In just two minutes, a cell from the immune system immobilizes, involves, swallows and destroys a particle that is alien to the organism, such as a bacterium or a parasite. The phenomenon is known as phagocytosis, from the Greek phagein (eat) and the Latin Cyta (cell) – and derives from a specific kind of fluctuations on the surface of the cell classified as large, as they form waves like the sea, with a height of a few micrometers (one micrometer is a millionth part of a meter). This was recently proved by a series of experiments done recently by researchers from the Statistical Physics and Biophysics Laboratory of the Federal University of Minas Gerais (UFMG).
The result changes one of the theoretical models proposed almost 30 years ago by American researchers to explain phagocytosis, the zipper model, according to which normal small vibrations – also called random- which permeate the whole surface of the cell and are like those on a swimming pool on a day with little wind – set off the process used by the cells to fight agents foreign to the organism, like bacteria and fungi. In the future, the discovery may lead to the development of more efficient drugs in fighting infections.
The road to this conclusion was a long one. First, the researchers from Minas Gerais developed an innovative technique, described in a scientific article that is about tobe published, which made it possible to observe and film details of the two kinds of movement on the surface of the cell with an optical microscope. Called defocusing microscopy, it modifies the focus of the image by distances of less than one micrometer and so makes it possible to see and to measure the curvature of the surface of the cell, an important parameter for assessing the quantity of energy spent by the defense cells, like the macrophages, to engulf the pathogen.
Tweezers of light
The method only worked when it was coupled with another one, created in the 70’s by American physicist Arthur Ashkin, of Bell Laboratories, in the United States. Known as optical tweezers – concentrated laser beams capable of catching and keeping in suspense atoms, molecules and cells -, this technique, previously applied to physics, has been adapted to studies in biology in recent years. With the help of these tweezers of light, physicist Oscar Nassif de Mesquita, from UFMG, and his doctorate student Ubirajara Agero successfully isolated a single mouse macrophage, without destroying it, and fed it with particles of zymosan, yeast used in experiments on phagocytosis in the laboratory.
In the analysis of this phenomenon, both big waves and small ones appeared – although only the big ones took an active part in the emission of prolongations to engulf the particle. “Our objective was to understand how the mechanical properties of the surface of the cell affect the process of phagocytosis”, explains Mesquita, the coordinator of this work, which is being carried on in collaboration with Catherine Ropert, from the René Rachou Research Center, at the Oswaldo Cruz Foundation (Fiocruz), in Belo Horizonte, and Ricardo Gazzinelli, who works at Fiocruz and at UFMG’s Immunology and Biochemistry Department. By measuring the size of the waves, the researchers have managed to calculate the energy spent by the cells to generate the movements. While the slight fluctuations, that occur all over the surface of the cell, practically spend no energy (the body temperature of 37° C is sufficient to produce them), the big undulations consume 100 times more energy.
Prospects
Proof of the new mechanism of phagocytosis was, however, missing. The researchers added to the macrophages’ culture medium a drug, cytochalasin D, which modifies the consistency of the cells’ skeleton and inhibits the formation of big undulations. Measuring once again the duration of the process, they found that the time spent to annihilate the invader increased 60 times, from two minutes to two hours, confirming the importance of the big waves for phagocytosis. “These results open the way for the study of medicines that act on the structure that sustains the cells and on the production of energy to increase the effectiveness of the phagocytosis”, comments Mesquita. Three years ago, he set off to analyze biological systems, and this year he has published two articles on the use of optical tweezers – one in the Physical Review E magazine, and the other inApplied Physics Letters, both from the American Physical Society.
Optical tweezers have also been assisting the team from Minas Gerais to study biological phenomena on the molecular scale, more specifically the elasticity of the DNA molecule. This physical property guarantees the genetic material the capacity for folding itself to fit into the nucleus of the cell, which has only a few micrometers – the DNA contained in a single cell can be as much as 2 meters long if stretched out.
Interaction
After isolating a single segment of DNA with the help of the tweezers, Mesquita and his student Nathan Viana measured the molecule’s flexibility using a method that they had created. Called dynamic light scattering and described in Viana’s doctorate, concluded in May: the method assesses the intensity of a laser reflected by polystyrene spheres linked to the DNA.
The experiment made it possible to validate the technique that before the end of this year should be used to analyze the interaction of genetic material with proteins and medicines, substances that alter the elasticity of the molecule. This is the way how the researchers hope to understand better DNA’s replication process. Recently, the team started to assess the damage caused in a single DNA molecule by ultraviolet radiation, of the kind that causes sunburn. “We intend to see the evolution in the course of time of the damage caused to a single molecule”, Mesquita explains. “We still do not know all the possible implications of this, but I believe that it will be important for assessing the risk of skin cancer, caused by this kind of damage”.
Another line of research in which the researcher’s team is investing is the study of water transport in kidney cells, more specifically the water loss process from the cells to the outside medium, more concentrated, for example, in salt, the substance used in the experiment. As a result of this process, called osmosis, the cell begins to wither and immediately activates the mechanism to regulate its volume, so as to go back to its original state – this is osmoregulation, another essential mechanism.
In work done in partnership with Robson dos Santos, from the hypertension group at UFMG’s Physiology Department, Mesquita and his doctorate pupil Aline Duarte Lúcio measured, with greater precision than in other methods, the volume of water lost in the process, by observing a dog kidney cell isolated and suspended in a culture medium by means of optical tweezers. They also calculated the speed of the osmosis, which the specialists call permeability, and it proved to be far lower than previous estimates: only 5 micrometers a second – the studies that had assessed osmosis by indirect observation, in groups of cells, put this speed at between 0.5 and 50 micrometers a second. “The possibility of error was much reduced with the elimination of interference from other cells and of the contact with the glass slide of the microscope, which deforms the cell and prevents direct measurement”, comments Mesquita.
Permeability
The researchers followed the osmosis in real time and quantified, as a result of this time, the permeability of the membrane of the cell in relation to the water, which is due to the creation of channels in the cell membrane by specialized proteins, called aquaporins. In the experiments, varied quantities of NaCl (sodium chloride, or table salt) in the external medium, to force the water to leave the cell, which withers rapidly, in a few seconds; but straight afterwards, it activates its volume regulating mechanism, the process of osmoregulation, to return to practically the original volume, in about ten minutes.
This stage takes more time because the solute – the compounds dissolved in the insides of the cells, which include minerals like potassium – have to migrate to specific, osmotically active, regions in the cell, so as to reverse the flow of water. The addition of the vasopressin hormone, which is produced naturally in the body and is related to hypertension, increases permeability, because it causes the migration of new aquaporins to the surface of the cell. The next step, comments Mesquita, is to carry out experiments on kidney cells in two groups of animals, hypertensive and normal. More details may arise from this about alterations in the water transport mechanisms, potentially useful in the quest for new drugs to treat high pressure and kidney problems.
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