**2. Lipid rafts and membrane heterogeneity**

Even though the Cell Theory started to be developed in the nineteenth century [10], it was not until the first quarter of the twentieth century that the idea of a membrane encompassing the cell was experimentally observed. In 1924, the Dutch physiologists Gorter and Grendel elegantly demonstrated, for the first time, the existence of a lipid bilayer surrounding red blood cells of various animals [11]. They isolated erythrocytes from humans and different mammals (rabbit, dog, guinea pig, sheep, and goat), extracted their lipids using acetone, and let those lipids spread on an air-water interface of a Langmuir-Adam apparatus. By knowing the number and area of the erythrocytes used in their experiment, they concluded that those cells were surrounded by a layer of lipids whose thickness was equivalent to two molecules [11], hence a lipid bilayer. The Gorter and Grendel model for cellular plasma membrane considered only the lipid nature of this cellular component and, because of that and due to other experimental and theoretical inaccuracies, it failed in explaining satisfactorily experimental results for membrane thickness [12], membrane tension [13], membrane electrical capacitance [14], and membrane permeability [15].

In order to explain those membrane properties, another model, called the paucimolecular model, was proposed by Danielli and coworkers in 1935 [12, 16, 17]. By examining the surface tension of a single drop of mackerel egg oil, Danielle and Harvey found that the value they measured was lower than the equivalent obtained for nonliving pure water-oil systems. They hypothesized that the difference observed for surface tension in living versus nonliving water-oil systems was due to the fact that the plasma membrane not only contained lipids but also proteins adsorbed in the lipid bilayer [16]. In the same year, Danielli and Davson [17] extended the paucimolecular model in order to explain permeability experiments. In that model, the layer of proteins adsorbed on top of a lipid film was able to discern size of molecules and charge of ions that were penetrating the membrane. This lipid film containing adsorbed proteins was considered to be relatively stable with mosaics consisting of practically impenetrable regions and hydrated areas where anions could move through [17].

For the next 30 years, the paucimolecular model was the most accepted one among the scientific community. However, with the advancement of microscopy techniques and structural studies, a new and more robust model, named the fluid mosaic model, was proposed in 1972 by Singer and Nicolson [18]. According to that model, integral transmembrane proteins are arranged in the plasma membrane of living cells such that the polar regions are facing the aqueous phase and the hydrophobic regions are embedded on a viscous phospholipid bilayer and those proteins are able to move freely on that two-dimensional, approximately homogeneous fluid "sea" of phospholipids [18]. One year later, in 1973, Bretscher published a Science paper in which he discusses overall membrane organization based on evidences collected from experiments performed in red blood cells [19]. According to that paper, the plasma membrane of mammalian cells was not as simple as depicted by the fluid mosaic model. Some of the integral proteins span the membrane and their glycosylation is responsible for locking them at the membrane impeding their migration to the cytoplasm. Another important contribution from this paper is that proteins not only interact with the outer layer of the plasma membrane but also with the inner layer, and membrane proteins are a subtype of cytoplasmic proteins that are not secreted [19]. In the same year, Yu and collaborators, also performing experiments in red blood cells, showed that when those cells are incubated with the nonionic detergent Triton X-100, there are some fractions of the cellular proteins that are resistant to the detergent extraction and seem to form oligomeric complexes with some of the lipid components, which were preferentially composed by nonglycosylated proteins and sphingolipids [20].

#### **2.1. Lipid rafts**

that contain sphingolipids and cholesterol and are responsible for compartmentalizing and regulating several intracellular signaling events [4–6]. One way of studying the importance of cholesterol for a specific cellular function is to decrease its concentration by either interfering directly with its synthesis, through the mevalonate pathway, or by chelating the molecule directly through the use of cyclodextrins [7]. However, depending on how one does the cholesterol depletion, the effects on cellular contractility can be opposite. Differences in muscle versus nonmuscle cell contractile behavior are observed upon cholesterol depletion using cyclodextrins. Muscle cells get impairment in their contractile machinery [8] whereas nonmuscle cells get more contractile [9]. This book chapter gives an overview about how cholesterol is organized at

Even though the Cell Theory started to be developed in the nineteenth century [10], it was not until the first quarter of the twentieth century that the idea of a membrane encompassing the cell was experimentally observed. In 1924, the Dutch physiologists Gorter and Grendel elegantly demonstrated, for the first time, the existence of a lipid bilayer surrounding red blood cells of various animals [11]. They isolated erythrocytes from humans and different mammals (rabbit, dog, guinea pig, sheep, and goat), extracted their lipids using acetone, and let those lipids spread on an air-water interface of a Langmuir-Adam apparatus. By knowing the number and area of the erythrocytes used in their experiment, they concluded that those cells were surrounded by a layer of lipids whose thickness was equivalent to two molecules [11], hence a lipid bilayer. The Gorter and Grendel model for cellular plasma membrane considered only the lipid nature of this cellular component and, because of that and due to other experimental and theoretical inaccuracies, it failed in explaining satisfactorily experimental results for membrane thickness [12], membrane tension [13], membrane electrical capacitance

In order to explain those membrane properties, another model, called the paucimolecular model, was proposed by Danielli and coworkers in 1935 [12, 16, 17]. By examining the surface tension of a single drop of mackerel egg oil, Danielle and Harvey found that the value they measured was lower than the equivalent obtained for nonliving pure water-oil systems. They hypothesized that the difference observed for surface tension in living versus nonliving water-oil systems was due to the fact that the plasma membrane not only contained lipids but also proteins adsorbed in the lipid bilayer [16]. In the same year, Danielli and Davson [17] extended the paucimolecular model in order to explain permeability experiments. In that model, the layer of proteins adsorbed on top of a lipid film was able to discern size of molecules and charge of ions that were penetrating the membrane. This lipid film containing adsorbed proteins was considered to be relatively stable with mosaics consisting of practically

impenetrable regions and hydrated areas where anions could move through [17].

For the next 30 years, the paucimolecular model was the most accepted one among the scientific community. However, with the advancement of microscopy techniques and structural studies, a new and more robust model, named the fluid mosaic model, was proposed in 1972 by Singer and Nicolson [18]. According to that model, integral transmembrane proteins are

the plasma membrane and how its depletion changes cellular contractile properties.

**2. Lipid rafts and membrane heterogeneity**

34 Cholesterol - Good, Bad and the Heart

[14], and membrane permeability [15].

The idea of possible membrane microdomains started to be speculated in the early 1970s [20, 21] and experimentally demonstrated in 1982 by Karnovsky [22], who showed that there were multiple phases in the lipid environment of a membrane. One type of microdomain can be formed by cholesterol and sphingolipids [23]. These microdomains were already shown to be present in cell membranes [24]. In 1988, after several experimental demonstrations, Simons and van Meer called these microdomains as lipid rafts [25]. Thus, lipid rafts are defined as small, heterogeneous, and highly dynamic microdomains enriched in cholesterol, glycosphingolipids, and proteins that are much more organized than the surrounding lipid bilayer [26]. These membrane microdomains serve as organizing clusters capable of influencing several cellular processes such as membrane trafficking and neurotransmission [26].

The most striking difference between lipid rafts and the plasma membrane from which they are derived from is the lipid composition. Experiments have shown that rafts contain much more cholesterol than the surrounding bilayer [27, 28]. Cholesterol, therefore, works as a sort of "dynamic glue" that maintains the raft together [29], serving as a molecular spacer and filling the empty spaces between sphingolipids [30]. One of the main challenges when studying lipid rafts in living cells is their size. They are small microdomains ranging from 10 to 200 nm, below the classical diffraction limit of the optical microscope [28]. The first studies in the field considered methods to extract and separate rafts from the surrounding membrane. The procedure would take advantage of lipid raft resistance to nonionic detergents. When detergents are added to cells, the fluid membrane will dissolve while the lipid rafts may remain intact and could be extracted [31]; however, the validity of this methodology has been called into question due to ambiguities in the lipids and proteins obtained after extraction [32]. Other methods, based on synthetic membranes, were also used, however with many drawbacks. Firstly, synthetic membranes either lack or have lower protein concentration when compared to cell membranes [26]. Secondly, it is very difficult to simulate, in synthetic membranes, the membrane-cytoskeletal interactions that occur in cell membranes, although some recent studies have been able to overcome these limitations [33–35]. Finally, another problem includes the lack of natural asymmetry between the bilayer leaflets [36].

expressed in various cell types such as smooth muscle, fibroblasts, endothelial cells and adipocytes, among several others. Their functions are diverse and include endocytosis, calcium

Role of Membrane Cholesterol in Modulating Actin Architecture and Cellular Contractility

http://dx.doi.org/10.5772/intechopen.76532

37

The major constituent of caveolae is caveolin1 [46], followed by two other isoforms: caveolin2 [47] and the muscle-specific caveolin3 [48]. All three caveolin proteins share a common topology with both their N and C terminal domains in the cytoplasm and a long hairpin transmembrane domain. All three types of caveolin are formed inside the cells, more precisely in the Golgi apparatus, as monomers [49]. However, as soon as they enter in the secretory pathway, they start to be structured as oligomers [50]. For caveolin1, for example, its exit from the Golgi apparatus is accelerated upon addition of cholesterol [49]. The oligomerization ability of caveolin1 is crucial for caveolae formation [51]. Caveolin2 has also been implicated in caveolae formation [52], and although caveolin1 null mouse shows a significant decrease in caveolae assembly, they are still present in the caveolin2 mouse [53]. In muscle, caveolin3 is crucial for caveolae formation. Mutations or loss of caveolin3 result in dystrophic phenotypes [54, 55].

Caveolin expression at the plasma membrane is not the only inducer of caveolae formation. Cholesterol extraction has been extensively shown to disrupt caveolae at the plasma membrane [46] since it is required for caveolin incorporation into raft domains at the plasma mem-

Although caveolins and cholesterol were initially thought to be necessary and sufficient for caveolae formation, several studies have shown additional molecular players called cavins. This protein family has four different members already described: cavin-1 (also called PTRF) [57], cavin-2 (also called SDPR) [58], cavin-3 (also called SRBC) [59], and the muscle-specific cavin-4 (also called MURC) [60]. These four proteins are essential to caveolae formation and functions. Thus, caveolae formation is a highly complex and regulated cellular process. It has been estimated that ~150–200 caveolin monomers are necessary to associate with ~50–60 cavins in order to form a single caveola [61, 62]. Moreover, caveolae architecture was recently proposed to be a dodecahedron formed by cavins aligned with their vertices and also in the

As already mentioned, caveolae represent a subdomain of lipid rafts [43]. Confocal microscopy has shown that the distribution of GM1, a well characterized raft marker, do not merge with caveolin1 [63]. Another raft marker, flotillin, defines noncaveolar rafts and merges with GM1 [63]. Thus, rafts exhibit a heterogeneous distribution over the plasma membrane chang-

**3. Actomyosin cytoskeleton: the contractile machinery of muscle and** 

The cytoskeleton constitutes a dynamic network of filaments that exists in the inner space of a cell. This network not only provides scaffolding but is also responsible for transporting organelles, generating and transducting mechanical forces. The cytoskeleton maintains cellular organization by linking together several cellular components in such a way that it

signaling as well as regulation of various cell signaling pathways [45].

brane, a critical event for caveolae formation [56].

**nonmuscle cells**

caveolin oligomers located at each of the pentagonal faces [61, 62].

ing between caveolar (invaginated) and noncaveolar (planar) regions.

Although lipid rafts present sizes below the classical diffraction limit of the optical microscope, fluorescence microscopy has been extensively used in the field. For example, fluorophores conjugated to cholera-toxin B-subunit, which binds to the raft constituent ganglioside GM1, is used. Also, lipophilic membrane dyes (such as Laudran) that either partition between rafts and the surrounding membrane or change their fluorescent properties in response to membrane phase are used. Finally, lipid rafts can also be fluorescently labeled in cells after genetic expression of fluorescent fusion proteins [35].

Another methodology, which has been widely used in the study of lipid rafts, is the manipulation of cholesterol contents in membranes. Sequestration (using filipin, nystatin, or amphotericin), depletion and removal (using methyl-β-cyclodextrin, MβCD), or inhibition of cholesterol synthesis (using 3-hydroxy-3-methyl-glutaryl-coenzyme A, HMG-CoA, reductase inhibitors) are great examples of how cholesterol can be manipulated in lipid raft studies [26]. Several questions, however, have been raised against the effectiveness of the experimental design when disrupting lipid rafts. Acute methods of cholesterol depletion, which disrupt the rafts, can also disrupt another lipid, called PI(4,5)P2, which plays an important role in cytoskeletal regulation [37]. Thus, the loss of a particular cellular function after cholesterol depletion cannot necessarily be attributed only to raft disruption, since other processes are also being affected.

Despite these limitations, more sophisticated methods have been applied in order to fight against the problems of small size and dynamic nature of lipid rafts. These methods include single particle and molecule tracking using very sensitive CCD cameras together with total internal reflection microscopy. These combined techniques provide information of the diffusion coefficient of particles in the membrane and also reveal membrane corrals, barriers, and sites of confinement [38]. Finally, other optical techniques have been used to elucidate other features of lipid rafts: fluorescence correlation spectroscopy, to gain information of fluorophore mobility in the membrane [39]; fluorescence resonance energy transfer, to detect when fluorophores are in close proximity [40], and optical tweezers, to give information about the membrane mechanical parameters [8, 41]. In the future, it is expected that other super-resolution microscopy techniques, such as stimulated emission depletion microscopy [42] or various forms of structured illumination microscopy may overcome the problems imposed by the diffraction limit.

Apart from the different imaging methods, research over the last decades have demonstrated the existence of two types of rafts: (1) planar lipid rafts (also known as noncaveolar or glycolipid rafts) and caveolae. Planar rafts are known to be continuous with the plane of the plasma membrane (not invaginated) and contain flotillin proteins. Caveolae are flask shaped invaginations of the plasma membrane that contain caveolin proteins. Both types are enriched in cholesterol and sphingolipids. Flotillin and caveolins can either recruit or separate other molecules from lipid rafts and caveolae, respectively, thus playing an essential role in signal transduction [43].

#### **2.2. Caveolae**

Caveolae are plasma membrane invaginations with a diameter ranging from 60 to 80 nm and were first identified in the early 1950s by electron microscopy [44]. These invaginations are expressed in various cell types such as smooth muscle, fibroblasts, endothelial cells and adipocytes, among several others. Their functions are diverse and include endocytosis, calcium signaling as well as regulation of various cell signaling pathways [45].

some recent studies have been able to overcome these limitations [33–35]. Finally, another

Although lipid rafts present sizes below the classical diffraction limit of the optical microscope, fluorescence microscopy has been extensively used in the field. For example, fluorophores conjugated to cholera-toxin B-subunit, which binds to the raft constituent ganglioside GM1, is used. Also, lipophilic membrane dyes (such as Laudran) that either partition between rafts and the surrounding membrane or change their fluorescent properties in response to membrane phase are used. Finally, lipid rafts can also be fluorescently labeled in cells after

Another methodology, which has been widely used in the study of lipid rafts, is the manipulation of cholesterol contents in membranes. Sequestration (using filipin, nystatin, or amphotericin), depletion and removal (using methyl-β-cyclodextrin, MβCD), or inhibition of cholesterol synthesis (using 3-hydroxy-3-methyl-glutaryl-coenzyme A, HMG-CoA, reductase inhibitors) are great examples of how cholesterol can be manipulated in lipid raft studies [26]. Several questions, however, have been raised against the effectiveness of the experimental design when disrupting lipid rafts. Acute methods of cholesterol depletion, which disrupt the rafts, can also disrupt another lipid, called PI(4,5)P2, which plays an important role in cytoskeletal regulation [37]. Thus, the loss of a particular cellular function after cholesterol depletion cannot necessarily

Despite these limitations, more sophisticated methods have been applied in order to fight against the problems of small size and dynamic nature of lipid rafts. These methods include single particle and molecule tracking using very sensitive CCD cameras together with total internal reflection microscopy. These combined techniques provide information of the diffusion coefficient of particles in the membrane and also reveal membrane corrals, barriers, and sites of confinement [38]. Finally, other optical techniques have been used to elucidate other features of lipid rafts: fluorescence correlation spectroscopy, to gain information of fluorophore mobility in the membrane [39]; fluorescence resonance energy transfer, to detect when fluorophores are in close proximity [40], and optical tweezers, to give information about the membrane mechanical parameters [8, 41]. In the future, it is expected that other super-resolution microscopy techniques, such as stimulated emission depletion microscopy [42] or various forms of structured

problem includes the lack of natural asymmetry between the bilayer leaflets [36].

be attributed only to raft disruption, since other processes are also being affected.

illumination microscopy may overcome the problems imposed by the diffraction limit.

Apart from the different imaging methods, research over the last decades have demonstrated the existence of two types of rafts: (1) planar lipid rafts (also known as noncaveolar or glycolipid rafts) and caveolae. Planar rafts are known to be continuous with the plane of the plasma membrane (not invaginated) and contain flotillin proteins. Caveolae are flask shaped invaginations of the plasma membrane that contain caveolin proteins. Both types are enriched in cholesterol and sphingolipids. Flotillin and caveolins can either recruit or separate other molecules from lipid rafts and caveolae, respectively, thus playing an essential role in signal transduction [43].

Caveolae are plasma membrane invaginations with a diameter ranging from 60 to 80 nm and were first identified in the early 1950s by electron microscopy [44]. These invaginations are

genetic expression of fluorescent fusion proteins [35].

36 Cholesterol - Good, Bad and the Heart

**2.2. Caveolae**

The major constituent of caveolae is caveolin1 [46], followed by two other isoforms: caveolin2 [47] and the muscle-specific caveolin3 [48]. All three caveolin proteins share a common topology with both their N and C terminal domains in the cytoplasm and a long hairpin transmembrane domain. All three types of caveolin are formed inside the cells, more precisely in the Golgi apparatus, as monomers [49]. However, as soon as they enter in the secretory pathway, they start to be structured as oligomers [50]. For caveolin1, for example, its exit from the Golgi apparatus is accelerated upon addition of cholesterol [49]. The oligomerization ability of caveolin1 is crucial for caveolae formation [51]. Caveolin2 has also been implicated in caveolae formation [52], and although caveolin1 null mouse shows a significant decrease in caveolae assembly, they are still present in the caveolin2 mouse [53]. In muscle, caveolin3 is crucial for caveolae formation. Mutations or loss of caveolin3 result in dystrophic phenotypes [54, 55].

Caveolin expression at the plasma membrane is not the only inducer of caveolae formation. Cholesterol extraction has been extensively shown to disrupt caveolae at the plasma membrane [46] since it is required for caveolin incorporation into raft domains at the plasma membrane, a critical event for caveolae formation [56].

Although caveolins and cholesterol were initially thought to be necessary and sufficient for caveolae formation, several studies have shown additional molecular players called cavins. This protein family has four different members already described: cavin-1 (also called PTRF) [57], cavin-2 (also called SDPR) [58], cavin-3 (also called SRBC) [59], and the muscle-specific cavin-4 (also called MURC) [60]. These four proteins are essential to caveolae formation and functions. Thus, caveolae formation is a highly complex and regulated cellular process. It has been estimated that ~150–200 caveolin monomers are necessary to associate with ~50–60 cavins in order to form a single caveola [61, 62]. Moreover, caveolae architecture was recently proposed to be a dodecahedron formed by cavins aligned with their vertices and also in the caveolin oligomers located at each of the pentagonal faces [61, 62].

As already mentioned, caveolae represent a subdomain of lipid rafts [43]. Confocal microscopy has shown that the distribution of GM1, a well characterized raft marker, do not merge with caveolin1 [63]. Another raft marker, flotillin, defines noncaveolar rafts and merges with GM1 [63]. Thus, rafts exhibit a heterogeneous distribution over the plasma membrane changing between caveolar (invaginated) and noncaveolar (planar) regions.
