</p

Establishment of the chemical equilibrium.

<p>Variation of the concentration of the intermediate states during the phosphorylation process. The curves were obtained by solving equations (7) to (11) assuming a 10 nM concentration of kinase and using the parameters that allow deducing a k_on close to the ones used in the rest of text: k₁ = 0.1 nM⁻¹.s⁻¹, k₋₁ = k₋₃ = 1s⁻¹, k₂ = k₄ = 10 s⁻¹, k₃ = 1 nM⁻¹.s⁻¹. The lifetime of an active molecule should not be less than 1 s for it to be efficient and therefore: k_off>1 s⁻¹. We chose k_off = 0.5 s⁻¹ in order to obtain a characteristic reaction time larger than reality.</p