5.2.2.

2 Movements from microvilli

Microvilli are permanent, small extensions at the surface of different types of cells: enterocytes, hepatocytes, epithelial cells from proximal renal contort tube etc. In most of the cases microvilli have important roles in absorption processes. The absorption is enhanced both through a passive and an active mechanism. The passive mechanism consists in an increased absorption surface (in enterocytes the apical surface is increased of about 20 times by microvilli as compared to the basal surface of the cell). The active mechanism is achieved by the interaction between actin microfilaments and myosin molecules. About 20-30 microfilaments linked by fimbrin and villin are organized in parallel fascicles in the middle of intestinal microvilli. They are anchored to plasmalemma at the +end through a protein region and lateral by lateral arms consisting of myosin I. At their end, the microfilaments link to a perpendicular network made of spectrin and intermediate filaments. The actin-myosin interaction changes the shape of the microvillus that shortens and the absorbed material is pushed into the cytoplasm of the enterocyte.

5.2.2.3 Cytoplasmic currents

Cytoplasmic currents are intracellular movements that move organelles within the cell (jumping movements of mitochondria, cyclosis continuous movement of cytoplasm and chloroplasts in vegetal cells). The cytoplasmic currents are biological phenomena that ensure contact of every organelle with other organelles. Apparently these currents are irregular but they mix the cytoplasm. Light, pH and temperature influence them.

5.3. Movements based on microtubule-dynein mechanism 5.3.1 Axoplasmic transport

Axoplasmic transport is a particular type of cytoplasmic current responsible for transport along the axons of neurons. It has two directions, anterograde (forward), from perikaryon to the end of the axon, and retrograde (backward). By the anterograde transport materials are transported for axonal growth during morphogenesis, and later vesicles with neurotransmitters, mitochondria, vesicles with lipids and proteins necessary to keep constant the membrane surface, and soluble proteins from cytosol
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(actin, tubulin, MAPs etc.). The retrograde transport brings back to perikaryon old organelles and vesicles with neurotransmitters to be recycled. The anterograde transport is classified into: fast transport (3 m/sec, for synaptic vesicles), intermediate transport (for mitochondria), and slow (less than 1 mm/day, for cytoskeleton molecules). The mechanism of axoplasmic transport involves interactions of cellular motors with microtubules and microfilaments (near the terminal end). For the anterograde transport the most important motor protein is kinesin. It is a dimer made of 2 heavy chains and 2 light chains. Each heavy chain has a globular region (head), a central coiled-coil region (-helix), and a tail where the light chains are linked. The heads interact with the microtubule, and because they have ATPase activity they change their conformation and make steps of 8 nm on microtubule, moving from the end to the +end of microtubule. At the level of the light chains cargo is attached. When the kinesin arrives at the +end of the microtubule, the transport is continued on the peripheral actin microfilaments by myosin molecules that are also attached to the various vesicles or to the other materials to be transported to the proximity of plasma membrane. The retrograde transport is performed by dynein molecules that step from the +end to the end of microtubules. 2 classes of dynein are known: cytosolic dyneins and dyneins from axoneme. The dyneins are made of 2-3 heavy chains, and several intermediate and light chains. Each heavy chain has a head that is a globular region with ATPase function and which interacts with the microtubule. In the cytosolic dyneins the tail interacts with the transported material.

5.3.2 Movements of cilia and flagella

Cilia and flagella are considered as varieties of the same cellular structure. The both have an internal structure named axoneme with the same molecular organization and similar mechanisms of movement. They are permanent extensions, longer and bigger than microvilli. In humans ciliated cells are present in bronchia and oviduct (these cells display a high number of cilia on their surface). On the other hand, sperm cells are the only cells with flagellum (the flagellum is unique for a sperm cell). The movement of cilia has two steps: an active step in which the cilium is straight and has a beating movement like a whip (power stroke), and a passive step, when the cilium is bend (recovery stroke). All the cilia move in a coordinated manner, in successive waves. This movement displaces the mucus containing foreign materials like dust and bacteria that are
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eliminated. This way the epithelia are cleaned and the infections are prevented. The flagellum has a continuous helical movement. A cilium consists of 3 parts: free part, roots and basal body. The free part has a complex of microtubules named axoneme surrounded by cytoplasm and plasmalemma. The axoneme is made of 9 peripheral doublets of microtubules surrounding 2 central microtubules. Each peripheral doublet has a complete microtubule (with 13 protofilaments), named A subfiber and an incomplete microtubule (with 11 protofilaments) named B subfiber. On the A subfiber two protein arms made of dynein are linked with their light chains, while the globular heads interact with the adjacent doublet. The dynein arms are always arranged in a clockwise direction. The peripheral doublets are linked together at some intervals by another protein named nexin. The microtubules of the central doublet, connected by a bridge, are also linked to the peripheral doublets through radial spokes. The axoneme in a flagellum has an identical structure, but it is surrounded by mitochondria, arranged in a spiral shape; they provide energy necessary for movement. The flagellum needs more energy than the cilium and it is also much longer so ATP molecules cannot diffuse from cytoplasm. The mechanism of movement is represented by the formation and the disruption of lateral bridges between the dynein arms of a doublet and the tubulins of the adjacent doublet. These bridges disrupted and restored at the next level, drive the doublets to a reciprocal movement. Because they are linked by nexin, they dont slide one on another, but the axoneme bends. If the axonemal dynein is absent or deficient, the cilia and flagella are immotile leading to repeated respiratory infections and sterility. This is immotile cilia syndrome (or primary cilliary dyskinesia) and sometimes it may be associated with an anatomic variant known as situs inversus in which the internal organs are inversed; this association is named Kartagener syndrome.

5.4 Microtubule-organizing centers: centrioles and basal bodies of cilia and flagella

Centrioles and basal bodies are identical structures in terms of molecular organization and they can change their roles. The centrosome, or cell centre, can bee seen next to nucleus in interphase in most of the animal cells. It consists of 2 L-shaped centrioles surrounded by a dense pericentriolar material that contains a specific protein named pericentrin and -tubulin. They are cylindrical structures made of 9 triplets of microtubules, and with an empty central region. In each triplet one
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complete (13 protofilaments) and two incomplete (11 microfilaments) microtubules are present. In interphase the centrosome co-ordinates the dynamic network of microtubules. During cellular division the two centrioles replicate and form the two poles of the mitotic spindle. The microtubules have the end in the pericentriolar material and the +end distal. On the other hand, axoneme continues with the basal body. The axonemal doublet becomes triplet, and the central microtubules stop at the level of basal body. As the cytoplasmic microtubules, the microtubules from axoneme have their end in the basal body and the +end distal. The basal bodies coordinate the movements of cilia, flagella and the polymerization of tubulin within axoneme. The nexin, the spokes and the internal sheath also contribute to the coordination of movements of cilia and flagella. In particular cases the basal bodies can take the functions of centrioles. For example, the unicellular alga Chlamydomonas has 2 flagella and no centrosome. When this alga begins mitosis, the flagella retract, the basal bodies migrate next to the nucleus and become centrioles. It was also observed that if one of the 2 flagella is cut off, the remaining flagellum shortens and the first one reassembles, using the tubulin molecules obtained from the depolymerization of the remaining flagellum. The polymerization and depolymerization processes are coordinated by the basal bodies. Later new tubulin molecules will be synthesized and both flagella will be rebuilt to the normal length.

Chapter 6. Molecular biology of cell membranes

6.1 Definition and functions

Biological membranes are two-dimensional structures made of proteins and lipids, which separate the living matter and have selective permeability. The main functions of cell membranes are: a) barrier: they separate the content of cells from the environment or separate different cell compartments b) to make compartments within cells: membranes of cell organelles c) metabolic functions (by membrane enzymes): the most complex metabolic processes in the nature (oxidative phosphorylation and photosynthesis) are membrane processes d) control of information flow: they receive information from environment, or from other cells through specific receptors. They also emit information as chemical or electrical signals, thus having role in the intercellular communication e) transduction of signals received from environment or from other cells f) role in immunity, in defence against infections g) membranes can undergo adaptive functional modifications Types of membranes. In the eukaryotic cell there are the following main types of membranes: 1. cell membrane (plasmalemma): has a barrier role, providing cell individuality, but it is also the membrane that establishes communication with other cells; 2. membranes of cell organelles (nuclear membranes, of the endoplasmic reticulum, Golgi apparatus, mitochondria, peroxysomes, lysosomes), and of other vesicles from cytoplasm. They have both compartmentalization role and also metabolic functions through their membrane enzymes and receptors; 3. special membranes: the myelin sheath of nerves (which has mainly a barrier role), the membrane of the discs in the rod cells of retina

6.2 Chemical composition of biological membranes

The main components of membranes are proteins and lipids. Glucidic (carbohydrate) groups are also attached to proteins and lipids. The proteins : lipids ratio depends on membrane functions: the higher the metabolic function of a membrane, the higher is its protein percentage even up to 75% in the inner mitochondrial membrane (where the oxidative phosphorylation
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occurs), in the inner membrane of chloroplasts (where photosynthesis takes place), and the bacterial membrane. In membranes with barrier function, the amount of lipids is higher (80%) than of proteins. Lipids The membrane lipids are of three categories: phospholipids, glycolipids and cholesterol. They were briefly presented in the chapter 3. Lipids from membranes are organized in a lipidic bilayer, which has the property of fluidity. Phospholipids are phosphoglycerides and sphingolipids. Phosphoglycerides consist of a glycerol molecule in which two hydroxyl groups are esterified with fatty acids, and the third position is occupied by a polar group. Usually, at the opposite end to the polar group there is a saturated acid, and in the middle, an unsaturated one (with one or more double bonds). The length of fatty acid chains can vary between 12 to 24 C atoms. The polar group of phosphoglycerides has a phosphoric acid residue on which is bound one of the following: choline (in lecithin or phosphatydilcoline), ethanolamine (in phosphatydilethanolamine), serine (in phosphatydilserine), inositol (in phosphatydilinosytol). Sphingolipids have a sphingosine molecule, an amino-alcohol with a long chain of C atoms. The structure of sphingolipids is generally the same like in phosphoglycerides, having a polar group and two hydrophobic chains. As compared to phosphoglycerides they always have the same first long chain, while the second chain (of the fatty acid) is bound by an amide linkage. Sphingolipids have a similar three-dimensional structure with phosphoglycerides. The most important sphingolipid is sphingomyelin (with the same polar group as lecithin). Glycolipids in animal cells are also based on the structure of sphingosine as in sphingomyelin, but instead of the phosphorylcholine polar group, they have glucidic residues. In the simplest glycolipids, called cerebrosides, the polar group is only such a residue, for example glucose or galactose. Galactocerebroside is a major component of myelin, and is present in a ratio of 40% in the outer layer of the Schwann cell plasmalemma. The most complex glycolipids are gangliosides, which contain one ore more sialic acid residues, conferring a negative electrical charge. Gangliosides represent about 6% of the total amount of lipids in plasmalemma of neurons, and they are also present in small amounts in most of the cells. Cholesterol is another major lipid in the eukaryotic cell membranes. The cholesterol ratio is higher in plasmalemma and in myelin (where the barrier function prevails), and lower in intracellular membranes. The lipid composition of cell membranes is different from one type to another, even in the
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same cell, from one species to another, and from one cell to another (for the same type of membrane), with consequences upon the physical characteristics of the membrane, as well as upon the activity of membrane proteins. There are four major phospholipids in all cell membranes: phosphatydilcoline, phosphatydilethanolamine, phosphatydilserine, and

sphingomyelin. Phosphatydilinosytol must be also mentioned as a major lipid in certain membranes, and cardiolipin in the inner mitochondrial membrane. A common property of all these lipid molecules is their amphiphile structure. They have a hydrophilic head formed by the polar group, and a hydrophobic tail, formed by the nonpolar fatty acid chains. As consequence, when placed in water they can spontaneously form micelle, but the most stable shape is lipid bilayer, which represents the structural basis of the biological membranes. Thus, formation of the lipid part of a membrane is an auto-assembling process. Finally, the lipid bilayer has another characteristic fluidity, which makes it adequate for the membranes structure. An artificial lipid bilayer made by only one type of phospholipid can pass from a gel state (crystalline) into a fluid state (liquid crystal) at a specific temperature. This change was called phase transition. As the fatty acids in the lipid bilayer are shorter and more unsaturated, as the fluidity of the bilayer is higher, and the transition temperature is lower. The transition temperatures are characteristic for each membrane. In the physical chemistry, the fluidity is the opposite of viscosity, and is applicable to the isotropic liquids (with identical properties in all three directions of the space). Biological membranes are not isotropic, and the term of fluidity concerns many factors among which the mobility of lipids is an essential one. There are several types of movements. 1. Movements inside the phospholipid molecule: a) flexion movements of C atoms in methylene groups (-CH2-) of the fatty acid chains (more mobile to the middle of the bilayer and more rigid towards polar group); b) movements of atoms of the polar group; 2. Movements of entire molecule of phospholipid: a) movement of lateral diffusion; b) rotation movement around the longitudinal axis of the molecule; c) movement of transversal diffusion from one layer to another, named flip-flop; The lateral diffusion of phospholipids is performed very rapid, thus a molecule can cover a distance of around 2 m (equivalent to the length of a bacterium) in 1sec. The rotation movement is also rapid, while the transversal diffusion requests the intervention of certain proteins called flipases, and it is performed very slow.
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The membrane fluidity is strongly influenced by cholesterol. When is added in artificial lipid bilayers containing saturated lipids, cholesterol increases the membrane fluidity, while in the case of unsaturated lipids it decreases the membrane fluidity. It must be also mentioned that the membrane lipids from all cells are present in liquid crystal state, therefore in a fluid state at physiological temperatures. The membrane fluidity is an essential condition for the deployment of all functions of biological membranes. If the temperature at which cells are cultured is modified in vitro, the fatty acids composition of their membranes is also changed in order to keep the fluid state. Proteins While the lipids provide mainly a barrier function of membranes, the membrane proteins confer functionality of membranes: they achieve transport of particles across membranes, accomplish enzymatic functions or are receptors. This is why the protein ratio is higher in the membranes with more functions as compared with those with barrier role. Sizes of proteins are also bigger than of the lipids; for a percentage of 50% proteins in a membrane 1 protein molecule corresponds to 50 lipid molecules. Some proteins can be easily extracted by changing the ionic strength of solutions; these are called peripheral proteins (extrinsic), and are attached at the external side of the lipid bilayer. They interact by electrostatic forces with the polar heads of membrane lipids, or even with the integral proteins (intrinsic). The latter represent a second category of membrane proteins; they have a very hydrophobic part included in the lipid bilayer and tight linked to the hydrophobic region of lipids. The integral proteins can be extracted from membranes only after the destruction of the membranes with detergents. A third category of proteins, were described. They are proteins anchored in plasmalemma by lipid anchors (fatty acyl chains) myristate, palmitate, or by farnesyl residues. Identification and characterization of membrane proteins is performed by polyacrylamide gel electrophoresis in presence of sodium-dodecylsulphate (SDS-PAGE).

6.3 Molecular organization of the biological membranes

Fluid mosaic model, formulated by Singer and Nicholson (1970) is now accepted for the structure of biological membranes. In this model the peripheral proteins are attached to the lipid bilayer and the integral proteins are inserted into the bilayer. Electron microscopy images obtained by freeze-fracture of membranes showed these integral proteins.
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Because the lipid bilayer is fluid, proteins can also move. The membrane proteins have movements of rotation around their longitudinal axis (perpendicular on the membrane), and of lateral diffusion. The lateral diffusion was demonstrated by fusion between a human cell and a mouse cell under the action of the Sendai virus. The membrane proteins were previously labelled with two different fluorochromes, and after one hour at 37C the mixed colours of the fluorochromes indicated reciprocal protein diffusion. The biological membranes are asymmetric. The asymmetry concerns both the protein and lipids. The proteins are amphipatic, very big molecules having only a small intramembrane domain. For glycoproteins, the glucidic residues are always located opposite to the cytoplasm (either outside the cell, or inside the organelles). The phospholipids have a specific distribution in the two layers of a membrane. The erythrocyte has in the outer monolayer lecithin and sphingomyelin (75%) and in the inner monolayer cephalin and phophatidylserine prevail. The glucidic groups of glycolipids are also asymmetric: they are located always on the exoplasmic surface of membranes (opposite to the cytoplasm).