Traduction technique français anglais de la thèse de doctorat d’Amar TAMRA intitulée “Spectroscopie diélectrique HyperFréquence des cellules biologiques soumises à l’électroporation”
Université Toulouse 3 Paul Sabatier; Unité de recherche : LAAS-CNRS/IPBS
Copyright : Amar TAMRA 2017
French english scientific translation series : Phd thesis entitled : “Microwave dielectric spectroscopy of biological cells under electroporation”.
Phd Student : Amar TAMRA
University : Toulouse 3 Paul Sabatier University
Research Units : The Systems Analysis and Architecture Laboratory (LAAS) & the Institute of Pharmacology and Structural Biology (IPBS) of Toulouse.
Chapter I: Introduction and research objectives
The work presented in this manuscript treats the study of cells subjected to an electric treatment (electroporation), with or without the administration of a cytotoxic molecule, using a new analysis method: microwave dielectric spectroscopy. It was carried out thanks to a joint collaboration between the Cellular Biophysics team of the Institute of Pharmacology and Structural Biology, which studies vectorization mechanisms for molecules in the cells using electroporation and the Fluidic/Microwave Micro/Nanosystems team of the LAAS-CNRS, Which develops microsystems for microwave analysis. Our project is then halfway between biology, microtechnology and electronics and draws upon research findings in these different fields in order to develop new promising tools for cellular biology.
In order to delineate the scope of our thesis, we will divide this first chapter into three parts, which essentially reflect the scientific disciplines at the basis of our work on this thesis.
Before we embark on an explanation of the electroporation phenomenon, we have to understand the motivations behind the use of this technique. We will then describe, in the first part, the plasma membrane with its different constituents and functions, as well as the principle of electroporation and its various resulting applications.
Research at the single cell level can contribute to a better understanding of the underlying mechanisms of the phenomena under investigation. As such, we shall dedicate the second part of this chapter to a review of recent applications of microtechnologies in the fields of biology and medicine and the significance of research at the micrometric scale, also known as « Lab-on-chip ».
The third and last part of this chapter deals with microwave dielectric spectroscopy: we turn our attention to the principles of interaction between the biological substance and electromagnetic waves as a function of frequency.
The cellular membrane
Before the advent of electron microscopy at the beginning of the fifties, the cellular membrane was rarely mentioned in the scientific literature. Neither its organization, nor its composition were known. In order to better understand the concept of a cell and that of its membrane, researchers resorted to the use of indirect methods to help them create an image of this membrane before being able to visualize it. Their work enabled to establish the current model of the cellular membrane.
At the end of the 19th century, Charles Ernest Overton (1885) studied the osmotic properties of cells and concluded that the membrane around the cell had properties similar to those of oil: meaning essentially that it contains phospholipids, which regulate the permeability of the membrane . In 1925, Gorter and Grendel studied the surface of a monolayer of lipids extracted from a known number of red blood cells. They concluded that the cellular membrane is a lipid bilayer composed of two phospholipid layers, molecules made of hydrophilic heads and hydrophobic tails. However, the model proposed by Gorter and Grendel, while being correct, was not sufficient to answer questions regarding permeability, surface tension and electrical resistance of the membranes. For this, Davson and Danielli proposed ten years later (1935), the presence of protein layer associated with the lipid bilayer. In the nineteen fifties, Robertson confirmed the presence of the plasma membrane under the form proposed by Davson and Danielli thanks to electron microscopy. Nevertheless, it was not before 1972 that Singer and Nicholson developed new ideas concerning the structure of the modern cellular membrane: the fluid mosaic model. According to this model, proteins are incorporated in the lipid bilayer and the components of the latter (phospholipids and proteins) exhibit a free rotational and lateral diffusion in the plan of the membrane, which makes for their random and heterogeneous distribution. However, a significant body of research suggests that the movement of most of the proteins in the plasma membrane is partially limited at the nanometric scale. This lateral heterogeneity in the organization of the membrane is in fact the result of protein-protein, protein-lipid and lipid-lipid interactions. These interactions form the basis of a functional compartmentalization of the plasma membrane: some specific groups, called micro-domains, form in the membrane. These micro-domains facilitate cellular processes, such as signaling, adherence, and membrane traffic. A multitude of micro-domains can form: short-lived micro-domains created out of a few proteins and lipids, or stable well-organized micro-domains with a diameter ranging from a few tens to a few hundred nanometers.
The cellular membrane: the fluid mosaic model
All cell types, whether prokaryotes or eukaryotes, animal cells or plant cells, are delimited by a cytoplasmic cellular membrane which turns out to be essential to life, due to its ability to separate the intracellular medium from the extracellular one, thereby ensuring cellular integrity. Although the presence of a cellular membrane is common to all cell types, there are a few differences: unlike animal cells, plant and bacterial cells have a rigid cell wall associated with the plasma membrane.
Structure and composition
Electron microscopy analysis of membrane sections reveals that the cytoplasmic membrane (7-8 nm thick) has a continuous bilayer phospholipid structure arranged in a head to tail pattern. Every lipid is composed of two hydrophobic hydrogen carbonate chains (the tail) and a hydrophilic head: this is precisely what gives the membrane its amphipathic property thus leading to the bilayer configuration. The fundamental composition of the plasma membrane associates the lipid bilayer to two categories of molecules: lipids and proteins. Phospholipids are amphiphilic: they have a polar component that is attracted to water (hydrophilic) and an apolar component repelled by water (hydrophobic). When diluted in water, amphiphilic molecules adopt spontaneously the most thermodynamically stable molecular structure. Under biological conditions, phospholipids form a double layer in which the hydrophobic tails face each other at the heart of the structure, while the hydrophilic heads interact with the surrounding water (figure 1).
The plasma membranes of animal cells contain four major types of phospholipids (phosphatidylcholines PC, the phosphatidylethanolamines PE, phosphatidylserines PS and sphingomyelins). These phospholipids are distributed in an asymmetric way between the two external and internal leaflets of the membrane bilayer (figure 2).
The external leaflet of the plasma membrane is mainly composed of phosphatidylcholine and sphingomyelin, while the internal leaflet is predominantly composed of phosphatidylethanolamine and phosphatidylserine. A fifth type of phospholipid, the phosphatidylinositol, is also present. Besides, the cellular membrane of animal cells contains glycolipids (carbohydrate chains linked to lipids) and cholesterol. The former can only be found on the external leaflet of the membrane with the carbohydrated part exposed to the cellular surface.
They only represent 2% of all the lipids in the plasma membrane and constitute a hydrophilic protection layer of the extracellular side of the membrane. As for cholesterol, it is a major component of the membrane of animal cells present in both leaflets. This asymmetric distribution characterizes a healthy cell. Externalization of PS is one of several precocious signals of apoptosis, in other words, a certain type of cell death.
Although lipids are the fundamental structural elements of membranes, proteins are responsible for the execution of very specific functions within the membrane: receivers, carriers, intracellular messengers, enzymatic catalysis, cell adhesion, etc. Most cytoplasmic membranes are composed, in terms of weight, of about 50% lipids and 50% proteins. Given that proteins are much bigger than lipids, this percentage corresponds to approximately one protein molecule for every 50 to 100 lipid molecules. According to the model of Singer and Nicholson, the proteins are anchored in various ways in the fluid lipid bilayer. We may distinguish three classes of proteins:
(a) Extrinsic or peripheral proteins: these proteins are located outside the membrane. They are indirectly associated with the membrane through hydrogen electrostatic bonds or protein-protein interactions. They are either entirely intracellular or entirely extracellular.
(b) Intrinsic or integral proteins: these proteins interact strongly with the membrane. We can distinguish two classes: Proteins anchored in fatty acids and transmembrane proteins.
- The proteins that are anchored in fatty acids: they can be anchored on glycophosphatidyl-inositol (GPI) and are then present on the extracellular side of the membrane. On the other hand, if they are anchored in the membrane through fatty acids, they will be present on the intracellular side of the membrane.
- The transmembrane proteins: these proteins cross the two membrane leaflets and are linked in a stable way to the membrane with its internal hydrophobic part.
Interactions take place between the different proteins and lipids of the plasma membrane, which leads to the creation of what we call micro-domains. These micro-domains, while rich in cholesterol, lipid rafts, and aveolae, are particularly interesting given their participation in several cellular processes (figure 3).
In 1997, Simons and Ikonen conjectured the presence of structures called lipid rafts, responsible for the lateral heterogeneity of the plasma membrane. These dynamic sets of proteins and lipids float in the bilayer membrane, but might also gather to form large well-organized structures. They are composed of cholesterol molecules and sphingolipids from the external leaflet, which are connected to cholesterol, and phospholipids from the internal leaflet of the plasma membrane. These structures are fluid but more organized and packed than the rest of the membrane. They received increasing attention as structures that regulate the function of the membrane in eukaryotic cells. Lipid rafts take part in various cellular processes such as the assembly of signaling molecules, membrane trafficking, neurotransmission and trafficking of receivers.
The caveolae are invaginations of the cellular membrane, 50-70 nm in diameter, and were identified for the first time in 1955 with electron microscopy. Their biochemical composition is similar to that of lipid rafts (glycosphingolipids and cholesterol), hence their classification as a subtype of rafts. Nevertheless, the presence of a protein, the caveolin, is a defining characteristic. Recently, another type of proteins, the cytoplasmic cavins, seem to have a critical role in maintaining the structure and function of the caveolae.
Their relative abundance depends on cell type. Despite the fact that they represent up to 35% of the cellular surface of adipocytes as well as endothelial and muscular cells, they are absent from lymphocytes and from most neurons.
Although the cellular membrane plays a fundamental role in ensuring cellular integrity, another component is essential to the life of the cell. It is a complex and dynamic network of regulatory protein filaments extending across the cytoplasm called cytoskeleton. It enables the cell to adapt and respond to a wide variety of intracellular and extracellular stimuli, maintain cellular morphology, perform coordinated movements and control intracellular trafficking. The cytoskeleton is composed of actin microfilaments, intermediate filaments and microtubules (figure 4). Each one of these components is made of a different type of protein, having its own specific diameter, length and flexibility level.
Functions of the plasma membrane
The essential role of the cytoplasmic membrane is to delimit cell boundaries and preserve its integrity. It enables the recognition of specific cells and transmits information on the cellular environment to the cell interior. Besides, the membrane control molecular and particle exchange between the cell and the surrounding environment: a cell needs nutrients to grow, multiply, and move, and it has to evacuate its metabolic waste. Some molecules such as water, urea, oxygen and carbon dioxide are able to cross the membrane passively thanks to their biochemical properties. Other however, need channels and protein pumps located in the membrane to be able to do the same thing. It is precisely this property of selective permeability that enables controlled entrance of certain molecules while blocking the way for others.