ABSTRACT
Unfertilized sea urchin eggs, centrifuged into nucleate and enucleate fragments,
were activated by ammonia and time-lapse videotaped on a light microscope to
record move ments of the membrane surface. These fragments, unable to divide,
exhibit wiggling" and contractile movement that corresponds in time with the
nor mal first and second mitotic cycles, peaking in intensity with expected
cytokinesis. Movement then declines in intensity, corresponding in time with
nor mal interphase. Cytoskeletal isolations performed at times of intense
move ment show astral organization of microtubules in both nucleate and enucleate
fragments.
INTRODUCTION
It has been hypothesized by Koki Hara, Peter Tydeman, and Marc Kirshner
that control of the cell cycle is driven by an "autonomous oscillator" (Murray and
Kirshner, 1991). Three main experimental ideas, concerning the critical event of a
cell entering mitosis, have led to the formation of this concept.
First, premature chromosome condensation (PCC) occurs in an interphase cell
when fused with a mitotic cell; cell in Gl will prematurely condense its
chromosomes into single strands, a cell in S will prematurely condense its
chromosomes partially into single and partially into double strands, and a cell in G2
will prematurely condense its chromosomes into double strands (Johnson and Rao,
1970).
Second, unfertilized sea urchin eggs activated by ammonia-treated sea water
will begin successive cycles of DNA synthesis and chromosome condensation, but do
not divide (Mazia, 1974). These eggs will cause synchronous PCC in as many as 50
polysper mically fertilizing sperm nuclei. PCC also occurs in these sperm nuclei
when polysper mically fertilizing am monia-activated nucleate and enucleate cell
fragments formed from a whole cell by centrifugation or bisection (Poccia et al.,
1978).
Cell cycle control, therefore, appears to pervade the cell and be cytoplasmic
as opposed to being discretely contained in an organelle, such as the nucleus.
Third, cyclin, a protein component of MPF (maturation promoting factor or
mitosis promoting factor), has been shown to oscillate in amounts critical to the
onset of mitosis. Since enzymes are closely linked to protein levels, MPF defines
what is controlling the cell cycle closer to something that is inherent in the make
up of the cell, possibly a series of self-perpetuating chemical reactions (Murray and
Kirschner, 1991).
These studies that have shown unfertilized, enucleate fragments of sea
urchin eggs capable of controlling the onset of mitosis, or chromosome
condensation. By observation of membrane surface events, the present study
investigates the ability of these fragments to assemble a mitotic apparatus (MA),
that is, to proceed even further into mitosis.
MATERIALS AND METHODS
Eggs and sperm of Strongylocentrotus purpuratus were used. Animals were
induced to shed by hand-shaking and/or intracoelomic injection of 0.5 ml KCl. Eggs
were shed directly into filtered sea water (FSW), passed through a 130 um Nitex
cloth filter, and maintained at approx. 170C in a waterbath shaker. Sperm was
stored dry at 40C and diluted at least 3000X just before use.
Centrifugal production of fragments
Nucleate and enucleate fragments from S purpuratus were prepared in a
ultracentrifuge (Nishioka and Mazia, 1974). One molal sucrose was made with
distilled water and 0.25 molal sucrose was made by diluting the 1.0 molal sucrose
with FSW. Both sucrose solutions were used to prepare six 35 ml 0.25 molal to 1.0
molal linear sucrose gradients by hand and were allowed to stand for at least 10
min or overnight. Two ml aliquots of a 42 egg suspension were layered over each
gradient and centrifuged at 7,000 rpm for 10 min and 9,000 rpm for 10 min in a
Beck man ultracentrifuge with a SW-27.1 rotor. Immediately after centrifugation,
the nucleate fragments (upper band), whole eggs (middle band), and enucleate
fragments (lower band) were collected separately with a pipette into an equal
volume of sea water. Immediately after collection, the fragments and whole eggs
were resuspended in FSW.
Generally, three layers-a thick layer of nucleate halves, a thick layer of
enucleate halves, and a thin layer of whole eggs-were obtained, although on
occassion 1 or 2 additional layers of nucleate and enucleate quarters were
obtained. Whole eggs stratified into a centripetal oil cap, a large clear zone
containing the nucleus just underneath the oil cap, and yolk. Fragments were split
equally in size, with nucleate halves containing the oil, clear zone, and nucleus, and
the enucleate halves containing mostly yolk. Variations in size and stratification
did occur in eggs from female to female.
Manual production of fragments
Unfertilized eggs were passed, by mouth suction, through a 23 um Nitex
fitler fitted to the end of a small cylinder. Irregularly shaped fragments were
produced which eventually reassumed a round shape.
Egg sample preparations for time-lapse video recording
Fragments, centrifuged whole eggs (control for fragmentation by
centrifugation), and uncentrifuged whole eggs (control for centrifugation) were
combined together for ammonia activation as described below. Samples also
included control fertilized eggs obtained as described below. Egg samples were
prepared on chlorofluorocarbon slides and placed on a Zweiss WL microscope for
video recording by a Panasonic time-lapse system.
Chlorofluorocarbon (CFC) slides: Depression slides were filled with some CFC
FC-43. A normal microscope slide was tightly moved over the depression slide,
flattening a drops of egg sample. Petroleum jelly was applied to the edges of the
slides to ensure the tight seal between the depression and covering slides.
Ammonia activation
Eggs were activated by culturing for 18 min in FSW titrated to pH 9.4 with
NHAOH (Mazia, 1974). The culture was maintained in a waterbath shaker at
approx. 170C. Eggs were immediately resuspended in FSW (approx. pH 8).
Activation was considered to have occurred at initial exposure to pH 9.4.
Fertilized control eggs
Uncentrifuged, untreated whole eggs were fertilized at the same time eggs
were activated by ammonia, followed by 3 washes with FSW to eliminate sperm in
egg sample.
Time-lapse video recording
A Zweiss WL microscope fitted with a Panasonic WV350A camera was used
to record onto a Panasonic time-lapse video recorder. Recordings were taken at
time-lapse mode 180 hr/Ihr real time onto a videotape and played back at real
time speed. A Blue M cooling probe was layed flat on the microscope stage to
maintain a stage temperature of approx. 170C.
Note: Due to sudden difficulty in playback imaging, selected still images from the
time-lapse videotape were converted by a Megavision image processor.
Cytoskeletal isolations
A solution of 10 mM dithiothreotol (DTT) in sea water was calibrated to pH
9.0 with NaoH. Unfertilized eggs were treated with DTT for 5.5 sec with agitation
in a waterbath shaker. Eggs were resuspended in sea water before centrifugation.
collection, and ammonia activation, as described previously, to obtain fragments
and centrifuged whole eggs. Isolation medium was calibrated to pH 5.9-6.0 with
HCl. At times corresponding to expected mitotic apparatus assembly for the first
cleavage (1.75 -2.25 hrs), 3 ml samples in 15 ml rounded-bottom glass test tubes
were resuspended in KGE. Immediate resuspension again followed, with 200 ul
Triton X-100 pipetted into the suspension and covering with parafilm for gentle
mixing by inversion of the test tube. Observations were made by a Zweiss WL
microscope. Twenty ul of DAPI was added to each 3 ml sample for observations on
an Olympus BH2 microscope with fluorescence attachments.
RESULTS
Time-lapse video recording
Successive patterns of membrane surface movement were observed in both
nucleate and enucleate fragments and both centrifuged and uncentrifuged controls,
with increasing intensity corresponding to the onset of mitosis and cytokinesis of
the first and second cleavages in fertilized controls (Fig. 1). Decreasing intensity of
movement corresponded to post-cleavage events in fertilized controls, often
decreasing to the point of very little movement ('calm" period) or no movement at
all (’quiet" period). Furthermore, these events corresponded to the cell cycling of
uncentrifuged controls, apparent by the disappearance and reappearance of a
visible nucleus. In general, the fragments and controls exhibited the following
relative intensity of movement: nucleate » enucleate » centrifuged control »
uncentrifuged control.
On occasion, at times beyond the second 'calm/quiet" period, both nucleate
and enucleate fragments and both centrifuged and uncentrifuged controls, were
observed to undergo violent distortions of the the membrane surface to the extent
that the cell would ’explode", or fall apart. These movements would begin at 3.5-
6.5 hrs and would take place over the course of a few hours (Fig. 2 and Fig. 3).
TIME-SCALE OF MEMBRANE SURFACE MOYEMENTS:
TIME“
PERIOD
1:15 - 1:45 Movement
1:45 -2:45 First Cleavage
2:00 - 2:30
Calm/Quiet
2:15 -3:00
Movement
2:45 - 3:45
Second Cleavage
3:00 - 4:00
Calm/Quiet
Preparations for time-lapse video recording consisted of assorted ceil fragments and controis. Most
fragments and controls exhibited these movements. The time-scale of events was taken from four recordings.
These recordings were made from CFC stide preparations of egg samples that were temperature controlled
(approx. 170C), because it was found that fertilized controis were more successful at completing two
divisions at cosder temperatures. However, it did not appear that the cosder temperatures greatsy affected
the presented data.
Times (hrs: min) indicate the range of times the specific events were observed to occur. Note:
slight movement was often observed before the first movements (1:15-1:45).
Note: Slight movement was often observed before the first movements occurred (1:15-1:45).
Cytoskeletal Isolations
Cytoskeletal isolations of both nucleate and enucleate fragments at times
corresponding to expected MA assembly for the first cleavage showed an astral
microtubule arrangement in each type of fragment. DAPI staining confir med
distinction between nucleate and enucleate fragments (Fig. 4 and Fig. 5).
Note: Isolations of bisected eggs by Daniel Mazia were used to illustrate these
results. Isolations of centrifuged eggs were unsuccessfully photographed and time
constraints disallowed further isolations.
DISCUSSION
It is shown from this study that patterns of membrane surface events in
activated, but unfertilized, sea urchin egg fragments can be defined.
These
patterns can provide alternative means of analyzing the occurence of cell cycle
events by observing the outside of the cell, as opposed to the inside. These results
are supported by three different controls--centrifuged, uncentrifuged, and
fertilized eggs. Time-lapse video recording of bisected, rather than centrifuged,
fragments could provide further evidence. Slightly flattening these fragments by
the CFC preparation would facilitate the visibility of nuclei and, therefore, the
distinction between nucleate and enucleate fragments. The flattening, however,
would probably alter nor mal development somewhat.
The isolation of microtubule structure underlying the movements observed
also supports the findings of this experiment. An ammonia-activated sea urchin
egg will not divide, requiring the bipolarity the sperm provides with its
centrosome. Debate exists as to whether the unfertilized egg contains a centrosome
at all or whether it contains an inactive centrosome. Nonetheless, the egg is
capable of assembling microtubule structure, which has been described as "non-
polar" (Mazia, 1988). This experiment shows that this structure is at least capable
of moving the membrane surface. In some instances, mostly later in the cell's life
this movement is sufficient to destroy the membrane. Further studies on the
correlation between types of surface movement and types of cytoskeletal
arrangement in these fragments would provide a better understanding of assembly
and use of organized microtubule structure, such as in cytokinesis.
The correlation found between the time of the most active movement and
the isolation of asters may support hypotheses about communication of
intracellular elements with the surface. It is known that vesicles are transported
bidirectionally in microtubules. This experiment also has implications for models
of maternal centrosome arrangement. The linear model proposed by Mazia (Mazia
1987) is supported by this study in that an unfolded linear structure split by
centrifugation could explain the finding of astral organization in both nucleate and
enucleate fragments.
This study has addressed the idea of endogenous oscillators, or "clocks", in
biological systems. Endogenous control of behavioral, physiological, and
bioche mical patterns have been uncovered in most organisms that have been the
pursuit of such studies. For example, the hypothalamus in the brain of humans is
regulates cycles of rest, body temperature, metabolism.
What is the cellular clock?" is a question that has been the inquiry of
previous experiments. Enucleate cells and the discovery of MPF have provided a
clearer picture. The questions asked by this investigation are "Where is the
cellular clock?" and "How long will it run (under experimental manipulation)?
appears that it runs quite successfully throughout the cell, so that both nucleate
and enucleate fragments are capable of attempting cytokinesis, sometimes even to
the point of self-destruction.
ACKNOW LEDGEMENTS
l’d like to express my gratitude to Dr. Mazia for his guidance, support,
friendship, and a great dinner. Working with you has undoubtedly been a unique
experience for which I am very greatful. Special thanks goes to Chris Patton for
assistance both in the Mazia lab and photo lab. I am also indebted to Dr. Stuart
Thompson and Megavision Studios" for recovering my time-lapse videos/ data
from the dead. Emile, you deserve a good portion of recognition for cleaning my
dirty glassware as fast as I produced it. John, your popcorn, coffee, and company
kept me sane that day my data was destroyed. S. Purp, thank you for the results I
wanted. 663 Spencer Street *1, you treated me very well; I will not ever forget
the good times, recipe swapping. Simpsons Nights, pillow fights, and the people I
lived there with. And everyone else-the Spring students and those that chatted
with me in the halls and labs-you are also in every bit of this paper. Thank you.
I had a fun time.
BIBLIOGRAPHY
Johnson, R. T., Rao, P. N. (1970). Nature 226, 717.
Mazia, D. (1974). Proc. Natl. Acad. Sci. USA 71, 690.
Mazia, D. (1987). Intl. Rev. of Cytology 100, 49 -91.
Mazia, D. (1988). Zoological Sci. 5. 519 - 527.
Murray. A. W. and Kirschner, M. W. (1991). Sci. Amer. March, 56 - 63.
Poccia, D., Krystal, G., Nishioka, D. and Salik, J. (1978). Cell Repro. 17,197 - 206.
Nishioka, D. and Mazia, D. (1977). Cell Bio. Intl. Rep. 1, 23.
FIGURES
Figure 1. First Cleavage." Ammonia-activated centrifuged fragments. Approx.
activation/fertilization time (hrs:min) (a-d) 1:20-2:05. EH, enucleate half; NH
nucleate half; FC, fertilized control; UC, uncentrifuged control. Arrows
indicate fragments that exhibited the most movement. (inverse Megavision
image)
Figure 2. Explosions.“ Ammonia-activated centrifuged fragments.
Approx. activation/fertilized time (hrs:min) (a) 4:57 (b) 6:36. EH,
enucleate half; EQ, enucleate quarter; CC. centrifuged control. Arrows
indicate fragments observed to "explode" (not all shown).
Figure 3. Explosion. Ammonia-activated centrifuged fragments. Approx.
activation/fertilization time (hrs:min) (a-d) 5:34-7:14. EH, enucleate half; EO,
enucleate quarter; FC, fertilized.
Figure 4. Astral microtubule structure (Daniel Mazia). Ammonia-activated
bisected fragments. Isolation time 1:45 (hrs:min). (a) light microscopy
(b) DAPI staining and fluorescence microscopy. Arrows indicate fragments
with astral microtubule structure. EH, enucleate half; NH, nucleate half.
Figure 5. Astral microtubule structure (Daniel Mazia). Ammonia-activated
bisected fragments. Isolation time 1:45 (hrs:min). (a) light microscopy (b)
DAPI staining and fluorescence microscopy. EH, enucleate half.
EH
UC
Figure
Figure


a)
Figure 4
Figure 5