טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKreiserman Roman
SubjectA New Method to Measure Close Range Forces and Membrane
Fusion using Dual Trap Optical Tweezers
DepartmentDepartment of Physics
Supervisor Professor Ariel Kaplan
Full Thesis textFull thesis text - English Version


Abstract

Fusion of lipid membranes occurs every moment in every living cell and involved in numerous vital extracellular processes. Due to its importance, membrane fusion is a highly regulated mechanical process composed of several intermediates, hence initiation and advancing of the fusion process requires overcoming several energy barriers. Theoretical models predict that the elastic properties of the membrane play an important role in fusion regulation since each fusion intermediate involves a rearrangement of the lipids bilayers into highly curved structures. However, the energy landscape for fusion has not been measured experimentally. Our aim was to measure the forces and to quantify the energy landscape of membrane fusion using optical tweezers. Additionally, we aimed to resolve how the elastic properties of the membrane, as controlled by the integration of cholesterol, affect the energetic cost of fusion.

Membrane fusion is associated with close range interactions at few tens of nanometers. Such short-range forces are particularly difficult to characterize using the conventional measurement modes of a dual-trap optical tweezers. Therefore, we have developed a new method, using the measurement of the coupled fluctuations of two trapped spheres, in order to calculate short-range forces. Using our method, we have measured and calculated short-range forces, hydrodynamic forces and surface forces between two silica microspheres. We find that the hydrodynamic interaction between the microspheres extends for distances of hundreds of nanometers. These hydrodynamic interactions mask the measurement of the actual short-range forces using conventional force measurement protocol. Our method of measurement decouples hydrodynamic interaction from conservative force measurements, thus allowing to reliably measure surface forces. We validated our method by fitting force-distance curves to the well-established DLVO theory using two fitting parameters, Debye length and the Gouy-Chapman surface potential. Good agreement to the DLVO theory and Gouy-Chapman theories is found over a wide range of salt concentrations. Moreover, we report the first experimental verification of the hydrodynamic interactions at far and close sphere surface separations. Our measurements of the drag coefficients agree with the theoretical calculations of the resistance for two rigid spheres in low Reynolds numbers flow without any fitting parameters.

Lastly, we use our newly developed method to induce membrane fusion of two opposing spherical-supported-lipid-bilayers and measure the forces involved in such process as a function of the membranes’ separation. We found that force-distance curves typically display two “jumps” in distance, corresponding to the thickness of one bilayer. We interpret each jump as corresponding to a different fusion intermediate; the first jump to the hemifusion intermediate and the second one to full fusion. Using the force-distance measurements, we are able to produce the first reported experimental calculation of the energy landscape for membrane fusion. We assign the energetic cost of the fusion process and the energy required for propagation from one intermediate to the other. By adjusting the amount of cholesterol in the membrane, we found that the elastic properties of the membrane have a profound effect on the energy landscape of fusion. Our findings support the existence of highly curved intermediates during membrane fusion.