Also, other substances water-soluble molecules like salt are able to transport through the membrane by osmosis. Just before it reproduces, Amoeba proteus retracts most of its pseudopods and rounds up into a ball.
After replicating its genetic material DNA in the nucleus, the original nucleus of the Amoeba divides to form two daughter nuclei by the process of Karyokinesis. In this process, the long DNA molecules condense into chromosomes rod-liked shape to facilitate the separation.
After the nucleus has divided into two, the process of Cytokinesis takes place in which the cytoplasm in the mother cell pinches in and divides into two daughter cells. This leads to the formation of the two daughter Amoebae cell, having a nucleus and its own cell cytoplasm and organelles. Usually, the entire process may last anywhere from 30 minutes to an hour. Most of the time, amoebas reproduce by the binary fission.
When the environment is turning harsh, amoebas adapt to the multiple fission to increase the chance to survive. There is another rare way for Amoeba to reproduce, called Encystment or Multiple Fission. When amoeba senses the environment become unfavorable eg. This cyst is able to survive in much harsher conditions. At the same time, mitosis occurs many times inside the cyst, producing more than two daughter cells.
When the cyst wall ruptures when the condition turns favorable , these daughter cells are then released to become several new amoebas. When the environment of habitation becomes extremely unfavorable, Amoebas will reproduce through spores. This sexual reproduction can create genetic diversity and increase its chance to survive in a harsh conditions. Amoeba proteus likes to stay at the bottom of clean freshwaters. It is found feeding on decaying substances on the bottom of freshwater streams and stagnant ponds.
You can use a transfer dropper to collect the bottom sediments to look for Amoeba proteus. Amoeba proteus can also be ordered from science supply companies and is the classic specimen used in the classroom to demonstrate the pseudopods in action. Here are some pictures of habitations where I recently spot Amoeba proteus. A-C Amoebas like to hide in the bottom sediments like leaves of clear water ponds. D-E I used the forceps to collect some decaying leaves and water with sediments into my sample vial.
I will bring it home to look for amoebas and other pond lives under my microscope. Amoebas can be directly observed under an optical microscope without additional stains. It takes patience to locate Amoebas under the microscope because they are transparent color-less , slow-moving, and like to cover themselves under debris or bottom sediments. Use a transfer pipette to get a drop of water with some bottom sediments onto a microscopic slide.
Gently cover the sample with a coverslip and mount it on the microscope stage for viewing. Wait minutes to allow the microorganisms adapting to the new environment amoebas like to adhere to the surface of the glass. Gradually increase the illumination Amoebas are sensitive to bright light and scan the field by low magnification 5x or 10x. Looking for the tiny crystal-liked particles inside the cells of Amoebas may help you locate them.
If you have the phase contrast or polarized light filters, you may want to use them. Amoebas can also be studied by dye staining to visualize cellular organelles. However, this requires the chemicals and equipment to fix and mount the dead Amoebas. If you want to know the detail, check out this link. A stained Amoeba proteus slide. Direct observation of the Amoeba proteus has a significant advantage because the Amoeba proteus is still alive and active moving when being viewed under the microscope.
This allows you to see the finger-like projections pseudopods elongate and shorten as the Amoebas move or engulf food particles.
The color of the food vacuoles inside the Amoebas can also indicate the nutrient sources in the habitation. For example, I noticed that Amoebas collected in the late spring contain more green particles could be green algae and Amoebas from the early spring are more brownish engrafted brown diatoms.
Sometimes, you may see Amoebas in rest and stay motionless with an oval shape. If you have a camera or cell phone mounted on your microscope, the slow-moving Amoebas are great models to practice your microphotography and video-making skills. Another feature that you can easily observe is the abundance of crystal-liked inclusions inside Amoeba proteus. Most crystals of Amoeba proteus are in a bi-pyramidal shape. These crystals are contained in vacuoles and composed of triuret, a nitrogen waste product.
Other species of Amoebas have their crystals in different shapes, like spheres, sheets, and even croissant-shaped crystals. Here are some examples of crystals in different species of Amoebas. Some large Amoebas also have glycogen bodies to store their nutrient reserve.
These glycogen bodies are glossy spheroids and vary in size. Glycogen is a form of sugar and in our body, we store glycogen in the liver and muscle. When the amoeba digests large amounts of diatoms, you can even see the oil droplets inside the cell of amoeba. This is because some diatoms are tiny oil producers! Some large amoebas contain bacteria and small green algae inside their cytoplasm. For example, green algae live inside can provide additional energy to their host the amoeba , making the amoeba can live in nutrient-poor environments.
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Four cells in this scenario exhibited eventual displacements towards both sides without any preference, and therefore were included in the unconditioned group.
Since 43 cells persisted in the migration towards the anode until the end of the galvanotaxis, we used 50 cells for the next step. When an Amoeba proteus is placed in an electric field for long periods of time, the probability of dying or, at least, detaching from the substrate and adopting a spherical shape increases sharply.
By inverting the electric field, we demonstrated that the amoebae were neither directed to a specific point in the space nor they associated a specific point in the space to the peptide. The comparison between the cosines of displacements in the galvanotaxis without induction Fig. Since 16 cells maintained the migration towards the anode until the end of the galvanotaxis, we used 25 cells for the next step.
In addition, the test comparing the cosines during the third scenario Fig. One cell presented an atypical behavior characterized by immobility, and therefore was included in the unconditioned group. Note that these behaviors were never observed in any of the five galvanotactic experiments Figs.
Galvanotactic control of the cells that responded to the cathode during the induction process. All the amoebae migrated towards the cathode, confirming that these cells were unconditioned, and that their natural behavior was not altered by the induction process. The new emergent systemic behavior is also characterized by a limited duration through time.
Figure 7a is an illustrative example of the loss of conditioning in 15 induced cells experimental replicates: 4, number of cells per replicate: 3—6 as time goes on. To quantify this phenomenon, we have measured the duration time of the conditioned behavior in the conditioned cells in the three scenarios previously described. Persistence time in the conditioned motility patterns of Amoeba proteus.
The colors of the trajectories represent the duration of the conditioned behavior, as is indicated in the top of the figure. The cells that lost the persistent conditioned behavior at the beginning of the tests were not represented. The three scenarios were shaded in different green tones for better comprehension. Despite all the perturbations introduced in the experiments, the whole analysis indicates that the average time of the cells that lost the acquired motility pattern during any of the three scenarios was In order to examine the robustness of the observed conditioned behavior we have performed a preliminary study on another unicellular species, Metamoeba leningradensis , under the same induction process as the Amoeba proteus.
The metamoebae were exposed to the same intensity of the electric field and to the same peptide nFMLP concentration, and therefore, values of amperage or optimal concentration of peptide were not adapted to generate more efficient responses of these organisms to such stimuli.
The quantitative analysis showed that the values of the cosines of displacements were distributed between 0. Conditioning process in Metamoeba leningradensis. They were exposed simultaneously to chemotactic and galvanotactic stimuli, placing the peptide on the anode side Fig. Hence, the result of the induction process indicated that two fundamental cellular migratory behaviors had emerged in the experimental system, one towards the anode and another towards the cathode.
Induction process in Metamoeba leningradensis. The tracking has been represented up to the maximum value obtained towards the positive or negative pole. Finally, to verify whether the cells that moved towards the anode during the induction process presented some kind of conditioning in their migratory trajectories, we performed a conditioned behavior test Fig. Then, in a similar way that we did in the Amoeba proteus experiments, the metamoebae were placed usually in groups of 1—6 on a new identical glass and block set-up that had never been in contact with the chemotactic peptide.
This result supported mathematically that the majority of cells moved towards the anode in the absence of peptide, thus corroborating that a new locomotion pattern had appeared in the metamoebae cells. Here, using an appropriate direct-current electric field galvanotaxis and a specific peptide nFMLP as a chemoattractant chemotaxis we have addressed essential aspects of the Amoeba proteus and Metamoeba leningradensis migration.
More precisely, we have found that these cells can link two different past events, shaping an associative conditioning process characterized by the emergence of a new type of systemic motility pattern.
This behavior consists in a persistent migration towards the anode when these cells typically migrate to the cathode. After an induction process, most of amoebae seem to associate food with the anode and, consequently, modify their conduct, behaving against their known tendency to move to the cathode.
Strikingly, this induced association of anode and food can be maintained for relatively long periods of time. We have also observed that, after the induction process, a small subset of the amoebae was not conditioned. In our experiments, some cells probably were unconditioned or weakly conditioned due to their intrinsic physiological peculiarities, and in addition, some kind of cellular damage caused by the experimental process may have occurred.
To test the robustness of the conditioned behavior we have performed a preliminary study in Metamoeba leningradensis under the same strict conditions that we set up for Amoeba proteus. Despite these restrictive conditions, most metamoeba cells were able to link two different past events, same as Amoeba proteus , shaping an associative conditioning process characterized by the emergence of a new type of systemic motility pattern which consists in a persistent migration towards the anode when, in the absence of previous induction, these cells also typically migrate to the cathode Fig.
The controls carried out during the research indicated that cells exposed independently either to galvanotaxis or chemotaxis, did not present any observable atypical behavior Fig. In conclusion, the work we have performed here shows that most of the conditioned Amoeba proteus and Metamoeba leningradensis exhibited the ability to preserve the relationship between the two stimuli, acquiring a new type of systemic behavior via conditioning.
Noteworthy, the fact that individual cells are able to generate associative conditioned behaviors to guide their complex migration movements has never been verified so far. Our experimental results may allow another possible explanation. The exposure to nFMLP triggers a sub-population of cells to change the character of their migration in an electric field, making them to migrate towards the anode rather than the cathode. A notable number of cells belonging to both species can persistently change their migration pattern by these two external, simple and independent stimuli, when both are simultaneously applied.
Amoebae and metamoebae cells seem to associate the anode with the peptide in the induction process. After the conditioning, both stimuli seem to remain linked in these cells for a relatively long period of time, and consequently, the systemic movement of amoebae and metamoebae responded to the presence of an electric field by migrating towards the anode instead of the expected migration to the cathode. In brief, we have observed a systemic cell behavior that can be modified by two simple external and independent stimuli, when they are simultaneously applied.
Pavlov studied four fundamental types of persistent behavior provoked by two stimuli. However, in a strict sense, we cannot conclude that our findings represent the classical Pavlovian conditioning since complete controls and parametric analyses for classical conditioning studies have not been performed yet The experiments we show here were inspired by numeric predictions based on computational modeling that we published in dealing with complex metabolic networks Thus, analyzing complex enzymatic processes under systemic conditions using Statistical Mechanic tools and advanced Computational and Artificial Intelligence techniques, we were able to verify numerically that self-organized enzymatic activities in modular metabolic networks seem to be governed by Hopfield-like attractor dynamics similar to what happens in neural networks A key attribute of the analyzed metabolic Hopfield-like dynamics is the presence of associative memory.
This quantitative study showed that the associative memory in unicellular organisms is possible 24 , Such memory would be a manifestation of emergent properties underlying the complex dynamics of the systemic cellular metabolic networks. It is still too early to delineate the molecular mechanisms supporting this cellular associative conditioning.
However, there are evidences of a functional memory, which can be embedded in multiple stable molecular marks during epigenetic processes Likewise, long-term correlations mimicking short-term memory in neuronal systems have also been analyzed in experimental calcium-activated chloride fluxes in Xenopus laevis oocytes On the other hand, different studies have described several molecular processes in which both prokaryotic and eukaryotic cells show chemotactic memory.
For instance, changing dynamics in specific methylation-demethylation patterns in prokaryotes seem to be involved in molecular memory processes related to chemical gradient adaptation 27 — Besides, phosphotransfer processes and other post-translational modifications seem to be involved in chemostatic cellular persistence of eukaryotic cells 31 — In this paper, we have addressed essential aspects of the Amoeba proteus and Metamoeba leningradensis migration.
The mechanisms underlying amoeba locomotion are extremely complex and the ability to direct their movement and growth in response to external stimuli is of critical significance for its functionality; in fact, cellular life would be impossible without regulated motility. Although some progress is being made in the understanding of cellular locomotion, how cells move efficiently through diverse environments, and migrate in the presence of complex cues, is an important unresolved issue in contemporary biology.
Free cells need to regulate their locomotion movements in order to accomplish critical activities like locating food and avoiding predators or adverse conditions.
In the same way, cellular migration is required in multicellular organisms for a plethora of fundamental physiological processes such as embryogenesis, organogenesis and immune responses. In fact, deregulated human cellular migration is involved in important diseases such as immunodeficiencies and cancer 11 , Neoplastic progression invasion and metastases , for example, can be regarded as a process in which the survival of tumor cells depends also on their ability to migrate to obtain additional resources in a general context of scarcity Here, we have verified that two unicellular organisms such as Amoeba proteus and Metamoeba leningradensis are able to modify their systemic response to a determined external stimulus exclusively by associative conditioning.
This fact opens up a new framework in the understanding of the mechanisms that underlie the complex systemic behavior involved in cellular migration and in the adaptive capacity of cells to the external medium. They were cultured in the same conditions as Amoeba proteus. All the experiments were performed in a specific set-up Fig. The first electrophoresis block was directly plugged into the power supply while the other was connected to the first via the two agar bridges, which allowed the current to pass through and prevented the direct contact between the anode and cathode and the medium where the cells would be placed later.
In the center of the second electrophoresis block we placed the experimental chamber that allowed us to obtain a laminar flux when it was closed and the addition and extraction of cells when it was open. These three glasses were for only one use. This glass structure Fig. It was reusable after cleaning. The modified slide was placed in the central platform of the second electrophoresis block. To avoid medium going across the modified slide from below, we placed an oil drop in the central platform of the block of electrophoresis, on which the modified slide was placed.
It is very important that the oil drop expands to cover the entire width of the experimental chamber. In the center of the modified slide, without any glue, we placed the central piece and the two sliding lateral pieces leaving short distance between all of them. To note, it is crucial that the amoebae do not remain for more than a few seconds in the micropipette tip to avoid the adhesion of the amoeba to the inner surface of the tip.
Once the amoebae were placed under the central piece of the chamber, we waited for two minutes to allow the cells to stick to the surface of the modified slide. Then, we filled the wells of the electrophoresis blocks with simplified Chalkley medium up to the level necessary to contact with the base of the modified slide, but not the two sliding lateral pieces.
Later, the two sliding lateral pieces were pushed with two micropipette tips until they contact with the liquid in the wells. Next, the two sliding lateral pieces are pushed to contact the central piece in the chamber. This way, a laminar flux can be established throughout the inner space of the experimental chamber. Considering that the amoebae that had shown a specific behavior were needed to perform further experiments, the cells were collected opening the sliding lateral pieces with the tip of a micropipette.
Set of videos intended only for didactical purposes. They are merely descriptive, to make easier the understanding of our experimental procedure and the reproducibility of our studies. Note that steps 5 to 8 are performed directly under the microscope, and we have not filmed them under the microscope for better visualization.
In summary, the experimental chamber consisted in a sliding glass structure. The sliding lateral pieces could be displaced in the longitudinal direction. This way, when the sliding pieces were closed an inner laminar flux was available in the chamber and, when they were open, the placement and collecting of the cells were possible easily. Movies showing the main experimental procedures have been deposited in figshare Once starved, only the cells that were strongly attached to the substrate, actively moving through it and showing a little amount of thin pseudopodia were used in the experiments.
The experiments were always made with small groups of cells. For instance, in Amoeba proteus along the induction process, we analyzed a total of cells that were studied in 32 different times experimental replicates analyzing them in groups of 4—10 cells each number of cells per replicate. Scenario 1 was repeated 32 times. Scenarios 2 and 3 were performed 27 and 9 times, respectively.
The induction process was usually performed using around 7 cells per experiment, sometimes as few as 4 or 5 and other times as many as 9 or 10, the average being 6—8 cells.
The number of cells analyzed in the scenarios depended on how many cells appeared to be conditioned in the first step, so that the number of cells per experiment is lower each time, for instance, in scenario 1, the number of cells was usually between 2 and 4.
Finally, in scenarios 2 and 3 the experiments were performed using fewer amounts of cells per experiment, usually 1—3 which were the cells that migrated towards the cathode during the conditioning process. Compared to Amoeba proteus , the Metamoeba leningradensis showed a more varied array of behaviors and shapes.
These cells were also more difficult to handle, as they were more prone to strongly stick to the micropipette tips, while usually showing a weaker attachment to the glass chambers. An electric field was applied to the first electrophoresis block, which was then conducted to the second by the two agar bridges. Direct measurements taken with a multimeter in the second block where the cells were placed showed that the strength of the electric current oscillated between All the experiments where the only stimulus was an electric field were performed in an electrophoresis block that had never been in contact with any chemotactic substance.
Groups of 4 to 10 amoeba were placed in the experimental chamber.
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