Team:Paris/Production modeling2

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== DryLab - Vesicles biophysics model==
== DryLab - Vesicles biophysics model==
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/DryLab#bottom"> Main </a>|
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/DryLab#bottom"> Main </a>|
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<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Modeling#bottom"> Introduction</a>|
 
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Production_modeling2#bottom"> Vesicle model</a>|
 
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Production_modeling#bottom"> Delay model</a>|
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Production_modeling#bottom"> Delay model</a>|
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Production_modeling2#bottom"> Vesicle model</a>|
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Transduction_modeling#bottom"> Fec simulation</a>
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Transduction_modeling#bottom"> Fec simulation</a>
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Production_modeling2#bottom">Intro</a>|
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Production_modeling2#2">Membrane model</a>|
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Production_modeling2#3">Tol/Pal model</a>|
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Production_modeling2#4">Bleb initiation</a>|
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Production_modeling2#5">Tol/Pal accumulation</a>|
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Production_modeling2#6">Vesiculation</a>
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===<u>[[Team:Paris/Production modeling versions 2#top|'''Pour Samuel et gregory''']]</u>===
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<span/ id="1">
 +
This part aims at investigating an important issue regarding the creation of vesicles:
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===Unequal pressures create small blebbing:===
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<center>
 +
'''What is the link between Tol/Pal expression and vesicle formation?'''
 +
</center>
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'''Simplified approach: without brownian motion of the intermembrane Tol-Pal links'''
 
 +
Or, more precisely, '''can we explain vesicle formation solely by the diffusion of the doubly anchored Tol/Pal complex in the membrane''' ?
 +
Understanding this connection is instrumental, since '''our project relies on the hypothesis that an increased rate of vesicle formation can be obtained simply by destabilization of the Tol/Pal complexes'''.
-
::In the following we first consider a model without brownian movement of the Tol-Pal links that are considered immobilized and randomly distribute over E.coli membrane. We look also here at a simplified one dimensional membrane. Our aim is to get a visual representation of the equilibrium shape of the outer membrane for a given osmotic pressure difference. In one dimension and in polar coordinates the membrane equation of the membrane conformation given by Ou-Yang and Helfrich [Phys. Rev. Lett. 59 (1987) 2486] simplifies well.
 
 +
To answer this question, we developed a '''biophysical model of the cellular membranes and Tol/Pal complexes''', that incorporates '''outer membrane deformation''' and '''Tol/Pal diffusion''' in membranes by Brownian motion.
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[[Image:Diferential System.png|300px|center]]
 
 +
Our initial '''simulation results suggested a three-stage process''' for vesicle formation. 
 +
Thus, in what follows, we decomposed the original main question in three parts:
 +
* Can we explain the formation of small blebs by differences of osmotic pressures between intra- and extra-cellular environments?
 +
* Can we explain the accumulation of Tol/Pal molecules at the basis of nascent blebs?
 +
* Can we explain vesiculation by constriction of blebs basis by accumulated Tol/Pal molecules?
-
::Where H is the mean gaussian curvature and K is the Gaussian curvature :
 
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[[Image:Final Theorical Curvature.png|100px|center]]
+
Before answering these questions, we start by describing how we modeled '''membrane deformation''' and '''Tol/Pal diffusion'''.
-
::The principal curvatures of a surface on the point M are defined as the minimum and the maximum curvature at this position of the curves described by cuting the surface with plans containing the normal direction at this point.
+
The main features of our membrane model is that we take into account the presence of '''osmotic pressure differences''' between intra and extra cellular environments and the existence of an outer '''membrane intrinsic preferred curvature'''.
 +
The main feature of our Tol/Pal diffusion model is that we take into account that '''diffusion''' may happen '''on non-flat surfaces''', and that the '''Tol/Pal complex is anchored''' both in the '''intracellular''' (Tol) and '''extracellular''' (Pal) '''membranes'''.
 +
We assumed that the intracellular membrane position is fixed (pushed against the peptidoglycan layer).  
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<center>
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<span/ id="2">
 +
===Modeling membrane at steady state===
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[[Image:Principal_curvature.png|500px|center]]
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The objective of this part is to discuss the existence of an intrinsic curvature of the outer membrane at steady state, and to obtain an estimate of its characteristic parameter.
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Principal curvature on a given surface
+
-
<u>[http://en.wikipedia.org/wiki/Principal_curvature#top| extracted from an article on Wikipedia]</u>
+
-
</center>
+
-
::We first simplified the membrane equation to a 2 dimensional equation system based on a polar curvature simplification approximation :
 
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:*the curvature in polar is [[Image:Curvature.png|120px|center]] where
+
Lipopolysaccharides (LPSs) are important constituents of the ''E. coli'' outer membrane.
 +
They possess long polycaccharide chains pointing outwards.
 +
These extracellular sugar extensions create mutual attraction forces that curb the membrane.
 +
This situation is depicted in the figure below.  
-
[[Image:derivative.png|60px|center]]
 
-
::Due to cylindrincal symetry the principal curvatures are always equal to the curvature in the perpendicular plane of the cylinder and  the curvature in his axis so the mean and gaussian curvatures can be written as:
+
[[Image:LPS_CLUSTER_Wiki.png|250px|center]]
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[[Image:Curvatures.png|40px|center]]
 
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::indeed we have in those hypothesis:
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From a physical point of view, the lipid bilayer behaves like a liquid at 37°c.
 +
To each membrane conformation, one can associate an energy, called the bending energy [1].
 +
This is given by :
-
[[Image:Principal curvatures.png|50px|center]]
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[[Image:Bending energy.png|220px|center]]
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::the osmotic pressure can be written in this way:
+
where E is the energy of the membrane, K<sub>b</sub> and K<sub>g</sub> are Bending and Gaussian moduli, H<sub>g</sub> is the Gaussian curvature and c<sub>0</sub> is a parameter describing the intrinsic curvature of the outer membrane at steady state (ie, minimal energy). The shape of the membrane is described locally by the principal curvature variables c<sub>1</sub> and c<sub>2</sub> (see [http://en.wikipedia.org/wiki/Principal_curvature here] for details). dS is an infinitesimal surface element.
-
[[Image:Osmotic pressure.png|100px|center]]
 
-
::And by concidering that we have low concentrations we can concider a simpliest formula analogue to the perfect gases law:
+
Under simple assumptions on the shape of ''E. coli'' (seen as a cylinder) and assuming that on average the size of the vesicles is the one requiring a minimum of energy, we can using the above relation relate the radius of ''E. coli'' r, the mean radius of vesicles r', and the γ<sub>0</sub> parameter:
-
[[Image:Osmotic pressure Analogue.png|80px|center]]
+
[[Image:Relations.png|180px|center]]
 +
-
::Finaly we can write:
+
Using r= 300 nm and r’=100 nm (the vesicle size ranges from 25 nm to 175 nm), we estimate the outer membrane intrinsic curvature as
 +
-
[[Image:Delta pressure.png|150px|center]]
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[[Image:gamma0.png|150px|center]]
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::Here we can see that the model is depending of the volume V which will stabelized the equation and define totaly the whole parameters of the model dynamic.
 
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::With these relations the membrane equation becomes:
+
Obtaining an estimation of this parameter is essential to model and simulate the membrane dynamic deformation under osmotic pressure differences as we will see.
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[[Image:Systeme_Membrane.png|250px|center]]
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===Modeling membrane deformation===
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::In addition, '''we can translate the role of the Tol/Pal System as boundary condition on the whole system : cluster of Tol-Pal can be considered as a point with a radius equal to the peptidoglycan ones plus the length ot the protein. ''' futhermore As the surface is closed, we must impose the fact that r(0)=r(2&pi;) to account for closing the vesicle.  
+
To obtain a reasonably simple model, we assumed that ''E. coli'' cells can be represented as cylinders, as a first approximation.
 +
This way, we could develop a 2D model of a transversal section of cells, using cylindrical coordinates.
-
::the integration of the polar membrane equations gives results of the following type:
 
-
<center>
+
Because of osmotic pressure differences, we assumed that the intracellular membrane is pushed against the -perfectly circular- peptidoglycan layer. So, only outer membrane deformations have to be modeled.
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[[Image:Vesicle wiki.jpg|400px|center]]
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The difference of osmotic pressure between the intra-cellular environment and the periplasm, and between the periplasm and the extra-cellular environment comes notably from the turnover of peptidoglycan molecules <sup>[[https://2009.igem.org/Team:Paris/Production_modeling2#References 1]]</sup>. This hypothesis has been made by  Zhou & Doyle <sup>[[https://2009.igem.org/Team:Paris/Production_modeling2#References 3]]</sup>
-
::Equilibrium shape of the outer membrane presenting blebbing (red) with immobile Tol-Pal links fixed to the inner membrane (green) assumed cylindrical obtain with an increase of the periplasm pressure.
+
-
</center>
+
-
==The lipid surface conformation==
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To model membrane deformation, we use the equation proposed by Ou-Yang and Helfrich <sup>[[https://2009.igem.org/Team:Paris/Production_modeling2#References 4]]</sup>:
-
:*The Lipid conformation of the outer membrane is a well known problem: at 35°c the lipid bilayer behaves like a liquid which '''conformation character is ruled by an energy called the bending energy''' (R. Lipowsky, The Conformation of Membranes, Nature 349 (1991) 475-481). This energy represents the fact that the lipid bilayer will search a special conformation depending on the shape and the chemical properties of its constituents. In order to explain the way lipids organize together we need an expression for the membrane bending energy. This is given by :
+
[[Image:Diferential System.png|300px|center]]
-
[[Image:Bending energy.png|200px|center]]
+
After simplifications, we obtain the following equality. 
 +
 
 +
[[Image:Simplified_Zhong_can.png|400px|center]]
   
   
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:*E is the energy of a whole lipid bilayer (or monolayer). Kb and Kg are Bending and Gaussian moduli which can be obtained by experiments. γ0 is the intrinsic curvature of the outer membrane which describes the local form of a lipid bilayer when it is at is lowest state of energy ,the more stable. γm  and Hg are the ''mean curvature'' and the ''gaussian curvature''( see Helfrich [Z. Naturforsch. C 28 (1973) 693] ). dS is a surface infinitesimal element and the previous formula relates how the local surface energy varies with the local mean curvature and the ''Gaussian curvature''. Let us first calculate the energy of two different shapes of membranes,  (i) a model of the shape of E. coli before budding, and (ii) after budding of a vesicle.
 
 +
In this equation, &gamma; is the variable membrane curvature, &Delta;p is the osmotic pression difference, c<sub>0</sub> is the membrane intrinsic curvature, and other parameters are as described in Ou-Yang and Helfrich <sup>[[https://2009.igem.org/Team:Paris/Production_modeling2#References 4]]</sup>.
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:::(i) We consider the shape of E.coli as a cylinder of radius r =0.3.
 
 +
A formal connection between the membrane curvature &gamma; and the cylindric coordinate variables ''r'' and &theta; can be obtained by the following polar curvature simplification approximation:
-
::This is a simplified shape based on the heuristic fact that in septa where the vesicle will be constituted principally with lipids of higher bending energy.  In fact this model is just a first approach to determine range of parameters and could be developed in a more sophisticated way later.The aim of this first representation is to estimate this energy in the division region of E.coli before division. '''With this approximation γm = 1/2r'''.
 
-
::Thus for the E.coli lipids membrane the bending energy per aera is:  
+
[[Image:Curvature.png|120px|center]]
 +
where
 +
[[Image:derivative.png|60px|center]]
-
[[Image:dEnergyEcoli.png|150px|center]]
 
-
::(ii) For the vesicles we consider their basic shape as a sphere of rayon r’ so the bending energy by lipid area units is:
+
Lastly, by combining the two equations given above, we obtain the following set of '''differential equations describing membrane deformations''':  
-
+
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[[Image:dEnergy vesicle.png|200px|center]]
+
 +
[[Image:Systeme_Membrane.png|300px|center]]
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::Thus as the area of a sphere is known and is independent of the location on the surface we can write:  
+
Considering that we have low concentrations we can obtain a simpler formula analogue to the perfect gases law:
 +
[[Image:Osmotic pressure Analogue.png|80px|center]]
-
[[Image:EnergyDif.png|400px|center]]
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Finaly we have:  
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::The energy of the same membrane area in E.coli is:
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[[Image:Delta pressure.png|150px|center]]
 +
Here we observe that the model is depending of the volume V which will stabilized the equation and totally define  all the parameters of the system.
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[[Image:EnergyEcoli.png|150px|center]]
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In addition, '''we decided to model the role of the Tol/Pal system as boundary condition for the system of differential equations : cluster of Tol-Pal are considered as a point with a radius equal to peptidoglycan's one plus the length of the protein. ''' Furthermore, as the surface is closed, we must impose the fact that r(0)=r(2&pi;) to account for closing the vesicle.
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::The relations can provide a basic vision of the statistical repartition of vesicles in case of absence of integrity control system in the outer membrane.
+
<span/ id="3">
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::Ee is the potential energy of the lipid area in E.coli outer membrane before construction of a vesicle and Ev is the energy of the same lipid area but in the conformation after budding including a vesicle shape. So ''the energy which must be given to the whole system to create a vesicle is'':
+
===Modeling Tol/Pal diffusion===
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[[Image:EnergyDif.png|400px|center]]
 
 +
Tol and Pal are membrane proteins which are located respectively in the outer and the inner membrane.
 +
Tol and Pal proteins interact with each other, forming Tol/Pal complexes.
 +
By acting like press studs binding the inner and outer membranes, thus stabilizing the outer membrane using the peptidoglycan rigidity, the Tol/Pal complexes play a central role to preserve membrane integrity <sup>[[https://2009.igem.org/Team:Paris/Production_modeling2#References 5]]</sup><sup>[[https://2009.igem.org/Team:Paris/Production_modeling2#References 6]]</sup>.
 +
[[Image:TolPal Ancored.png|300px|center]]
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''Mettre une courbe expliquant ceci''
 
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The diffusion of proteins in these lipid bilayers can be modeled by '''Brownian motion'''. This diffusion model gives the probability law for the location of Tol and Pal in the membranes.
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The following three equations model the Brownian motion of Tol, Pal and Tol/Pal complexes, represented as particles.
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::We can suppose that ''the most easily created vesicles will be the ones which require a minimum energy''. By derivation we find that the minimum is obtain for:
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[[Image:Diffusion.png|400px|center]]
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[[Image:Relations.png|400px|center]]
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In the above equations, C<sub>X</sub> denotes the concentration of protein X, and D<sub>X</sub> denotes the constant kinetic coefficient associated to X. The '''k'''<sub>TolPal</sub> and '''k'''*<sub>TolPal</sub> denote the reaction constants for Tol-Pal complexation.
 +
In the last equation, &Phi; is an unknown function representing the Tol-Pal complex motility.
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However, because the Tol-Pal complex is significantly larger than the other proteins, and is doubly anchored in membranes, we made the assumption that '''the mobility of Tol-Pal complexes is negligable in comparison to the mobility of isolated Tol and Pal molecules'''. Stated differently, we set &Phi; to zero.
 +
Lastly, additional constraints on Tol/Pal diffusion have been added to enforce a constant distance between Tol and Pal proteins.
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::Hence as we know that the range of created vesicles radii is 25 nm to 175nm we can suppose that the r’ is somehow about 100 nm. This leads us to an estimation of the outer membrane intrinsic curvature:
 
-
 
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[[Image:gamma0.png|150px|center]]
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In the above equations, '''the laplacians capture the non-homogeneous molecular diffusion on non-flat membranes'''.
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Indeed, they link the evolution of protein concentrations with the local shape of the membranes, since we know that on a two dimensional space:
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::Which is realistic in the order of magnitude
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[[Image:Laplacian.png|100px|center]]
 +
where x and y are the coordinate of the surface and where the different partial derivatives are depending on the cartesian coordinates of the surface we are evolving on.
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::In fact we know that the E.coli lipid bilayer is built of distinct types of lipids: '''Lippopolysacharides (LPS) and simple phospholipids'''. LPS are located in the exterior lipid layer of the outer membrane. The others are located in the interior lipid bilayer. Moreover, those '''LPS present a sugar extension toward the medium'''. Those sugars can bind to each other. So we can assume that they are going to create clusters and to curve the membrane toward the exterior of E.coli.
+
<span/ id="4">
 +
===Unequal osmotic pressures create small blebbing===
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[[Image:LPS_CLUSTER_Wiki.png|300px|center]]
 
-
 
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::This part was dedicated to a first approach of the membrane shape and its characteristics introducing some concept of lipid membrane physics. It enable us to find a range value for one important parameter.
 
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==''Travail  à faire''==
 
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<font color=red>Reprendre pourquoi cette modélisation et introduire ce quel'on fait ici. Par ex.
 
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In this section we are going to develop an original biophysical model developed to explain the formation of vesicles. This study aims at a better understanding of the mechanisms leading to the production of vesicles by the cell a process still very poorly described [Références] despite the natural occurrence of this phenomenon in gram-negative bacteria. Knowledge of the underlying processes can prove valuable in the perspective of optimizing inter-cellular communication by vesicle transport.
 
-
</font>
 
 +
Using the equations for membrane deformation, we can compute numerically the shape of the outer membrane at steady state under various assumptions.
 +
If we assume that Tol/Pal complexes acts like '''press studs''' locally imposing a fixed distance between the two membranes, and that their initial distribution  in the membrane is '''not totally homogeneous''', then we obtain results of the following type.
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<font color=red>ON PASSE DANS UNE AUTRE PARTIE:  petite conclusion de ce qui précède et introduction de la suite</font>
 
-
==="Tol-Pal proteins diffusion"===
+
<center>
 +
[[Image:Vesicle wiki.jpg|400px|center]]
 +
::Shape at equilibrium of the outer membrane presenting blebs (red) resulting from osmotic pressure differences and with Tol/Pal complexes locally imposing a fixed distance between the inner (green) and outer membranes.
 +
</center>
-
Tol and Pal are membrane proteins which are located respectively in the outer and the inner membrane. The diffusion of proteins in those lipid bilayers can be modelled by a probabilistic Brownian movement. This diffusion model gives us the law of probability for the location of Tol and Pal on the membranes. It has been observed that the Tol and Pal proteins interact with each other, which is linked to the membrane stability: indeed the Tol and Pal will bind inner and outer membrane and furthermore stabilize the outer membrane using the peptidoglycan rigidity. <font color=red>FAIRE UN RENVOIT A LA PARTIE DU WIKI QUI PRESENTE LE SYSTEME TOL-PAL OU INCLURE ICI UN SCHEMA</font>
+
<span/ id="5">
 +
===Tol/Pal accumulates at the basis of nascent blebs===
 +
The value of the Laplacian operators used to model Tol and Pal diffusions in the membranes (see equations above) depends on the membrane curvature: in non-flat membranes, the "efficiency" of the diffusion depends on the direction.
 +
In fact, '''molecules anchored in the outer membrane''', like Pal, '''diffuse less efficiently in regions of negative curvature, and consequently tend to accumulate in these regions'''. 
 +
Using our computer program, we have been able to experimentally demonstrate this accumulation.
 +
The bottom plot presents the accumulation of Pal proteins in the membrane represented on the top plot.
 +
In the bottom plot, the left/right axis represents coordinates along the membrane, whereas the other horizontal axis represent the time evolution of protein concentrations (from time 70 to 100, instead of from 0 to 25 as indicated)
 +
One can show the protein accumulation at coordinates 11 and 43 approximately, corresponding to the regions of negative curvature of the membrane.
-
'''Treatment including  brownian motion of the intermembrane Tol-Pal links'''
 
 +
[[Image:Accumulation2.png|600px|center]]
-
<font color=red>We will now let the Tol-Pal links between the cell outer and inner membranes move freely with a brownian motion.</font> This motion links the local concentration of proteins with the shape of the membrane they are included in. <font color=red>The diffusion of this links is described by:</font>
 
 +
[[Image:Accumulation3.png|600px|center]]
-
<center>
 
-
{|
 
-
|- style="background: #0d3e99; text-align: center; color:white;"
+
This result is corroborated by recent experimental findings showing that “negative membrane curvatures [act] as a cue for sub-cellular localization of a bacterial protein” <sup>[[https://2009.igem.org/Team:Paris/Production_modeling2#References 7]]</sup>.
-
|[[Image:EquationEinstein.jpg|200px]]
+
-
|}
 
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</center>
+
Most notably, we can thus assume that Pal molecules accumulate at the basis of blebs.
 +
This naturally leads to an increase of Tol-Pal complexes formation in these regions.
 +
Lastly, because we additionally assumed that these large complexes diffuse less efficiently than Tol and Pal alone, we finally obtain that '''Tol-Pal complexes accumulate at the basis of the blebs'''.
 +
This is depicted in the following picture.
-
where P is the proteins presence density of probability .
 
-
<font color=red>PIERRE, JE ME RAPPELLE QUE TOL ET PAL DIFFUSAIENT INDEPENDEMMENT ET POUVAIENT QUAND ILS SE RENCONTRAIENT FORMER DES LIENS. C'EST CE QUI SE PASSE ICI OU BIEN CE SONT DE LIENS TOL-PAL DEJA FORMES QUI DIFFUSENT? ETRE PLUS CLAIR ICI</font>
+
[[Image:Shema_Curvature.png|400px|center]]
-
The Laplacian of P explains the fact that the concentration of proteins like Pal increased in regions of negative curvature. Such a model can explain some recent observations [Kumaran & Losick] “negative membrane curvatures as a cue for sub-cellular localization of a bacterial protein”.
+
<span/ id="6">
-
According to this we can assume that the Pal will be confined to region with negative curvature. In those regions the probability of linking between Pal and Tol is increased which enhances the local membrane stability and contributes to  enlarge  these regions.
+
-
<font color=red>PAS CLAIR POURQUOI LA VESICULE EST MURE.... Then the vesicle is mature.</font>  
+
 +
===Rings of accumulated Tol/Pal constricts blebs basis===
-
<font color=red>::So Brownian motion is a good explanation for the proteins migration toward the septa during division. PAS CLAIR SI GARDER </font>
 
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::But this Brownian motion can explain the vesiculation too.  
+
As explained above, Tol-Pal complexes tend to accumulate at the basis of the blebs, creating a ring of Tol-Pal (if seen from above).
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<font color=red> This reinforcement process driven by brownian motion can explain the vesiculation.</font>When there are too few proteins we can assume the creation of a vesicle at germinal states (called bleb). At the border of the blebs an amount of Pal protein prevent the whole membrane to swallow and keep the vesicle in form. But at the border between the blebs and the protein rich area curvature is highly negative and so the quantity of proteins in those regions increases enlarging the region toward the centre of the blebs.
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[[Image:Shema_diffusion.png|400px|center]]
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<font color=red>UN SCHEMA IMPERATIF</font>
 
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::Then two different options are expectable: the blebs is too small and then the proteins are “zippering” back the blebs. But if the bleb is big enough the zippering became a separation between the vesicle and the bacteria. The good vision of this blebs forming can be understood in this way: the proteins rich region on the border pass from an annular form toward a circular one.
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When Tol-Pal molecules move to the interior of the ring, due to Brownian motion, they narrow the two membranes because they impose to have a constant distance between the two membranes as explained in the picture below: the proteins act like zippers on membranes, as depicted below. This leads to vesicle formation.  
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[[Image:zip.png|400px|center]]
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====References====
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{{Template:Paris2009_guided|Production_modeling|Transduction_overview}}
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<ol class="References">
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<li> [[Team:Paris/Production_modeling2#1 | ^]]1991 - Lipowsky - The conformation of membranes, ''Nature'', 349(6309):475-481</li>
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<li> [[Team:Paris/Production_modeling2#2 | ^]]2008 - Park & Uehara - How bacteria consume their own exoskeletons, ''Microbiol Mol Biol Rev'', 72(2):211-227</li>
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<li> [[Team:Paris/Production_modeling2#3 | ^]]1998 - Zhou ''et al''  - On the origin of membrane vesicles in gram-negative bacteria, ''FEMS microbiology letters'', 163(2):223-228 </li>
 +
<li> [[Team:Paris/Production_modeling2#4 | ^]]1987 - Ou-Yang & Helfrich - Instability and deformation of a spherical vesicle by pressure, ''Phys. Rev. Lett.'', 59:2486-2488</li>
 +
<li> [[Team:Paris/Production_modeling2#5 | ^]]2009 - Deatherage ''et al''  - Biogenesis of bacterial membrane vesicles, ''Mol Microbiol'', 72(6):1395-1407 </li>
 +
<li> [[Team:Paris/Production_modeling2#6 | ^]]2005 - Kuehn & Kesty      - Bacterial outer membrane vesicles and the host pathogen interaction, ''Genes & Dev'', 19:2645-2655 </li>
 +
<li> [[Team:Paris/Production_modeling2#7 | ^]]2009 - Kumaran & Losick  - Negative membrane curvature as a cue for subcellular localization of a bacterial protein. ''PNAS USA'', 106(32):13541-13545
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</li>
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</ol>

Latest revision as of 03:55, 22 October 2009

iGEM > Paris > DryLab > Vesicle biophysics Model (vesicle model)





Contents

DryLab - Vesicles biophysics model

This part aims at investigating an important issue regarding the creation of vesicles:


What is the link between Tol/Pal expression and vesicle formation?


Or, more precisely, can we explain vesicle formation solely by the diffusion of the doubly anchored Tol/Pal complex in the membrane ? Understanding this connection is instrumental, since our project relies on the hypothesis that an increased rate of vesicle formation can be obtained simply by destabilization of the Tol/Pal complexes.


To answer this question, we developed a biophysical model of the cellular membranes and Tol/Pal complexes, that incorporates outer membrane deformation and Tol/Pal diffusion in membranes by Brownian motion.


Our initial simulation results suggested a three-stage process for vesicle formation. Thus, in what follows, we decomposed the original main question in three parts:

  • Can we explain the formation of small blebs by differences of osmotic pressures between intra- and extra-cellular environments?
  • Can we explain the accumulation of Tol/Pal molecules at the basis of nascent blebs?
  • Can we explain vesiculation by constriction of blebs basis by accumulated Tol/Pal molecules?


Before answering these questions, we start by describing how we modeled membrane deformation and Tol/Pal diffusion.


The main features of our membrane model is that we take into account the presence of osmotic pressure differences between intra and extra cellular environments and the existence of an outer membrane intrinsic preferred curvature. The main feature of our Tol/Pal diffusion model is that we take into account that diffusion may happen on non-flat surfaces, and that the Tol/Pal complex is anchored both in the intracellular (Tol) and extracellular (Pal) membranes. We assumed that the intracellular membrane position is fixed (pushed against the peptidoglycan layer).

Modeling membrane at steady state

The objective of this part is to discuss the existence of an intrinsic curvature of the outer membrane at steady state, and to obtain an estimate of its characteristic parameter.


Lipopolysaccharides (LPSs) are important constituents of the E. coli outer membrane. They possess long polycaccharide chains pointing outwards. These extracellular sugar extensions create mutual attraction forces that curb the membrane. This situation is depicted in the figure below.


LPS CLUSTER Wiki.png


From a physical point of view, the lipid bilayer behaves like a liquid at 37°c. To each membrane conformation, one can associate an energy, called the bending energy [1]. This is given by :

Bending energy.png

where E is the energy of the membrane, Kb and Kg are Bending and Gaussian moduli, Hg is the Gaussian curvature and c0 is a parameter describing the intrinsic curvature of the outer membrane at steady state (ie, minimal energy). The shape of the membrane is described locally by the principal curvature variables c1 and c2 (see [http://en.wikipedia.org/wiki/Principal_curvature here] for details). dS is an infinitesimal surface element.


Under simple assumptions on the shape of E. coli (seen as a cylinder) and assuming that on average the size of the vesicles is the one requiring a minimum of energy, we can using the above relation relate the radius of E. coli r, the mean radius of vesicles r', and the γ0 parameter:

Relations.png


Using r= 300 nm and r’=100 nm (the vesicle size ranges from 25 nm to 175 nm), we estimate the outer membrane intrinsic curvature as


Gamma0.png


Obtaining an estimation of this parameter is essential to model and simulate the membrane dynamic deformation under osmotic pressure differences as we will see.

Modeling membrane deformation

To obtain a reasonably simple model, we assumed that E. coli cells can be represented as cylinders, as a first approximation. This way, we could develop a 2D model of a transversal section of cells, using cylindrical coordinates.


Because of osmotic pressure differences, we assumed that the intracellular membrane is pushed against the -perfectly circular- peptidoglycan layer. So, only outer membrane deformations have to be modeled. The difference of osmotic pressure between the intra-cellular environment and the periplasm, and between the periplasm and the extra-cellular environment comes notably from the turnover of peptidoglycan molecules [1]. This hypothesis has been made by Zhou & Doyle [3]


To model membrane deformation, we use the equation proposed by Ou-Yang and Helfrich [4]:

Diferential System.png


After simplifications, we obtain the following equality.

Simplified Zhong can.png


In this equation, γ is the variable membrane curvature, Δp is the osmotic pression difference, c0 is the membrane intrinsic curvature, and other parameters are as described in Ou-Yang and Helfrich [4].


A formal connection between the membrane curvature γ and the cylindric coordinate variables r and θ can be obtained by the following polar curvature simplification approximation:


Curvature.png

where

Derivative.png


Lastly, by combining the two equations given above, we obtain the following set of differential equations describing membrane deformations:

Systeme Membrane.png

Considering that we have low concentrations we can obtain a simpler formula analogue to the perfect gases law:

Osmotic pressure Analogue.png

Finaly we have:

Delta pressure.png

Here we observe that the model is depending of the volume V which will stabilized the equation and totally define all the parameters of the system.

In addition, we decided to model the role of the Tol/Pal system as boundary condition for the system of differential equations : cluster of Tol-Pal are considered as a point with a radius equal to peptidoglycan's one plus the length of the protein. Furthermore, as the surface is closed, we must impose the fact that r(0)=r(2π) to account for closing the vesicle.

Modeling Tol/Pal diffusion

Tol and Pal are membrane proteins which are located respectively in the outer and the inner membrane. Tol and Pal proteins interact with each other, forming Tol/Pal complexes. By acting like press studs binding the inner and outer membranes, thus stabilizing the outer membrane using the peptidoglycan rigidity, the Tol/Pal complexes play a central role to preserve membrane integrity [5][6].

TolPal Ancored.png


The diffusion of proteins in these lipid bilayers can be modeled by Brownian motion. This diffusion model gives the probability law for the location of Tol and Pal in the membranes. The following three equations model the Brownian motion of Tol, Pal and Tol/Pal complexes, represented as particles.

Diffusion.png

In the above equations, CX denotes the concentration of protein X, and DX denotes the constant kinetic coefficient associated to X. The kTolPal and k*TolPal denote the reaction constants for Tol-Pal complexation. In the last equation, Φ is an unknown function representing the Tol-Pal complex motility. However, because the Tol-Pal complex is significantly larger than the other proteins, and is doubly anchored in membranes, we made the assumption that the mobility of Tol-Pal complexes is negligable in comparison to the mobility of isolated Tol and Pal molecules. Stated differently, we set Φ to zero. Lastly, additional constraints on Tol/Pal diffusion have been added to enforce a constant distance between Tol and Pal proteins.


In the above equations, the laplacians capture the non-homogeneous molecular diffusion on non-flat membranes. Indeed, they link the evolution of protein concentrations with the local shape of the membranes, since we know that on a two dimensional space:

Laplacian.png

where x and y are the coordinate of the surface and where the different partial derivatives are depending on the cartesian coordinates of the surface we are evolving on.

Unequal osmotic pressures create small blebbing

Using the equations for membrane deformation, we can compute numerically the shape of the outer membrane at steady state under various assumptions. If we assume that Tol/Pal complexes acts like press studs locally imposing a fixed distance between the two membranes, and that their initial distribution in the membrane is not totally homogeneous, then we obtain results of the following type.


Vesicle wiki.jpg
Shape at equilibrium of the outer membrane presenting blebs (red) resulting from osmotic pressure differences and with Tol/Pal complexes locally imposing a fixed distance between the inner (green) and outer membranes.

Tol/Pal accumulates at the basis of nascent blebs

The value of the Laplacian operators used to model Tol and Pal diffusions in the membranes (see equations above) depends on the membrane curvature: in non-flat membranes, the "efficiency" of the diffusion depends on the direction. In fact, molecules anchored in the outer membrane, like Pal, diffuse less efficiently in regions of negative curvature, and consequently tend to accumulate in these regions.

Using our computer program, we have been able to experimentally demonstrate this accumulation. The bottom plot presents the accumulation of Pal proteins in the membrane represented on the top plot. In the bottom plot, the left/right axis represents coordinates along the membrane, whereas the other horizontal axis represent the time evolution of protein concentrations (from time 70 to 100, instead of from 0 to 25 as indicated) One can show the protein accumulation at coordinates 11 and 43 approximately, corresponding to the regions of negative curvature of the membrane.


Accumulation2.png


Accumulation3.png


This result is corroborated by recent experimental findings showing that “negative membrane curvatures [act] as a cue for sub-cellular localization of a bacterial protein” [7].


Most notably, we can thus assume that Pal molecules accumulate at the basis of blebs. This naturally leads to an increase of Tol-Pal complexes formation in these regions. Lastly, because we additionally assumed that these large complexes diffuse less efficiently than Tol and Pal alone, we finally obtain that Tol-Pal complexes accumulate at the basis of the blebs. This is depicted in the following picture.


Shema Curvature.png

Rings of accumulated Tol/Pal constricts blebs basis

As explained above, Tol-Pal complexes tend to accumulate at the basis of the blebs, creating a ring of Tol-Pal (if seen from above).

Shema diffusion.png


When Tol-Pal molecules move to the interior of the ring, due to Brownian motion, they narrow the two membranes because they impose to have a constant distance between the two membranes as explained in the picture below: the proteins act like zippers on membranes, as depicted below. This leads to vesicle formation.


Zip.png

References

  1. ^1991 - Lipowsky - The conformation of membranes, Nature, 349(6309):475-481
  2. ^2008 - Park & Uehara - How bacteria consume their own exoskeletons, Microbiol Mol Biol Rev, 72(2):211-227
  3. ^1998 - Zhou et al - On the origin of membrane vesicles in gram-negative bacteria, FEMS microbiology letters, 163(2):223-228
  4. ^1987 - Ou-Yang & Helfrich - Instability and deformation of a spherical vesicle by pressure, Phys. Rev. Lett., 59:2486-2488
  5. ^2009 - Deatherage et al - Biogenesis of bacterial membrane vesicles, Mol Microbiol, 72(6):1395-1407
  6. ^2005 - Kuehn & Kesty - Bacterial outer membrane vesicles and the host pathogen interaction, Genes & Dev, 19:2645-2655
  7. ^2009 - Kumaran & Losick - Negative membrane curvature as a cue for subcellular localization of a bacterial protein. PNAS USA, 106(32):13541-13545