Pinocytosis There are two types of pinocytosis: receptor-independent and receptor mediated pinocytosis.

The receptor-independent pinocytosis is also called fluid-phase pinocytosis or constitutive pinocytosis and is present in many cells. For example, macrophage ingests in 30 min the equivalent of the entire plasmalemma, the fibroblasts a third part of plasmalemma, and amoebas have an even greater speed of pinocytosis. As consequence, great amounts of macromolecules in solution are introduced in the cell. But, because the cell surface and volume must be constant, it means that an equivalent amount of materials is added to plasmalemma, simultaneously with the endocytosis, through vesicles that come from the Golgi apparatus. The receptor-mediated pinocytosis is performed with participation of membrane receptors, which recognize specific macromolecules from extracellular fluid. The link between ligand and receptor induces diffusion of the ligand-receptor complex in special areas of plasmalemma, known as caveolae. On the cytoplasmic face these depressions are covered by a network made of a protein named clathrin. A clathrin molecule consists of 2 polypeptide chains: a heavy, long chain with a globular head and a tail, and a light, short one attached at the terminal region of the heavy chain tail. Three clathrin molecules interact at the level of their tails and light chains to form a structure with two changes of directions called triskelion (three feet in Greek). The triskelions polymerize and form a network like pentagons or hexagons. The caveolae become, in the next step, vesicles that will lose their clathrin coat. The receptor-mediated endocytosis concentrates certain ligands in endosomes, and this is an important difference as compared to the receptor-independent endocytosis, in which the concentration of substances from vesicles is equal with the extra-cellular fluid. An important process performed by receptor-mediated endocytosis is the capture of cholesterol in animal cells. The majority of cholesterol molecules are transported in blood as lipid-protein complexes named Low Density Lipoproteins (LDL), which are spherical particles of 22 nm that have a core of around 1,500 cholesterol molecules esterified with fatty acids, surrounded by a peripheral lipidic bilayer with a protein. When a cell needs cholesterol for membrane synthesis, it produces receptors for LDL. These receptors are next inserted in plasmalemma, bind LDL with high specificity, and then the LDL-receptor complexes move in membrane in certain regions where clathrin-coated caveolae are formed. They are included in vesicles that rapidly loss their clathrin network, and fuse with early endosomes. Here the LDL particles dissociate from receptors, and the receptors accumulate at one pole of endosome. A vesicle separates with receptors and goes back to the plasmalemma. The receptors are included in
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plasmalemma and are recycled in this manner. The early endosome fuses with a late endosome, or with a lysosome, to form a digestive vesicle where the cholesterol esters are hydrolyzed. The free cholesterol passes into cytosol where inhibits the synthesis of receptors for LDL and the synthesis of cholesterol. There are persons with a genetic disease with abnormal receptors for LDL, that cannot bind LDL or cannot go to caveolae, and therefore the cells cannot capture cholesterol. As consequence, these persons synthesize cholesterol in great amounts, followed by enormous hiper-cholesterolemia. The massive presence of cholesterol in blood induces the premature formation of atherosclerotic plaques on the blood vessel wall. In most of the cases such patients die at young ages due to complications of atherosclerosis, especially myocardial infarct. After the elucidation of this process a new class of substances statines (inhibit the synthesis of cholesterol) was discovered and introduced in the medical practice for the treatment of hypercholesterolemia and in the treatment of the above mentioned genetic disease.

6.6.5.3 Transcytosis

Transcytosis is a trans-cellular transport of materials included in vesicles that are not intercepted by lysosomes, and pass unmodified through cell, to be released at the opposite pole. By electron microscopy George Palade (1953) described in the cytoplasm of endothelial cells vesicles crossing the cells, and suggested that they had an important role in the exchange of macromolecules between plasma and interstitial fluid. Ion Baciu (Cluj-Napoca) demonstrated high speed of this transport; the vesicles of 40-200 nm appeared in the first 30s-2 min after China ink injection. Nicolae and Maya Simionescu (Bucharest) and George Palade demonstrated that the vesicles could be independent (moving from the lumenal face exposed to blood to the interstitial face exposed to the interstitial liquid), or rarely, the vesicles could fuse and form a continuous channel within the cell. The transcytosis was studied later in other cells. An important process is the transit of antibodies by transcytosis through the enterocyte of newborns from the mothers milk. For this reason the naturally feed newborns are resistant to infections.

6.7 Cell junctions There are stable, permanent structures of plasmalemma, specialized for the intercellular communication. There are 3 types: adherens junctions, tight junctions and gap junctions. 6.7.1 Adherens junctions: desmosomes Desmosomes are junctions that keep the cells together, thus conferring mechanical strength to the tissues that are subject to severe mechanical stress: epithelia, heart muscle, or uterine cervix during delivery. There are three different types of desmosomes: a) Belt desmosomes consist of a continuous ring of actin filaments at the apical end of the interacting cells in an epithelium. The belts from all neighboring cells are placed at the same level, and they are inter-connected by integral proteins molecules of cell adhesion. b) Spot desmosomes are made of two dense disks placed under the plasma membranes of the neighboring cells, inter-connected by integral proteins crossing the membranes and the intercellular space. They also have anchoring sites for intermediate filaments. c) Hemidesmosomes have only a half of the structure of the spot desmosomes and attach the cytoplasmic plaque with the basal lamina. 6.7.2 Tight junctions Tight junctions are regions where the intercellular space disappears, and the adjacent membranes are connected by junction proteins. The junction proteins are arranged in rows and they prevent the leaking of fluids between cells. Thus all the substances must cross the plasma membrane (absorption process). For example, glucose from the intestine lumenus is actively transported at the apical side of cells and then will pass in blood by passive transport at the basolateral side of the plasma membrane. The distribution of these transporters and the membrane domains as well are also maintained by the tight junctions. Another example of tight junction is blood-brain barrier, made of tight junctions between endothelial cells in brain blood vessels. The function of the blood-brain barrier is to protect the brain against certain toxins, but allows glucose, oxygen and carbon dioxide to pass. However there are toxic substances that cannot be stopped by this barrier, such as ethanol, or morphine. In the new-born children the myelin are able to cross the blood-brain barrier, while proteins and the antibiotics cannot cross it.
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6.7.3 Communication junctions: gap junctions and synapses

The gap junction is the most spread junction that can be found in all animal cells. In this type of junction the communication between cells is achieved through protein structures called connexons, that cross the plasmalemma and are prominent on both parts of the lipidic bilayer. Each connexon is made of 6 proteic subunits (connexins). By sticking the ends of connexons, the gap junction with the shape of a tube is formed. The channel is completely isolated from the extracellular fluid and makes possible a direct communication between cells without the influence of the extracellular space. Each junction consists in fact of hundreds of connexons arranged after a hexagonal pattern. Ions and small molecules, under 1,000-1,500 Da (such as glucides, amino acids, nucleotides, hormones, vitamins) can pass through the gap junctions. The electric synapses allow the nervous influx to pass without any intermediates; they are 10,000 times more permeable for metallic ions that the rest of membrane. They are responsible for the electric coupling in myocardium and in the smooth muscle cells of intestine. These channels are dynamic. If the cell is damaged, the Ca2+ enters in the cell and the gap junctions will be closed in the neighboring cells in order to prevent the spreading of the lesions. After the healing processes, the junctions are restored and the communications resumed.

6.8 Implications of membranes in pathology

In any disease changes at the level of membranes appear. a) Genetic diseases due to certain defects in membranes, localized in a single cell type. For example: the genetic diseases of erythrocyte in which certain proteins in membrane are absent or modified. This defect is followed by changes in the shape and size of cells and an increased fragility that results in hemolytic anemias: spherocytosis, eliptocytosis. b) Genetic diseases due to certain generalized membrane defects. In these cases proteins from different types of cells are absent or modified, but the disease is manifested only in organs or tissues where these proteins have important roles. As example, in muscular dystrophies is absent or modified dystrophin, a peripheral protein in muscle cell membrane (related proteins are found in red blood cells and neurons). This disease is manifested in muscles because in muscle cells dystrophin is the main protein involved in the normal permeability of the plasma membrane. When this protein is absent, the permeability of plasma membrane for cytosolic proteins is increased and the cell becomes unable to perform its functions. Even though the
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muscles could be hypertrophiated, the muscular force is reduced. Another example is represented by channelopathies, in which ionic transporters have modified structures. These proteins could be present in all cells, but the diseases appear mainly in the nervous system (some genetic epilepsies) because the inefficient activity of the channel in the nervous cells has higher effects upon the properties of these cells as compared to other cell types. c) Genetic diseases of cell organelles, in which the defect is the absence or modification of a membrane protein. d) Implication of membranes in the infectious diseases. The cell membranes are involved in all infectious diseases. Viruses infect cells after by introducing their genetic material trough the plasma membrane of the host cells. Other pathogens (bacteria, parasites) enter the cell through an entrance gate and must avoid the defense systems of the organism (like phagocytosis). On the other hands, the anti-bodies act upon the membranes of pathogen agents. e) Implication of membranes in autoimmune diseases. In this case, the modifications in the membrane structure are achieved during the cells life. As result, they are recognized as foreign by the organism, which will produce anti-bodies against them. The phagocytes will next attack and destroy these cells and also all cells of the same type in the organism, resulting in auto-immune diseases such as anemias, hepatitis, etc. f) Implication of membranes in cardio-vascular diseases. Is well-known the involvement of ion channels in arterial hypertrension, and also in atheromatosis, besides the defects in the LDL receptors. g) Implication of membranes in cancer. In different malignant cells many changes of the plasma membrane structure were describe, as well as of certain intracellular membranes (i.e of rough endoplasmic reticulum). h) All drugs and therapeutic procedures presume interactions with cell membranes. Most of the drugs act by their binding on membrane receptors. Others pass through plasma membranes and sometimes through other cell membranes.

Chapter 7. NUCLEUS

7.1 Roles and components

Nucleus has two functions: 1) it stores the most of genetic material of a eukaryotic cell (in chromosomes), and 2) it coordinates all cellular functions. The nuclear structure is different in interphase and in cell division. In interphase nucleus has all the components: envelope, chromatin, nucleolus and nuclear matrix and is known as metabolic nucleus. During the cell division, the nuclear envelope disassembles, chromatin condenses and forms chromosomes (genetic nucleus).

7.2 Structure of nucleus in interphase

7.2.1 Nucleus in the living cell

Interphase nucleus can be studied in the living cell or after fixation and staining. In order to study the nucleus in the living cell special techniques of light microscopy are used: phase contrast microscopy or interferential contrast microscopy. The nuclear envelope is observed at the periphery while inside nucleus there is a granular structure (chromatin) and a denser and darker nucleolus that contains dry substance up to 80%. If the nuclear envelope is perforated or removed from the cell using micro-manipulation techniques, the cell dies; if only the plasmalemma is perforated, the cell survives. By video-microscopy, and micro-cinematography movements of nucleus may be observed: rotation, lateral translation and expulsion of degenerated nuclei from the old cells. Nuclei of the cells in the same tissue can be separated by differential centrifugation and studied with physical-chemical methods. Nucleus is denser than cytoplasm and it separates first using at a relative low centrifugal acceleration; it also has a more alkaline pH than the cytoplasm.

7.2.2 Morphology and ultrastructure of nucleus after fixation

7.2.2.1 Position, number, shape and size

Nucleus usually occupies a central position in cells, for example in young spherical cells. In the differentiated cells the nucleus may have a peripheral location (in adipose cells and in striated muscle cells) or close to plasma membrane (in secretory cells).
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As a rule, the nucleus is unique, but there are cells with two nuclei like hepatocytes and chondrocytes and bone cells with maximum 100 nuclei, named osteoclasts. There are also cytoplasm territories with hundreds of nuclei, named sincitia like striate muscle fibers. The shape of nucleus generally corresponds to the shape of cell: it is spherical in round cells, ovoid in cylindrical or prismatic cells; it may be like a spindle or a stick in long cells, or flat in endothelial, adipose and secretory cells. In the polymorphonuclear leukocytes, the nucleus has more lobes. Malignant cells have also nuclei with aberrant shapes. The size of nucleus depends on the tissue and is in average 5-12 m, in gametes is 4 m and in ovocyte is 25 m. The nucleo-plasmatic ratio ranges between 1/3 - 1/20. The young cells with intense metabolism have a big nucleus and high nucleo-plasmatic ratio (1/3); the malignant cells have a high nucleo-plasmatic ratio too, at it is observed during microscopic examination. The adult and old cells have a smaller nucleo-plasmatic ratio.

7.2.2.2 Nuclear envelope

Nuclear envelope has two special features: it consists of two membranes and has pores that are visible on electron micrographs. The nuclear envelope is made of: - Outer nuclear membrane continues with the rough endoplasmic reticulum (RER) membrane; it also may have ribosomes attached on its cytoplasmic side. Inner nuclear membrane adheres to the nuclear content. Between the nuclear membranes lies the perinuclear space that continues with the RER lumen. The two concentrically membranes continue with each other at the level of nuclear pores. - Nuclear lamina is a network of fibrous proteins attached under the inner nuclear membrane, and between pores. It is made of intermediate filaments: tree lamins named A, B and C. The nuclear lamina is responsible for the nuclear shape, and for the organization of chromatin in interphase. It also is crucial for the disassembling of the nuclear envelope in the beginning of the cellular division (in prometaphase) and its reassembling at the end of the cellular division (in telophase). A point de novo mutation in position 1824 of gene LMNA (citosine replaced by timine) on chromosome 1 results in HutchinsonGilford Syndrome (progeria premature ageing). - Nuclear pores (pore complexes or porosomes) have a complex structure and occupy about 10% of the nuclear envelope surface. The nuclear pores are formed in many regions where the
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two nuclear membranes come in contact. The space between the membranes is occupied by the pore complex made of around 100 different proteins. By X-ray diffraction was observed the structure of the nuclear pore complex. It consists of 8 subunits like columns that cross the entire thickness of the nuclear envelope, from the nuclear side to the cytoplasmic side. Each subunit has fibrilar prolongations extended both to the nuclear matrix and to cytoplasm. The inner fibrils are connected by a ring to form the nuclear basket. In the center of the pore was observed a central granule. The pores contribute to the temporal and spatial separation of nuclear and cytoplasmatic processes, for example DNA and RNA synthesis (inside nucleus) and protein synthesis (outside nucleus in cytoplasm). The nuclear pores ensure the communication between nucleus and cytoplasm as they are the most complex transporters of eukaryotic cell. Proteins such as DNApolymerases, RNA-polymerases, histones, ribosomal proteins etc. are imported into the nucleus through the nuclear pores. On the other hand, RNA molecules and the ribosomal precursors are transported from nucleoplasm to cytoplasm. In order to accomplish these transport processes, the nuclear pores have a selective permeability. Ions, water and small molecules can pass rapidly through the nuclear pore, but there is a membrane potential, and the ions pass through a controlled manner. Na+ is present in a lower concentration inside nucleus and K+ has a higher concentration inside nucleus as compared to its cytoplasmic concentration. The potential difference is E = -12 mV. Transport of macromolecules through the nuclear pore is performed by specialized transporters called nuclear transport proteins. They are different according to the direction of transport, and they specifically recognize the molecules to be transported. Proteins that must enter the nucleus have specific sequences of amino-acids known as nuclear localization signals (NLS) and the transporters involved in this process are called nuclear import receptors. The proteins having NLS are attached to the transporter, and the complex is guided to the pore complex by the cytoplasmic fibrils. The pore opens for the import and also the conformation of macromolecule is changed in order to allow the molecule to pass through pore. The both processes use GTP as source of energy. The proteins that are transported towards cytoplasm have different specific sequences recognized by receptors for nuclear export. Gnter Blobel (Nobel Prize in Physiology or Medicine, 1999) discovered the amino-acid sequences called signal sequences and proved that all proteins receive such signals during their synthesis in ribosomes. The signal sequences indicate the final destination of proteins in the different compartments of the cell (nucleus, endoplasmic reticulum, mitochondria, peroxisomes).
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