Team:Paris Bettencourt/Modeling/Assisted diffusion

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=Assisted diffusion=
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== Introduction to the model ==
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<h1>Assisted diffusion</h1>
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The diffusion through the nanotubes is a fast process. This speed can be partially explained by the passive diffusion through the tubes. But what if it is faster?
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<h2>Introduction to the model</h2>
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The article from Dubey and Ben-Yehuda suggests that the diffusion is an active process. Several points can be opposed to this statement:
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* First, the diffusion is happening with molecule of different natures, that have nothing to do with the natural compoments of a cell
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* Unlike the mamalian cells, the tube seems not to have no "railroad" design for such a transport
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The question is: can we imagine a process that is faster than passive diffusion but does not rely on specific interractions?
 
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The answer is probably yes, and in this page we propose a new model, really challenging for the mind, but that can play a role in the diffusion process through the nanotubes.
 
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== General physical concepts and hypothesis ==
 
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=== How a connection between two bacteria might be established ===
 
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For different reasons the inner pressure of a bacterium can grow locally. That can cause the tube growth (if the pressure is big enough). If two bacteria are close to each other, their tubes could meet and even touch each other on the extremities. In this situation the phospholipids on the ends of the tubes are rather instable. Their hydrophobic part is more exposed to water because of the high curvature on the extremities. So the energy-preferable state is to fuse. In this case there is no specific protein involved to do the fusion, so it seems to be the explanation of the process closest to reality.
 
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In further paragraphes we will be more interested in what happens after the membrane fusion.
 
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=== Starting with a physical analogy ===
 
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Imagine two bottles of gaz connected by a tube. The first one have a higher pressure than the second one. In the first one, there are a few molecules of another nature diluted in the gaz. We follow these molecules.
 
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When you open the tape, the bottle with a higher pressure will equilibrate with the other one my moving a certain quantity of its particles through the tube in the direction of the second bottle. These moving molecules will drag with them the components diluted in the gaz and a few of these molecules will be transported to the other bottle.
 
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=== From the analogy to the biology ===
 
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Of course the cell is not a bag of liquid under pressure. The water is equilibrated at both sides of the exterior membrane. The pressure, we are dealing with, is not related to water or osmotic pressure (that is a "passive diffusion thermodynamical pressure"). There is a part of the cell we are not used to think about that undergo a certain variablilty of pressure: the phospholipid membrane!
 
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Let's evaluate the constraints that impact the membrane. As it is a Gramm positive bacteria, the external sugar envelope impose the shape of the bacteria. On the other hand, the osmotic pressure is pushing the membrane against the sugar wall. At every moment inside the membrane the number of phospholipids is fixed, but this number can fluctuate depending on the phospholipid production. So we can pretend that our system is evoluating through a serie quasiequilibrium states.
 
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<html>
 
<table>
<table>
<tr>
<tr>
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<td><center><img src="https://static.igem.org/mediawiki/2011/8/8a/MembraneCompression.png" height=200px></center></td>
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<td>
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<td><center><img src="https://static.igem.org/mediawiki/2011/d/d3/MembraneExtension.png" height=250px></center></td>
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<img style="width:150px; margin-top:20px;" src="https://static.igem.org/mediawiki/2011/b/b9/Active-diff-button.png">
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<tr>
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</td>
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<tr>
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<td>
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<td><p><center><u><b>Fig2:</b></u> If there is an excess of phospholipids,  the membrane is under compression<p></td>
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<p> Inspired by the experiments of Dubey and Ben-Yehuda we asked ourselves several questions.
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<td><p><center><u><b>Fig3:</b></u> If there are not enough phospholipids, the membrane is under extension<p></td>
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<tr>
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</table>
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</html>
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When the cell "start the communication", a flow of phospholipids of the membrane can pass from the cell that have the highest membrane tension to the other one. To pass from one cell to another, phospholipids run around the tube.
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What kind of process could do this molecular transfer? How can we characterize it?
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The newly arrived phospholipids change the membrane tension of the bacterium and, under our hypothesis of constant volume, which is justified by the sugar layer that "forbids" the bacterium to grow. As a consequence they change the internal Laplacian pressure of the bacterium.
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It could be an active process, a passive diffusion or something else.
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[[File:Laplacian_pressure.png|center|Laplacian pressure]]
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Several arguments can be opposed to the <em>active process</em> hypothesis:
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<ul>
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where R is the radius of bacterium, τ is a membranian tension.
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<li>During the process, an exchange of <em>molecules of different natures</em> takes place. These molecules have nothing to do with the natural components of a cell (GFP, calcein, etc.). Thus there is <em>no specificity</em> of transport, and there should be no specific mechanism of active transport.</li>
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<li>Unlike the mammalian cells, the bacterial tubes seem to have no "railroad" to guide the transported molecules.</li>
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A simple analogy of clothesline can help to understand what is happening. You need to pull more your clothesline to put more clothes on it. When you pull the rope by two ends you create a tension. More this tension is more weight (pressure) you can put on the rope.
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</ul>
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&nbsp; &nbsp; &nbsp; &nbsp;
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All this will lead to establish the pressure difference at the tube extremities and we will get a Poiseuil flow. Constituents diluted in water will move from one cell to another unidirectionally and faster than simply diffusing. This is a fast process that we have named the "assisted diffusion"
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</td>
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<td>
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=== When membranes behave like a 2D Van der Waals fluid ===
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Trapped between the osmotic pressure and the cell wall, phospholipids particles are moving in a double layered environment. Their motion is constricted into a two dimensional motion, following the shape of the membrane. At the scale of the phospholipid, the motion can be approximated to a motion in 2D. Using statistical physics approach, we can say that the phase space has 4N dimensions: two dimensions of impulsion and two dimensions of coordinate for each particle(N is the number of particles in the system).
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Can we describe the interaction between two phospholipids in the membrane? The question is complicated but we will demonstrate that by describing properly the phospholipid, it is reasonable to fit a Lennard-Jones potential energy to this interaction. The energy of binding is smaller and the speed of sliding of one phospholipid against another is slower than the internal vibration of the chemical bounds and the internal conformers rotation of the CH2 tails. Though, in ''Intermolecular Forces'' by Israelachvili(to be completed) the author shows that a phospholipid trapped in a bilayer can be aproximated by a section of cone. The section of cones is an individual, and the others are section of cone joining the previous one. The shape of the sections are giving the shape of the global structure.
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<html>
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<br/>
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<table>
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<tr>
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<td align="center"><img src="https://static.igem.org/mediawiki/2011/e/e9/Image_phospholipide.png" height=200px></td>
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<td align="center"><img src="https://static.igem.org/mediawiki/2011/8/82/Mod%C3%A8le_Phospholipide.png" height=200px></td>
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</tr>
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<tr>
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<td align="center"><center><b><u>Fig5:</u></b> Chemical structure of one pholpholipid</center></td>
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<td align="center"><center><b><u>Fig6:</u></b> Schematics of the cylindrical approximation made for the phospholipid</center></td>
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</tr>
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</table>
</table>
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<br/>
 
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</html>
 
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Speaking in term of energy, once we have these cones, it is reasonable to fit a Lennard-Jones potential to this interaction. If the cones are interpenetrating, a sterical repulsion keeps the molecules apart, and the hydropatic interaction that joins one lipid with the other acts as an attractive force keeping the coherence of the membrane. We will discuss later about the value of the two coefficients.
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<p>The question is :</p>
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<p><center><b>Can we develop a theoretical model of "active" transfer that can justify what was observed in the orignal article?</p>
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</b></center>
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[[File:LJpot.jpg|center|Lennard-Jones potential]]
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<p>We need to know if such a model can be designed starting from physical laws and if this model can explain quantitatively the transfer through the nanotubes. Due to its purely physical nature, our model can also shed some light as to the nanotube formation.</p>
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== Is this phenomenon important? Back-of-the-envelope calculation ==
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<p>We managed to come up with an idea for such a process, and in this page <em>we propose a new model</em> that can possibly explain the speed of the molecule exchange through the nanotubes.</p>
 +
<p>As a matter of fact, <em> the difference in membrane tension</em> between two bacteria could lead to a pressure difference. That could induce a small cytoplasmic transfer to reach internal pressure equilibrium. </p>
 +
<h2>Summary</h2>
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Let's start with a rough estimation of the flux transmitted through a nanotube. We assume that 5% of the surface area of one bacterium could be transmitted through the nanotube to a second bacterium. An order of magnitude of the transmitted surface is 10<sup>-14</sup> m<sup>2</sup>. A nanotube of the radius 100 nm (that is the mean value of radius taken from the article) with this surface would be 0.5 µm long. The nanotubes observed in Dubey and Ben-Yehuda experiments were about 100 nm to 1 µm.
 
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Let's take for example a tube 0.2 µm long. That means that 2.5*of the surface of the tube will be used in the process. We can imagine it splited in two stages: the first is the establishment of contact between two bacteria, for that 1 surface of the tube will be used. The remaining 1.5 surface of the tube will be used to transport the liquid inside the tube. As the diameter of the tube is of order of 100 nm, we can neglect the complexity of the fluid movement inside. In our model we will consider the fluid moving with the membrane. So the volume of transmitted liquid is about 10<sup>-20</sup> m<sup>3</sup>, whereas the cell volume is 5*10<sup>-18</sup> m<sup>3</sup>.
 
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We consider then that some molecules are produced in the first cell and they are uniformally distributed. With 10'000 molecules in the first cell, we can expect about 200 molecules transmitted through one nanotube to another cell. That number of molecules seems to be sufficient to be detected.
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<div id="assisted_diff" style="margin-left:50px;">
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<div class="assisted_diff_link" style="position:relative; left:80px; top:30px;"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/Assisted_diffusion/Membrane_tension"><img src="https://static.igem.org/mediawiki/2011/7/7b/Select_bilayer.png" /></a></div>
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<div class="assisted_diff_link" style="position:relative; left:375px; top:19px;"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/Assisted_diffusion/Tube_formation"><img src="https://static.igem.org/mediawiki/2011/8/8f/Select_formation.png" /></a></div>
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<div class="assisted_diff_link" style="position:relative; left:339px; top:94px;"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/Assisted_diffusion/From_membrane_tension_to_liquid_flux"><img src="https://static.igem.org/mediawiki/2011/4/47/Select_pressure.png" /></a></div>
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</div>
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<center><h4>Click on the circles on the above picture to discover our assisted diffusion model in details</h4></center>
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== The membrane tension calculation ==
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<p>
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Model description in few words :
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</p>
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Considering one membrane a sphere of the radius R, on which the phospholipid double layer is uniformally distributed, we can find approximately a part of the whole sphere surface per one phospholipid :
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<p>
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[[File:Surface_per_phospholipid.png|center|Surface per phospholipid]]
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We do not know what is the mechanism behind nanotube formation. We can suppose they are made of lipid membrane within a cell-wall like matrix, as suggested by the original electron microscopy experiment. When the membrane of the two cells fuse, there might be a <em>difference of tension between the two phospholipid bilayers</em>. This phenomenon might lead to a <em>movement of lipids</em> from one membrane to the other. The newly arrived phospholipids change the membrane tension of the bacterium. As a consequence, they change the internal Laplace pressure <i>ΔP<sub>Lap</sub></i> of the bacterium.
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</p>
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The caracteristic distance between two neighbor phospholipids on the membrane can be written like this :
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<p>
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<center>
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<img src='https://static.igem.org/mediawiki/2011/7/7d/Laplacian_pressure.png' style="height:45px">
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</center>
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</p>
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[[File:Characteristical_length_between_2_phl.png‎|center|Characteristical distance between 2 phospholipids]]
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<p>
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where τ is membrane tension, R is a radius of bacterium.
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</p>
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where N is the number of phospholipids on the membrane.
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<p>
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A simple analogy of clothesline can help to understand what is happening. You need to pull more your clothesline to put more clothes on it. When you pull the rope by two ends you create a tension. The bigger the tension, the more weight (pressure) you can put on the rope.
 +
</p>
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From this moment on we will work in canonical ensemble. The temperature is fixed and so is the number of particles in the system N. We also consider that the phospholipids can move only on the sphere surface that means that we will study the evolution of our system in 2N dimentional phase space.
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<p>
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All of this will lead to establishing the pressure difference at the tube extremities and we will get a Poiseuille's flow. Constituents diluted in water will move from one cell to another unidirectionally and faster than by simple diffusion. This is a fast process that we have named the <em>"assisted diffusion"</em>.
 +
</p>
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The partition function will look like :
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<div style="margin-left:50px; margin-right:50px; padding: 5px; border:2px solid black;"><b><p>The assisted diffusion model in 3 bullet points:
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<ul>
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    <li>Characteristic time of the process is about 100 ns</li>
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    <li>The effect strongly depends on the initial phospholipid distribution on the membrane of two connected bacteria</li>
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    <li>The phenomenon predicts the flux of only 0.1 % of the cytoplasme, not enough to explain the GFP experiment of the original paper</li>
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</ul></p></b></div>
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[[File:Partition_function.png‎|center|Partition function in canonical ensemble]]
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<html>
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<p>
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We have done a <a href='https://2011.igem.org/Team:Paris_Bettencourt/Modeling/Assisted_diffusion/Back_of_the_envelope_calculation'>back of the envelope calculation</a> to see whether an order of magnitude is acceptable.</p>
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</html>
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where the Hamiltonian is :
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<html>
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<div id="citation_box">
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[[File:Hamiltonian.png‎|center|Hamiltonian of the system]]
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<p id="references">References</p>
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<ol>
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Knowing the partition function is a great deal. We can derive a free energy
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<li><i>Intercellular Nanotubes Mediate Bacterial Communication</i>, Dubey and Ben-Yehuda, Cell, 2011, available <a href="http://bms.ucsf.edu/sites/ucsf-bms.ixm.ca/files/marjordan_06022011.pdf">here</a></li>
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<li><i>BioNumbers</i> <a href="http://bionumbers.hms.harvard.edu/">here</a></li>
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[[File:Free energy.png‎|center|Free energy]]
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</ol>
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</div>
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of each sphere, total free energy and the membranian tenstion from it :
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</html>
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[[File:Free energy total.png‎|center|Total free energy of the system]]
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[[File:Tension.png‎|center|Membranian tension]]
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For two spheres we get :
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[[File:Tension 1.png‎|center|Tension 1]]
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and
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[[File:Tension 2.png‎|center|Tension 2]]
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== Back to the classical physics ==
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To deduce the force exercising on the tube from membranian pressure, please refer to an explanation figure below :
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<html><a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/Assisted diffusion"><img style="width:350px" src="https://static.igem.org/mediawiki/2011/f/f3/Tension-force_explanation.jpg"></a></html>
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<html><a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/Assisted diffusion"><img style="width:350px" src="https://static.igem.org/mediawiki/2011/5/58/Geometry.png"></a></html>
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[[File:Geometry.png|center|Geometry of the system]]
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Here T is a surface tension, N is a resulting force of the area of the tube attachment to the bacterium, Δα or α is the half of the angle with a vertex in the center of the bacterium and between the tube extremities. Only this zone is giving a non-compensated contribution to the resulting force.
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So the force exercising on the tube is given by the formala :
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[[File:Tension-force.png|center|The force exercising on the tube ]]
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Now that we can calculate the tension of the membrane (i.e. the pressure of the Wan der Waals fluid), we can start calculating the equential equation of the relaxation of the membrane. As it is a fluid with an intrinsic viscosity, we will use the Navier-Stokes equation.
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[[File:NavierStokes.jpg|center|Navier Stokes equation]]
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Let's first choose a point of view of considering the flux. There is the Bernouilli point of view in which we track the fluid particule destiny and the stokes point of view in which we are fixed in one point and watch the flux of fluid particle. The second point of view is easier for us, because, we want to count in the end the a certain quantity of phopholipids that have passed through the tubes, and link that to the quantity of water.
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Let's imagine that we are an observer sitting at the entrance of the nanotube. He watch the liquid passing between his legs, and canlculate the flux.
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...
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...
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Your job agains Oleg, the games get it's tracks!!
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...
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...
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Finally with a difference of pressure P2-P1, we can estimate the caracteristical time of the relaxation to be:
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where...
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== Getting back to the quantity of water ==
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We are in the situation where a tube, that have good hydrophilic properties is moving, the tube is small and the liquid viscuous (the density of the cytoplasm over the tensity of water is about 3.2). Water will be dragged in the tube.The fluid will undergo a Poiseuille flow, but this time the tube is moving and the water following.
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[[File:Poiseuille.jpg|center|a Poiseuille flow inside a tube]]
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...
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...
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Going down the Poiseuille equation you will estimate the total flux dragged for a certain quantity of phospholipids that have passed bhy depending on the size of the tube, the viscosity of water...
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...
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...
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== Getting the good parameters ==
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It will take some time, but once you have the equation, from the size of a vesible in equillibium, when we know the number of phospholipids. The viscosity has probably been estimated by FRAP techniques. For the rest, we will find out.
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== Analysing the results ==
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== Can this process be an efficient way for passing molecules?? ==
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== Conclusion ==
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<html>
<html>
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<br>
<br>

Latest revision as of 02:14, 29 October 2011

Team IGEM Paris 2011

Assisted diffusion

Introduction to the model

Inspired by the experiments of Dubey and Ben-Yehuda we asked ourselves several questions. What kind of process could do this molecular transfer? How can we characterize it? It could be an active process, a passive diffusion or something else. Several arguments can be opposed to the active process hypothesis:

  • During the process, an exchange of molecules of different natures takes place. These molecules have nothing to do with the natural components of a cell (GFP, calcein, etc.). Thus there is no specificity of transport, and there should be no specific mechanism of active transport.
  • Unlike the mammalian cells, the bacterial tubes seem to have no "railroad" to guide the transported molecules.
       

The question is :

Can we develop a theoretical model of "active" transfer that can justify what was observed in the orignal article?

We need to know if such a model can be designed starting from physical laws and if this model can explain quantitatively the transfer through the nanotubes. Due to its purely physical nature, our model can also shed some light as to the nanotube formation.

We managed to come up with an idea for such a process, and in this page we propose a new model that can possibly explain the speed of the molecule exchange through the nanotubes.

As a matter of fact, the difference in membrane tension between two bacteria could lead to a pressure difference. That could induce a small cytoplasmic transfer to reach internal pressure equilibrium.

Summary

Click on the circles on the above picture to discover our assisted diffusion model in details

Model description in few words :

We do not know what is the mechanism behind nanotube formation. We can suppose they are made of lipid membrane within a cell-wall like matrix, as suggested by the original electron microscopy experiment. When the membrane of the two cells fuse, there might be a difference of tension between the two phospholipid bilayers. This phenomenon might lead to a movement of lipids from one membrane to the other. The newly arrived phospholipids change the membrane tension of the bacterium. As a consequence, they change the internal Laplace pressure ΔPLap of the bacterium.

where τ is membrane tension, R is a radius of bacterium.

A simple analogy of clothesline can help to understand what is happening. You need to pull more your clothesline to put more clothes on it. When you pull the rope by two ends you create a tension. The bigger the tension, the more weight (pressure) you can put on the rope.

All of this will lead to establishing the pressure difference at the tube extremities and we will get a Poiseuille's flow. Constituents diluted in water will move from one cell to another unidirectionally and faster than by simple diffusion. This is a fast process that we have named the "assisted diffusion".

The assisted diffusion model in 3 bullet points:

  • Characteristic time of the process is about 100 ns
  • The effect strongly depends on the initial phospholipid distribution on the membrane of two connected bacteria
  • The phenomenon predicts the flux of only 0.1 % of the cytoplasme, not enough to explain the GFP experiment of the original paper

We have done a back of the envelope calculation to see whether an order of magnitude is acceptable.

References

  1. Intercellular Nanotubes Mediate Bacterial Communication, Dubey and Ben-Yehuda, Cell, 2011, available here
  2. BioNumbers here



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