The cell membrane is a special casing necessary to keep homeostasis for cells survival. It is a two-dimensional agglomeration of lipids that holds a large number of membrane proteins with diverse vital functions. The fluid nature of the membrane makes it difficult to be handled, and requires the development of ad hoc techniques to investigate its properties and composition. In this PhD thesis, I developed a method to isolate the apical cell membrane of single cells. Taking advantage of the Atomic Force Microscope (AFM), I imaged and probed the mechanical properties of these isolated patches of membranes. I also extensively performed AFM-based single-molecule force spectroscopy to unfold the membrane proteins from the native membranes, collecting hundreds of thousands of unfolding curves. I analyzed these data with a custom software able to find the recurrent pattern of unfolding in the data, and I developed a Bayesian inference method to assign these unfolding curves to a limited number of membrane proteins. The underlying motivation of these experiments is to bring AFM technologies a step closer to an application in biomedicine. This work demonstrates that i) the cell membrane can be reliably isolated from single cells; ii) AFM can be used to characterize the membrane topography and mechanical properties of the cells of interest (e.g. I found that neural cell membranes are thicker and stiffer than membranes of brain cancer cells); iii) it is possible to record the unfolding pathways of the membrane proteins contained in the cell membranes and to identify them with the cross-matching of proteomic databases, and iv) the population of unfolding curves obtained with SMFS reflects the actual population of membrane proteins obtained with Mass Spectrometry.

AFM applications to native cell membranes and membrane proteins / Galvanetto, Nicola. - (2019 Oct 28).

AFM applications to native cell membranes and membrane proteins

Galvanetto, Nicola
2019

Abstract

The cell membrane is a special casing necessary to keep homeostasis for cells survival. It is a two-dimensional agglomeration of lipids that holds a large number of membrane proteins with diverse vital functions. The fluid nature of the membrane makes it difficult to be handled, and requires the development of ad hoc techniques to investigate its properties and composition. In this PhD thesis, I developed a method to isolate the apical cell membrane of single cells. Taking advantage of the Atomic Force Microscope (AFM), I imaged and probed the mechanical properties of these isolated patches of membranes. I also extensively performed AFM-based single-molecule force spectroscopy to unfold the membrane proteins from the native membranes, collecting hundreds of thousands of unfolding curves. I analyzed these data with a custom software able to find the recurrent pattern of unfolding in the data, and I developed a Bayesian inference method to assign these unfolding curves to a limited number of membrane proteins. The underlying motivation of these experiments is to bring AFM technologies a step closer to an application in biomedicine. This work demonstrates that i) the cell membrane can be reliably isolated from single cells; ii) AFM can be used to characterize the membrane topography and mechanical properties of the cells of interest (e.g. I found that neural cell membranes are thicker and stiffer than membranes of brain cancer cells); iii) it is possible to record the unfolding pathways of the membrane proteins contained in the cell membranes and to identify them with the cross-matching of proteomic databases, and iv) the population of unfolding curves obtained with SMFS reflects the actual population of membrane proteins obtained with Mass Spectrometry.
Torre, Vincent
Galvanetto, Nicola
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11767/103861
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