Discovery Talk Questions and Answers: The Dynamic Mitotic Spindle

Shinya Inoue 
Assessments created by Dr. Delquin Gong

Questions

1. In his talk and in the paper, Dr. Inoue introduces the concept of birefringence. In simple terms, what is birefringence and why did Dr. Inoue need to use it for his cell division experiments?
2. Dr. Inoue and his colleague used several methods to perturb the dividing cells to measure the changes in birefringence of the spindle fibers.

   Which method was used in the paper but not mentioned in his talk?

   1. Alternating temperatures from high to low.
   2. Substitution of heavy water (D2O) for normal water (H2O).
   3. Use of drugs that affect spindle birefringence, namely colchicine.
   4. None of the above.
3. Dr. Inoue and his colleague perform an experiment in which they continually drop and then raise the temperature of the dividing cells. What was the result of this experiment, and what model did they come up with to explain their observation?
4. In his talk, Dr. Inoue presents an experiment that he performed using colchicine-treated marine worm oocytes, which have built-in spindles because they are arrested in metaphase.
   1. In addition to the changes in birefringence, what else did he observe about the spindle?
   2. How was this observation determinant for understanding the mechanics of the mitotic spindle?
5. After Dr. Inoue and colleagues performed their initial experiments on changes in birefringence of the spindle fibers, they came up with a model to explain their observations.

   Which of the following are true statements about the model? Select all that apply.

   1. Their model assumes that polymers, which are in equilibrium with dissociated molecules, have no orientation.
   2. Their model explains how spindle birefringence changes based on experimental alterations.
   3. Their model explains that spindle fibers are in dynamic equilibrium.
   4. Their model states that polymerization is temperature-sensitive, with polymers dissociating at higher temperatures, up to a maximum.
   5. None of the above.
6. In the paper, Dr. Inoue and his colleague make use of heavy water (D2O) for their birefringent studies. [Paper]
   1. What is the basis of this type of perturbation, and what does the model predict for mitotic spindles in heavy water?
   2. Did Dr. Inoue’s results match the prediction?
7. In his talk, Dr. Inoue describes an experiment that Howard Fuhr carries out, in which he uses a UV laser beam to generate an area of reduced birefringence (Arb). Strikingly, the Arb migrates along the spindle while the birefringence remains unchanged.
   1. Which direction does the Arb migrate?
   2. Why doesn’t the Arb change in birefringence? What does this tell us about how the spindle is organized?
8. In the paper, Dr. Inoue and his colleague notice that the spindle can recover fairly rapidly after disruption, and they wonder whether these molecules are synthesized rapidly each time, or whether there is an available pool of these molecules from which to draw upon. How do they go about testing each possibility? Select all that apply. [Paper]
   1. They measure the amount of protein in isolated spindles from heavy water compared to total cellular levels of protein.
   2. They use drugs such as Colcemid that change the birefringence of the spindle fibers.
   3. They use protein synthesis inhibitors such as actinomycin D, puromycin and chloramphenicol.
   4. None of the above.
9. In the paper, Dr. Inoue and his colleague emphasize that contraction should be brought about by slow removal of molecules from the polymerized filaments.
   Why is a slow rate so important for this process? [Paper]
10.  In the paper, which technique does Dr. Inoue and his colleague use to state the case that microtubule filaments underlie the spindle birefringence? [Paper]
    1. a. Electron microscopy
    2. b. Polarized light microscopy
    3. c. Video microscopy
    4. d. None of the above

Answers

1. Birefringence means that the object under surveillance has different optical properties when viewed using polarized light. These optical properties give insight on how molecules are organized, and changes in birefringence point to changes in the structure and organization of the observed molecules. Dr. Inoue used birefringence to observe the mitotic spindle because previous microscopy methods produced images that were too dark for live cell imaging, while taking photos of fixed cells introduced the possibility of artifacts from the fixation process.Video (4:38): “The microscope that is shown here is what I built when I came to Princeton in 1948 as a graduate student. And with the microscope, I was able to show what part of the cell was birefringent. Birefringence is a word I’ll keep on using that means it has different optical properties than the rest, which you can see by using a polarized light microscope. And by using polarized light, not only can you visualize a structure that is birefringent, but birefringence tells us how molecules are lined up, how they change, and so on. So, this is the special kind of microscope that I built for that purpose.”
2. b.
   268, “5. EFFECT OF HEAVY WATER ON SPINDLE BIREFRINGENCE” and Figs. 4-7, 9 and Table I.p. 268: “In the dynamic equilibrium model, structured water is believed to dissociate from the protein molecules upon polymerization. Substitution of heavy water (D20) for normal water (H20) might then be expected to alter the equilibrium. (…) When 45% D2O was applied at the appropriate stage of mitosis, the spindle birefringence was found to increase at least 2-fold, and the volume occupied by the birefringent spindle, to increase as much as 10-fold at times. Fig. 7 illustrates the rapid change in the responsiveness of the spindle to D2O (and temperature treatment) during mitosis in developing sea urchin eggs.”
3.  Dr. Inoue and colleagues found that dropping the temperature did away with the birefringence of the spindle fibers, but raising the temperature brought the birefringence back. This points to a dynamic system, in which molecules can associate and dissociate in a reversible fashion in some form of equilibrium. They then came up with the dynamic equilibrium model to explain the dynamic nature of the spindle fiber.p. 266: “3. DYNAMIC NATURE OF SPINDLE FIBERS – Their birefringence is abolished in a matter of seconds by treatment with low temperature in Chaetopterus oocytes (Inoue, 1952 a) and in Lilium pollen mother cells (Inoue, 1964). Upon return to normal temperature, they recover in the course of a few minutes, after which chromosome movement can continue (Inou6, 1964). Low temperature disintegration of the spindle can be repeated for as many as 10 times in the same cell. In Chaetopterusoocytes, Lilium pollen mother cells, Halistaura developing eggs, and Dissosteira spermatocytes, recovery from low temperature treatment is not affected by the duration of chilling, even up to several hours or longer (Inoue, 1952 b, and unpublished data).”

   p. 267: “4. THE DYNAMIC EQUILIBRIUM MODEL – In order to explain the dynamic nature of the spindle fiber and the response of the spindle birefringence and structure to various experimental alterations, Inoue (1959, 1960, 1964) earlier proposed a dynamic equilibrium model. In the model, spindle fibers are made up of oriented polymers, which are in an equilibrium with dissociated molecules. The equilibrium is temperature-sensitive, the polymers dissociating at lower temperatures and the molecules associating to form oriented polymers at higher temperatures, up to a maximum.”

   Video (7:05): “What was really interesting to me was to find out that these birefringent structures were not just there to move chromosomes and so on, but had some very intriguing properties. They would come and go. Here’s a dividing sea urchin egg, which is about to divide, and you see the birefringent spindle in the middle. But now I’m going to drop the temperature, and raise it again, drop it, and raise it, and as you see, this is a time-lapse movie, each time I drop the temperature, then the birefringence just disappears. What it means is that the molecules are not bound there together tightly, but can fall apart very easily. Again, dropping the temperature, and this doesn’t affect what the cell does. It’s perfectly happy and keeps on going. Drop the temperature, raise again. So, we can keep on doing this experiment over and over again. This tells us that the molecules that make up these fibers are in a dynamic equilibrium with something… they can either fall apart or be put together again (…)”

4. a. When the spindle lost its birefringence, the spindle became shorter and shorter, pulling the chromosomes to the cell surface. When the colchicine was washed out, the birefringence returned, and the chromosomes were pushed back toward the center of the cell.p. 273: “As described in the case of Chaetopterus oocytes (Inoue, 1952 a), when low concentrations of colchicine or of Colcemid are applied, the chromosomal spindle fibers contract and pull the chromosomes to the cell surface to which they are closest. Fig. 9 D shows an electron micrograph of a spindle in the process of dissolution in Colcemid. The cell was fixed by the method described in section 10. Remnants of short fragments of spindle filaments could still be seen around the chromosome, and the chromosomes moved closer to the cell surface during contraction of the spindle (compare with control spindle, Fig. 9 A). Fig. 9 C shows a similar situation in a cell treated with cold. When cells treated with Colcemid are washed with normal seawater, their spindle recovers in the course of 45-60 min. The recovery is not shortened or prolonged by washing with heavy water.”

   Video (8:54): “Now, what happened with colchicine is, when I applied colchicine to living cells… In this case, it’s a oocyte — a cell that forms an egg — of a marine worm, Chaetopterus. These are my favorite material because the cell stays in metaphase unless you stimulate it to go further. So, it has a metaphase spindle built in. But, then when you apply colchicine, then as you see in the lower row, the birefringence gradually disappears in a few minutes. And then, if you use a lower concentration, then as you see in the upper row, then not only does the birefringence disappear, but the spindle gets shorter and shorter and shorter, and at the same time, it pulls the chromosomes to the cell surface. So, from this, I concluded that colchicine, just like cold, is one of the agents that makes the spindle material fall apart. But what’s interesting is that, as the molecules are falling apart, they can generate force for pulling. This is a very strange concept that you can generate pulling force that will pull chromosomes and the inner spindle pole to the cell surface. And then if you take the colchicine away, the reverse happens — the molecules come back together, and it pushes the chromosomes and the inner pole towards the middle.”

   4. b. This provided the fodder for the idea that pulling forces can be generated from dynamic molecules, and could explain the mechanism of sister chromatid separation in mitosis.

   Video (10:38): “So, this gave rise to the whole concept that molecules that are falling apart can actually generate pulling forces. Now, this seems so strange. It took 20 years until, in the test tube, this was proven to be correct.”

5.  b and c.(a) incorrect because the polymers are oriented, which is the basis of birefringence.

   (d) incorrect because polymers associate rather than dissociate at higher temperatures.

6.  a. The molecules of deuterated water pack in more tightly than normal water, so the higher percentage of D2O mixed in with normal water, the more the D2o will pack with itself and exclude other molecules like proteins. This creates higher local densities of proteins, and in the case of the mitotic spindle, creates more stability of the spindle and thus higher birefringence of the spindle fibers.p. 273: “As described earlier, heavy water increases the birefringence retardation and the volume of the birefringent spindle material (Figs. 4-7). It also increased both of these parameters in isolated spindles, provided the cells were treated with heavy water while still intact (Fig. 11). The amount of the major spindle (22S) protein in the isolated spindle increases approximately proportionately with the amount of birefringent material, as shown in Table I, while the total 22S protein per cell remains unchanged (Sato et al., 1966).

   The rapid rise of birefringence and volume of the spindle in D20-sea water, and the parallel increase in the spindle protein content without increase in the same protein in the whole cell, suggests that a sizable pool of protein must preexist in the cell. Kane (1967) has, in fact, found a large quantity of the major spindle protein already present in the unfertilized sea urchin egg. The amount of 22S protein in the unfertilized whole egg is some 20 times greater than that extracted from the first mitotic metaphase spindle.”

   6. b. Dr. Inoue and colleagues found that treatment with deuterated water increased polymerization of the spindle fibers, with sensitivities similar to that of temperature shifting. This result is consistent with the dynamic equilibrium model that explains the dynamic nature of spindle fibers.

   p. 273: “These observations fit our working hypothesis that there exists in the cell a large quantity of pool material, which can be associated to form the filaments and fibers of the mitotic spindle.”

7.  a.  The Arb migrates toward the spindle pole.Video (10:57):“One other graduate student working with us, Howard Fuhr, did another experiment. He used a small spot of ultraviolet light, as you see in the top, second to the left panel… a bright spot of ultraviolet light. When you shine a bright microbeam of ultraviolet light on the spindle and watch the birefringence, then you see that spot itself loses birefringence, and you develop an area of reduced birefringence — Arb as you see there. What was really surprising is that this Arb then gradually migrates to the spindle pole and then disappears, which means that there must be some movement of the spindle material from the chromosomes towards the spindle pole. And while this is going on, the part between the chromosomes and the Arb, and between the Arb and the pole, the birefringence doesn’t change which meant that there must be a microtubule-organizing center both at the chromosomes and at the spindle poles. So, this was quite a revolution… a revelation. And, when we put all of this together, then as Ted and I put together the summary article later on, the spindle has organizing centers both at the spindle poles and right at the chromosome kinetochore. And then those are both responsible for lining up microtubule material, tubulin, but the tubulin is constantly flowing from the chromosomes towards the spindle pole. And as Tim Mitchison and others showed later on, this occurs very, very rapidly.”

   7. b. The birefringence does not change at the spindle area that is on both sides of the Arb – which suggests that there is a microtubule-organizing center at both extremities. The fact that Arb migrated toward the spindle pole and disappeared indicated that there is movement of materials up and down the spindle.

8. a and c.b is incorrect because it does not provide any extra information on whether there is a soluble pool of protein or whether the protein is synthesized de novo.
9.  According to the model proposed by Dr. Inoue and colleagues, the spindle is in dynamic equilibrium such that both polymerized filaments co-exist with a pool of readily available molecules. If the rate of contraction is too fast, the system will not be in equilibrium and the spindle structure will fall apart. The authors have seen this with fast removal of colchicine and rapid cooling. Only removal from initially low concentrations of drug, or a slowed rate of cooling maintained an intact, contracting, spindle structure.p. 277: “Slow removal of the molecules from the filaments allows the filaments to reach a new equilibrium position rather than falling apart, thereby effectively shortening the fibers and resulting in a slow contraction. If the molecules were pulled out too fast, then the filaments would fall apart, the structure would simply collapse, and one would not achieve contraction. This was observed with rapid cooling or in the presence of higher concentrations of colchicine (Inoue, 1952 b).It is proposed, in anaphase movement of chromosomes, that the slow removal of the material from the chromosomal spindle fibers, particularly toward the spindle pole region, is responsible for the shortening of the chromosomal spindle fibers and, thus, for the movement of the chromosomes (outside of the movement contributed by the elongation of the central spindle). The reason for the belief that depolymerization takes place primarily toward the pole arises from the observation of the distribution of birefringence during the normal course of division, and from the results of UV microbeam irradiation of the chromosomal spindle fibers (Forer, 1965, 1966; Inoue 1964; also see interpretation of Forer’s work by Wada, 1965).”
10.  a See p. 278: “10 – RELATION OF SPINDLE FIBER BIREFRINGENCE TO MICROTUBULES AND TO FILAMENTS OBSERVED BY ELECTRON MICROSCOPY”

 

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