Molecular organization of the nuclear pore complex. Nuclear pores: description, structure and functions. Properties of nuclear pores
3.2.1. Impairment of the barrier function of biological membranes
Their processes play an important role in membrane damage. mechanical stretching as a result of disturbance of osmotic balance in cells. If you place red blood cells in a hypotonic solution, water will enter the cells, they will take a spherical shape, and then hemolysis will occur. Mitochondria also swell in hypotonic environments, but only the outer membrane ruptures; the inner one remains intact, although it loses its ability to retain small molecules and ions. As a result, mitochondria lose their ability to undergo oxidative phosphorylation.
Similar phenomena are observed in whole cells and tissues under pathological conditions. Thus, as a result of activation of phospholipase A 2, mitochondrial membranes during hypoxia become permeable to potassium ions. If tissue oxygenation is restored under these conditions, then the membrane potential on the mitochondrial membranes will be restored (with a minus sign inside) and the mitochondria will “pump” potassium ions, after which phosphate enters the matrix. The concentration of ions inside the mitochondria increases, and the organelles swell. This leads to membrane stretching and further damage.
Molecular mechanisms of increasing the permeability of the lipid layer of membranes for ions. When studying the molecular basis of the permeability of the lipid layer, model membrane systems are widely used: isolated membrane structures (erythrocytes, mitochondria, vesicles of the sarcoplasmic reticulum), as well as artificial phospholipid membranes (bilayer lipid membranes and phospholipid vesicles - liposomes). The study of such systems has shown that the lipid layer itself is practically impermeable to ions. Under the influence of various chemical and physical factors it becomes permeable for one of three reasons (or combinations thereof):
1. In the lipid bilayer, the microviscosity of which is close to the viscosity of olive oil, a fat-soluble substance appears that can bind ions. The mechanism of ion transfer in this case resembles “transporting passengers in a boat from one shore to
another" and is called "shuttle", or transfer with the help of a movable carrier. An example of a mobile carrier is the ionophore antibiotic valinomycin, which forms a complex with potassium ions, soluble in the lipid phase of the membrane. Mobile carriers may include water-soluble products of lipid peroxidation, the presence of which, as it turns out, increases the permeability of the membrane to hydrogen ions.
2. Substances appear in the lipid layer, the molecules of which, when collected together, form a channel through the membrane. Through such a channel, ions can pass from one side of the membrane to the other. The channels are formed by certain antibiotic molecules, such as gramicidin A and polymyxin. Lipid peroxidation products can also form channels in the lipid layer if there are calcium ions in the solution. The products of the cleavage of some phospholipids (in particular, cardiolipin) by phospholipase A 2 form channels for monovalent cations.
3. The electrical strength of the lipid layer of the membrane decreases, and its section is destroyed by electric current, which occurs under the influence of the potential difference existing on the membrane. This phenomenon is called “electrical breakdown” (see below). The formation of “pores” in the membrane with the induction of membrane breakdown underlies violations of the barrier function of the membrane during the adsorption of polyelectrolytes, proteins foreign to the cell, and antibodies on the lipid bilayer.
Free radicals. Free radical (lipid peroxidation). It is well known that in organic molecules (including those that make up our body), electrons in the outer electron shell are arranged in pairs: one pair in each orbital. Free radicals differ from ordinary molecules in that they have an unpaired (single) electron in their outer electron shell. This makes them chemically active as they seek to regain the missing electron by taking it away from the surrounding molecules and thereby damaging them. Free radicals react with inorganic and organic compounds - proteins, lipids, carbohydrates, nucleic acids, and initiate autocatalytic reactions, during which the molecules with which they react also turn into free radicals. Thus,
Free radicals are highly active molecules that can destroy cell structures.
The main source of radicals is molecular oxygen. TO oxygen radicals include: NO* (nitric oxide or nitroxide), RO* (alkoxy radical), RO* 2 (peroxide or peroxide radical), O* 2 - (superoxide anion radical or superoxide), HO* 2 (hydroperoxide radical, HO* (hydroxyl radical).
In general, all radicals formed in the human body can be divided into natural and foreign. In turn, natural radicals can be divided into primary, secondary and tertiary (Fig. 3-2).
Primary radicals- those radicals whose formation is carried out with the participation of certain enzyme systems (NADPH oxidase, NO synthase, cyclooxygenase, lipoxygenase, monooxygenase, xanthine oxidase, etc.). First of all, the primary radicals include semiquinones formed in the reactions of such electron carriers as coenzyme Q (we denote the radical as Q*) and flavoproteins, O* 2 -, NO*.
Rice. 3-2. Classification of radicals in the human body
From the primary radical - O* 2 -, as well as as a result of other reactions in the body, very active molecular compounds are formed: hydrogen peroxide (H 2 O 2), hypochlorite (HOCl), lipid hydroperoxides. Under the influence of metal ions of variable valency, primarily Fe 2 +, these substances form secondary radicals(HO*, lipid radicals), which have a destructive effect on cellular structures.
To protect against the damaging effects of secondary radicals, the body uses a large group of substances called antioxidants (see below), which include “traps” (“interceptors”) of free radicals. Examples of the latter are alpha-tocopherol, thyroxine, reduced ubiquinone (QH 2) and female steroid hormones. Reacting with lipid radicals, these substances themselves transform into antioxidant radicals, which can be considered as tertiary radicals.
Along with these radicals, which are constantly formed in varying quantities in the cells and tissues of the human body, radicals that appear under influences such as ionizing radiation, ultraviolet irradiation, or even illumination with intense visible light, such as laser light, can have a destructive effect. Such radicals can be called foreign. These also include radicals formed from foreign compounds and xenobiotics that enter the body, many of which have a toxic effect precisely due to free radicals formed during the metabolism of these compounds.
However, one should not assume that free radicals are only a cell-damaging factor. An example of the positive role of these compounds is the cellular immune system. For example, phagocytic leukocytes (which include granulocytes and monocytes of the blood and tissue cells - macrophages), in contact with the surface of bacteria at the site of inflammation, are activated and, with the help of NADPH oxidase, an enzyme built into the membrane of cells and intracellular vesicles-phagosomes, generate from O 2 superoxide anion radical, which has a bactericidal effect (Fig. 3-3). Nitroxide (NO *), released by phagocyte cells along with superoxide radicals, is used to combat fungal microbes. To carry out their killer functions, phagocytes also use hypochlorite (OCl -) formed from hydrogen peroxide. Reaction
Rice. 3-3. Superoxide radical reactions
The formation of hypochlorite is catalyzed by a special enzyme - myeloperoxidase: H 2 O 2 + Cl - - H 2 O + OO - . Hypochlorite itself is not a free radical (belongs to the group of active oxygen metabolites of a non-radical nature), but interacts with organic molecules through radical mechanisms. With the participation of hypochlorite, highly active molecules such as hydroxyl radical (Fe 2 + + OCl - + H+ - Fe 3 + + HO "+ Cl -), singlet oxygen (IO 2) are formed. In activated leukocytes, hydroxyl radical (HO") can be formed also during the decomposition of hydrogen peroxide in the presence of divalent iron ions (H 2 O 2 + Fe 2 + - Fe 3 + + HO "+ HO"). The cytotoxic effect of OCl - and HO" lies in their ability to destroy SH groups and other amino acid residues of proteins, induce DNA and RNA strand breaks, enhance the activity of lipid peroxidation, proteinases, complement system proteins, and inhibit fission proteins and bacterial enzymes.
Free radicals also perform other functions, including regulatory ones. Thus, some tissues, in particular the brain, are characterized by increased synthesis of prostaglandins, thromboxanes and leukotrienes, formed from arachidonic acid during the induction of lipid peroxidation with the participation of superoxide anion. The ubiquinone radical (coenzyme Q) - semiquinone (HQ") is involved in the electron transport chain; if the respiratory chain is disrupted, it can become a source of other radicals, primarily oxygen radicals.
In addition, free radicals actively participate in the processes of cell signal transmission and can act as secondary
ric messengers in signaling cascades triggered by angiotensin II, endothelin, etc. Thus, “NO”, formed by the cells of the walls of blood vessels (endothelium) with the participation of the heme-containing enzyme NO synthase, plays a key role in the regulation of vascular tone and blood pressure: its deficiency leads to hypertension, excess leads to hypotension.Impaired NO metabolism causes diseases associated with changes in blood pressure.Radicals formed in the cytosol of the cell in response to stimulation by growth factors are involved in the regulation of the proliferative process.
Under normal conditions, oxygen radicals do not accumulate in cells. The condition of cells characterized by an excess content of oxygen radicals in them is called oxidative stress. Oxidative stress develops when redox homeostasis (redox homeostasis or balance) in a cell is disrupted. This imbalance may be due to overproduction of reactive oxygen species or insufficiency of the antioxidant defense system, which includes low molecular weight compounds of plant and animal origin (contained in the blood plasma, cytoplasm or cell membranes). There are several main groups of antioxidants:
1) enzymatic - superoxide dismutase, catalase, glutathione cycle enzymes (glutathione peroxidase, glutathione reductase, glutathione-S-transferase);
2) phenolic - vitamin E, coenzyme Q, flavonoids (quercetin, rutin, hesperetin, etc.);
3) carotenoids - fat-soluble plant pigments that are part of vegetables and fruits (carrots, spinach, mango, apricot, etc.);
4) ascorbic acid (vitamin C) - found in fresh vegetables, fruits and berries (parsley, young cabbage, rose hips, black currants, lemon, orange, papaya, apple, etc.), in the body it is found in large quantities in the adrenal glands, pituitary gland , thymus gland;
6) chelators of metal ions of variable valence - transferrins, ferritin, ceruloplasmin, metallothioneins, uric acid, etc.
Based on the principle of antioxidant action, antioxidants of direct (directed) and indirect (mediated) action are distinguished. The effectiveness of the latter is manifested only in living systems (in vivo), while targeted compounds can suppress oxidative processes involving active oxygen metabolites such as in vivo, so and in vitro.
Under natural conditions, antioxidants (superoxide dismutase, catalase, taurine, etc.) protect phagocytes from autodestruction by their own radicals (superoxide, hypochlorite, hydroxyl radical), coordinate the generation of inflammatory mediators by neutrophils and macrophages (prostaglandins, IL-6, TNF-α, etc.) . The effects of some antioxidants are presented in table. 3-5.
Table 3-5. The most famous antioxidants
|
Antioxidant |
Characteristic |
|
Ceruloplasmin |
Oxidizes Fe 2+ to Fe 3+ with molecular oxygen |
|
Transferrin apo protein |
Binds Fe 3+ |
|
Ferritin |
Oxidizes Fe 2 + and deposits Fe 3 + |
|
Carnosine |
Binds Fe 2+ |
|
Superoxide dismutase |
Superoxide is removed to form hydrogen peroxide |
|
Catalase |
Decomposes hydrogen peroxide releasing oxygen |
|
Glutathione peroxidases |
1. Hydrogen peroxide is removed due to the oxidation of glutathione 2. Remove lipid hydroperoxides |
|
Glutathione reductase |
Restores oxidized glutathione |
|
Tocopherol, thyroxine, steroids |
Intercepts lipid radicals |
|
Ascorbic acid |
Regenerates oxidizing tocopherol and ubiquinone |
|
Glutathione |
Used to reduce peroxides |
The main stages of chain oxidation. The reaction of chain oxidation of lipids plays an exceptional role in cellular pathology. It occurs in several stages: initiation, continuation, branching and chain termination (Fig. 3-4).
Rice. 3-4. Chain reaction of lipid peroxidation: 1 - old oxidation chain, 2, 3 - new oxidation chains
The initiation of a chain reaction begins with the introduction of a free radical into the lipid layer of membranes or lipoproteins. Most often this is a hydroxyl radical. Being a small uncharged particle, it is able to penetrate into the thickness of the hydrophobic lipid layer and enter into chemical interaction with polyunsaturated fatty acids (usually referred to as LH), which are part of biological membranes and blood plasma lipoproteins. In this case, lipid radicals are formed:
HO" + LH - H 2 O + L".
The lipid radical (L) reacts with molecular oxygen dissolved in the medium, resulting in the formation of a new free radical - lipid peroxide radical (LOO):
This radical attacks one of the neighboring phospholipid molecules to form lipid hydroperoxide LOOH and a new radical L:
LOO"+ LH - LOН + L"
The alternation of the last two reactions is precisely chain reaction FLOOR (see Fig. 3-4).
A significant acceleration of lipid peroxidation is observed in the presence of small amounts of ferrous iron ions. In this case, chain branching occurs as a result of the interaction of Fe 2 + with lipid hydroperoxides:
Fe 2 + + LOOH - Fe 3 + + HO - + LO"
The resulting LO radicals initiate new chains of lipid oxidation (see Fig. 3-4):
LO" + LH - LOН + L"; L"+ O 2 - LOO" - etc.
In biological membranes, chains can consist of a dozen links or more. But, in the end, the chain breaks as a result of the interaction of free radicals with antioxidants (InH), metal ions of variable valency (for example, the same Fe 2 +) or with each other:
LOO" + Fe 2 + + H+ - LOOH + Fe 3 +
LOO" + InH - In"+ LOOH
LOO + LOO - molecular products
Damaging effects of lipid peroxidation. In Fig. Figures 3-5 show the main targets of LPO in the membrane structures of cells. Either protein structures or the lipid bilayer as a whole are damaged. IN Lately Scientists are paying increasing attention to the interaction of membranes with nucleic acids in the nucleus and mitochondria. Apparently, one of the results of lipid peroxidation may be damage to these molecules with all the ensuing consequences.
The most sensitive to lipid peroxidation are the sulfhydryl, or thiol, groups (SH) of membrane proteins: enzymes, ion channels and pumps. Oxidation of thiol groups produces radicals (S), which then either react with each other to form disulfides (SS) or combine with oxygen to form sulfites and sulfates (SO 3 and SO 4). Also plays a major role in cell pathology
Rice. 3-5. Damaging effect of lipid peroxidation on biological membranes
damage to ion-transporting enzymes (for example, Ca 2 +, Mg 2+ -ATPase), the active center of which includes thiol groups (Fig. 3-5, 1). Inactivation of Ca 2 + -ATPase leads to a slowdown in the pumping of calcium ions from the cell and an acceleration of their “leakage” into the cell (where their concentration is lower). This causes an increase in the level of calcium ions in the cytoplasm and damage to cellular structures.
Oxidation of thiol groups of membrane proteins leads to the appearance of defects in the membranes of cells and mitochondria. Under the influence of an electric field, sodium ions enter the cells through such defects, and potassium ions enter the mitochondria. As a result, there is an increase in osmotic pressure inside cells and mitochondria and their swelling. This leads to even greater damage to membrane structures.
Along with proteins and nucleic acids, the lipid bilayer itself serves as a target for the damaging effects of LPO. It has been shown that LPO products make the lipid phase of membranes permeable to hydrogen and calcium ions (Fig. 3-5, 2-3). This leads to the fact that oxidation and phosphorylation in mitochondria are uncoupled, and the cell finds itself in conditions of energy starvation. At the same time, calcium ions are released from the mitochondria into the cytoplasm, which damage cellular structures.
Perhaps the most important result of peroxidation is a decrease in the electrical stability of the lipid layer, which
This leads to electrical breakdown of the membrane by its own membrane potential (Fig. 3-5, 4). Electrical breakdown causes the membrane to completely lose its barrier functions.
Stability of the lipid layer of membranes and the phenomenon of electrical breakdown. As is known, membranes have a certain resistance R to electric current I, which, with a small potential difference φ between the two sides of the membrane, is a constant value. In other words, Ohm’s law is observed for the membrane: I = φ / R. This means that the relationship between the voltage across the membrane φ and the current through the membrane I is linear. However, this dependence remains at relatively small values of |φ|: no higher than 200-300 mV. At a certain critical potential difference, the current increases sharply, which can cause destruction of the membrane. This phenomenon is called electrical breakdown.
The electrical breakdown of the membrane is based on the spontaneous (due to the thermal movement of molecules) generation of defects in the lipid bilayer - pores through which water-soluble molecules and ions can pass. In the absence of a potential difference on the membrane, an increase in the size of spontaneously formed pores does not occur, since this process is accompanied by an increase in the area of the lipid-water interface and requires energy expenditure to overcome the surface tension forces at the interface. However, as the potential difference across the membrane increases, the amount of energy required to form and increase the size of the pore decreases, which contributes to its further growth, which, after overcoming a certain energy barrier, becomes spontaneous and leads to complete destruction of the membrane (Fig. 3-6). At the small membrane potentials that exist in a living cell (-70 mV on the cytoplasmic membrane and -175 mV on the inner mitochondrial membrane), this does not happen because the energy barrier is quite high. Moreover, under normal conditions, under the action of surface tension forces, the resulting defect is “tightened” and the membrane remains intact. The magnitude of the barrier decreases with increasing membrane polarization. The potential at which electrical breakdown begins is called breakdown potential and is usually denoted as U* or φ*. The breakdown potential is different for membranes with different compositions of proteins and lipids and can serve as a quantitative measure of electrical
Rice. 3-6. Electrical breakdown of membranes: A - appearance in the lipid bilayer of the membrane of a pore filled with water; B - the size of the internal surface of the pore is proportional to its radius; B is the energy of the membrane with the pore depending on its radius (the magnitude of the potential barrier decreases as the pore grows); G - increase in current depending on the breakdown potential
membrane stability. The more stable the membrane, the higher its breakdown potential (i.e. |φ*|).
In living cells, the breakdown potential is higher than the membrane potential (|φ*|>|φ|), otherwise the membranes would be breached by their own potential and the cell could not exist. However, the electrical safety margin is small - 20-30 mV. This means that for |φ*|<|φ|, т.е. при снижении электрической прочности, может произойти «самопробой» мембраны.
As mentioned above, the main reasons for the violation of the barrier properties of membranes in pathology are their mechanical (osmotic) stretching, activation of lipid peroxidation, hydrolysis of phospholipids and adsorption of polyelectrolytes on the surface. A study of the influence of these factors on the electrical strength of membranes showed that they all reduce the surface tension forces at the interface between the lipid layer of the membrane and the surrounding aqueous solution, and, consequently, the magnitude of the breakdown potential (Fig. 3-7). Thus, Electrical breakdown is a universal mechanism for disrupting the barrier function of membranes in pathology.
Membrane systems for protection against electrical breakdown. There are two known factors by which living cells increase the electrical stability of their membrane structures:
Rice. 3-7. Reduction in the electrical strength of a lipid bilayer membrane (BLM) upon exposure to ultraviolet radiation(UV), phospholipase A2, peptides, during membrane stretching caused by a difference in hydrostatic pressure (ΔΡ)
1. Asymmetric surface potential. Surface potential occurs on the membrane when charged chemical groups, such as carboxyl or phosphate, appear on the surface of the lipid layer. The lipid bilayer is directly affected by a potential equal to the difference between the membrane potential (i.e., the potential between aqueous media washing the membrane) and the surface potential (Fig. 3-8). Due to the unequal charge density on the membrane surface, the actual potential difference applied to the lipid bilayer differs from the transmembrane potential difference. This reduces the likelihood of membrane breakdown by its own potential.
2. Cholesterol. It has been shown that the inclusion of cholesterol molecules in the phospholipid bilayer significantly increases the electrical strength of membranes, i.e. increases the breakdown potential (see Fig. 3-6, D). The effect of cholesterol on damaged membranes is especially noticeable. The protective properties of cholesterol against electrical breakdown of the membrane can be explained by its effect on the viscosity of the lipid bilayer. It is known that the introduction of cholesterol into the phospholipid bilayer increases the viscosity of the latter by 2-3 times. This leads to a slowdown in the formation and growth of defects (pores) in the lipid bilayer of membranes, which underlie the phenomenon of electrical breakdown.
Rice. 3-8. The influence of surface potential (cp S) on the potential difference on the lipid layer of membranes (cp L) at the same membrane potential (φ)
Criteria for assessing violations of the barrier function of the cytoplasmic membrane. The main criteria for judging a violation of the barrier properties of the cytoplasmic membrane and an increase in its permeability are: a decrease in the electrical resistance of the tissue, the penetration of a water-soluble dye into the cytoplasm, a decrease in the resting membrane potential, an imbalance in the ion balance, the release of intracellular metabolites into the environment, and cell swelling.
Reducing the electrical resistance (impedance) of the tissue. A method for assessing the state of both plasma and intracellular membranes can be the measurement of electrical resistance - tissue impedance, which includes ohmic and capacitive components, since each cell is like a system of capacitors (biological membranes) and resistors (biological membranes, intercellular fluid and cytoplasm). When cells are damaged or aged, a decrease in tissue capacitance is recorded, which is mainly associated with a violation of the condition of cell membranes. When cells undergo swelling, or striction, the ohmic (high-frequency) component of the impedance changes. To quantify these violations, B.N. Tarusov proposed a definition of the cell viability coefficient (K) as the ratio of tissue resistance to alternating current with a frequency of 104 Hz (R104) to tissue resistance when exposed to current with a frequency of 106 Hz (R106): K = R104 / R106.
Staining of the cytoplasm with various dyes. Water-soluble dyes do not penetrate well through the membranes of intact cells, bind weakly to intracellular structures and therefore stain them weakly. An increase in the permeability of the plasma and intracellular membranes leads to an increase in the amount of dye that enters the cell and binds to the components of the cytoplasm. Consequently, cell staining with dyes increases when it is damaged. This is the basis for many histochemical methods for determining cell viability (using neutral blue, eosin, etc.).
Decreased resting membrane potential. The difference in electrical potential between the contents of the cell and the environment (resting membrane potential), as is known, is created mainly by the diffusion of potassium ions from the cell into the environment. Uneven distribution of ions between the cell and the surrounding
The environment underlying the generation of electrical potentials on the membrane is ensured by the constant operation of a molecular ion pump (Na + /K + -ATPase) built into the plasma membrane of cells.
Thus, inside cells the content of potassium ions is 20-40 times higher, and sodium ions - 10-20 times lower than in the extracellular fluid. Due to the difference in the concentration of ions in the cell and the environment on the plasma membrane, there is a potential difference with a minus sign inside the cell (about -70 mV for nerve and muscle cells). A decrease in membrane polarization under the influence of damaging factors occurs both as a result of a nonspecific increase in ion permeability and as a result of a decrease in ion concentration gradients due to the shutdown of ion pumps.
The latter occurs both with direct damage to Na+/K+- ATPase, and with a decrease in ATP levels due to disruption of bioenergetic processes in mitochondria. For example, a decrease in the resting membrane potential of liver cells in laboratory animals during asphyxia has been established. A decrease in membrane potential is also observed during cold, radiation, allergic, toxic and other damage to cells and subcellular structures.
Release of potassium ions from cells. Due to the potential difference between the internal contents of the cell and the surrounding fluid, potassium ions enter the cell. This constant flow of K+ into the cell compensates for the spontaneous release of potassium outward, which occurs due to the diffusion of these cations from an area with a higher potassium concentration to an area with a lower concentration. Damage to the cell is accompanied by a decrease in the ATP content in it, inhibition of Na + /K + -ATPase, a drop in the electrical potential on the plasma membrane, an increase in the content of intracellular Ca 2 + and the release of potassium from the cells. The release of potassium from cells has been described during mechanical trauma, various intoxications, allergic conditions, hypoxia, hypothermia and many other damage to organs and tissues. A decrease in the K+ content in the cell can also occur under the influence of large doses of mineralocorticoid hormones, under the action of certain drugs, such as cardiac glycosides. In turn, an increase in potassium concentration in the extracellular environment leads to a decrease in membrane potential
la neighboring intact cells, which in the case of electrically excitable tissues can cause the generation of action potentials. Thus, an increase in potassium concentration at the site of myocardial infarction can be one of the causes of cardiac fibrillation.
Accumulation of calcium ions in the cytoplasm. In normal cells, the concentration of calcium ions in the cytoplasm is extremely low: 10 -7 M or even 10 -8 M, while the environment surrounding the cell contains 10 -3 M calcium ions. It should be borne in mind that calcium ions pass into the cell not only spontaneously (the process of “leakage” through the membrane), but also in some cells through calcium channels in the membrane. These channels can open in response to membrane depolarization (voltage-gated calcium channels) or the binding of hormones and transmitters to membrane receptors (receptor-gated calcium channels). The entry of Ca 2+ into the cell is compensated by the work of three types of calcium transport systems: the calcium pump (Ca 2+ /Mg 2+ -ATPase) in the membrane of the sarcoplasmic reticulum and plasmalemma, the accumulation of Ca 2+ in mitochondria and in some cells of the Na+/Ca 2+ exchanger. , built into the plasmalemma.
When the cell is damaged, the functioning of mitochondria is disrupted: the membrane potential of the inner mitochondrial membrane decreases and oxidative phosphorylation stops. As a consequence of a decrease in membrane potential, the absorption of calcium ions by mitochondria decreases. A decrease in the concentration of ATP in the cell leads to inhibition of Ca 2+ /Mg 2+ -ATPase of the plasma membrane and the sarcoplasmic reticulum membrane. An increase in the concentration of Na+ in the cell due to inhibition of the sodium pump with a lack of ATP leads to the shutdown and even reversal of the direction of Na+ /Ca 2+ exchange through the plasma membrane. As a result, the calcium concentration increases from 10 -8 M - 10 -7 M to 10 -6 M - 10 -5 M, which leads to the activation of a large number of calcium-dependent enzymes (protein kinases, phosphatases, phospholipases, cyclic nucleotide phosphodiesterases, etc.) , cytoskeletal disorders (see section 3.4), the formation of insoluble calcium inclusions in the mitochondrial matrix, damage to intracellular membranes and general disorganization of metabolism. Morphologically, this manifests itself in a slowdown in the Brownian motion of various inclusions inside the cell (an increase in the “viscosity of protoplasm”) and an increase in light scattering; dyes begin to penetrate more easily
cell and bind in large quantities to intracellular structures. All these signs are typical for a “nonspecific cell response to damage” according to D.N. Nasonov and V.Ya. Alexandrov (see above).
Release of metabolites. An increase in the permeability of the cell membrane and a deterioration in the functioning of ion pumps lead to the release of cytoplasmic components into the environment. Substances released from cells are by no means indifferent to other cells, tissues and organs. Thus, among the substances released from cells damaged as a result of ischemia (impaired blood flow) or burn, there are polypeptides that have the ability to cause cardiac arrest (ischemic, burn toxins). Detection of these substances is carried out by various methods, including measurement of chemiluminescence of blood plasma, the intensity of which decreases in the presence of polypeptide toxins.
Increase in volume (swelling) of cells. An increase in cell volume is one of the earliest signs of cell damage, which manifests itself, for example, when there is a lack of oxygen in the tissue - tissue hypoxia. The preservation of the normal shape and volume of cells is associated with the state of the cytoskeleton and the maintenance of a certain ratio between the osmotic pressure of proteins and electrolytes inside and outside the cell. In this case, the shape of the cell is determined to a greater extent by the cytoskeleton, while the volume is determined by the maintenance of osmotic balance. Since all biological membranes are well permeable to water, but poorly permeable to most substances dissolved in water, including salts, cells, as well as intracellular structures, such as mitochondria, have the property of an osmometer: their volume changes with changes in the concentration of ions and molecules inside and outside cells or organelles. Under normal conditions, the ratio of concentrations of all ions and molecules inside and outside the cell is strictly maintained. As soon as the concentration of ions or molecules in the cytoplasm begins to increase, the volume of the cell increases as water enters. The pumping out of ions by membrane pumps and exchangers is accompanied by restoration of its volume due to the release of excess water following the ions.
Cell swelling is associated with a violation of the regulation of its volume on the part of the plasma membrane. In normal cells, the protein concentration is higher than outside cells, as a result of which mammalian cells have a higher intracellular
colloid osmotic (oncotic) pressure than extracellular fluid. This would inevitably lead to an increase in cell volume if, to balance this “excess” pressure, sodium ions were not removed (pumped out) from the cell due to the work of the energy-dependent Na + /K + -ATPase. Since the cell membrane is highly permeable to chlorine ions, chlorine also comes out along with sodium due to the potential difference across the membrane. In other words, the sodium pump removes NaCl from the cell and reduces the concentration of ions in the cytoplasm, which leads to a decrease in cellular volume. This process is opposed by the process of spontaneous entry of sodium into the cell through defects in the lipid bilayer, sodium channels, transporters that couple the entry of sodium with the transport of sugars and amino acids into the cell, Na + /H + - and Na + /Ca 2+ exchangers, as well as Na + / K + /2C1- cotransporter.
Thus, a living cell is in a state of dynamic equilibrium, in which the “leakage” of the cell membrane is compensated by the constant operation of the ion pump (this is the so-called leak and pump hypothesis).
In pathology, either an increase in the ionic permeability of the cell membrane (an increase in “leakage”) or a disruption in the functioning of ion pumps can occur, for example, with a lack of energy supply due to hypoxia, the action of cyanide or oxidative phosphorylation uncouplers (dinitrophenol). In experiments with isolated cells of the liver, kidneys and brain, it was shown that poisoning with mercury salts or other heavy metals leads to an increase in the ionic permeability of the cell membrane (increased “leaking”), disruption of ATP-dependent transport and an increase in cell volume (i.e. swelling fabrics).
The second mechanism of cell swelling during hypoxia is an increase in intracellular osmotic load caused by the accumulation of metabolites (catabolites), such as inorganic phosphate, lactate and purine nucleosides.
Core(Latin nucleus, Greek karion-core) is an essential component of eukaryotic cells. It is clearly visible in non-dividing cells and performs a number of important functions:
1) storage and transmission of hereditary information in the cell;
2) creation of a protein synthesis apparatus - synthesis of all types of RNA and formation of ribosomes.
Loss or disruption of any of these functions leads to cell death.
Fig.24. Scheme of the ultramicroscopic structure of the nucleus.
The cell contains, as a rule, one nucleus, but there are binucleate and multinucleate cells.
Interphase nuclei consist of: nuclear envelope, nuclear sap (karyoplasm, karyolymph or nucleoplasm), nuclear protein backbone, chromatin and nucleoli.
Nuclear envelope(karyolemma) consists of two membranes, between which there is a perinuclear space 10-40 nm wide, filled with an electron microscopically loose substance. The outer membrane of the nuclear envelope on the cytoplasmic side in a number of areas passes into the membranes of the endoplasmic reticulum, and polyribosomes are located on its surface. The inner membrane of the nuclear membrane is involved in ensuring internal order in the nucleus - in fixing chromosomes in three-dimensional space. This connection is mediated by a layer of fibrillar proteins similar to the intermediate filaments of the cytoplasm.
The nuclear envelope has pores with a diameter of about 90 nm. In these areas, along the edges of the holes, the membranes of the nuclear envelope merge. The holes themselves are filled with complexly organized globular and fibrillar structures. The set of membrane perforations and structures filling them is called pore complex.
Along the edge of the pore opening, granules are located in three rows (8 granules in each row). In this case, one row lies on the side of the cytoplasm, the other - on the side of the internal contents of the nucleus, and the third - between them. Fibrillar processes extend radially from the granules of these layers, forming a kind of membrane in the pore - a diaphragm. Fibrillar processes are directed towards a centrally located granule.
Fig.25. Structure of nuclear pores (pore complex).
Pore complexes are involved in the reception of macromolecules (proteins and nucleoproteins) transported through the pores, as well as in the active transfer of these substances through the nuclear envelope using ATP.
The number of nuclear pores depends on the metabolic activity of the cells. The more intense the synthesis processes occur in the cell, the more pores there are. On average, there are several thousand pore complexes per core.
Main functions The nuclear envelope is as follows:
Barrier (separation of the contents of the nucleus from the cytoplasm and restriction of free access to the nucleus of large biopolymers);
Regulation of transport of macromolecules between the nucleus and cytoplasm;
Participation in the creation of intranuclear order (fixation of the chromosomal apparatus).
Karyoplasm(nuclear sap, or nucleoplasm, or karyolymph) is the contents of the nucleus, which has the appearance of a gel-like matrix. It contains various chemicals: proteins (including enzymes), amino acids and nucleotides in the form of a true or colloidal solution.
Nuclear or protein backbone (matrix). In interphase nuclei, non-histone proteins form a network—the “protein matrix.” It consists of a peripheral fibrillar layer lining the nuclear envelope (lamina) and an internal network to which chromatin fibrils are attached. The matrix is involved in maintaining the shape of the nucleus and organizing the spatial position of chromosomes. In addition, it contains enzymes necessary for the synthesis of RNA and DNA, as well as proteins involved in DNA compaction in interphase and mitotic chromosomes.
Chromatin– a complex of DNA and proteins (histone and non-histone). Chromatin is an interphase form of chromosome existence.
1. Euchromatin; 2. Heterochromatin
Fig.26. Chromatin of interphase chromosomes.
During this period, different sections of chromosomes have unequal degrees of compaction. Genetically inert regions of chromosomes have the greatest degree of compaction. They stain well with nuclear dyes and are called heterochromatin. Distinguish constitutive And optional heterochromatin.
Constitutive heterochromatin formed by non-transcribed DNA. It is believed that it is involved in maintaining the structure of the nucleus, attaching chromosomes to the nuclear envelope, recognizing homologous chromosomes during meiosis, separating neighboring structural genes and in the processes of regulating their activity.
Optional heterochromatin, in contrast to constitutive one, can become transcribed at certain stages of cell differentiation or ontogenesis. An example of facultative heterochromatin is the Barr body, which is formed in organisms of homogametic sex due to the inactivation of one of the X chromosomes.
Decompacted regions of chromosomes that are poorly stained with nuclear dyes are called euchromatin.This is functionally active, transcribed chromatin.
Nucleoli– compacted bodies, usually round in shape, less than 1 micron in diameter. They are present only in interphase nuclei. Their number varies in diploid cells from 1 to 7, but in some types of cells, for example, micronuclei of ciliates, nucleoli are absent.
Ministry of Education of the Republic of Belarus
Educational institution
“International State Ecological University named after A.D. Sakharov"
Faculty of Environmental Medicine
Department of Biochemistry and Biophysics
Molecular organization of the cell nucleus
Performed:
4th year student
MBD specialties
92062-2 groups
Shilova Anastasia
Minsk 2012
Content:
Introduction………………………………………………………3
- Nuclear envelope (karyolemma)…………………………….4
- The structure of the nuclear membrane……………………………...4
- Structural organization of nuclear pores……………….5
- Properties of nuclear pores………………………………….8
- Nucleoporins……………………………………………………..8
- Assembly and disintegration of the nuclear envelope…………………...8
- Chemistry of the nuclear envelope……………………………...10
- Nuclear - cytoplasmic transport…………………..11
- Regulation of molecular transport through the nuclear pore..12
- Nuclear matrix………………………………………….17
- Chromatin……………………………………………………..18
- Chromatin DNA………………………………………………………...20
- Chromatin proteins……………………………………………………….21
- Chromosomes………………………………………………………..23
- Nucleolus……………………………………………………...24
- Number of nucleoli in a cell………………………...24
- Physiology and chemistry of the nucleolus………………………...25
- Nucleolar RNA……………………………………………………………...26
- Nucleolar DNA……………………………………………………..26
- Ultrastructure of nucleoli…………………………….27
- The fate of nucleoli during cell division………………...28
- Karyoplasm………………………………………………….28
- The role of the core……………………………………………………...30
Conclusion……………………………………………………...32
References……………………………………………………………..33
Introduction:
When we talk about the cell nucleus, we mean the actual nucleus of eukaryoticcells. Their nuclei are built in a complex way and differ quite sharply from“nuclear” formations, nucleoidsprokaryotic organisms. The latternucleoids (nucleus-like structures) include a single ringa DNA molecule practically devoid of proteins. Sometimes such a DNA moleculebacterial cells are called bacterial chromosome, or genophore(carrier of genes). The bacterial chromosome is not separated by membranes fromthe main cytoplasm, however, is collected into a compact nuclear zone - the nucleoid,which can be seen in a light microscope after special staining(Fig. 1).
Figure 1. Structure of the nucleus of eukaryotic and prokaryotic cells.
The term “core” was first used by Brown in 1833. to indicate spherical permanent structures in plant cells.Later the samethe structure was described in all cells of higher organisms.
The nuclei are usually spherical or ovoid; the diameter of the first is equalapproximately 10 microns, and the length of the second is 20 microns.The nucleus is necessary for the life of the cell, since it is it that regulates its entireactivity. This is due to the fact that the nucleus carries the genetic(hereditary) information contained in DNA.
- Nuclear envelope (karyolemma)
Karyolemma is a nuclear membrane that separates the contents of the nucleus from the cytoplasm (barrier function), while at the same time ensuring regulated metabolism between the nucleus and the cytoplasm. The nuclear envelope takes part in chromatin fixation. The karyolemma consists of two bilipid membranes, the outer and inner nuclear membranes, separated by a perinuclear space 20 × 100 nm wide. The karyolemma has pores with a diameter of 80 × 90 nm. In the pore region, the outer and inner nuclear membranes pass into each other, and the perinuclear space becomes closed. The pore lumen is closed by a special structural formation, the pore complex, which consists of fibrillar and granular components. The granular component is represented by protein granules with a diameter of 25 nm, located along the edge of the pore in 3 rows. Fibrils extend from each granule and unite in a central granule located in the center of the pore. The pore complex plays the role of a diaphragm, regulating its permeability. The pore sizes are stable for a given cell type, but the number of pores can change during its differentiation. There are no pores in the sperm nuclei. Attached ribosomes can be localized on the outer surface of the nuclear membrane. In addition, the outer nuclear membrane may continue into the ER channels. In general terms, the nuclear envelope can be represented as a hollowa two-layer sac that separates the contents of the nucleus from the cytoplasm. Of allintracellular membrane components with this type of membrane arrangementonly the nucleus, mitochondria and plastids have. However, the nuclear membrane hasa characteristic feature that distinguishes it from other membrane structurescells. This is the presence of special pores in the shell of the core, which are formed due tonumerous zones of fusion of two nuclear membranes and represents, as it were,rounded perforations of the entire nuclear envelope.
- Structure of the nuclear envelope
The outer membrane of the nuclear envelope, directlyin contact withcytoplasm of the cell, has a number of structural features that make it possible to attributeit to the membrane system of the endoplasmic reticulum itself. Yes, onThe outer nuclear membrane usually contains a large number of ribosomes. Uof most animal and plant cells, the outer membrane of the nuclearthe shell does not represent a perfectly flat surface - it can form protrusions of varying sizes or outgrowths to the side cytoplasm.
The inner membrane is in contact with the chromosomal material of the nucleus.The most characteristic and conspicuous structure in the nuclear envelopeis the nuclear time. Pores in the shell are formed due to the fusion of twonuclear membranes in the form of rounded through holes or perforations withwith a diameter of 80-90 nm. Rounded through hole in the nuclear envelopefilled with complex globular and fibrillar structures.The collection of membrane perforations and these structures is called a pore complexkernels. This emphasizes that the nuclear time is not just a through hole innuclear membrane, through which the substances of the nucleus and cytoplasm directlymay be reported.
The complex complex of pores has octagonal symmetry. Along the border of the roundholes in the nuclear envelope, there are three rows of granules, 8 pieces pereach: one row lies on the side of the nucleus, the other on the side of the cytoplasm,the third is located in the central part of the pores. The size of the granules is about 25 nm. Fromfibrillar processes extend from these granules. Such fibrils extending fromperipheral granules, can converge in the center and create, as it were,partition, diaphragm, across the pore. In the center of the hole you can often seethe so-called central granule.The number of nuclear pores depends on the metabolic activity of cells: the highersynthetic processes in cells, the more pores per unit surfacecell nucleus.
- Structural organization of nuclear pores
Nuclear pores are not just perforations, but complex, multifunctional, regulated structures organized by approximately 30 proteins, nucleoporins. The protein component of the nuclear pore is referred to as the “nuclear pore complex” ( English nuclear pore complex, NPC ). The mass of the nuclear pore complex ranges from ~44 MDa in yeast cells to ~125 MDa in vertebrates.According to electron microscopy data, nuclear pores in cross section have the shape of an “eight-spoke cart wheel,” that is, they have an eighth-order symmetry axis. These data are confirmed by the fact that nucleoporin molecules are present in the nuclear pore in an amount that is a multiple of eight. A channel permeable to molecules is located in the center of the structure. The nuclear pore complex is anchored to the nuclear envelope by a transmembrane part, from which structures called spokes face the lumen of the channel, analogous to the spokes of a cart wheel. This core part of the pore, built of eight domains, is bounded on the cytoplasmic and nuclear sides by the cytoplasmic and nuclear rings, respectively (in lower eukaryotes they are absent). Attached to the nuclear ring are protein strands directed into the nucleus (nuclear filaments), to the ends of which a terminal ring is attached. This entire structure is called the nuclear basket. Strands directed into the cytoplasm, called cytoplasmic filaments, are also attached to the cytoplasmic ring. At the center of the nuclear pore is an electron-dense particle, a “sleeve” or transporter (English, plug)(Fig. 2) .
Fig. 2 Structure of a nuclear pore.
Many individual components of the NPC have a subunit structure, which ensures its high plasticity in the process of molecular transport. Two peripheral rings with a diameter of about 120 nm - cytoplasmic and intranuclear - limit the central part of the nuclear pore, consisting of two mirror-symmetrical sections.Each of these sections includes 3 interconnected rings: an internal one, in contact with the central conveyor; the middle one, penetrating the lateral portion of the nuclear membrane that forms the pore, and the radial one, located in the lumen between the outer and inner nuclear membranes. The middle and radial rings ensure strong anchorage of the pore in the nuclear envelope, and the inner ring plays the role of the main frame around and inside which the remaining components of the pore are assembled. The central channel of the pore has a variable internal diameter (changing from 10 to 26 nm) and is located inside a transporter consisting of 4 interconnected parts: 2 symmetrical thin-walled cylinders and 2 identical peripheral granules, which are attached by 8 fibrils to the peripheral pore rings and close both entrances to the central canal. The transporter occupies the central part of the inner ring of the pore.
The peripheral sections of the NPC are asymmetrical, which is probably due to different mechanisms of nuclear-cytoplasmic transport of molecules through the pore at the initial stages of their import and export. On the cytoplasmic side, the pore has 8 granules located on the cytoplasmic ring, like beads on a string, and containing short fibrils, and on the nuclear side, 8 fibrils extending from the intranuclear ring and forming a structure similar to a basketball basket (called a basketball). In an inactive pore, basket fibrils close the entrance to the pore, and in an active pore they form an additional ring with a diameter of about 50 nm.
The structural organization of the NPC in all higher organisms, including humans, birds, amphibians, insects and higher plants, is similar and highly conservative. The density of pores in the nuclear envelope (NE) varies on average from 13 to 30 pores per 1 μm 2 surface of the nucleus, reaching 5000 pores per nucleus in frog oocytes and early Drosophila embryos. It is assumed that all nuclear pores are universal and can ensure the transport of molecules both into the nucleus and into the cytoplasm. A change in the number of pore complexes in the nuclear complex of higher eukaryotes can occur with a change in the functional state of cells, probably due to their formation de novo . At the same time, due to the close connection with the lamina, a fibrillar mesh structure located on the inside of the nuclear membrane. Unlike higher organisms, lower eukaryotes (for example, yeast) do not have a lamina, due to which their nuclear pores can move freely along the nuclear shell, and their density in different parts of the shell can vary significantly. The structure of nuclear pores in yeast has not yet been studied in detail, although it has been shown that their diameter (~100 nm) is smaller than that of the pores of higher organisms (~120 nm), and some nucleoporins are absent in them. Instead of 50 nucleoporins, the yeast pore contains only 30. This is consistent with the yeast NPC model, which demonstrates its simpler structure compared to the cells of higher eukaryotes. For example, in yeast NPC there is no radial ring in the central component of the pore. However, the peripheral sections in the pores of yeast are also asymmetrical, and the central canal has the same dimensions as a similar canal in higher eukaryotes. The observed universality of the NPC organization suggests that precisely this structure is necessary to ensure the possibility of bidirectional transport of molecules through the NPC.
- Properties of nuclear pores
The number of nuclear pores per nucleus can vary from 190 in yeast, 3000-5000 in human cells to 50 million in mature oocytesclawed frog(Xenopus laevis ). This indicator may also vary depending on cell type, hormonal status and cell cycle stage. For example, in vertebrate cells the number of nuclear pores doubles during S phase, simultaneously with chromosome doubling. When the nuclear envelope is disassembled during mitosis, vertebrate nuclear pores break up into subcomplexes with masses of about a million daltons. It has been shown that disassembly of the nuclear pore complex is initiated by cyclin B-dependent kinase,phosphorylatingnucleoporins. After completion of cell division, nuclear pores assemble de novo . Nuclear pores of the interphase nucleus move in large arrays, rather than independently of each other, and these movements occur synchronously with the movements nuclear lamina . This serves as evidence that nuclear pores are mechanically interconnected and form a single system (NPC network).
- Nucleoporins
Nucleporins, the proteins that make up nuclear pores, are divided into three subgroups. The first group includes transmembrane proteins that anchor the complex in the nuclear envelope. Nucleporins of the second group contain a characteristic amino acid motif - FG, FXFG or GLFG sequences repeated several times (the so-called FG repeats, where F is phenylalanine, G is glycine, L is leucine, X is any amino acid). The function of FG repeats appears to be to bind transport factors necessary for nuclear-cytoplasmic transport. Proteins of the third subgroup have neither membrane domains nor FG repeats; they are the most conserved among all nucleoporins; their role, apparently, is to ensure the binding of FG-containing nucleoporins to transmembrane ones. Nucleoporins also differ in their mobility within the nuclear pore. Some proteins are associated with a specific pore throughout the cell cycle, while others are completely turned over in just a few minutes.
- Assembly and disintegration of the nuclear envelope
Until now, the question of the formation and disintegration of the nuclear envelope (NE) and NPC in the process of mitosis in vivo remains insufficiently studied. However, recent experiments on the incubation of cytoplasmic extract from amphibian oocytes with sperm chromatin in vitro made it possible, using high-resolution scanning electron microscopy, to obtain new data on the regulation of this process. It was shown that within 1.5-2.5 hours of incubation in such a system, functionally active (capable of replication and transcription) nuclei with mature NPCs are formed. In the first stage, smooth and rough endoplasmic reticulum vesicles bind to the surface of decondensing chromatin and fuse together to form the inner and outer nuclear membranes. To carry out this process, Ca 2 ions are required+ and large amounts of energy supplied by GTP and ATP. It was shown that two types of endoplasmic reticulum vesicles, differing in protein composition, participate in the formation of the nuclear envelope.After the formation of a closed nuclear complex around chromatin, the assembly of nuclear complexes begins. First, small pits appear in various places of nuclear weapons, which then turn into 10-20 nm empty pores. After this, the pore size increases to 40 nm and then the sequential formation of first internal and then peripheral components of the pore begins. It has been established that the assembly of the components that make up the pore occurs in fragments; first, one component subunit is formed, then the second, etc. In this case, the pore gradually increases in size and in 4-6 minutes turns into a mature pore with a diameter of 110-120 nm.It is still not clear what the sequence of protein assembly is during the formation of NPC de novo . It is assumed that specific proteins, binding to the membranes of the nuclear membrane, stimulate their gradual approach and fusion, after which the integral proteins POM121 and gp210 attach to this part of the membrane, which stabilize the formed hole. Then other nucleoporins necessary for the formation of the central (p62 complex) and peripheral components (Nup 358, Nup 214, Nup 153, etc.) of the pore are delivered here, and, finally, the mature pore is additionally fixed in the nucleotide with the help of lamina proteins.
Using mitotic extract in experiments in vitro , it was shown that the disintegration of the NPC occurs due to the splitting off of endoplasmic reticulum (ER) vesicles from it, and the disassembly of the NPC occurs through intermediate structures similar to the intermediates that are observed during the assembly of the NPC. In this case, the peripheral and then the central components of the JPC are disassembled first. Results of experiments to study the assembly and decay of nuclear pores in vitro have been confirmed in experiments in vivo when studying nuclear division in early Drosophila embryos. It has been demonstrated that pore disassembly occurs in prophase of mitosis, first disassembling the central and then the peripheral components of the pore. The assembly of new pores begins in telophase after the formation of NR and passes through the same intermediate forms that were observed during the formation of pores in experiments in vitro. Interestingly, the most pore formation in experiments in vivo is initiated predominantly in areas where the membranes of the endoplasmic reticulum vesicles merge with the outer nuclear membrane.
In addition to pore complexes in the RN, similar structures were also found in the cytoplasm, as part of the ER membranes. The protein composition of these pore-like complexes is similar to the JPC proteins. However, unlike NPCs, their formation in the system in vitro occurs in the absence of chromatin. These specific ER membranes have been called perforated or fenestrated membranes (AL-annulate lamellae). Interestingly, perforated membranes with pores very similar to NPCs are absent in somatic cells, but are detected in large quantities in rapidly dividing cells, such as eggs, embryonic and tumor cells. The functional role of these structures is still poorly understood. It is assumed that these structures represent a depot of nuclear pore proteins and can participate in the assembly of nuclear pores in the case of rapid mitosis, but the mechanisms of regulation of this process, as well as the features of the use of these structures in the formation of nuclear pores and nuclear cells in vivo almost not studied.
- Chemistry of the nuclear envelope
Small amounts of DNA (0-8%), RNA (3-9%) are found in the nuclear membranes, but the main chemical components are lipids (13-35%) and proteins (50-75%), which is the same for all cell membranes.The lipid composition is similar to that of microsomal membranes or membranes
endoplasmic reticulum. Nuclear membranes are characterized relativelylow cholesterol and high- phospholipids enrichedsaturated fatty acids.The protein composition of membrane fractions is very complex. Found among proteinsa number of enzymes common to the ER (for example, glucose-6-phosphatase, Mg-dependentATPase, glutamate dehydrogenase, etc.) RNA polymerase was not detected. HereThe activities of many oxidative enzymes (cytochrome oxidase, NADH-cytochrome c reductase) and various cytochromes.Among the protein fractions of nuclear membranes there are main proteins of the typehistones, which is explained by the connection of chromatin regions with the nuclear envelope.
- Nuclear-cytoplasmic transport
Nuclear-cytoplasmic transport is the material exchange between the cell nucleus and cell cytoplasm . Nuclear-cytoplasmic transport can be divided into two categories: active transport, which requires energy, as well as special receptor proteins, and passive transport, which occurs by simple diffusion of molecules through the nuclear pore channel.
Small molecules (ions, metabolites, mononucleotides etc.) are able to passively diffuse into the nucleus. The conductivity of nuclear pores is different for molecules of different sizes. Proteins weighing less than 15 kDa quickly penetrate the nucleus, while proteins weighing more than 30 kDa require some time. Protein molecules weighing more than 60-70 kDa apparently cannot passively pass through nuclear pores at all. However, the capacity of nuclear pores for passive diffusion may vary depending on the cell type and cell cycle stage.
By active transport, much larger molecules and entire supramolecular complexes can pass through nuclear pores. Thus, ribosomal subparticles up to several megadaltons in size are transported from the nucleus to the cytoplasm through nuclear pores, and there is no reason to assume that the transport process is accompanied by partial disassembly of these subparticles. Active transport systems provide all macromolecular exchange between the nucleus and the cytoplasm. RNA molecules synthesized in the nucleus enter the cytoplasm through pores, and proteins involved in nuclear metabolism enter the nucleus. Moreover, some proteins must enter the nucleus constitutively (for example, histones), while others must enter in response to certain stimuli (for example, transcription factors). Special sequences responsible for their localization have been identified in nuclear proteins. The most common of them, the so-called “classical” nuclear localization signal NLS (from English, Nuclear Localization Signal), represents one or two sections of positively charged amino acids, arginine and lysine . Translocation of proteins into the nucleus, in contrast to translocation into mitochondria and the endoplasmic reticulum, is not accompanied by the cleavage of this signal sequence and the unfolding of the polypeptide chain. NLS-containing proteins, like all other substrates of nuclear transport systems, are transported into the nucleus in complex with special proteins, transportins or karyopherins. Each transportin or transportin complex must have three activities to carry out its function: firstly, it must recognize and bind the transported substrate, secondly, it must be anchored at the nuclear pore, and thirdly, it must bind a small protein called GTPase Ran, which belongs to the Ran family of proteins. Ras-like GTPases and serves to couple transport with GTP hydrolysis, which makes the process irreversible (supplies it with energy). The actual act of GTP hydrolysis is carried out directly by this protein. The nucleotide exchange factor (GTPase Exchange Factor, GEF) for Ran, the chromatin-binding protein RCC1, is localized strictly in the nucleus, and the activators of GTPase activity (GTPase Activation Protein, GAP) RanGAP1 and some other proteins are strictly in the cytoplasm . This asymmetric localization leads to the formation of a gradient: the GTP-bound form of Ran is predominantly found in the nucleus, and the GDP-bound form is found in the cytoplasm, on the contrary. Ran is used to supply energy to both the import and export processes of various substrates, and the entire circuit is called the Ran-cycle. The Ran cycle powers both export and import using a common principle mechanism, the key steps of which are the hydrolysis of GTP in the cytoplasm and the exchange of GDP for GTP in the nucleus.
- Regulation of molecular transport through the nuclear pore
Since the active transport of molecules between the nucleus and the cytoplasm, carried out by the NPC, is vital for various intracellular processes, it is controlled by many factors. They include 3 interacting systems: 1) a complex of biochemical regulators located in the nucleus or cytoplasm and binding to the signal sequences of the transported molecule and nuclear pore proteins; 2) a complex of nucleoporins that form the NPC and are capable of interacting with each other and biochemical regulators, and 3) a structural complex of the pore, consisting of a set of individual components that specifically change the spatial organization during the transport of molecules and thus ensure their more efficient transfer in the desired location direction. Let us briefly consider how transport is regulated by these three systems.
First system . Biochemical regulators include 5 main types of proteins involved in both the import and export of molecules: 1) transportins (importin a, importin b and a number of other factors); 2) Ran protein (guanosine triphosphatase), 3) GTP (guanosine triphosphate), 4) p10 protein, and 5) a set of additional proteins that provide activation, inhibition or change in the structural conformation of the above proteins, as well as their transport between the nucleus and the cytoplasm. The functional role of each of the listed regulators was established in studies conducted either in the system in vitro (using extracts from amphibian oocytes), or in vivo (mainly in experiments with yeast cells).Transportins play the role of receptor proteins that, through intermediary proteins (adapter proteins) or directly bind to the signaling regions of the transported molecule. Ran is a protein that is capable of utilizing GTP energy. It can have two states: associated with either GTP (Ran-GTP) or GDP (Ran-GDP). Ran does not hydrolyze GTP well, and to change its state, additional proteins located in the nucleus (RCCI) and in the cytoplasm (RanGAP1, RanBP1, and RanBP2) are required. Both forms of Ran are present in both the nucleus and the cytoplasm, but the concentration of Ran-GTP is higher in the nucleus, while Ran-GDP is found predominantly in the cytoplasm.
It is assumed that the p10 protein regulates the access of transported complexes into the central channel of the pore from the cytoplasm, possibly due to its interaction with nucleoporins that form the peripheral components of the pore (the transporter locking granule, internal filaments, and others). However, the main function of this protein is that it is able to bind to the Ran protein (in its various forms) and transport it to the nucleus or cytoplasm.
The process of import of molecules into the nucleus has now been studied in more detail than their export. The first requirement for a transported molecule is the presence of a signal sequence in its structure. It is assumed that the process of protein import into the nucleus includes several successive steps: first, importin b binds to importin a, which then, directly or through adapter proteins, recognizes the signal sequence in the transported molecule and binds to it. This ternary complex, due to the interaction of importin b with one of the peripheral nucleoporins, is anchored on the peripheral component of the pore, possibly on the cytoplasmic fibril. In parallel with this, the Ran protein binds to GTP in the cytoplasm, after which this complex is also fixed on the cytoplasmic fibril, due to the interaction of the Ran protein with nucleoporin, not far from the first complex. All these processes occur without consuming energy.Then the two complexes interact with each other and the p10 protein, which ensures the preparation of the peripheral part of the central channel for transport (it is assumed that p10 can open the entrance to the central channel of the pore from the cytoplasm). In this case, GTP hydrolysis occurs, and the entire formed complex moves from the cytoplasmic fibril to the central part of the pore and is further transported into the nucleus. The cytoplasmic entrance to the central channel of the pore then closes, and the complex that has moved into the nucleus is separated from the transferred molecule and disintegrates into a dimer consisting of importins a and b, and Ran-GDP. The latter complex, with the help of a specific factor, is again converted into Ran-GTP, which then separates importin a and importin b.Recently, evidence has emerged that many molecules can be imported into the nucleus without the participation of Ran and, accordingly, without energy consumption. In this case, the corresponding transportins and adapter proteins bind to the imported substrate in the cytoplasm, and this complex passes through the nuclear pore into the nucleus. In the nucleus, the transport factors of this complex interact with Ran-GTP, which leads to the release of the imported substrate due to the conversion of Ran-GTP to Ran-GDP. Then transport factors again bind to Ran-GTP and this complex returns to the cytoplasm.
It is assumed that many of the biochemical factors listed above may be involved in the regulation of not only the import, but also the export of proteins, as well as RNA from the nucleus to the cytoplasm. However, the process of export of mRNA from the nucleus is more complex than the import or export of proteins, since it is under the control of many additional factors, including various RNA-binding proteins. For example, it has been shown that when mRNA is exported from the nucleus, it is directed into the central channel of the pore at the 5 end, and cap-binding proteins located at the 5 end of the mRNA probably play an important role in this process. It has also been shown that for each class of RNA (mRNA, tRNA, rRNA, spliceosomal RNA) there are specific carrier proteins, some of which are detached from the RNA during its transport through the pore and remain in the nucleus, while others accompany the RNA molecule into the cytoplasm(Fig. 3).
Fig.3. Scheme of protein import into the nucleus.
1. Formation of the cargo-receptor complex (importin). 2. Anchoring of the complex on nuclear pore proteins and the translocation itself. 3. Dissociation of the cargo-importin complex under the influence of Ran-GTP, release of cargo, formation of the Ran-GTP-importin complex. 4. Re-export of the formed complex into the cytoplasm. 5. Hydrolysis of GTP and dissociation of the complex.
During the export of Ran-GTP molecules, it forms a complex with transportins, corresponding adapter proteins, and the exported substrate. This entire complex complex passes through the pore into the cytoplasm. Here, the cytoplasmic factors RanGAP1, RanBP1, and RanBP2 stimulate the hydrolysis of GTP, which causes the breakdown of the transported complex with the release of Ran-GDP. That is, the energy released in this case is used to free the transported molecules from their carriers. The p10 protein, which due to its small size (mw 10 kDa) can freely diffuse between the nucleus and the cytoplasm, binds to Ran-GDP in the cytoplasm and transports it to the nucleus. The nucleus contains the chromatin-bound factor RCCI, which causes the release of GDP and the conversion of Ran to the GTP-bound form. The process of Ran circulation between the nucleus and the cytoplasm is called the Ran GTPase cycle. Thus, it can be assumed that the Ran concentration gradient, constantly maintained between the nucleus and the cytoplasm, represents a mechanism that determines the direction of transport(Fig. 4).
Rice. 4. Scheme of protein export from the nucleus.
1. Formation of the cargo-exportin-Ran-GTP complex. 2. Anchoring of the complex on nuclear pore proteins and the translocation itself. 3. Hydrolysis of GTP, dissociation of the complex and release of cargo. 4. Re-import of released exportin.
Second system . Of the 50 putative nucleoporins (Nup) that are part of the nuclear pore of higher eukaryotes, about 40 proteins have now been described, 25 of which have already been sequenced. Almost all nuclear pore proteins have been characterized in yeast (30 proteins), and experimental data obtained in higher organisms are sparse. The distribution of many nucleoporins on various structural components of the pore has been studied immunohistochemically using antibodies to these proteins. It has been established that NPC proteins can be divided into 3 groups: the first contains proteins with specific repeating sequences (such as FXFG, etc.), which are recognized by biochemical factors; the second contains proteins that do not have such sequences, and the third includes the so-called integral proteins, localized either in the nuclear membrane membrane that forms the pore, or in the pore region located in the lumen between the nuclear membranes. A comparative analysis of nucleoporins in higher and lower eukaryotes showed the presence of 30-50% homology for 4 protein pairs: Nup62/Nsp1p; Nup107/Nup84; Nup155/Nup170; Nup98/Nup116 (the first in pairs are the proteins of higher eukaryotes, the second are the proteins of lower eukaryotes; the names of the proteins are given according to the classification generally accepted in the literature). Recently, it was found that nucleoporins can form complex complexes consisting of 5-7 proteins, which probably reflects their participation in the formation of individual pore components. Some of the nucleoporins, such as Nup188, Nup170, Nup157, Nic 96, POM152, account for up to 25% of the mass of nuclear pores and are present in 10-20 copies per pore.Evidence has been obtained that nucleoporins are directly involved in the regulation of the transport of molecules through the NPC. Thanks to their mutual contact, as well as interaction with biochemical factors carrying the transported molecule, they can ensure its sequential transmission, like a relay baton, from one section of the nuclear pore to another. Some of the nucleoporins can apparently bind directly to the transported molecule. For example, Nup153 and Nup98, which are part of the basket fibrils, contain RNA-binding domains, and Nup358 and CAN/Nup214, located on the cytoplasmic fibrils of the pore, recognize the signal sequences of some proteins. The transport of molecules through the central components of the pore is controlled by the Nup62 protein, which is the most representative and distributed along the entire central channel.
Third system . The use of a high-resolution scanning electron microscope made it possible for the first time to detect conformational changes in individual components of the NPC during the process of molecular transport. It was shown that the export of giant mRNA synthesized by the Balbiani ring genes in Chironomus is accompanied by a cyclic reorganization of the basket and transporter, which function as a system of opening and closing diaphragms.According to our observations made in a scanning electron microscope, in an inactive pore, both entrances to the central channel of the pore are closed by peripheral transporter granules. In addition, the entrance to the pore from the side of the nucleus is additionally closed by basket fibrils. In the first stage of export, the RNA molecule, packaged during transcription with proteins into a 50 nm RNP particle, moves inside the nucleus to the pore and attaches to the top of the basket. It is assumed that Nup153 and Nup98, who are part of the basketball, are actively participating in this event. Basket fibrils form a ring that increases in size, which gradually captures the particle, and it sinks into the basket. Since the maximum diameter of the central channel of the NPC is only 26 nm, the RNP particle inside the basket is decompacted into a 26 nm fibril. It was also found that the RNP particle rotates inside the basket, which is probably due to the need for its transport into the pore by the 5-end. Thus, the basket structure acts as a “customs house,” checking and preparing the RNP molecule for transport through the pore.
At the next stage, a hole opens in the peripheral granule of the transporter from the side of the nucleus and the RNP fibril begins to move inside the pore. The inner diameter of the central cylinders of the transporter, which previously had a size of 10 nm, expands to 26 nm, and the fibril is transported further through them, towards the cytoplasm. The peripheral granule of the transporter on the cytoplasmic side also forms a hole with a diameter of 26 nm, and the RNP fibril gradually completely enters the cytoplasm, where the translation process begins. After the end of transport, all components of the nuclear complex quickly return to their original state. It was found that during transport, the peripheral granules of the transporter can move in the vertical direction by 5 nm, and the time itself can be flattened or elongated, thus facilitating more efficient movement of the transported molecule. All these data indicate that the JPC is a very plastic and dynamic structure directly involved in the regulation of transport. However, it should be noted that in recent years, evidence has emerged that pora can actively transport up to 300 or more small molecules per second. This suggests the presence of some additional and still unknown to us mechanisms that ensure such a high rate of movement of molecules through the pore. Since the pore, on the one hand, is closely connected with the lamina and, therefore, with the nuclear matrix, and on the other hand, through the nuclear envelope with the cytoskeleton, the process of transport through the NPC can also be regulated at the level of these intracellular structures.
- Nuclear matrix
This complex does not represent any pure faction; it includescomponents of the nuclear membrane, nucleolus, and karyoplasm. With nuclearBoth heterogeneous RNA and part of DNA were bound to the matrix. Theseobservations have given reason to believe that the nuclear matrix plays an important role notonly in maintaining the general structure of the interphase nucleus, but can alsoparticipate in the regulation of nucleic acid synthesis.Nuclear matrixsome researchers call the insoluble intranuclear framework. It is believed that the matrix is built primarily from non histone proteins that form a complex branched network communicating with nuclear lamina . Perhaps the nuclear matrix takes part in the formation of functional chromatin domains. IN genome cells have special insignificant A-T-richsites of attachment to the nuclear matrix(eng. S/MAR M atrix/ S caffold A ttachment R egions), which are thought to serve to anchor chromatin loops to nuclear matrix proteins. However, not all researchers recognize the existence of the nuclear matrix.
- Chromatin
Chromatin is a substance that accepts dye well (chromos), hence its name. Chromatin consists of chromatin fibrils with a thickness of 20 × 25 n m, which can be located loosely or compactly in the core. On this basis, we can distinguish euchromatin - loose (or decondensed) chromatin, weakly stained with basic dyes, and heterochromatin - compact (or condensed) chromatin, well stained with basic dyes. When a cell prepares for division, chromatin fibrils spiral in the nucleus and transform chromatin into chromosomes. After division, chromatin fibrils despiralize in the nuclei of daughter cells, and the chromosomes are again converted into chromatin. Thus, chromatin and chromosomes are different states of the same substance. The ratio of euchromatin and heterochromatin is an indicator of the synthetic activity of the cell. DNA reduplication occurs on chromatin fibrils during the S-period of interphase. These processes can also occur in heterochromatin, but much longer. When observing some living cells, especially plant orcells after fixation and staining, zones of densesubstances. Chromatin consists of DNA in complex with protein. In interphaseIn cells, chromatin can evenly fill the volume of the nucleus or be locatedseparate clots (chromocenters). Often it is especially clearly visible onperiphery of the nucleus (parietal, near-membrane chromatin) or forms insidethe cores of the weave are quite thick (about 0.3 µm) and long strands,forming something like an intranuclear chain.Chromatin of interphase nuclei is a DNA-carrying body(chromosomes), which at this time lose their compact shape,loosen and decondense. The degree of such chromosome decondensation canbe different in the nuclei of different cells. When a chromosome or a region of it is completely decondensed, then these zones are called diffuse chromatin.
With incomplete loosening of chromosomes in the interphase nucleus, areas
condensed chromatin (sometimes called heterochromatin). Shownthat the degree of decondensation of chromosomal material in interphase may reflectfunctional load of this structure. The more diffuse the chromatininterphase nucleus, the higher the synthetic processes in it. Synthesis FallRNA in cells is usually accompanied by an increase in condensed zones chromatin. Chromatin is maximally condensed during mitotic cell division,when it is found in the form of dense bodies - chromosomes. In this periodchromosomes do not carry any synthetic loads;inclusion of DNA and RNA precursors.Based on this, we can assume that cell chromosomes can be intwo structural and functional states:
In working condition, partially or completely decondensed, when with them
participation in the interphase nucleus processes of transcription and
reduplication;
Inactive - in a state of metabolic rest at maximum
condensation when they perform the function of distribution and
transferring genetic material to daughter cells.
Chemically, chromatin preparations are complexcomplexes of deoxyribonucleoproteins, which include DNA andspecial chromosomal proteins - histones. Found in chromatinalso RNA. Quantitatively, DNA, protein and RNA are found as1:1.3:0.2. There is still not enough information about the importance of RNA in the composition of chromatinunambiguous data. It is possible that this RNA represents an accompanyingthe drug functions as a synthesized RNA and therefore partially associated with DNAor is it a special type of RNA characteristic of the chromatin structure.Huge length of molecules DNA eukaryotes predetermined the emergence of special mechanisms for the storage, replication and implementation of genetic material. Chromatin called molecules chromosomal DNA in complex with specific proteins necessary to carry out these processes. The bulk consists of “storage proteins”, the so-called histones . Nucleosomes are built from these proteins - structures on which strands of DNA molecules are wound. Nucleosomes are arranged quite regularly, so that the resulting structure resembles beads. The nucleosome consists of four types of proteins: H2A, H2B, H3 and H4. One nucleosome contains two proteins of each type; a total of eight proteins. Histone H1, larger than other histones, binds to DNA at its entry site into the nucleosome. The nucleosome together with H1 is called a chromatosome. The DNA strand with nucleosomes forms an irregular solenoid -like structure with a thickness of about 30 nanometers , the so-called 30 nm fibril. Further packaging of this fibrils may have different densities. If chromatin is packed tightly, it is called condensed or heterochromatin, and it is clearly visible under a microscope. DNA found in heterochromatin is nottranscribed, this condition is usually characteristic of insignificant or silent areas. In interphase, heterochromatin is usually located along the periphery of the nucleus (parietal heterochromatin). Complete condensation of chromosomes occurs before cell division. If chromatin is loosely packed, it is called eu- or interchromatin. This type of chromatin is much less dense when observed under a microscope and is usually characterized by the presence of transcriptional activity. The density of chromatin packing is largely determined by histone modificationsacetylation And phosphorylation.
It is believed that in the nucleus there are so-called functional chromatin domains (DNA of one domain contains approximately 30 thousand base pairs), that is, each section of the chromosome has its own “territory”. Unfortunately, the issue of spatial distribution of chromatin in the nucleus has not yet been sufficiently studied. It is known that telomeric (terminal) and centromeric (responsible for linking nursing chromatid in mitosis ) sections of chromosomes are attached to nuclear lamina proteins.
- DNA chromatin
In a chromatin preparation, DNA usually accounts for 30-40%. This DNAis a double-stranded helical molecule. DNA chromatinhas a molecular weight of 7-9*106. Such a relatively small massDNA from drugs can be explained by mechanical damage to DNA inprocess of chromatin separation.The total amount of DNA included in the nuclear structures of cells, in the genomeorganisms varies from species to species. Comparing the amount of DNA per celleukaryotic organisms, it is difficult to discern any correlations betweenthe degree of complexity of the organism and the amount of DNA per nucleus. About the sameThe amount of DNA is found in various organisms, such as flax, sea urchin, perch (1.4-1.9 pg) or char and bullfish (6.4 and 7 pg).Some amphibians have more DNA in their nuclei than human nuclei.10-30 times, although the human genetic constitution is incomparably more complex,than frogs. Therefore, it can be assumed that “excessive”the amount of DNA in lower organized organisms is either not related tofulfilling a genetic role, or the number of genes is repeated this or that number of times. Satellite DNA, or DNA fraction with frequently repeatedsequences, may participate in the recognition of homologous regionschromosomes during meiosis. According to other assumptions, these areas play a roleseparators (spacers) between different functional unitschromosomal DNA.As it turned out, the moderately repeating fraction (from 102 to 105 times)sequences belongs to a motley class of DNA regions that playimportant role in metabolic processes. This fraction includes ribosomal DNA genes,repeatedly repeated sites for the synthesis of all tRNAs. Moreover,some structural genes responsible for the synthesis of certain proteins,can also be repeated many times, represented by many copies (genesfor chromatin proteins - histones).
So, the DNA of eukaryotic cells is heterogeneous in composition, containsseveral classes of nucleotide sequences:
Frequently repeated sequences (>106 times) included in the fractionsatellite DNA and non-transcribed;
Fraction of moderately repetitive sequences (102-105),representing blocks of true genes, as well as shortsequences scattered throughout the genome;
A fraction of unique sequences that carries information for
most cell proteins.
The DNA of a prokaryotic organism is one giant
cyclic molecule. The DNA of eukaryotic chromosomes islinear molecules consisting of tandem (one after another) locatedreplicons of different sizes. The average replicon size is about 30 microns. Themthe human genome itself should contain more than 50,000 replicons,sections of DNA that are synthesized as independent units. These repliconshave starting and terminal points of DNA synthesis.Let us imagine that in eukaryotic cells each of the chromosomal DNA,like bacteria, it is one replicon. In this case, at speedsynthesis 0.5 µm per minute (for humans) reduplication of the first chromosome fromDNA about 7 cm long should take 140,000 minutes, or about three months.In fact, due to the polyreplicon structure of DNA molecules, the entirethe process takes 7-12 hours.
- Chromatin proteins
These include histones and non-histone proteins.
Histones are strongly basic proteins. Their alkalinity is related to their enrichmentbasic amino acids (mainly lysine and arginine). These proteinsdo not contain tryptophan. The total histone preparation can be divided into 5 factions:
H1 (from English histone) - lysine-rich histone, they say. Weight 2100;
H2a - moderately lysine-rich histone, weight 13,700;
H2b - moderately lysine-rich histone, weight 14,500;
H4 - arginine-rich histone, weight 11,300;
H3 - arginine-rich histone, weight 15,300.
In chromatin preparations, these histone fractions are found inapproximately equal quantities, except for H1, which is approximately 2 timesless than any of the other factions.Histone molecules are characterized by an uneven distribution of the mainamino acids in the chain: enriched with positively charged amino groupsobserved at the ends of protein chains. These histone regions bind tophosphate groups on DNA, while comparatively lesscharged central regions of molecules ensure their interaction betweenyourself. Thus, the interaction between histones and DNA, leading tothe formation of a deoxyribonucleoprotein complex is ionic in nature.Histones are synthesized on polysomes in the cytoplasm, this synthesis beginsslightly earlier than DNA reduplication. Synthesized histones migrate fromcytoplasm into the nucleus, where they bind to sections of DNA.The functional role of histones is not entirely clear. At one time it was believed thatHistones are specific regulators of DNA chromatin activity, butthe similarity of the structure of the bulk of histones indicates a low probabilitythis. More obvious structural role histones, which ensuresonly the specific folding of chromosomal DNA, but also plays a role in the regulation
transcriptions. Non-histone proteins are the most poorly characterized fraction of chromatin.In addition to enzymes directly associated with chromatin (enzymesresponsible for repair, replication, transcription and modification of DNA,enzymes modifying histones and other proteins), this fraction includesmany other proteins. It is very likely that some of the non-histone proteinsare specific proteins - regulators that recognize certainnucleotide sequences in DNA.
Chromatin RNA makes up 0.2 to 0.5% of the DNA content. This RNArepresents all known cellular types of RNA that are in processsynthesis or maturation in connection with chromatin DNA.Lipids up to 1% by weight can be found in chromatinDNA content, their role in the structure and functioning of chromosomes remains
unclear.
- Chromosomes
The primary degree of folding of DNA molecules is chromosomal fibril. Observationschromatin structure using an electron microscope showed that inIn the composition of the nucleus, fibrillar elements are always visible on ultrathin sections.They were first discovered by H. Rees (1957), who gave them the name elementarychromosomal fibrils.
Chromosome morphology
The morphology of chromosomes is best studied at the time of their greatestcondensation, in metaphase and at the beginning of anaphase. Chromosomes of animals and plants inin this state they are rod-shaped structures of different lengths withfairly constant thickness; most chromosomes can be easily foundthe primary constriction zone, which divides the chromosome into two arms.Chromosomes with equal or almost equal arms are called metacentric, withshoulders of unequal length - submetacentric. Rod chromosomes
with a very short, almost invisible second shoulder - acrocentric.
The centromere, or kinetochore, is located in the region of the primary constriction. Thisdisc-shaped lamellar structure. It is connected by thin fibrilswith the chromosome body in the constriction region. Tufts grow from itmicrotubules of the mitotic spindle running towards the centrioles.They take part in the movement of chromosomes to the poles of the cell during mitosis.Usually one chromosome has only one centromere (monocentricchromosomes), but dicentric andpolycentric.Some chromosomes have a secondary constriction. The latter is usuallylocated near the distal end of the chromosome and separates the smallsite, satellite Secondary constrictions are also called nucleolar constrictionsorganizers, since it is on these chromosome regions in interphase
the formation of a nucleolus occurs. The DNA responsible for rRNA synthesis. The chromosome arms end in telomeres, the terminal regions. Telomericthe ends of the chromosomes are not able to connect with other chromosomes or theirfragments, in contrast to the ends of chromosomes lacking telomeric regions,which can attach to the same broken ends of other chromosomes.
The size of chromosomes varies widely among different organisms. So,chromosome length can vary from 0.2 to 50 microns. The smallest chromosomesfound in some protozoa and fungi. The longest ysome orthopteran insects, amphibians and lilies. Chromosome lengthhuman is in the range of 1.5-10 microns.
The number of chromosomes in different objects also varies significantly, but
characteristic of each species. Some radiolarians have the number of chromosomes
reaches 1000-1600. The record holder among plants for the number of chromosomes (about 500) is the grass fern, the mulberry tree has 308 chromosomes, and the river grass fern has 196 chromosomes. Lowest number of chromosomes (2 per diploid set)observed in one of the races of roundworm, in the Asteraceae Haplopappus gracilic - only 4 chromosomes (2 pairs).The totality of the number, size, size and morphology of chromosomes is calledkaryotype of this species. Even closely related species have different chromosome setsfrom each other or by the number of chromosomes, or by the size of at least one orseveral chromosomes. Therefore, the karyotype structure may betaxonomic feature.
- Nucleolus
In almost all living cells of eukaryotic organisms, the nucleus is visibleone or more usually round bodies that strongly refract light, -these are nucleoli, or nucleoli.The nucleolus is not an independent structure or organelle. It is derivativechromosomes, one of its loci, actively functioning in interphase.In the processes of synthesis of cellular proteins, the cell nucleus is the placeformation of ribosomal RNA and ribosomes on which synthesis occurspolypeptide chains.The nucleolus is located inside the nucleus and does not have its own membrane shell, but clearly visible under light and electron microscope. The main function of the nucleolus is the synthesis ribosomes In the genome cells have special areas, the so-called nucleolar organizers, containing genes ribosomal RNA (rRNA), around which the nucleoli are formed. rRNA synthesis occurs in the nucleolusRNA polymerase I, its maturation, assembly of ribosomal subunits. The proteins involved in these processes are localized in the nucleolus. Some of these proteins have a special sequence called the Nucleolus Localization Signal (NoLS). It should be noted that the highest concentration of protein in the cell is observed in the nucleolus. About 600 types of different proteins were localized in these structures, and it is believed that only a small part of them is actually necessary for the implementation of nucleolar functions, and the rest get there nonspecifically.
- Number of nucleoli in a cell
Starting from green algae, fungi and lower protozoa to higherorganisms, all cells have obligatory intranuclear structures -nucleoli. This rule has a large number of exceptions, which are onlyemphasize the importance and necessity of the nucleolus in the life cycle of the cell. TOsuch exceptions include cells of cleavage eggs, where nucleoli are absentin the early stages of embryogenesis, or cells that have completed development and are irreversiblyspecialized, for example, some blood cells.The number of nucleoli in a cell may vary, but their number per nucleusdepends on the gene balance of the cell. It was found that in the formation of nucleolicertain places on some chromosomes are involved, the connection of which with the nucleoluscan be clearly observed in telophase and prophase. These chromosomes are usuallyhave secondary constrictions, the zones of which represent the places where it goesdevelopment of nucleoli. McClintock (1934) called these chromosome regions“nucleolar organizers”.Places of secondary constrictions are especially characteristic of the location of the nucleolarorganizers, but the latter can sometimes be located at the ends of chromosomes orin several places along the length of the chromosome.The total number of nucleoli per nucleus is determined by the number of nucleolar organizers
and increases according to the ploidy of the nucleus. However, often the number of nucleoliThere are fewer nucleolar organizers per nucleus. It has been shown thatnucleoli may fuse; in addition, in the formation of one nucleolus sometimesSeveral organizers are involved.
Even in the works of M.S. Navashin (1934) it was shown that the chromosomal locus,which under normal conditions forms a large nucleolus, becomesinactive when, after hybridization, a “stronger” one appears in the nucleuslocus on another chromosome. The fact that under certain conditions maythe activity of some nucleolar organizers is suppressed or increasedthe activity of others who were previously in a latent, hidden state,indicates that a certain balance is maintained in the cellsamount of nucleolar material or, in other words, is regulated“gross” production produced by nucleoli.
Based on the facts listed above, the following conclusions can be drawn:
The formation of nucleoli and their number are associated with the activity of certain
sections of chromosomes - nucleolar organizers, which are located
mostly in areas of secondary constrictions;
Changes in the number of nucleoli in cells of this type can occur overdue to the fusion of nucleoli or due to shifts in the chromosomal balance of the cell.
- Physiology and chemistry of the nucleolus
The nucleolus, in comparison with other components of the cell, is characterized asthe densest structure with the highest concentration of RNA, with extremelyhigh activity in relation to RNA synthesis.The concentration of RNA in nucleoli is always higher than the concentration of RNA in othercomponents of the cell, so the concentration of RNA in the nucleolus can be 2-8 timeshigher than in the nucleus, and 1-3 times higher than in the cytoplasm. Attitudethe concentration of RNA in the nucleus, nucleolus and cytoplasm of mouse liver cells is1:7.3:4.1, in pancreatic cells - 1:9.6:6.6.DNA is not detected in the nucleolus, but still, when examining fixedCells around the nucleolus always have a zone of chromatin. Thisperinucleolar chromatin, according to electron microscopy,appears to be an integral part of the complex structure of the nucleolus.The nucleolus is one of the most active places in the cell for inclusionprecursors in RNA. Nucleolar RNA ispredecessorcytoplasmic RNA. Cytoplasmic RNA is synthesized in the nucleolus.
- Nucleolar RNA
Evaluating general content in the nucleolar fractions of proteins, RNA and DNA, it is possiblesee that RNA accounts for about 10% of the total mass of the nucleolus.Since the bulk of cytoplasmic RNA is ribosomal RNA,then we can say that nucleolar RNA belongs to this class.Confirming the idea that the nucleolus is the placerRNA synthesis and ribosome formation, was due to the fact that from the nucleoluspreparations, RNP particles were isolated, which, both in RNA composition (insedimentation properties), and by size can be characterized asribosomes or their precursors with different sedimentation coefficients.
- Nucleolar DNA
Biochemical studies found in isolated nucleolia certain amount of DNA that can be identified with the perinucleolarchromatin or with nucleolar chromosome organizers. DNA content inisolated nucleoli - 5-12% of dry weight and 6-17% of the total DNA of the nucleus.The DNA of the nucleolar organizer is the same DNA on which thesynthesis of the nucleolus, i.e. ribosomal, RNA.Thus, from biochemical works, ideas emerged that inNumerous identical genes for synthesis are localized on the DNA nucleolusrRNA. rRNA synthesis proceeds through the formation of a huge precursor andits further transformation (maturation) into shorter RNA molecules forlarge and small ribosomal subunits.While studying the nucleoli of newt oocytes, researchers came across an interestingphenomenon - supernumerary nucleoli. In X. laevis, during oocyte growthUp to 1000 small nucleoli appear, not associated with chromosomes. Exactly thesenucleoli were isolated by O. Miller. at the same time, the oocyte nucleus increasesamount of rDNA. This phenomenon is called amplification. Itis that superreplication of the nucleolar zone occursorganizer, numerous copies leave the chromosomes and becomeadditionally working nucleoli. This process is necessary for the accumulationa huge (1012) number of ribosomes per egg cell, which will providefuture embryo development early stages even in the absence of synthesisnew ribosomes. Supernumerary nucleoli after egg cell maturation disappear.
- Ultrastructure of nucleoli
When studying a large number of different cells of animals and plants, it was notedfibrous or reticulate structure of the nucleoli, enclosed in more or lessdense diffuse mass. Names have been suggested for these parts:fibrous part - nucleoneme and diffuse, homogeneous part amorphoussubstance or amorphous part. Made almost simultaneously with thiselectron microscopic studies also revealed the fibrous-filamentous structure of the nucleoli.
However, this filamentous structure of the nucleolus is not always clearly expressed. U
In some cells, individual strands of nucleonemes merge, and the nucleoli can becompletely homogeneous.Upon closer examination of the nucleolus, one can notice that the mainthe structural components of the nucleolus are dense granules with a diameter of about 15 nm andthin fibrils 4-8 nm thick. In many cases (oocytes of fish and amphibians,meristematic cells of plants) the fibrillar component is assembled into a densecentral zone (core), devoid of granules, and granules occupyperipheral zone of the nucleolus. In some cases (for example, root cellsplants) in this granular zone there is no additional structuring. It was found that the amorphous areas of the nucleoli are heterogeneous. In their structureslightly colored zones - fibrillar centers - and surrounding areas are revealeddarker areas, also having a fibrillar structure.In addition to these two components of the nucleoli, recently there has been much attentionfocused on the structure of perinucleolar chromatin. This chromatin andintranucleolar network of DNA are a single system and representintegral component of the nucleolus.The granules and fibrillar part consist of ribonucleoproteins.It was shown that it is the light fibrillar centers that contain rDNA.
- The fate of the nucleolus during cell division
It is known that the nucleolus disappears in prophase and appears again in middle telophase. As rRNA synthesis decays in the middle prophase, loosening occursnucleolus and release of finished ribosomes into the karyoplasm, and then into the cytoplasm. Atcondensation of prophase chromosomes, fibrillar component of the nucleolus and partgranules are closely associated with their surface, forming the basis of the matrixmitotic chromosomes. This fibrillar-granular material,synthesized before mitosis, it is transferred by chromosomes to daughter cells.In early telophase, as chromosomes decondense, release occursmatrix components. Its fibrillar part begins to gather into smallnumerous associates - prenucleoli, which can combine with each otherfriend. As RNA synthesis resumes, the prenucleoli undergorestructuring, which is expressed in the appearance of RNA granules in their structure, and thenin the formation of the definitive form of a normally functioning nucleolus.
- TOarioplasm
The cells of all organisms have a single structural plan, which clearly shows the commonality of all life processes. Each cell includes two inextricably linked parts: the cytoplasm and the nucleus. Both the cytoplasm and the nucleus are characterized by complexity and strict orderliness of structure, and, in turn, they include many different structural units that performabsolutelycertainfunctions.Karyoplasm (nuclear juice, nucleoplasm) in the form of an unstructured mass surrounds the chromosomes and nucleoli. The viscosity of nuclear juice is approximately the same as the viscosity of the main substance of the cytoplasm. The acidity of nuclear juice, determined by microinjection of indicators into the nucleus, turned out to be slightly higher than that of the cytoplasm. In addition, nuclear sap contains enzymes involved in the synthesis of nucleic acids in the nucleus, andribosomes.Nuclear juice is not stained with basic dyes, so it is called achromatin substance, or karyolymph, in contrast to areas that can be stained, chromatin. Karyoplasm is the main internal environment of the nucleus; it occupies all the space between the nucleolus, chromatin, membranes, all kinds of inclusions and other structures. Under an electron microscope, karyoplasm appears as a homogeneous or fine-grained mass with low electron density. It contains ribosomes, microbodies, globulins and various metabolic products in suspension.Karyoplasm is characterized by special structural and functional properties. The functions of karyoplasm are extremely diverse, since the colloidal properties of the nucleus are associated with it, as well as the phenomena of growth, DNA synthesis, various RNA and proteins, transmission of irritation, etc. Physicochemical characteristics karyoplasma is due to its colloidal nature. They are determined by the presence of many particles in it, which together form a huge surface of interaction with the environment, which ensures the passage of variousphysico-chemicalprocesses.
Thanks to the force of surface tension that arises on a microscopic lump of karyoplasm, the process of adsorption occurs - the concentration of one substance on the surface of another. Depending on the magnification given by the microscope, karyoplasm appears homogeneous or granular, granular. The size of the granules is close to the size of macromolecules. The viscosity of karyoplasm, measured in centipauses, can change significantly under the influence of external or internal factors (the viscosity of water at a temperature of 20 degrees is taken as the unit of measurement). The viscosity of karyoplasm, measured in centipoise, can change significantly under the influence of external or internal factors (the viscosity of water at a temperature of 20 degrees is taken as the unit of measurement). The viscosity of the karyoplasm of a plant cell reaches 3-4 cP. In particular, it depends on temperature and concentration: hypotonic solutions cause it to decrease, hypertonic solutions cause it to increase. During mitotic divisioncellsherviscositycontinuouslyincreases.Karyoplasm-
the least dense part of the core, while membrane systems have a denser structure. The density of karyoplasm ranges from 1.025 to 1.055. Chemical composition it is extremely complex and is represented by organic and inorganic substances. The main organic substances are proteins, carbohydrates, deoxyribonucleic and ribonucleic acids, fat-like substances (lipids).
Of simple proteins (proteins), karyoplasm contains histones, protamines, albumins and globulins, and of proteids - lipoproteins, glucoproteins and nucleoproteins. Most proteins belong to globular structures, and a smaller part belongs to fibrillar structures. Proteins of globular shape that can turn into fibrillar are calledstructural.
To study the ultrastructure of the nucleus, a method is used that is based on tissue homogenization or destruction of nuclear walls and subsequent separation of subnuclear structures (fractionation). The main ones in it are enzymes that take part in the processes of activation of amino acids during protein synthesis. This fraction also includes enzymes that catalyze many reactions that require ATP energy.
Of the inorganic substances, karyoplasm usually contains a large amount of water (80-85%), which plays an important role in the life of both the nucleus and the cell. Karyoplasmic water can be in a free state (in the form of a solvent) and be bound by hydrogen bonds to polargroupsproteinmolecules.
Other inorganic substances of karyoplasm are contained in the form of salts, ions or in combination with proteins, amino acids, carbohydrates and lipids. Highest value in the construction of karyoplasm the elements are calcium, phosphorus, potassium and sulfur. Karyoplasm accounts for approximately 20% of the mass of the nucleus. In addition to the widespread elements (C, O, H, N, K, Ca, Mg, P, S, Fe, Na, Cl), Li, Ba, Cu, Zn, Si, F, Cr, Br are found in the cells of some organisms , J, Ag. Although many of them are found in very small quantities, they are essential for properfunctioningkernelsAndcells.It is assumed that metal ions play the role of cofactors of nuclear enzymes, factors of permeability and transfer of substances through the membrane and shell, complexing agents of the inorganic component of the karyoplasm itself, which maintains a certain ionic strength liquid phase. However, the function of each of these metals is strictly specific. This explains the importance of microelements in the life of organisms.
- Role of the kernel
The kernel performs two groups of general functions: one related to thestorage of genetic information, the other with its implementation, withensuring protein synthesis.The first group includes processes associated with the maintenance of hereditaryinformation in the form of an unchangeable DNA structure. These processes are associated with the presenceso-called repair enzymes that eliminate spontaneous
damage to the DNA molecule (break of one of the DNA chains, part of the radiationdamage), which keeps the structure of DNA molecules practically unchanged inover a number of generations of cells or organisms. Next, in the core there isreproduction or reduplication of DNA molecules, allowing twocells get exactly the same in both qualitative and quantitativemeaning volumes of genetic information. Processes occur in the nucleichanges and recombination of genetic material that is observed duringmeiosis (crossing over). Finally, kernels are directly involved in processes
distribution of DNA molecules during cell division.Another group of cellular processes provided by the activity of the nucleus isis the creation of the protein synthesis apparatus itself. It's not onlysynthesis, transcription on DNA molecules of various messenger RNAs andribosomal RNA. The formation of subunits also occurs in the nucleus of eukaryotesribosomes by complexing ribosomal RNA synthesized in the nucleolus withribosomal proteins that are synthesized in the cytoplasm and transported tocore.
Thus, the nucleus is not only the receptacle of geneticmaterial, but also the place where this material functions and is reproduced.Therefore, lil loss is a violation of any of the above functionsdetrimental to the cell as a whole. So the disruption of reparation processes will belead to a change in the primary structure of DNA and automatically changeprotein structure, which will certainly affect their specific activity,which may simply disappear or change in such a way that it will notprovide cellular functions, resulting in cell death.Disturbances in DNA replication will lead to a stop in cell reproduction or tothe appearance of cells with an incomplete set of genetic information, which alsoharmful to cells. Process disruption will lead to the same result.distribution of genetic material (DNA molecules) during cell division.Loss as a result of damage to the core or in case of violations of anyregulatory processes of synthesis of any form of RNA will automatically lead tostopping protein synthesis in the cell or causing severe disturbances.The importance of the nucleus as a repository of genetic material and its the main role Vdetermination of phenotypic traits have been established for a long time. Germanbiologist Hammerling was one of the first to demonstrate the crucial role of the nucleus. Hechose as the object of his experiments an unusually large
single-celled (or non-cellular) seaweed Acetabularia. Existstwo closely related species A. medierranea and A. crenulata, differingonly in the shape of a “hat”.In a number of experiments, including those in which the “hat” was separated fromthe lower part of the “stalk” (where the nucleus is located), Hammerling showed that fornormal development of the cap requires a nucleus. In further experiments, inwhich were connected by a lower part containing a core of one type with a coreless onestem of a different species, such chimeras always developed a cap, typicalfor the species to which the nucleus belongs.When evaluating this nuclear control model, one should, however, take into accountthe primitiveness of the organism used as an object. Methodtransplantations were used later in experiments conducted in 1952 by twoAmerican researchers, Briggs and King, with Rana frog cellspipenis. These authors removed nuclei from unfertilized eggs and replacedtheir nuclei from late blastula cells, already showing signsdifferentiation. In many cases, recipient eggs developednormal adult frogs.
Conclusion:
Core (lat.nucleus) this is one ofstructural componentseukaryoticcellscontaininggenetic information(moleculesDNA), performing the main functions: storage, transmission and sale of hereditary information with the provisionsynthesissquirrel. The core consists ofchromatin, nucleolus, karyoplasm (or nucleoplasm) and nuclear membrane. Occurs in the cell nucleusreplication(or reduplication) doubling of DNA molecules, as well astranscriptionsynthesis of moleculesRNAon a DNA molecule. RNA molecules synthesized in the nucleus are modified and then released intocytoplasm. Formation of both subunitsribosomesoccurs in special formations of the cell nucleusnucleoli. Thus, the cell nucleus is not only the repository of genetic information, but also the place where this material functions and reproduces.
Bibliography:
- Chentsov Yu.S., Polyakov V.Yu. “Ultrastructure of the cell nucleus.” M., Science,
1974
- Zegnbusch P. “Molecular and cell biology" M., Mir, vol. 1,2, 1982
- Leninger A. L. Fundamentals of biochemistry. In 3 T. M.: Mir, 1985. 1056 p.
- Reshetnikov V.N. Cell nuclei of higher plants. Composition, structure, functions. Minsk: Navuka i tehnika, 1992. 88 p.
- Harris G. Nucleus and cytoplasm. M.: Mir, 1973. 192 p.
- Davis L.I.The nuclear pore complex // Annu. Rev. Biochem. 1995. V. 64. P. 865-896.
- Ryan K.J. and Wente S.R. The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm.// Curr.Opin. Cell Biol.. 2000. P.12.P.361-371.
In preparing the abstract, materials obtained fromWorldwide Biological Network (BIOSCI) via the Internet.
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3. The nucleus, its structure and biological role.
The core consists of 1) surface of the core apparatus(it contains: 2 membranes, perinuclear spaces, pore complexes, lamina.) 2) karyoplasma(nucleoplasm) 3) chromatin(it contains euchromatin and heterochromatin) 4) nucleolus(granular and fibrillar components.)
The nucleus is a cell structure that performs the function of storing and transmitting information, and also regulates all the life processes of the cell. The nucleus carries genetic (hereditary) information in the form of DNA. The nuclei are usually spherical or ovoid in shape. The nucleus is surrounded by a nuclear envelope. The nuclear envelope is permeated with nuclear pores. Through them, the nucleus exchanges substances with the cytoplasm (the internal environment of the cell). The outer membrane passes into the endoplasmic reticulum and can be studded with ribosomes. The ratio of the sizes of the nucleus and the cell depends on the functional activity of the cell. Most cells are mononuclear. Cardiomyocytes can be binucleate. Ciliates are always binucleate. They are characterized by nuclear dualism (that is, the nuclei differ in structure and function). The small nucleus (generative) is diploid. It provides only the sexual process in ciliates. The large (vegetative) nucleus is polyploid. It regulates all other life processes. The cells of some protozoa and skeletal muscle cells are multinucleated.
4. Surface apparatus of the nucleus, its structure and functions. Structure of the nuclear pore complex. Import and export of proteins through nuclear pores.
P.A.Y. or karyoteka ) has a microscopic thickness and is therefore visible under a light microscope. The superficial apparatus of the nucleus includes:
a) nuclear membrane, or karyolemma;. b) steam complexes; c) peripheral lamina densa (LPD), or lamina .
(1) Nuclear envelope (karyolemma). consists of 2 membranes - outer and inner, separated by the perinuclear space. Both membranes have the same fluid-mosaic structure as the plasma membrane and differ in the set of proteins. Among these proteins are enzymes, transporters and receptors. The outer nuclear membrane is a continuation of the GR membranes and can be studded with ribosomes, on which protein synthesis occurs. On the cytoplasmic side, the outer membrane is surrounded by a network of intermediate (vi-mentin) fipaments. Between the outer and inner membranes there is a perinuclear space - a cavity 15-40 nm wide, the contents of which communicate with the cavities of the EPS channels. The composition of the perinuclear space is close to the hyaloplasm and may contain proteins synthesized by ribosomes. home karyolemma function - isolation of hyaloplasm from karyoplasm. Special proteins of nuclear membranes located in the region of nuclear pores perform a transport function. The nuclear envelope is penetrated by nuclear pores, through which the karyoplasm and hyaloplasm communicate. To regulate such communication, the pores contain (2) pore complexes. They occupy 3-35% of the surface of the nuclear envelope. The number of nuclear pores with pore complexes is a variable value and depends on the activity of the nucleus. In the region of nuclear pores, the outer and inner nuclear membranes merge. The set of structures associated with a nuclear pore is called nuclear pore complex. A typical pore complex is a complex protein structure - containing more than 1000 protein molecules. At the center of the pore is located central protein globule(granule), from which thin fibrils extend radially to peripheral protein globules, forming a pore diaphragm. Along the periphery of the nuclear pore there are two parallel ring structures with a diameter of 80-120 nm (one on each surface of the karyolemma), each of which is formed 8 protein granules(globules).
The protein globules of the feather complex are divided into central And peripheral . By using peripheral globules macromolecules are transported from the nucleus to the hyaloplasm. (fixed in the membrane by a special integral protein. From these granules they converge towards the center protein fibrils, forming a partition - pore diaphragm)
It involves special proteins of peripheral globules - nucleoporins. Peripheral globules contain a special protein - a carrier of t-RNA molecules
Central globule specializes in the transport of mRNA from the nucleus to the hyalopdasm. It contains enzymes involved in the chemical modification of mRNA - its processing.
Granules of pore complexes are structurally associated with proteins of the nuclear lamina, which is involved in their organization
Functions of the nuclear pore complex:
Ensuring regulation of selective transport between the cytoplasm and the nucleus.
Active transfer V protein core
Transfer of ribosomal subunits into the cytoplasm
(3) PPP or lamina
layer 80-300 nm thick. adjoins from the inside to the inner nuclear membrane. The inner nuclear membrane is smooth, its integral proteins are associated with the lamina (peripheral lamina densa). The lamina consists of special intertwined lamin proteins that form the peripheral karyoskeleton. Lamin proteins belong to the class of intermediate filaments (skeletal fibrils). In mammals, 4 types of these proteins are known: Lomima A, B, B 2 and S. These proteins enter the nucleus from the cytoplasm. Laminas different types interact between them and form a protein network under the inner membrane of the nuclear envelope. With the help of lamins “B”, the PPP is connected to the special integral of the protein nuclear membrane. The proteins of the peripheral holobules “inside the ring” of the pore complex also interact with the PPP. Telomeric sections of chromosomes are attached to lamin “A”.
Lamina functions: 1) maintain the shape of the core. (even if the membrane is destroyed, the core, due to the lamina, retains its shape and the pore complexes remain in place.
2) serves as a component of the karyoskeleton
3) participating in the assembly of the nuclear membrane (formation of the karyolema) during cell division.
4) in the interphase nucleus, chromatin is attached to the lamina. Thus, the lamina provides the function of fixing chromatin in the nucleus (ensuring the orderly laying of chromatin, participates in the spatial organization of chromatin in the interphase nucleus). Lamin A interacts with telomeric regions of chromosomes.
5) providing structures with the organization of pore complexes.
import and export of proteins.
To the core through nuclear pores enter: enzyme proteins synthesized by cytoplasmic ribosomes that participate in the processes of replication and repair (repair of damage in DNA); enzyme proteins involved in the transcription process; repressor proteins that regulate the transcription process; histone proteins (which are associated with a DNA molecule and form chromatin); proteins that make up the ribosomal subunits: nuclear matrix proteins that form the karyoskeleton; nucleotides; ions mineral salts, in particular, Ca and Mg ions.
From the core mRNAs are released into the cytoplasm. tRNA and ribosomal subunits, which are ribonucleoprotein particles (protein-linked rRNA).
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Nuclear pore complexes (NPCs) are symmetrical structures located at the junction of the outer and inner nuclear membranes
In human cells, each NPC has a mass of about 120 x 10 6 Da, which is 40 times the mass of a ribosome, and consists of many copies of molecules including 30 proteins
The NPC contains filaments extending into the cytoplasm and basket-like structures extending into the nucleus
Nuclear pore complex(NPCs) of the nuclear membrane are the only channels that connect the nucleus and cytoplasm. In human cells, NPCs have molecular weight about 120 x 106 Yes and the outer diameter is about 120 nm. The total mass of the NPC is 40 times greater than the mass of the eukaryotic ribosome. The nuclear pore complex consists of many copies of approximately 30 different polypeptides, nucleoporins. In contrast to the NPC, ribosomes contain one copy of four types of RNA, and about 80 different polypeptides.
Nuclear pore complex(NPC) are barrel-shaped structures that pass through the nuclear envelope and protrude somewhat beyond both membranes, forming ring-shaped structures. As shown in the figure below, most NPCs exhibit eighth-order symmetry. From the nuclear and cytoplasmic sides, the pore looks different. The parts of the NPC that protrude into the nucleoplasm and cytoplasm are called terminal structures.
From the cytoplasmic side JPC the terminal structures are eight relatively short fibrils that extend into the cytoplasm over a distance of about 100 nm. On the nuclear side, similar fibrils form a ring. This structure is called the nuclear basket or "top". In some cells of multicellular organisms, additional fibrils are directed from the nuclear basket deep into the nucleus. On the side of the cytoplasm and nucleus, the terminal structures are the places of contact of transported molecules at the entrance and exit from the NPC.
Models describing nuclear pore structure, were proposed based on the analysis of hundreds of electron micrographs of individual NPCs obtained at high resolution. To superimpose images and analyze them, mathematical methods were used, making it possible to obtain an averaged picture of the electron density distribution or a generalized structure of the NPC core (this method does not provide optimal resolution of terminal structures).
The figure below shows models of the core structure JPC yeast and Xenopus cells. The cell sizes of S. cerevisiae and other unicellular eukaryotes are about 60 x 106 Da - i.e., half smaller than the sizes of the NPCs of multicellular organisms. However, despite the difference in size, their general structure is the same. The size of the central pore channel, as well as its transport properties, are also similar in Metazoa and yeast. Currently, the best images of NPCs are obtained by cryoelectron microscopy.
The NPC is characterized by an eighth-order symmetry axis located perpendicular to the core shell.Sometimes there are pores with seventh or ninth order symmetry.
The eighth-order symmetry is easily visible in enlarged images of individual NPCs (photos below).
An average electron micrograph obtained from several hundred individual photographs (bottom right).
As shown in the picture below, in any position JPC The outer and inner membranes of the nucleus merge. We don't know how this happens, but most likely fusion is an integral part of the process of NPC assembly in the nuclear envelope. The complexes are anchored in the shell by integral membrane proteins that are part of the basic structure. These proteins pass into the perinuclear space. NPCs penetrate the nuclear lamina and also adhere to it.
Generalized YPC model, based on many studies, suggests that the nuclear pore consists of several ring and spoke-like structures. These structures are interconnected in a complex way. YPCs consist of modular components. Using a scanning electron microscope, various structures can be observed that support this point of view. Based on the data obtained, a model is proposed that describes the assembly of modular structures. However, we cannot yet verify whether they are actually connected in this way. We also know very little about the YPC assembly process.
Fixation of cells allows observing the stages of material movement via YPK channel. When examining preparations under an electron microscope, it is often seen that the cavity of the central canal is filled with a dense medium. There are different points of view regarding the composition of this environment. According to one of them, the medium is the part of the NPC that is most strongly associated with the cargo transported through the channel. Therefore, the term conveyor or bushing is used to designate it. An alternative view suggests that the electron-dense material is actually a cargo-receptor complex. Based on high-resolution electron microscope studies, this material appears to have variable sizes and variable localization within the NPC channel, which is more consistent with the view that it is composed of cargo-receptor complexes.
In some JPC cells are found not only in the nuclear envelope, but also in structures called fenestrated membranes, which are stacks of double membranes containing NPC and located in the cytoplasm. Often NPCs in the layers of fenestrated membranes are located as shown in the figure below. Fenestrated membranes are typically present in invertebrate and vertebrate oocytes, but can also be observed in other cell types. Their origin and functions remain unknown.
Nuclear pore complex (JPC) mammalian cells are difficult to separate from the nuclear envelope because they are usually associated with the lamina, which is an insoluble structure, and are therefore an inconvenient object of study. Since fenestrated membranes do not have an underlying lamina, they represent a valuable source of NPC isolation for subsequent biochemical and cytological studies. Probably, the NPCs of fenestrated membranes have the same structure and composition as the pore complexes of the nuclear envelope.
NPCs have different terminal structures. As electron microscope studies show,
from the core side they are shaped like a basket (left),
and on the cytoplasmic side they are represented by fibrils (right).
Cytoplasmic fibrils and nuclear baskets of nuclear pores, visible in a transmission electron microscope.
Three-dimensional computer models of the nuclear complex, illustrating the distribution of average electron density.
The models are presented from the side, along the plane of the nuclear envelope, and from above, perpendicular to the envelope.
The outer and inner membranes of the nuclear envelope join at the nuclear pore complex.
It is assumed that YPCs are assembled from modular components. Photographs of these components taken in an electron microscope at different stages of NPC assembly after mitosis are shown.
Fenestrate membranes in Xenopus oocytes. The photograph was taken using a transmission electron microscope.