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Author(s) of the publication: N. Nikolsky, E. Kornilova

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Scientists working in this field feel that, sooner or later, they will be able to produce a molecular "portrait" of the cell reflecting the orderly pattern of millions of its molecules of DMA, RNA, proteins, lipids, low-molecular substances, water, etc. And that will not be a flash photo of the statics of a given moment. It will be what one could call a computer biologo-mathematical model which will make it possible to observe its vital activities. And this is because a cell is a dynamic system with an on-going replacement of all the "working" structures. What remains unchanged is only the genome, but even in the DNA molecules there occur constant lesions which have to be "repaired" by a special mechanism keeping watch on their structure. All processes of renovation of cell elements, of the movement and transport of substances within it are "legalized" and organized. Their uninterrupted functioning is ensured by the vesicular (from Latin vesicle-small bladder, blister) transport.

Pages. 41

By Academician Nikolai NIKOLSKY, Director, RAS Institute of Cytology; Yelena KORNILOVA, Dr. Sc. (Biol.), senior researcher of the same Institute

How does this vesicular transport operate? To illustrate the point, let us consider two examples. Let us imagine people moving across a market place of a small town crammed with carriages and merchants' stands. From a bird's-eye view these movements seem to be disorderly. In actual fact each of the participants, having spent a certain amount of time and efforts, finally reaches his destination. And, a second example-lots of rush-hour traffic at a multi-level road-crossing in a big city. "Streams" of cars and trucks traveling in opposite directions, on top of one another, merging and halting in traffic jams, but ultimately delivering passengers and freight to their destinations. And it was not accidental that the English synonym of vesicular transport is "traffic" (movement of vehicles).

These examples illustrate the movement of "cargoes" in the cell: newly synthesized proteins from one of the organelles-endoplasmatic reticulum (network) (EPR)-move towards the cysside of the Golgi's apparatus*, pass through it and, on the trans-side are packed into new vesicles, spreading out in different directions. This is what we call the biosynthetic route. If after the Golgi's apparatus proteins move towards the plasmatic membrane, the distance covered by them is called exocytotic, or secretory. The endocytotic flux, or stream, is directed in the opposite direction-into the cell, carrying the contents of the external environment and proteins of the plasmatic membrane. Phagocytosis and pinocytosis, as described in textbook on biology, are varieties of endocytosis. More than 90 percent of the molecules absorbed by the cell in such situations are recycled, with only few types of them passing the whole route to the end: through what are called early endosomes into late ones (the latter differ from the former by the time of origin, morphology and functions, etc.), then into lysosomes (structures containing enzymes capable of splitting proteins).

Part of the vesicles can pass the cell through and deliver their contents untouched to the opposite side. This process is known as transcytosis. And, incidentally, there is a number of other ones capable of performing exchange of proteins practically among all of the cell organoids.

And, clear enough, such busy "traffic of goods" should somehow be regulated. During the past decade researchers have identified the principles of the "traffic" of substances within the cell.

At the molecular level, the process of vesicular transport can be divided into several stages. At the first, a bulge is formed at the expense of one organelle (donor); later on it "spins off' and turns into a transport vesicle or tubule. Then the latter is delivered to a certain organelle (acceptor). At the third stage there occurs their mutual recognition and interaction, after which they blend. As a result all of the regulatory proteins, which govern the proceeding events, return to their initial positions, i.e. recycle (also by means of vesicular transport) into the membrane of the donor-organelle so as to take part in new cycles.

At the present time many proteins are known already which determine to this or that extent the aforesaid processes. They are grouped into several classes and are regarded as universal elements of the cell transport system. For example, in the formation of a vesicle the key role belongs to the so-called margins-protein complexes performing two functions: first, they promote, directly or indirectly, the bending of a membrane section in the area of a vesicle (bubble) formation; secondly, they provide for the "sorting out" of proteins as a result of which the bubbles are filled with certain contents, although sometimes they remain empty, which happens very rarely. As a rule they are "loaded" with proteins: either directly incorporated in the membrane or soluble and capable of forming complexes with the intertied specific receptors. The latter recognize and "anchor up" margin proteins in the zone of formation of vesicles.

Thus, with the help of the latter, proteins connected with the membrane are transported. This provides for the first specific level of the transport under consideration which functions on all the routes interconnecting intracellular organelles or leading to the plasmatic membrane. In the opposite direction, during the formation of endocytosis vesicles on the plasmatic membrane, they can incorporate, apart from lig-and-receptor complexes, also substances of the extracellular medium which have no receptors of their own.

'We consider the problem of vesicular transport within the framework of studies of the mechanism of action of the epidermal growth factor (EOF), which acts as mitogen (i.e. stimulates mitosis-process leading to proportional distribution of genetic material in cell division) for some cell types and participates in the regulation of their embriogenesis, differentiation, mobility and apoptosis**, that is belongs to the range of very important growth factors.

Cells can respond to EGF only if they contain a highly specific receptor- transmembrane protein, whose cytoplasmatic part contains an area capable of attaching inorganic phosphate (-PC 3- 4 ) to the remainder of tyro-

* Golgi's apparatus-a specialized region of the cytoplasm, often close to the nucleus, which is composed of stacks of flattened cisternae, numerous vesicles and some larger vacuoles. In secretory cells it is concerned with packaging of secretory products.- Ed.

** Apoptosis-process in which DNA fragmentation takes place and the cell dies down, as if "cowling up", with the surrounding tissues remaining unaffected.- Ed.

Pages. 42

Dynamics of EGF endocytosis (image under fluorescent microscope): a- cells, incubated with EGF-rhodanine at 4 o C; growth factors are associated with their receptors on plasmatic membranes and therefore only cell contours are visible. b-same cells 10 min after stimulation of endocytosis by means of transfer into medium heated to 37 o C; brightly glowing EGF-rhodanine has been removed from the plasmatic membrane so that one can see less brightly colored dotted structures-early endosomes. c-after 30 - 60 min fine glowing dots disappear, EGF receptor complexes have shifted to near-nucleus area where brightly glowing dots have appeared-late endosomes.

sine. This activity is stimulated after the binding of EGF with the receptor. As a result part of the tyrosine residues in the receptor molecule is phosphorylated* and are recognized by proteins-substrates of the receptor which leads to their activation.

On the other hand, the formation of EGF-receptor complexes upon plasmatic membrane stimulates their endocytosis. And the question appears: is there a connection of the process of signal transfer and vesicular transport? This and other questions were before us at the start of our search.

As specialists know, after a long incubation of cells with growth factor upon the membrane the number of receptors diminishes. This led us to the following idea: as different from most of the "metabolic" receptors (carrying complex proteins- transferrin, lipoproteins of low density, etc.) which return to the surface after the complexes dissociation in early endosomes, the EGF receptors are delivered to lysosomes and degrade therein. Thus, in this particular case the essence of endocytosis is the termination of the signal being stimulated on the cell surface.

Our studies have confirmed that both EGF and the receptor get into lysosomes. But to get there the receptor passes first through the early and then gets into the late endosomes. These latter ones are located near the core and possess "multivesicular" morphology (in other words, contain different numbers of small inner bubbles). And it is within them that the receptors degrade. For a long time this was believed to be the only possible way.

But as far back as in 1987 - 1989 our institute researchers discovered that a considerable share of EGF receptor complexes after endocytosis can recycle, returning back into the plasmatic membrane. At first it was decided that this discovery concerns cells possessing extremely large numbers of EGF receptors, and this overabundance is being simply "non-specifically" involved into the back flow of membranes.

Subsequent studies demonstrated, however, that the same picture is observed in cells with low numbers of receptors. What is more, the ratio between the paths of recycling and degradation can change within one and the same cells. This led us to the conclusion that recycling is a universal stage in the process of endocytosis which can influence the length of the signal stimulated by the EGF receptor. But if the path into the lysosomes results in a weakening of the signal because of a reduction of the number of receptors due to their degradation, the recycling receptors, nevertheless, can participate in several signal stimulation cycles. In this way the cell responds to the signal length and intensity. And how regulated is the endocytosis path of EGF- receptor complexes? What universal proteins of the transport system are involved? And what is the role in the process of the receptor itself? As indicated by our studies, it is not just a passive passenger. Using sets of cells carrying different forms of EGF mutant receptors, we realized: the tyrosinkinasa activity of the receptor is necessary for its entry into late endosomes (which means its entering the path of degradation). In order to accomplish its first task, the receptor, must, by means of phosphorylation, activate a certain protein (or proteins) which we call "sorter". Thus it is the sorting out into late endosomes (from the early ones) that takes place under special control, and the recycling takes place "by silent agreement".

And although the sorter protein has not yet been identified, we do know something about it already. Our analysis, conducted in conjunction with our former co-worker, Professor A. Sorkin (now US citizen, working at the Colorado State University), proved that the domain of binding of this protein with the receptor is located not far from its cytoplasmatic end, and can modulate the effectiveness of its interaction with the sorter protein. Incidentally, an activated receptor generates such a strong sorting signal that even certain impacts (such as suppression of oxidation of endosomes) blocking the lysosomal transport of many substances, is

* Phosphorylation-chemical reaction leading to the introduction into molecule of organic or inorganic substance of phosphoric acids residues (- PO 3 H 2 ).- Ed .

Pages. 43

Localization of EGF receptor (green) and Rab7 (red) in cells (under fluorescent microscope): a-30 min after stimulation of EGF endocytosis its receptor is localized near nucleus same as Rab7 (yellow color). b-endocytosis stimulated in cells treated with nocodazol, but neither EGF receptor nor Rab7 collect in near- nucleus area.

incapable of preventing its delivery to lysosomes.

Obviously, the sorting protein, which represents the "receptor-specific" part of the endocytosis machine, must be interacting with some universal cell regulators of transport. Our attention was caught by Rab7 protein, traditionally regarded as the marker of late endosomes. But the data as to which section of the path it services was very contradictory.

In our initial experiments we established that after stimulation of EGF endocytosis Rab7 is found first in the early and then in late endosomes and is redistributed together with the receptor into the near-nucleus area which as much as attests of its participation in the regulation of the process of sorting out into the late endosomes- which is the stage of special interest to us. But on its way to lysosomes the receptor moves from the peripheral zone of the cell into the one near the nucleus, and this transition depends on microcanals-one of the elements of the cell cytoskeleton.

The obtained results made us turn to the hypothesis of G. Griffits and G. Greenberg (European Joint Lab of Molecular Biology in Heidelberg, Germany) predicting that the transition from the early to late endosomes takes place with the help of bubbles, like those which provide transport between EGF and the Golgi's apparatus. But when we treated cells with nocodazol- an agent which depolymerizes micro-canals, we saw that the receptor treated with endocytosis does not shift to the near-nucleus area-the receptor remains in vesicles scattered around the whole cell. Nevertheless, the dynamics of EGF-receptor complexes getting into late endosomes and lysosomes, and also their degradation were not upset. And that means that the transfer into the near- nucleus area is needed for the receptor to get into structures possessing the properties of late endosomes. Which means that the latter can appear from early ones by way of "maturation"-a process of gradual change of membrane composition. This results from the fact that proteins typical of early endosomes, gradually recycle, and those characteristic of membranes of late endosomes are constantly arriving from the trans- network of the Golgi's apparatus.

And one more big surprise-the behavior of Rab7 protein in these conditions. This, like the receptor, does not move towards the nucleus, but is bound with certain structures distributed all along the cytoplasm. However-and this is surprising! - they do not contain EGF receptor! So it turns out that the destruction of microcanals completely "divorced" Rab7 and EGF receptor, but did not obstruct the normal endosomal processing* of EGF-receptor complexes. This could mean that Rab7 has nothing to do with the processes of membrane fusion (if they do take place in general) in the process of the receptor entry into late endosomes and lysosomes. It is most likely that Rab7 "builds up the architecture" of the endocytosis path, providing for the interaction of endosomes with micro-canals and their shift to the near-nucleus area. Soon there appeared data from other labs confirming the above conclusion. Thus, the Bruno Goud team (Curie Institute, Paris, France) discovered that Rab6 interacts specifically with a protein which has the properties of the motor protein of kinesin, and Dr. Marino Zerial with his colleagues from the aforesaid lab in Heidelberg demonstrated that Rab5, being expressed in a cell in large quantities, is redistributed towards the nucleus by way of micro-tubules.

Thus it has been possible to demonstrate for the first time the new function of Rab- proteins-their interaction with cytoskeleton. And it turned out that they, thanks to the ability to interact with a broad spectrum of other proteins which are not bound between themselves either structurally, or functionally, regulate the whole transport process- from the formation of vesicle with the help of edging and up to its directional delivery along the elements of the cytoskeleton to its final destination and the provision of specific fusion of the transport vesicle with the target-compartment.

Analyzing experimental data and relevant publications, we came to the conclusion that the EGF-receptor is an active participant in the process of its own endocytosis. And in 2001 a team headed by Professor Ph. Stahl (USA) demonstrated that it is this factor that can practically directly activate RIN1 protein (factor of exchange of nucleo-tides for Rab5)-the main regulator of fusion of early endosomes. And although a direct interaction between the endocytized EGF receptor and Rab5 was not discovered, it is possible to assume that the EGF-activated by Rab5 gathers around itself proteins which ensure for the receptor the passage of all the following stages up to their entry into inner bubbles of late multivasicular endosomes, forming in this way a sorting platform. One of the components of the latter is phosphatidylinositol-3- kinase. Its product-phosphatidylinositol-3-phosphate-binds many proteins and par-

* Processing-set of reactions occurring with EGF and its receptor in endosomes and leading to their degradation.- Ed.

Pages. 44

Diagram of organization of endocytal route of EGF-receptor complexes.

ticipates in the formation of invaginations* of multivesicular endosomes.

Entry of the receptor into such platforms makes it possible to avoid recycling. What is more, an activated Rab5 stimulates the fusion of early endosomes into structures similar to tubular prominence oriented towards the nucleus. Moving along the membrane of such early endosomes are complex-platforms because of their interaction with microtubules also involving Rab5 and Rab7. But the destruction of micro-tubules causes no "catastrophe" since platforms themselves contain practically all proteins which are necessary so that in the final domains of "early-endosomal" tubules, where EGF receptors are gradually concentrated, there could begin the formation of invaginations with the subsequent separation of the already formed multivesicular late endosomes from the early ones. In a word, late endosomes appear "from" and "upon" the early ones, and the whole process is stimulated by EGF receptor.

It is interesting that although the transfer of EGF-receptor complexes into the near- nucleus area is not necessary for the receptor degradation in natural conditions, it does take place nevertheless. But why is it necessary if that is so? And here again there appears a supposition about a possible connection between endocytosis and signal passage. It is likely that the movement of an activated receptor from the periphery to the nucleus makes it possible to organize the stimulation of different signal routes in time and space.

Another apparently productive hypothesis concerns the transfer to the nucleus, together with the EGF receptor, of signal proteins which it activates. It has been demonstrated in our lab that such EGF-stimulated signal molecules (providing for signal transfer from receptor to cell genome and metabolic routes), as phospholipase Cy and transcription factor STAT1, can be connected with EGF receptor, localized in the membrane, and also with receptor-containing endosomes. And quite recently there came out a publication on studies conducted at several American universities under the direction of Professor Richard Jove which proved that the transfer of STAT3 into the nucleus is suppressed by the blocking of endocytosis of EGF receptor.

What one would like to note in conclusion: the role of endocytosis for a cell can be not only useful, but also harmful. It turns out that many hereditary ailments are caused by malfunctions in the mechanisms of delivery of certain proteins to the required address.

On the other hand, a number of health disorders are linked with mutations in genes which coordinate certain proteins-regulators of vesicular transport**. Many toxins, viruses and protozoa causing serious infectious diseases get into the cell by using the route of receptor-mediated endocytosis. That is why the effect of certain antiviral preparations is directed at the suppression of endocytosis.

Illustrations supplied by the authors.

* Invagination-formation of gastrula by an unfolding of part of the wall of the blastula leading to formation of internal bubbles in endosomes, which later turn into multi- vesiculas.- Auth.

** See: V. Baranov, "Predisposition Genes, or the Deseases We Take", Science in Russia, No. l,2003.- Ed.



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