Amoeboid LocomotionIncluding locomotion of white blood cells, and also structural cells of our body. Also including the mechanical forces by which amoebae and body cells crawl. And also including structure-creating functions of these cell forces ("traction") For this topic, you will be asked to use your computers to look at certain time lapse movie clips which I took and which my wife and I have posted on my departmental web site. For most of the clips we have provided two versions, one darker and one lighter overall. Use whichever one allows you to see more detail on your computer. We hope that the formats of these video clips will be compatible with your computers and software. If or when incompatibilities occur, then tell me and we will change the format or solve the problem some way.
1) In Amoeba proteus, cytoplasmic flow is the most dramatic aspect of the cell's locomotion.
Please look at the first video clip [or the lighter version of same clip], and notice carefully the details of cytoplasmic flow in Amoeba proteus. Can you find some examples where forward flow of liquid cytoplasm also occurs around the outside of the hollow cylinder of jelled cytoplasm?
2) In the next clip ,
notice whether a single amoeba cell can have more than one fountain zone at the same time.
What eventually happens in all but one of these fountain zones?
When A. proteus "eats" ( = phagocytizes) some food organism, then
the tip of its pseudopod changes shape from a convex bulge to a concave, cup-like shape, which surrounds and engulfs the food.
another engulfment sequence
3) INVENT SOME HYPOTHESES, about what forces move Amoebae forward. A very different (almost the opposite) kind of theory was proposed by Prof. Bob Allen in the late 1950s. His idea was that interior cytoplasm gets pulled forward by its own contraction at the front end (in the fountain zone, itself).
Can you invent some experiments to test these and other ideas? Perhaps by marking one part of a cell? As you probably guess, all these kinds of experiments have actually been tried.
4) Look at the next clip, which shows particles of soot attached to the outer surfaces of crawling Amoeba proteus. Notice the general pattern in which these particles are pulled around by the cell's plasma membrane, especially relative to the forward protrusion of pseudopodia, and the later retraction of these pseudopodia. To me, this seems to show that the plasma membrane slides forward independently of the gelled cortical cytoplasm.
5) Look at the next clip or its lighter version.
6) The next clip
is a completely different kind of amoeba, called Labyrinthula. Species of this group are very common in the ocean (salt water), especially in sea grass. They are believed to cause a disease in sea grass: a disease so serious that it produced a major change in coastal ecology during the 1930s, here in North Carolina.
The cellular structure of Labyrinthula is contrary to what we all learn in biology courses. Watch them carefully, and make hypotheses about where forces are being exerted. Textbooks never tell you about such creatures as this, but they are common in sea water. 7) Some kinds of amoebae live inside tiny shells that they either secrete, and/or make by gluing together the tiniest sand grains. Difflugia and Arcella are two genera in this category. They produce long, straight pseudopodia, which contract when they contact anything. Sometimes this contraction pulls the amoeba toward the direction of the contact; other times, contracting pseudopodia pull objects, including food, or more sand grains (and in these sequences, bits of carbon black). You need higher magnification to see it, but there seems to be a fountain zone pattern of cytoplasmic flow inside the pseudopodia of these amoebae (like the pattern in Amoeba proteus).
8) Dictyostelium discoideum is one of the most intensively studied species in the world. It was discovered by Kenneth Raper, who did his undergraduate studies in this department here at UNC, and was a professor at the University of Wisconsin nearly all his life. He wrote an excellent large book about these creatures, and other "cellular slime molds".
What makes these amoebae so interesting is that part of the time they live as separate individual cells, and at other times live as an aggregation of tens of thousands of cells, which crawls around as a multicellular unit , part of which then forms a stalk that sticks up into the air, with the rest of the cells somehow moving up to the top of the stalk and then differentiating into spores. The result is like a tiny mushroom. The spores later hatch to form amoebae, often after having blown away somewhere else. The multicellular "slugs" happen to look like miniature versions of the kinds of mollusks called slugs, but shouldn't be equated to them. Another word for these multicellular masses is "grex", which is Latin for "herd" (but is Russian for "sin"; which in turn is Spanish for "without"!). The process of aggregation of individual amoebae to form a grex is by chemotactic attraction of the amoebae toward a substance, which (in this one species: D. discoideum) was proven to be cyclic AMP. Other species within this group use other chemicals as their chemotactic attractant. John Bonner proved that attraction was by chemotaxis in the 1940s, but not until the 1970s was it discovered what the attractant substance is. Students often do not realize that experimenters usually have to prove the existence of signaling and other chemicals, and approximately how they work, and where they come from, before it is them possible for anyone to do the experiments to find out what actual chemical is being used.
(All?) other species of Dictyostelium besides discoideum immediately form a stalk and then "fruit) right at the location where their cells have aggregated, but D. discoideum grexes crawl around for hours, and over distances of centimeters (next clip), before fruiting. Incidentally, I have met a lot of researchers on this organism who don't know things like that! The locomotion of the individual Dictyostelium amoebae is more similar to that of tissue culture cells (i.e. the cells of our body) than any other kind of amoeba (in my opinion). Much excellent molecular genetic research has been done using this organism, because it is practical to create and isolate mutant strains in which a certain protein is altered or absent. For example, mutants were isolated that lack the myosin used to form the contractile ring in mitotic divisions. The resulting cells can still divide, however, but do so by an abnormal mechanism that consists of two halves of a cell crawling off in opposite directions. If such mutants don't have a solid floor to pull against, then they can't divide after mitosis, and therefore become more and more multinucleate. Computer simulations of this "pulling apart" process have been done in Munich, using a computer program that I wrote and sent them (I am proud to say). There are many unanswered questions about Dictyostelium. For example, no one knows whether the mechanism of locomotion of the multicellular grexes is the same or different from the mechanism of locomotion of the individual amoebae. Furthermore, it isn't known we should think of the grex as crawling up the outside surface of the stalk during fruiting, nor whether the force exerting mechanism is the same as in the locomotion of either the grex or the individual amoebae. Another advantage of Dictyostelium as a model research organism is that there is a consistent ratio of number of amoebae that differentiate into spores (during "fruiting"), in proportion to the number of amoebae that differentiate into stalk cells. I need to go look up this detail in Raper's book, but I think the normal ratio is approximately 85% spores and 15% stalk cells, or something like that. In addition, there slugs tend to have a consistent ratio of length to width, of about 8 to 1. We have some original time lapse videos to look at to decide whether we think this constancy is an exaggeration or not. To the extent that such ratios (of slug length to width; and of spore number to stalk cell number) remain approximately constant, whether a slug is made of 100 cells or made of 500,000 cells, then these can be regarded as examples of a very general phenomenon called developmental "regulation". The first example of regulation (in this sense of the word) to be discovered was Hans Driesch's observation that the first two cells of sea urchin embryos can be separated, and each will develop into a half-sized larva, with normal proportions. Later, he and others showed that the first 4 cells can also regulate to form normally-proportioned, quarter-normal size embryos. Furthermore, two embryos can be fused at around the one-celled stage, and will regulate to form a double sized embryo. Thus, one says that sea urchin embryos can regulate over a range of volumes of 8:1, whereas Dictyostelium can regulate over a range of nearly ten thousand to one. No one knows, whether the mechanisms of regulation are even remotely the same in animal embryos as compared with Dictyostelium slugs. On the other hand, hundreds of millions of dollars worth of research on Dictyostelium has been funded on the assumption that their cell properties are like cancer cells. Many people regard regulation as THE most important unsolved problem in developmental biology. It's sort of like Fermat's last theorem in math, or the Riemann conjecture, or the Gordian knot. Incidentally, embryos of Drosophila (flies) and C. elegans (nematode worms) are especially NOT capable of regulation. Forgive my cynicism if I suggest that so much developmental research is concentrated on those species as a means of avoiding the need to explain regulation, since they don't do it. Vertebrate embryos, in contrast, do undergo this kind of regulation. And mammal embryos are the most regulative of all. Imagine if scientists studying rain concentrated their research on deserts where it rains as little as possible. In fact, one might argue in favor of such a policy!
9) Physarum, eating a piece of oatmeal Physarum is an "acellular" slime mold. You may have seen these sorts of beasts in broken wood, like when you turn over a log. And if you haven't noticed them before, then maybe in future you will. They are multinucleate, with one long stringy network of cytoplasm containing thousands or millions of nuclei, all flowing actively back and forth in the cytoplasm. This cytoplasmic flow goes one way for a while, then reverses direction for a minute or two, then reverses again. They also contract for a while, then relax for a while. There must be a direct relation between these wave of contraction and the alternations of contraction, but the little guys hate to crawl on rubber, so I haven't found out whether they flow when they are contracting, or when they are relaxing, or when they are going in one direction, and then relax when they are going in the other direction, or what. 10) Nearly all the individual component cells of multicellular animals undergo something like amoeboid locomotion, not just white blood cells but also liver cells, heart cells, pigment cells, the precursors to muscle cells, and many others. Putting cells in tissue culture stimulates their active locomotion. In living sponges, all the cell types continually crawl around actively, as can be seen in time lapse movies of thin parts of sponges In the video sequence shown here, the cells are those that make up the outer layer. These would be called epidermal epithelial cells in other kinds of animals, but in sponges they are called "pinacocytes", and the layer they form is called the "pinacoderm". This is because they are believed to lack something called a basement membrane, which epithelial cells almost always have. In this video sequence, notice that one of the many pinacocytes actually pulls itself entirely loose from the sponge, and crawls a short way by itself. Later, the edge of the sponge touched this separated cell, and it quickly rejoined the rest of the surface cells. Mesenchymal and other cell types inside sponges also undergo constant active locomotion and rearrangement, and we have time lapse videos of their movements, but not in this sequence. Probably the most important, and certainly the most famous, scientific discovery ever made at UNC was H.V. Wilson's discovery that sponges can be dissociated into a random mass of separate individual cells, and then reform all their functional organs in two or three days. He later discovered that a kind of coral can also reform its anatomical structure after having been separated into its individual cells. Similar discoveries were made by others in embryos of some higher animals, including vertebrates. These experiments led eventually to the discovery of special cell-cell adhesion proteins, by which (for example) the surface of each cell of your liver sticks to the surface of other liver cells. Wilson himself advocated the hypothesis that dissociated cells switch from one differentiated cell to another, instead of cells of each differentiated cell type moving back to their correct relative locations (as is now believe to be the correct explanation). Others at UNC later discovered that sponge cells constantly rearrange according to their differentiated cell type, even when not dissociated or disturbed. If you disturb them, the cells rearrange even faster. Considerable amounts of active cell locomotion and rearrangement also occur inside the human body. This is a major part of wound healing. If we were transparent, we might be surprised that some of our cells constantly rearrange like those of sponges and some other lower animals. Random, uncontrolled "amoeboid" locomotion of cancer cells allows them to invade adjacent tissues and organs, and often to penetrate into blood vessels, flow through the blood to some other location, and again penetrate blood vessel walls into the surrounding tissues. Many people tend to assume that this capability for quasi-amoeboid locomotion is an abnormality of cancer cells. But it is not cell locomotion itself that is abnormal for cancer cells; what is abnormal and harmful is that the cancer cells' locomotion is uncontrolled, and even random. Their abnormally uncontrolled cell locomotion disturbs the normal geometric arrangements of cells in tissues and organs. These geometric arrangements are normally created and maintained by active cell locomotion, somewhat as in Dictyostelium. Much research has focused on these normal control mechanisms, that guide and restrict the "amoeboid" locomotion of body cells, but they are still not well understood. This subject is full of unsolved problems.
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11) The clip above shows tissue culture cells crawling on a thin sheet of rubber. These cells are from chicken embryos. But human cells in tissue culture behave exactly the same way. Notice that the wrinkling pattern of the rubber, and the changes on this pattern over time, show us approximately where the cells are exerting forces, and in what directions these forces are acting, and also that these "traction" forces seem to be excessively strong - stronger than needed to move the cells. Many laboratories now use variations on this method, in combination with computer calculations of force locations and strengths. That is called "Traction Force Microscopy". I did not invent this name, but I did invent the method (when I was a post-doctoral fellow in Cambridge!) and I published the first papers on the subject. The method has been used by others to discover many important facts. 12) Look at the next clip or the lighter version This was made by Professor Barbara Danowski, of Union College in New York State. Notice that about half-way through the sequence, something causes weakening of the traction forces exerted by these tissue culture cells. The change is so extreme and quick that you might have thought these were two different sequences. But really it's a continuous sequence, with the same cells, before and after treatment with "Phorbol Ester" a "Tumor Promoter" chemical that temporarily converts the cells from a non-cancerous to a cancerous pattern of behavior. That's an over-simplification, but it's fundamentally true. In this and all other cases that anyone has studied (that I know of) the cancerous cells exerted weaker traction forces than equivalent non-cancerous cells. Cancerous cells usually are morphologically and structurally abnormal, as compared with equivalent normal cells, especially in the irregular shapes of their nuclei, the over-activity of their locomotory cell surface movements along their edges, and other visible properties. Histological section through a cancerous tumor
Time lapse video sequence of the edge of a normal embryonic mesenchymal cell beside the edge of a sarcoma cell. Can you figure out which is which?
Time lapse video of a sarcoma cell crawling, and colliding with a normal mesenchymal cell. In many other cases, it has been discovered that the locomotion of normal cells is inhibited by contact with each other ("Contact Inhibition"), and that cancer cells are often less sensitive to contact inhibition than are normal cells, However, in this sequence, the sarcoma cell's locomotion gets inhibited by contact with the normal cell.
Time lapse videos of several sarcoma cells. Their vigorous ruffling and blebbing movements of their plasma membranes along their edges don't seem to be accomplishing much locomotion of the cells. Can you find abnormal irregularities in the shapes of these cells and their nuclei, similar to the abnormalities in the color slide of the section through a cancerous tumor? Some more videos: a leucocyte moving among red blood cells; lighter version a cell undergoing mitosis
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