Study of the RGD role in cells patterning onto a substrate in microfluidic devices

1.3. Protein Functions
The proteins play many important roles in a organism depending both  on 
their chemical and mechanical disposition. Since they are made of a  sequence 
of amino acids they can fold up in different three dimensional shapes showing 
different interactions on different binding sites 
1
The property of proteins to bind to other molecules enable them to act 
with different function such catalysts, signal receptors, switches, motors or 
tiny pumps [3].
All proteins stick to other protein in a organism. Sometimes this binds are 
very weak and short-lived, sometimes they are thigh and lasting, but every 
protein show a great specificity , that is every binding sites present on its 
structure can bind only to a specific chemical.
The fi gure 1.1 represent schematically the kind of interaction that usually 
happens between a protein and its ligand.
1 - Introduction
3
1
 The binding site is defined in biochemistry as the region of protein that bind to a specific 
molecule or ion called ligand. The binding sites exhibit a specificity that is related to their 
capability to bind only to certain types of molecules, and an affinity that measure the 
strength of the bind.
Fig. 1.1 The fi gure aims to explain the kind of interaction that happens 
between a molecule and its ligand. It’s possible to notice that binding is due to 
the capability of the molecule to fold itself, while its strength is determined by a 
large number of weak bounds to avoid the irreversibility of the attachment 
(Velcro effect ).
Every singles bond is weak though the ensemble of many of them, thanks 
to a specific configuration of the protein, can become a strong connection. 
The most common of these weak interactions are non-covalent bonds like 
hydrogen bonds, ionic bonds, van der Waals forces and they can be promote 
by hydrophobic interactions that just cause a repulsion from the water.
Another important parameters in the interaction of two molecules is the 
affinity. This value is determined by the dissociation constant and means, 
roughly speaking, how many things the protein and the ligand have in 
common, that is determining the probability they do have to interact 
accordingly to their sympathies/antipathies.
1.3.1. The dissociation constant
The aff inity/specifi city can be easily quantifi ed by means of a 
mathematical model called the dissociation constant.
In biochemistry the dissociation constant expresses the affinity between 
protein and its ligand, being itself a particular type of equilibrium constant. If 
we keep in consideration a reaction protein (P), the ligand (L) and the 
complex (C) that they are forming, we have:
1 - Introduction
4
with a dissociation constant that is:
The variable K
affinity 
is the concentration of ligands for which half of the 
protein are free ([L]=[PL]), let’s say the concentration of ligand, at which the 
concentration of protein with ligand bound, equals the concentration of 
protein with no ligand bound. This value is expressed in molar units and is 
inversely proportional to the tight of the binding, i.e. the smaller is the 
dissociation constant the higher is the affinity and the higher is the strength 
of the bond.
Once we know this parameter is easy to compute the binding energy  (G) 
between the two molecule that is:
where R is the ideal gas constant, T is the temperature, K is the 
concentration of which the association rate is equal to the dissociation rate 
(equilibrium). This number show us an overall idea of the binding strenght.
1.4. The Extracellular Matrix
The extracellular matrix (ECM) is defined as the extracellular part of 
animal tissue that provide support to the cells more than other important 
function including tissue repair, embryogenesis, blood clotting, and cell 
migration/adhesion. In some cases, the ECM accounts for more of the 
organism's bulk than its cells and it basically determine the physical behavior 
of the tissues.
The ECM of vertebrates is made up of:
Cnull P+L
K
affinity
=
[P][L]
[C]
nullG =nullRTln (K
equilibrium
)=1.425log(K
affinity
)
1 - Introduction
5
• Proteoglycans, that are molecules constituted by polysaccharide chains 
of the class of glycosaminoglycans (GAGs) that are usually covalently 
linked to protein
• Fibrous protein like collagen, elastin, fibronectin and laminin. These 
molecule help to determine the structure giving it the right elasticity 
and most of all they allow cells to attach in determined locations
• Mineral deposits (in the case of bone) 
This compounds are secreted locally mostly by fi broblast cells and 
assembled into an organized meshwork in close association with the surface of 
the cell that produce them. The structure of this matrix is rather complex 
and although the understanding of its organization is still incomplete the 
progress in its characterization is very rapid [3].
The most of the proteins are glycoproteins, that is having short chains of 
carbohydrate residues attached to them and are collagen, elastin, laminin 
and fi bronectin. The protein that, in our case, plays a crucial role in the 
binding of the cells in vivo and can be used as well for the immobilization of 
cells in any substrate is the Fibronectin. In fact this large protein found in all 
vertebrates is able to bind to transmembrane glycoproteins called integrin 
that is present in all the cells of all the animals tested so far. The morphology 
and the function of the fi bronectin is going to be discussed right now, as well 
as the structure of the integrin and the way in which they interact.
1.4.1. The Fibronectin Molecule
The above discussed high-molecular weight extracellular matrix 
glycoprotein
2
 consist in a dimer of two nearly identical polypeptide chains 
connected by means of a C-terminal disulfide bond with a total molecular 
weight of 450 kDa (g/mol). This protein is present in all the vertebrates and 
is composed by a dimer of homologous, repeating structural motifs (classified 
1 - Introduction
6
2
 A glycoprotein is a protein containing an oligosaccharide chain attached to its polypeptide 
side-chain. This oligosaccharide chain is composed by few (typically three to ten) simple 
sugar.
as fi bronectin repeats FN-I, FN-II, and FN-III) that are grouped together 
into functional domains. Every domain is specialized for binding to a 
particular molecule or to a cell, as indicated in fi gure 1.2 (only some of the 
binding site are shown in this fi gure). The main type and the most repeating 
module in the fi bronectin is the type III, containing a sequence of about 90 
amino acids. In this module we can fi nd the RGD sequence (Arg-Gly-Asp) 
that is able to “recognize” some of the proteins belonging to the integrins 
family. Since only few integrins can bind to a specific matrix molecule, we can 
think that the RGD sequence is not the only factor determining the 
interaction between fi bronectin and integrin, but as we have seen in the 
experiments we ran, even synthetic short peptides containing the this RGD 
sequence can compete with fi bronectin for the binding site on cells and be in 
some cases better [4]. It is possible to fi nd the fi bronectin both in the 
extracellular matrix as well as in the plasma, because this is used, in the 
second case, for the wound healing, that is the natural process of regenerating 
dermal tissue and for the blood clotting (coagulation of blood). The structure 
of such protein is really complex and it suggest us that not only chemical 
interactions are responsible for the interaction with cells but also a physical 
conformation of its domains. Being a so big protein, it is easily foldable in 
different configuration, but so far no studies show exactly how the spatial 
organization contributes to the binding.
1 - Introduction
7
Fig. 1.2 Representation of the ninth and tenth repetition of the module III in 
the Fibronectin. The picture is interesting in showing the sites of the RGD loop 
(sticking outside of the molecular profile) and synergy site, that site is required 
for optimum affinity since this site can increase the effectiveness of the adhesion. 
The whole molecule is composed by nearly 2500 amino acid grouped in fi ve/six 
domains. Every domain is responsible for binding to a particular molecule. 
Every arrow show a beta-sheet of the structure where two chains of amino acids 
are kept together by a series of hydrogen bonds. 
The fi bronectin that was used for the experiment is from bovine plasma, 
purchased from SIGMA-AlDRICH
®
 in a concentration of 1 mg/ml (in 0.5 M 
NaCl, 0.05 M Tris, pH 7.5).
1.5. The Cytoskeleton
In every eucaryotes we can fi nd the cytoskeleton. It’s that part of the cell 
contained within the cytoplasm
3
, responsible of many of its vital function, 
1 - Introduction
8
3
 The cytoplasm is the part of the cell encapsulated by the fragile plasma membrane, where 
all the organelle are playing a specific function.
especially related to the shape and conformation. The main functions of the 
cytoskeleton are to provide mechanical strength, enable cellular motility, 
control intracellular transport and support cell division.
Recent studies have shown that this dynamic three dimensional structure 
is present also in procaryotes [5]. This structure that provide support as both 
muscle and skeleton, is responsible for movement and stability. The long 
fibers of the cytoskeleton are polymers of subunits.
The cytoskeleton is basically made up of three families of protein 
molecules that are able to form three main types of filaments:
• The Actin filaments (also known as microfilaments) are composed 
mainly by the actin protein, a threated-like protein fi ber; these very 
small fi laments (3-6 nm in diameter) are also responsible for muscle 
contraction
• The Microtubules are long hollow cylinders made of protein;they are 
more rigid than the actin fi laments because of the biggest diameter 
(about 20-25 nm); they are composed of two subunits called alpha- 
and beta-tubulin
• Intermediate fi laments are around 10 nm thick and they determine cell 
tensile stress
For the sake of this research we have considered the cytoskeleton for the 
important role it plays in tying itself to the extracellular matrix. This is 
possible because of certain kind of proteins present in the cell membrane and 
that we are describing in the next paragraph. The integrins for example is 
responsible for binding the ECM to the cytoskeleton, in particular the 
microfilaments.
1.5.1. The Integrin
Integrins are transmembrane cell adhesion protein that act as matrix 
receptors. They belong to a large family of cell surface receptors which are 
involved in cell-matrix and cell-cell interaction. Therefore these are the 
principal receptors of the main extracellular matrix molecule as, for example, 
1 - Introduction
9
the fi bronectin. They also play an important role in intracellular 
communication. The fi gure 1.3 highlight how the integrin acts as mediator 
between the inner and the outer part of the cell.
Fig. 1.3 Schematic depiction of integrins molecule composed by alpha e beta 
unit. The important role of integrins molecule is highlighted in the picture. In 
fact, as we can see, it is the link between the extracellular matrix and the 
cytoskeleton of the cell containing on the two side a ECM and a cytosolic 
domain binding site. 
The integrins is an heterodimer since it’s assembled from two glycoproteins 
subunits called alpha and beta. So far 16 alpha and 8 beta have been 
identifi ed, being expressed by as many diff erent genes. Not all the 
combination are possible and only 22 different integrins are known nowadays. 
This is why the molecular mass of such a protein vary between 90 kDa to 160 
kDa.
The main difference between integrins and other cell surface receptors is 
that integrins are able to establish a huge number of weak bind with its 
ligand (let’s say the fi bronectin). In fact they have a low affinity with their 
ligands, in the order of 10
6 
- 10
9
 liters/mole. So that one protein alone is not 
enough to attach the cell to matrix or other cells and neither it is if they 
spread along the cell membrane. But when they reach a certain minimal 
1 - Introduction
10
concentration, called focal adhesion (FA), the hemidesmosomes
4
 (HD) have 
the capability to stick to the ECM in the way they keep this situation 
reversible. Cells can indeed have the opportunity to explore the surrounding 
environment and to keep their motility without losing all the binding 
because, as we discussed before, the attachment is due to multiple and 
dynamic connections. Consistent with the hypothesis that integrin-ligand 
binding provides a stable mechanical linkage, the unbinding requires a high 
force comparable to the peak force observed in the unfolding of the strongest 
FN-III modules.
Fibronectin is able to bind different types of integrins (almost ten) but the 
most common receptor for this ligand is the α 5
β 1
 [6]; it has been shown that 
in the case of fi broblast (the kind of cells we have used for our experiments) 
this common receptor collaborate with the α 4
β 1.
By the way both integrins subunits are required for the adhesion, as in the 
presence of cations. Integrins are composed of long extracellular domains 
which adhere to their ligands, and short cytoplasmic domains that link the 
receptors to the cytoskeleton of the cell [8].
1 - Introduction
11
4
 These are stud- or rivet-like structures on the inner basal surface of the cell, specialized for 
the cell adhesion.