J. Cell Sci. 39, 29-52 (1979)
Printed in Great Britain © Company of Biologists Limited
29
TEMPERATURE EFFECTS ON ANAPHASE
CHROMOSOME MOVEMENT IN THE
SPERMATOCYTES OF TWO SPECIES
OF CRANE FLIES {NEPHROTOMA SUTURALIS
LOEW AND NEPHROTOMA FERRUGINEA
FABRICIUS)
CATHERINE J. SCHAAP AND ARTHUR FORER
Biology Department, York University, Dovmsview, Ontario MJj, 1P3, Canada
SUMMARY
Using phase-contrast cinemicrography on living crane fly (Nepkrotoma suturalis Loew and
Nephrotoma ferruginea Fabricius) spermatocytes, we have studied the effects of a range of
temperatures (6-30 °C) on the anaphase I chromosome-to-pole movements of both autosomes
and sex chromosomes. In contrast to previous work we have been able to study chromosome-topole velocities of autosomes without concurrent pole-to-pole elongation. In these cells we
found that the higher the temperature, the faster was the autosomal chromosome movement.
From reviewing the literature we find that the general pattern of the effects of temperature on
chromosome movement is similar whether or not pole-to-pole elongation occurs simultaneously
with the chromosome-to-pole movement. Changes in cellular viscosities calculated from
measurements of particulate Brownian movement do not seem to be able to account for the
observed velocity differences due to temperature.
Temperature effects on muscle contraction speed, flagellar beat frequency, ciliary beat
frequency, granule flow in nerves, and chromosome movement have been compared, as have
the activation energies for the rate-limiting steps in these motile systems: no distinction
between possible mechanisms of force production is possible using these comparisons. The
data show that even the different autosomes within single spermatocytes usually move at
different speeds. These velocity differences cannot simply be related to chromosome size as
the autosomes are visually indistinguishable.
The sex chromosomes start their anaphase poleward movement after that of the autosomes,
and move more slowly (by a factor of about 4), but their velocities appear to be affected by
temperature in the same fashion as those of the autosomes. The interval between the onset of
autosome anaphase and sex chromosome anaphase is also affected by temperature: the higher
the temperature, the shorter the interval between the 2 stages. We have observed abnormalities
in sex chromosome segregation, which may be due to temperature, but have not determined
what the exact temperature shift conditions are that cause these abnormalities.
INTRODUCTION
The mechanisms which produce the movement of chromosomes during anaphase
are still unknown, and the hypotheses are subject to much debate (for reviews see
Schrader, 1953; Gruzdev, 1972; Nicklas, 1975). Anaphase movement can be subdivided into 2 distinct processes which may or may not occur simultaneously:
Send correspondence to C. J. Schaap.
3
CEL39
30
C.J. Schaap and A. Forer
chromosome-to-pole movement and spindle pole-to-pole elongation. These 2 processes may have different mechanisms (e.g. Ris, 1949; Oppenheim, Hauschka &
Mclntosh, 1973; McDonald, Pickett-Heaps, Mclntosh & Tippit, 1977). To understand better the mechanisms of anaphase chromosome movement, workers have
tried to alter chromosome-to-pole movement, or pole-to-pole elongation, or both,
by means of experimental treatments including altered temperature, increased pressure,
addition of drugs, ultraviolet microbeam irradiation, and micromanipulation of
chromosomes (e.g. Inoue", 1952; Taylor, 1959; Inoue", 1964; Forer, 1965; Nicklas,
1973; Oppenheim et al. 1973; Inoue & Ritter, 1975; Salmon, 1976). These studies
have led to the formulation of several models for anaphase chromosome movement
(e.g. Mclntosh, Hepler & Van Wie, 1969; Bajer, 1973; Forer, 1976; Inoue, 1976;
Margolis, Wilson & Keifer, 1978). Each model has faults, however, and there is no
universally accepted model for anaphase chromosome movement.
This paper represents the first part of a study of the effects of temperature on
anaphase chromosome movement in the first meiotic division of spermatocytes of
crane flies {Nephrotoma suturalis and Nephrotoma ferruginea). There are several
factors which make these animals suitable for studies of anaphase: (1) there are only 3
pairs of autosomes in large cells, making it possible to follow the movement of individual chromosomes; and (2) the major part of the chromosome-to-pole movement
precedes the pole-to-pole elongation (Forer, 1964, 1965, 1966). Thus we are able to
measure the effect of temperature on chromosome-to-pole movement separately from
the effect of temperature on pole-to-pole elongation, which was not possible in
previous studies (Barber, 1939; Ris, 1949; Fuseler, 1973, 1975). In this paper we
report the effects of a range of temperatures on anaphase velocities in crane-fly
spermatocytes: anaphase chromosome-to-pole velocities tend to increase with temperature within the range studied (6-30 °C) and at any given temperature chromosomes in N. suturalis spermatocytes tend to move faster than those in N. ferruginea
spermatocytes.
MATERIALS AND METHODS
Animals and living spermatocyte cell preparations
The 2 species of craneflies(Nephrotoma suturalis Loew and Nephrotoma ferruginea Fabricius)
used in these experiments are reared in the laboratory, using methods described in detail in
Forer (1964). The N. suturalis stock originated from North Carolina, while the N. ferruginea
stock originated from Toronto. Under our conditions spermatocytes enter prometaphase of
the first meiotic division about 10-12 days after the male larvae enter the fourth instar stage.
The testes are dissected out of the appropriate larvae while the larvae are immersed under
oil (Halocarbon Oil series 10—25: Halocarbon Products Corporation, Hackensack, New Jersey,
U.S.A.). The testes are then transferred to a drop of Voltalef oil (Huile 10 S: Ugine Kuhlmann,
Division Plastiques; II, bd. Pershing, 75017-Paris), where the surrounding fat is carefully
dissected away. (In this transfer and all subsequent steps, caution is exercised to avoid exposure
of the testes to the air.) Testes are then placed under Voltalef Huile 10 S on the bottom of a
holder designed to fit the temperature-control slide. The testes are punctured and the cells
spread out onto the glass surface of the bottom of the holder.
After the temperature-control slide is mounted on the microscope stage, the preparation is
scanned and aflatcell in metaphase I is located. In our experiments, all cells were at the experi-
Temperature and anaphase movement
31
mental temperature at least 10 min before the start of anaphase. Cells were photographed from
metaphase until at least the end of anaphase. Pictures were taken at intervals of 20—60 s
depending on the temperature (e.g. 60 s at 10 °C or 20 a at 25 °C).
Temperature-control slide and supporting apparatus
The temperature-control set up is similar to that used by Stephens (1973). Modifications to
the Stephens system were however necessary because we used an inverted microscope. We
used a Zeiss 40 x (N.A. 075) phase-contrast water-immersion lens with a long working
distance. To control the temperature of the coolant, 50 % ethanol, we used a Lauda K2/RD
(Brinkmann Instruments) constant temperature circulator: this both brings the solution to
the proper temperature and circulates it. Thermistor probes (Teflon Probe, Part no. 44104) connected to a YSI Tele-Thermometer (Model 43 TZ Yellow Springs Instrument Co., Inc.,
Yellow Springs, Ohio, U.S.A.) were used to monitor the temperature of coolant going into
and coming out from the temperature-control slide. When care was taken to dislodge air
bubbles from the thermistors, the incoming and outgoing temperatures differed by only about
0-5 °C or less. The Lauda circulator temperature varied by only ±o-oi to ±0-03 °C during
the course of an experiment.
Time-lapse apparatus and photography
The experiments were recorded on 16-mm film (Kodak Plus X Negative Film 7231). We
used a Bolex (H-16-J) with a time-lapse camera drive controlled by a time-lapse control panel
(R. J. Matthias and Associates, Houston, Texas, U.S.A.). The negatives were developed with
Diafine in a Cramer automatic processor (Sarasota, Florida, U.S.A.). Positive working prints
were made at a commercial laboratory.
Film analysis and statistics
The films were analysed frame by frame using an Athena projector (Model 224 by L-W
Photo Inc., Van Nuys, California, U.S.A.). The working prints were projected onto a table
via a mirror on a retort stand in front of the projector. For the analysis the final magnification
of the cells was 2000 times. The magnification of the film was calibrated using photographs of
a stage micrometer.
To determine the velocities of the separating autosomal chromosome pairs, we measured
interkinetochore distances parallel to the pole-to-pole axis in successive frames of the film as
anaphase proceeded. These measurements were plotted with respect to time, and a typical
result is given in Fig. 1. The chromosome separation velocity was determined by standard
least mean squares linear regression line (Sokal & Rohlf (1969), p. 419) through the points from
the linear portion of the distance-v.-time graph (e.g. the points between the arrows in Fig. 1).
To determine the velocities by which the sex chromosomes separate the distance between the
farthest kinetochores was measured in successive frames. Distance-v.-time graphs were
plotted and the slopes of the initial steep parts of the graphs were calculated by linear regression.
The slopes of the distance-v.-time curves are the velocities at which the autosomes or sex
chromosomes separate. These will be referred to as the chromosome velocities in the results.
Since there is no pole-to-pole elongation until at least the end of autosomal anaphase, the
chromosome velocities calculated for autosomes are directly proportional to the chromosome-topole velocities. Since the 2 separating autosome half-bivalents usually move at the same speed,
then their velocities to the poles are half the calculated autosomal chromosome velocities. On
the other hand, the pole-to-pole distances often increase as the sex chromosomes move poleward,
so that the calculated sex chromosome velocities include pole-to-pole elongation as well as
chromosome-to-pole movement.
By analysis of covariance (Sokal & Rohlf (1969), pp. 448-458) we compared the 3 autosome
velocities within individual cells. Thus we determined the frequency with which an autosome
pair within an individual cell has a velocity different from the other autosome pairs.
Viscosity values were computed from measurements of the movement of cytoplasmic particles. Equations and techniques developed by Fiirth (1917, 1930) and Pekarek (1930) enable
one to compute the viscosity of a Newtonian fluid from measurements of Brownian movement.
3-2
C. J. Schaap and A. Forer
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Fig. i. Typical graph of interkinetochore distances of autosomes (O, D, and <]) and
sex chromosomes ( • ) throughout anaphase. (N. ferruginea cell at25 °C). Chromosomeseparating velocities were determined by calculating the slopes using the points on the
graph between the arrows. The time labelled ' o ' on the abscissa indicates the start of
autosomal anaphase. The ordinate is the distance between chromosomes in microns.
The technique was later used by Taylor (1965), who extensively discusses the limitations of the
technique. The equation we used (Pekarek, 1930) is
1] =
(kTt)/(2naLhi)
(?/ is viscosity, k is the Boltzmann constant, T is the absolute temperature, t is the time it takes
the particle for n first crossings of parallel grid lines spaced L units apart, and a is the particle
radius). We controlled T, and we measured a and corresponding n and t values; from these
we calculated •>/ at various temperatures. Fig. 2 shows diagrammatically how the measurements
were made. As many 'first crossings' as possible were counted for each particle, but these were
limited due to particles leaving the focal plane, or single particles being mixed with other
particles.
RESULTS
General description of first meiotic division
The first meiotic division in N. suturalis spermatocytes appears to be similar to that
in N. ferruginea spermatocytes (Figs. 3, 4) in the following respects: there are 3 pairs
of autosomes and 2 sex chromosomes; all chromosomes can be seen clearly at metaphase in a flat cell; the 3 bivalents (autosome pairs) begin anaphase together, while
the sex chromosomes remain at the equator; the sex chromosomes begin to move
Temperature and anaphase movement
3f
L
Fig. 2. Schematic drawing of a hypothetical particle moving randomly in the cytoplasm
by Brownian movement.' Crossings' are counted only the first time a particle crosses a
particular grid line, until that particle has crossed a second grid line. After that if it
crosses the first grid line again, this may be counted as a crossing. The path of the
particle is represented by the 'jagged' series of lines. The particle starts at position l
which is the first crossing. Subsequent crossings are numbered 2-4, L is the distance
between grid lines and a is the particle radius (redrawn from fig. 1 of Pekarek, 1930).
Figs. 3, 4. Series of pictures from the anaphase of N. suturalis spermatocyte (Fig. 3)
and N. ferrugi-nea spermatocyte (Fig. 4) at 15 CC. The times, in min from the start of
anaphase were: 3A, B, C, D, — 22, +5, + io, +48; and 4A, B, c, D, — 10, +5, +21,
+ 55, respectively. The bars are 10/Jm apart.
34
C. J. Schaap and A. Forer
19)
polewards only when the autosomes have completed or almost completed their poleward movement; there is no pole-to-pole elongation until the autosomes are near the
poles; there may be pole-to-pole elongation during the poleward movement of the
sex-chromosomes; and the separating autosomes attain their maximum separating
velocities almost immediately (the time required depends upon the temperature).
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Fig. 5. Effect of temperature on the average velocities of separation for N. suturalis
(A) and N.ferruginea (B). The vertical bars represent the standard deviations from the
means. (All of the standard deviations were calculated assuming a normal distribution
about the mean using n— 1 degrees of freedom as described in Sokal & Rohlf (1969),
pp. 53-55.) The number of cells and the total number of autosome separating velocities
(n) which are part of the mean are also indicated. We thank A. Barkas for permission to
analyse his films in order to gain additional data for N. suturalis, which we have incorporated with our own. (Note that for various technical reasons it was not always possible
to get data from all 3 autosome pairs in each cell. A typical reason for this would be
that not all of the chromosome pairs were in the same focal plane.)
Temperature and anaphase movement
35
More detailed descriptions of meiosis in crane fly spermatocytes can be found in
Bauer (1931), Wolf (1941), Dietz (1956, 1959), Bauer, Dtetz & Robbelen (1961),
Dietz (1963), Forer (1964), Ullerich, Bauer & Dietz (1964), Forer (1965, 1966), Behnke
& Forer (1966) and LaFountain (1972).
Effect of temperature on autosomal chromosome velocities
As measured from our phase-contrast micrographs, the pole-to-pole distances do
not appear to increase until the autosomes are near the poles. Phase-contrast microscopy does not allow one to locate the positions of the poles accurately, to within more
than a few microns, but data from polarization microscopy confirm that pole-to-pole
distances do not increase until the autosomes are near the poles (see illustrations and
graphs in Forer, 1965, 1966, 1976 or Dietz, 1969). The pole-to-pole distances did not
appear to increase during autosomal anaphase at any of the temperatures studied.
Thus the autosomal chromosome velocities which we calculated really are proportional to velocities of chromosome-to-pole movements: increase of the pole-topole distance does not contribute to autosome velocity at this time.
Our results on autosomal velocities at different temperatures are illustrated in
Fig. 5. These figures demonstrate clearly that as the temperature increases the
autosomal velocities also increase. However, at any temperature the range of possible
velocities is very large, and these velocities often overlap with those seen at other
temperatures. A wide range of autosomal velocities was also observed by Forer (1964,
1965) for N. suturalis studied at room temperatures, though some of that spread may
have been due to variations in room temperature.
There are some differences between the cells of the 2 species. At any given temperature autosomes of N. suturalis spermatocytes tend to move faster than those of
N. ferruginea spermatocytes. Using Student's£test(Sokal&Rohlf(i969),pp. 143-145),
these differences are significant (a = 0-05) at 20, 15, and 10 °C but not at 25 °C. The
temperature ranges are also different. Autosome movement in N. ferruginea spermatocytes is limited to a range between about 6 and 25 °C: outside this the cells die,
spindles collapse and/or the chromosomes clump. The limits for N. suturalis spermatocytes have not been determined, but the upper limit is at least 30 °C. Our N.
ferruginea culture derives from flies caught in Toronto while the N. suturalis culture
derives from flies caught in North Carolina, which has a much warmer climate than
Toronto. This may have an effect on the range of temperatures at which normal
divisions can occur.
The different autosomes (half-bivalents) within a cell do not necessarily move
polewards at the same velocity. The chromosome velocities within cells were compared statistically by an analysis of covariance (Sokal & Rohlf (1969), pp. 448-458).
This test allowed statistical comparison of the velocities of autosomes within a cell,
while at the same time taking into account variations inherent in the distance-v.-time
measurements. In other words, were the differences in velocities calculated from
regression lines statistically significant (a = 0-05) or were they merely due to errors
in measuring? In the usual case it is clear that, in both species, the autosome pairs in
any given cell do not all move polewards at the same speed, as illustrated in Fig. 6.
C. J. Schaap and A. Forer
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Fig. 6. Summarized data of analysis of covariance on the separating velocities of
autosomes within the same cell (A, N. suturalis; B, N.ferruginea). As described in the
text this type of data analysis has allowed us to determine whether one or more pairs
of separating autosorr.es actually have the same separating velocities. Since the autosome pairs cannot be visually differentiated they are arbitrarily named aa', bb'', and
cc' as one moves from left to right along the metaphase plate of the cell (bb' is always the
centre autosome pair). The separating velocity of aa' is designated as a, of bb' as b and
of cc' as c. The shaded boxes mean that there were only 2 measurable autosome pairs
in that particular cell. (The data from the films of A. Barkas were not included in the
analysis of covariance and thus also not in Fig. 7 and Fig. 8, but we have no reason to
believe they would significantly alter the results.) Categories: A, a = b or b = c;
B, a = c; C, a — b = c; D, a 4= b 4= c; E, statistically strange, e.g. a = b, b = c,
a 4= c.
Usually at least one pair moves poleward at a different speed from the others. One
might argue that since the outside autosomes must travel a greater distance, they
should travel faster in order to get to the poles at the same time. One can rule this out,
however, because if 2 of the 3 autosome pairs move at the same speed, these are more
likely to be one middle autosome pair and an outside one, as compared to 2 autosomes
positioned on the outside (Fig. 6).
The variabilities of autosome velocities appear to be independent of temperature
(Fig. 6); therefore these differences are not merely temperature effects. This is
corroborated by comparing the percentage differences,
higher velocity — lower velocity
x ioo,
lower velocity
between chromosome pairs considered statistically different: the percentage differences
do not vary with temperature (Fig. 7), which they would do if the temperature changes
caused different autosomes in the same cell to move with different velocities. On the
other hand the differences in velocities between autosomal pairs in the same cell are
dependent on chromosome velocities (Fig. 8), and the shapes of these curves are very
Temperature and anaphase movement
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Temporature, °C
Fig. 7. The mean percentage differences between the velocities found to be statistically
different by the analysis of covariance are represented in relation to temperature
(A, N. suturalis; B, N.ferruginea). The vertical bars represent the standard deviations
from the means.
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Fig. 8. The relationship of mean absolute differences in autosome separating velocities
within cells to temperature (A, N. suturalis; B, N. ferruginea). The vertical bars
represent the standard deviations from the means.
similar to the curves of autosome velocity versus temperature (Fig. 5). Thus, the
differences in autosome velocities within cells are proportional to the autosome
velocities, and are not temperature-dependent effects.
In summary, increased temperature results in increased autosome velocities; at any
given temperature autosomes in N. suturalis spermatocytes tend to separate faster
than those of N'. ferruginea spermatocytes; and for both species temperature does not
38
C. J. Schaap and A. Forer
appear to control the observed differences in autosome velocities within individual
cells.
Temperature effects on sex-chromosome velocities
The sex chromosomes do not start their anaphase poleward movements until the
autosomes have reached or almost reached their respective poles. Although pole-topole elongation as well as chromosome-to-pole movement may occur simultaneously,
the sex chromosomes move much more slowly than the autosomes (Fig. 9), often
by a factor of about 3 or 4. The shapes of the autosome and sex chromosome velocityv.-temperature curves are comparable (cf. Fig. 9 with 5). Thus temperature appears
to affect the 2 types of movement similarly, and we would expect to be able to predict
the approximate separating velocity of the sex chromosomes if the average autosomeseparating velocity for that cell were known. This is roughly true, as illustrated in
Fig. 10: a plot of autosome velocity-v.-sex chromosome velocity in the same cell
is indeed linear, but there is considerable scatter. (In Fig. 10 there is one point which
seems particularly out of place; we have no explanation for this apparent misfit.)
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Fig. 9. The effects of temperature on the velocity of sex chromosome separation of
N. suturalis (A) and N. ferruginea (B). (Note that we could not get the sex chromosomeseparation velocity for each cell studied: for example if the 2 sex chromosomes \s'ere
not in the same focal plane.)
Relative timing of sex chromosome movements
In the first meiotic division of crane-fly spermatocytes the anaphase of the autosomal bivalents precedes that of the sex chromosomal univalents. How are the 2
anaphases linked? What is it that controls the time lag between them? Temperature
affects the mechanisms which control the lag time between the 2 anaphases (Fig. 11):
in both species the time between the start of autosomal anaphase and the start of the
sex chromosome segregation depends on temperature. This time difference seems to
Temperature and anaphase movement
39
depend more on temperature in N.ferruginea than in N. suturalis: in the former it is
clear that the lower the temperature the longer it takes for the sex chromosomes to
start their anaphase, but the trend is not as pronounced in N. suturalis (Fig. 11).
However, more data are needed in order to see whether or not there is a difference
between the spermatocytes of the 2 species in this regard.
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Average autosomal velocity of separation, ^m/min
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Fig. 10. A comparison of sex chromosome velocity of separation with the autosome
velocity of separation in the same cell (N. suturalis, # ; N.ferruginea, A): the autosome
velocities are averages of up to 3 velocities per cell, or are single velocities (when we had
only one autosome velocity for that cell).
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Fig. 11. The log of the time difference between the start of autosomal anaphase and
the start of the sex chromosome segregation is plotted on the ordinate and temperature
is plotted on the abscissa (N. suturalis: individual cells, O; mean for a given temperature, • ; N. ferruginea: individual cells, A; mean for a given temperature, A).
C. J. Schaap and A. Forer
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Fig. 12. Cytoplasmic viscosity, on the ordinate, is plotted versus temperature,
on the abscissa (N. suUiralis, O'» N. ferruginea, A)38
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I
I I I I I I I I I
10
15
20
Mean velocity of autosome separation, yni
I
I
I I
2-5
I
I
I )
30
Fig. 13. The measured cytoplasmic viscosity within a cell (ordinate) is compared with
the average velocity of autosome separation within that cell (abscissa) (AT. suturalis, # ;
N.femiginea, A).
Temperature and anaphase movement
41
Effect of temperature on cytoplasmic viscosity
In order to see if the observed differences in velocity might be due to temperatureinduced viscosity changes, cytoplasmic viscosity was calculated from measurements
of Brownian movement velocities of particles in the cytoplasm. The particles measured
were about 0-5-1 -o /im in size. The areas studied in these measurements were not
chosen at random, but rather had relatively few particles and were away from interferences of the spindle area and the cell membrane. Because it was very difficult to
find suitable cytoplasmic particles and because there is extensive literature on similar
measurements in other cells relatively few measurements were made. As expected,
the viscosity increases as the temperature decreases (Fig. 12). The range of viscosities
(about a factor of 6-7) is not as large as the range in velocities (about a factor of 8-10).
We have compared viscosities directly with autosome velocities measured in exactly
the same cells (Fig. 13), and while viscosity might influence chromosome velocities at
lower velocities (less than about o-6 /<m/min), there seems to be no effect at higher
velocities. Hence the effect of temperature seems to be primarily on the movement
mechanisms and not on the viscosity of the medium, at least at temperatures greater
than about 15 °C.
DISCUSSION
Autosomal velocities
We have shown that autosomal velocities in anaphase depend on external temperature: the higher the temperature the faster the movement (Fig. 5). In previous
work, Barber (1939), Ris (1949) and Fuseler (1973, 1975) have also shown that
chromosome velocities in anaphase, in various cell types, depend on temperature, and
we have summarized these data in Fig. 14. While these earlier data are similar to
ours in indicating that anaphase velocities increase as temperature increases, the
results are not directly comparable to ours, because our measurements are of chromosome-to-pole velocities. The previous measurements included both chromosome-to-pole
velocity and velocity of pole-to-pole elongation. In the previous experiments, for
example, it was possible that pole-to-pole elongation was greatly affected by temperature while chromosome-to-pole movement was unaffected, or the converse. As far as
we know, ours are the first data which directly measure the effect of temperature on
chromosome-to-pole motion during anaphase, separately from pole-to-pole elongation.
Other previous workers described the effect of temperature on anaphase, but these
data deal primarily with the duration of anaphase at different temperatures (Fig. 15).
These data might be compared with our data on velocity by assuming that total
anaphase distances are constant: in that case velocities would be inversely proportional
to anaphase durations. We have made such calculations from published data, and we
summarize these data in Fig. 15. These recalculated data agree with ours in showing
that anaphase chromosome velocities increase as temperature increases, but here too,
these data are of possible mixtures of both pole-to-pole elongation and chromosometo-pole motion.
C. J. Schaap and A. Forer
50 -
a
A
A
-
3 • ,
,
A •
A
A
A,'
10 • c
—
0 •
A
A
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0-5
A
—
A
—
f
B
'•
o
6
cf
i
o/
-
va
0-1
-
0-05
n-m
•
• /
1
i
i
10 15 20 25
Temperature, °C
i
i
30
35
Fig. 14. Mean anaphase velocities of separation from each of several systems are
plotted on the log scale of the ordinate to show their relationship to temperature
(abscissa) and to compare these data directly with ours (AT. suturalis: autosomes, A;
sex chromosomes, V; N. ferruginea: autosomes, • ; sex chromosomes, |£; Barber
(1939) Tradescantia virginiana, A; Ris(i949) Chorthophaga viridifasciata, • ; Fuseler
(1975) (estimated from fig. 7), Asterias forbesi, O, Tilia americana, • ) . The broken
lines show the trends in velocity changes due to temperature, of both autosomes (A)
and sex chromosomes (B).
Temperature and anaphase movement
10
43
r
0-1
c
1
t:
0-01
0001
10
15
20
25
30
Temperature, °C
35
40
45
Fig. 15. Review of data from the literature on the effects of temperature on the length
of anaphase in several systems. Since we assume that the duration of anaphase is inversely proportional to anaphase velocity, we have plotted the inverse of the time of
anaphase on a log scale on the ordinate and compared it with temperature on the
abscissa (Laughlin (1919), Allium cepa, @; Bucciante (1927), chick fibroblasts, • ;
Makino & Nakahara (1953), Yoshida sarcoma cells, A, MTK sarcoma I cells, • ;
Agrell (1958), Echinus esculentus, O,Psammeckinus miliaris, <^>; Evans & Savage (1959),
Vicia faba, (E; Dettlaff (1963), Acipenser gilldenstadti colckicus V. Marti, # ; Lopez-
Saez, Gim£nez-Martfn & Gonzalez-Ferndndez (1966), Allium cepa, A; Stephens
(1972), Strongylocentrottis droebachiensis, <*).
44
C. J. Schaap and A. Forer
Might the temperature dependence of chromosome-to-pole movements give a clue
to the mechanism of force production ? Some suggest that the forces for chromosometo-pole motion are produced by muscle proteins such as actin (e.g. Forer, 1976), while
others suggest that the forces arise from the action of dynein (e.g. Mclntosh et al.
1969; Mclntosh, 1974; Margolis et al. 1978); if the temperature dependence of
anaphase chromosome movement were like muscle contraction but not ciliary beating,
for example, this might suggest that chromosome movement was due to the action of
muscle proteins, or the converse. We have looked at the literature describing the
effects of changing temperature on various motile processes, such as muscle contraction, particle transport, ciliary beating and flagellar beating, and we summarize these
data in Fig. 16. These data show that these processes occur faster at higher than at
lower temperatures, but actin-based mechanisms (muscle, movements of cytoplasmic
particles) cannot be distinguished from dynein-based mechanisms (cilia, flagella).
Thus the data do not suggest that the force for chromosome movement arises from
one mechanism and not the other.
Another way to look at data on temperature effects is to calculate activation energies
(from the Arrhenius equation) for the rate-limiting step in the motile system in question. To do this one must assume that the same rate-limiting step applies at all the
temperatures considered. Activation energies calculated from data on temperature
dependence have been compiled in Table r. In our case, we plotted log velocityv.-1/absolute temperature, and calculated activation energy (Ea) from the slope of
the regression line.
If the activation energies of Table 1 are compared with activation energies characteristic of enzyme-catalysed reactions (Table 2), it appears that usually the ratelimiting step of these motile systems is not enzyme-catalysed. From the literature
reviewed by Lineweaver (1939) and Sizer (1943) it appears that the activation energies
of enzyme-catalysed reactions are commonly below about 13 kcal per mol (e.g. see
Table 2), while those of the motile systems discussed tend to be higher. Thus although
enzymes are probably involved in these processes they appear not to be catalysts in
the rate-limiting reaction. The rate-limiting step may however be catalysed by inorganic ions (e.g. hydrogen, calcium or platinum).
Our data are also relevant to all hypotheses of force production for chromosome
movement in which all chromosomes would be expected to move at the same speed
in anaphase: as a general rule, they do not move at the same speed (Figs. 6-8). This
implies that one cannot invoke a simple temperature-regulated microtubule
assembly—disassembly equilibrium as the motive force for chromosome movement (e.g.
Inoue, 1976), because that would imply that all chromosomes move at the same speed.
At the least one must superpose upon a mechanism such as this, and indeed upon the
other proposed mechanisms, some further control, such that some chromosomes can
move at a velocity up to 50 % different from that of other chromosomes in the same
cell (Figs. 6-8). These differences in velocities are not related to obvious differences in
chromosomal size, because the autosomes in crane-fly spermatocytes are not distinguishable by size.
We have calculated viscosity in spermatocytes at different temperatures from
Temperature and anaphase movement
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Fig. 16. Compilation of representative data showing the influence of temperature
(abscissa) on several types of biological movement.
A. Rate of ciliary movement on gills of Mytilus as determined by the rate of particle
transport (from the data of Gray, 1924).
B. Ciliary beat frequency of Stentor polymorphic (from the data of Sleigh, 1956).
C. Rate of retrograde intra-axonal organelle transport in Rana catesbeiana (redrawn
from Forman el al. 1977).
D. Rate of fast axonal amino acid transport in Anodonata cygnea (redrawn from Heslop
& Howes, 1972).
E. Flagellar beat frequency of Strigomonas oncopelti (from the data of Holwill &
Silvester, 1965).
F. Rate of heart beat in rabbit ( • ) and dog (O) (from data of Frank, 1907).
CEL 39
t
Process
Activation energy values of some motile systems
Autosome anaphase movement
Sex chromosome anaphase movement
Autosome anaphase movement
Sex chromosome anaphase movement
Anaphase chromosome movement
(Calc. from max. velocity)
Anaphase chromosome movement
Anaphase chromosome movement
Anaphase chromosome movement
Flagellar beat frequency
Ciliary beat frequency
Rate of particulate transport by cilia on
the gills
Rapid retrograde axoplasmic organelle
transport
Rapid anterograde axoplasmic transport
in C-fibres
Rapid anterograde axoplasmic transport
Rapid anterograde axoplasmic transport
Rapid anterograde axoplasmic transport
Velocity of contraction of heart muscle
Velocity of contraction of heart muscle
Velocity of contraction of heart muscle
Velocity of contraction of heart muscle
I.
Cited in Crozier (1926)
I
\Cited in Cosens er al. (1976)
Forman et al. ( 1 ~ 7 ) t
Reference
Calculated from tabular data by present authors (s.E. of regression line calculated as in Sokal & Rohlf (1969) pp. 417-436).
Estimated from graphed data by present authors.
Cat
Rabbit
Rana temporaria
Frog
Dog
Cat
Rabbit
Rana catesbeiana
Tilia americana
Asterias forbesi
Chorthophaga viridayasciata
Strigomonas oncopelti
Stmtor polymorphus
My tilus
N e p h r o t m a suturalis
System
Table
Temperature and anaphase movement
47
Table 2.* Activation energies of selected chemical reactions with and without enzyme
catalysts
Reaction
Hydrogen peroxide
decomposition
Catalyst
None (dust)
Fe(OH) 3 "1
IVInOs
^
I~
Ea (kcal/mol)
18-0
US
J
n-7
Colloidal Pt
Liver catalase
5'5
H+ion
Sucrose inversion
260
Yeast invertase
us
130
Malt invertase
HC1
206
Casein hydrolysis
144
Trypsin-kinase
Trypsin
1
12-0
Crystalline trypsin
,Crystalline chymotrypsin J
H+ion
Ethyl butyrate
13-2
4-2
Pancrease lipase
• Partial reproduction of table i Lineweaver (1939). Activation energies for other enzymecatalysed reactions can be found in Sizer (1943).
measurements on granule movements: while viscosity does change somewhat with
temperature (Fig. 12), the increase in viscosity as temperature is lowered cannot
account for the reduced chromosome velocity (Fig. 13), especially at temperatures
from 15 to 30 °C. The technique we used to calculate viscosities certainly has limitations (see discussions in Heilbrunn, 1928; Taylor, 1965; Heilbrunn, 1958), but our
results agree with those using other methods. We have summarized in Fig. 17 results
of other calculations of viscosity at different temperatures in various cells. One can
see from this that some other calculations give results similar to ours. Heilbrunn
(1958), in reviewing calculations of cytoplasmic viscosity at different temperatures,
suggests that there are 2 general groups of cells, (1) those in which ' the protoplasm...
shows a progressive decrease in viscosity as the temperature is raised', and (2) those
in which 'the viscosity goes through a maximum with rising temperature'. Our
results seem to fall into the first class, and we conclude that changes in viscosity are
not the main reason for the observed changes in chromosome velocity. Barber
(1939) reached a similar conclusion, although he himself made no viscosity measurements.
Movements of sex chromosomes
Sex chromosome velocities change with temperature in much the same way as do
autosomal chromosome velocities, but the sex chromosome velocities are considerably
slower than the autosomal velocities, by a factor of about 4. This difference in speed
might be a reflexion of the different orientations of the 2 kinds of chromosomes: the
autosomes are syntelically orientated while the sex chromosomes are amphitelically
4-2
C. J. Schaap and A. Forer
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Fig. 17. Compilation of data on the effects of temperature on cellular viscosity. (For
discussion of methods and data see Heilbrunn, 1958).
A. Relative viscosity of immature eggs of Nereis diversicolor as determined by centrifugation (redrawn from Pantin, 1924).
B. Relative viscosity of Cwningia eggs as determined by centrifugation (redrawn from
Heilbrunn, 1924, as quoted by Heilbrunn, 1928).
C. Viscosity of Spirogyra protoplasm as determined by studying particle Brownian
movement in the cytoplasm (redrawn from Bass Becking, Sande Bakhuyzen &
Hotelling, 1928).
D. Viscosity of Arbacia eggs as determined by centrifugation (redrawn from Costello,
1934, as quoted in Heilbrunn, 1958).
E. Relative protoplasmic viscosity of Ascaris megalocephala eggs as determined by
centrifugation (from data of Faur6-Fremiet, 1913, as quoted by Heilbrunn, 1958).
F. Protoplasmic viscosity of Amoeba dubia as determined by centrifugation (redrawn
from Murphy, 1940, as quoted by Heilbrunn, 1958).
G. Protoplasmic viscosity of Phaseolus multiflorus as determined by the time required
for a starch gTain to fall through the protoplasm (from data of Weber & Weber, as quoted
by Heilbrunn, 1958).
Temperature and anaphase movement
49
orientated (Bauer, Dietz & Robbelen, 1961). That is to say, the autosomes have one
chromosomal spindle fibre extending between autosome and pole; this spindle fibre
shortens as the autosomal half-bivalent moves poleward in anaphase. On the other
hand the sex chromosomes have chromosomal spindle fibres extending to both poles;
during anaphase one spindle fibre shortens and the other one elongates as any given
sex chromosome moves polewards. The different spindle fibre arrangements might
give rise to different poleward velocities.
Temperature affected not only chromosome velocities but also the time interval
between the onset of autosome anaphase and sex chromosome anaphase (Fig. n ) :
the higher the temperature the shorter the interval between the 2 stages. With regard
to a cellular signal giving rise to sex chromosome anaphase, one might guess that sex
chromosome anaphase begins after the autosomes reach the poles, or after the autosomes have moved a certain distance, but this seems not to be true: if one plots
autosomal velocity (from Fig. 5) versus the time interval between autosome and sex
chromosome anaphase at the same temperatures (from Fig. 11), one does not get the
straight line which would be expected if sex chromosome anaphase began after the
autosomes had moved a certain distance. The signal to start sex chromosome anaphase,
and how temperature affects this, remain mysteries.
We have observed abnormalities in sex chromosome segregation throughout these
experiments: for example, a single sex chromosome sometimes moved polewards
before autosomes, or together with the autosomes. Such abnormalities rarely if ever
occur in cells studied at room temperature without experimental treatment (e.g. Dietz,
1969; Forer & Koch, 1973), and we presume that the temperature shifts somehow
affected the sex chromosome segregation mechanism. However, we have not been
able to determine what, exactly, in our procedures might be causing alterations in sex
chromosome segregation.
We should like to thank Dr F. Eckhardt for aiding us in our understanding of the German
viscosity papers. We are also grateful to our colleagues in the Biology Department at York
University for useful discussions, Mrs D. Gunning for excellent secretarial assistance and the
National Research Council of Canada for financial support.
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C. J. Schaap and A. Forer
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