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190587695.Pdf Published Online: 1 June, 1980 | Supp Info: http://doi.org/10.1083/jcb.85.3.853 Downloaded from jcb.rupress.org on May 9, 2019 FUNCTIONAL IMPLICATIONS OF COLD-STABLE MICROTUBULES IN KINETOCHORE FIBERS OF INSECT SPERMATOCYTES DURING ANAPHASE E . D. SALMON and DAVID A. BEGG From the Marine Biological Laboratories, Woods Hole, Massachusetts 02543, the University of North Carolina, Chapel Hill, North Carolina 27514, and the University of Pennsylvania, Philadelphia, Pennsylvania 19104 . Dr . Begg's present address is the Department of Anatomy, Harvard Medical School, Boston, Massachusetts 02115 . ABSTRACT In normal anaphase of crane fly spermatocytes, the autosomes traverse most of the distance to the poles at a constant, temperature-dependent velocity. Concur- rently, the birefringent kinetochore fibers shorten while retaining a constant birefringent retardation (BR) and width over most of the fiber length as the autosomes approach the centrosome region . To test the dynamic equilibrium model ofchromosome poleward movement, we abruptly cooled or heated primary spermatocytes of the crane fly Nephrotoma ferruginea (and the grasshopper Trimerotropis maritima) during early anaphase. According to this model, abrupt cooling should induce transient depolymerization of the kinetochore fiber micro- tubules, thus producing a transient acceleration in the poleward movement of the autosomal chromosomes, provided the poles remain separated . Abrupt changes in temperature from 22°C to as low as 4°C or as high as 31 °C in fact produced immediate changes in chromosome velocity to new constant velocities . No tran- sient changes in velocity were observed . At 4°C (10°C for grasshopper cells), chromosome movement ceased . Although no nonkinetochore fiber BR remained at these low temperatures, kinetochore fiber BR had changed very little . The cold stability of the kinetochore fiber microtubules, the constant velocity character of chromosome movement, and the observed Arrhenius relationship between tem- perature and chromosome velocity indicate that a rate-limiting catalyzed process is involved in the normal anaphase depolymerization of the spindle fiber micro- tubules . On the basis of our birefringence observations, the kinetochore fiber microtubules appear to exist in a steady-state balance between comparatively irreversible, and probably different, physiological pathways ofpolymerization and depolymerization . A major proposition in the "dynamic equilibrium and disassembly ofthe microtubules that make up theory" of mitotic spindle assembly and function the spindle fibers . In his dynamic equilibrium (23-26, 51) is that pushing and pulling forces for theory, Inoué (23, 25, 26) proposes that the spindle moving chromosomes are generated by assembly fiber microtubules are in a continuous state of flux J . CELL BIOLOGY ©The Rockefeller University Press - 0021-9525/80/06/0853/13 $1 .00 853 Volume 85 June 1980 853-865 that he sees as the consequence of a simple, labile, 30-32, 46, 47) . The crane fly spermatocytes ap- physiochemical equilibrium between the polym- peared to be highly suitable for this study because erized microtubules and a cellular pool of tubulin (a) they have only three autosomes and two sex subunits . According to Inou6's view, changes in chromosomes, (b) both spindle poles are well an- the equilibrium constant or in the concentration chored at the cell surface (33), (c) the autosomes of activated tubulin subunits initiate microtubule move poleward at a nearly constant velocity (13, assembly or disassembly, thus generating forces 14), and (d) the interpolar spindle does not usually for mitotic movements . elongate (called "Anaphase B" by Inou6 and Bit- The major support for the dynamic equilibrium ter [25]) until the autosomes have nearly com- theory came from experiments with metaphase- pleted their movement to the poles ("Anaphase arrested oocytes of Chaetopterus in which the spin- A") (13, 14) . Consequently, for living crane fly dle microtubules were induced to disassemble by spermatocytes, observation of changes in chro- cooling (23, 25, 26), pressurization (50-52, 56), or mosome velocity could be related directly to the addition of colchicine (22) . As the microtu- changes in the assembly and length ofthe birefrin- bules depolymerized, the spindle shortened and gent kinetochore fibers by using sensitive polari- the chromosomes were moved from their meta- zation microscopy methods . Measurements of bi- phase-plate position toward the pole that anchored refringent retardation (BR) have been shown to be the spindle to the cell surface. When the depolym- good measures of the number and distribution of erizing conditions were reversed, the spindle re- spindle fiber microtubules in crane fly spermato- grew, moving the chromosomes away from the cytes (32, 33) and other cell types (58) . anchored pole . We felt that more convincing evi- To our surprise, unlike the results reported ear- dence for the dynamic equilibrium theory would lier for marine eggs and plant cells (18, 23, 27, 51, be provided if the same microtubule-depolymer- 61), extensive cooling of these spermatocytes did izing agents, when applied during anaphase, not cause appreciable reduction in the birefrin- would cause the chromosomes to accelerate as gence of the kinetochore fibers, nor did it induce they moved poleward . a transient acceleration of the chromosomes . In More specifically, we predicted on the basis of this paper we discuss the temperature dependence the dynamic equilibrium theory that abruptly of the poleward velocity of the chromosomes and cooling or pressurizing a cell during anaphase examine the implications of the striking differen- would cause the spindle fiber microtubules to de- tial stability that kinetochore and nonkinetochore polymerize and shorten rapidly until they reached fiber microtubules have to cooling . Results ofsim- a new equilibrium state . During this transitional ilar experiments using hydrostatic pressure to de- period of rapid microtubule depolymerization, we polymerize the microtubules will be reported in a expected to observe an increase in the chromo- subsequent paper . A preliminary report of our somes' poleward velocity, provided the spindle work has been presented previously (53) . poles remained separated . Then, as a new equilib- rium became established, we expected chromo- MATERIALS AND METHODS some velocity to decrease until it reached a new Primary spermatocytes of the crane fly Nephrotoma ferruginea steady-state rate . Steady-state chromosome veloc- Fabricius and the grasshopper Trimerotropis maritima Harris increases exponentially were used for these studies . Crane flies were reared in the ity with temperature, with laboratory according to the method described by Begg (4) and a Q1o of 2.6-4 .3 depending on cell type (18, 59) . grasshoppers were periodically collected during August through As illustrated in Fig. l for a hypothetical example, October from a wild population near Falmouth, Massachusetts . abrupt cooling should induce transient accelera- Spermatocyte smears were prepared as described previously (5, tion of the chromosomes (region í) followed by a 6) . Glass coverslips containing a spermatocyte smear were sealed to a temperature-controlled slide with valap (a l :1 :1 mixture of steady-state velocity slower than originally ob- Vasoline, parafm, and lanolin) . served (region ii) . For our studies we used primary spermatocytes from two insects, primarily the crane fly Nephro- Temperature Control toma ferruginea and, secondarily, the grasshopper The temperature of the spermatocyte preparation was regu- Trimerotropis maritima. These cells were chosen lated and monitored by a water-flow controlled-temperature slide, similar in because they contain large spindles with relatively construction and operation to that described by Stephens (61) (Fig . 2). Cell temperature was taken to be the few chromosomes and because they develop dis- mean of the influx and efflux water temperatures, as measured tinct chromosomal fibers during anaphase (13, 14, during the experiment by thermistor probes (61) . The water flow 854 THE JOURNAL OF CELL BIOLOGY " VOLUME 85, 1980 was driven by gravity and the slide temperature was controlled by switching between two 5-gallon insulated reservoirs, one maintained near 22°C and the other at the desired experimental temperature . Fogging of the slide at low temperatures was pre- vented by blowing dry air over both windows of the slide . Microscopy Phase contrast observations were made using a Nikon model S microscope equipped with a Zeiss 40x (NA 0.65) phase objec- tive and a Zeiss model IS long-working-distance phase condenser (NA 0.7; working distance, 1 I mm). Spindle fiber birefringence was observed with the high extinction, inverted polarization microscope of Inoué (23) (crane fly experiments) or a Zeiss WL stand (grasshopper experiments) equipped with a Nikon rectified 40x (NA 0 .65) objective and a Nikon 16-mm long-working- distance rectified condenser (NA 0 .52) (Fig. 2). The illuminating beam for both microscopes was filtered with a glass heat-cut filter and a 546-nm interference filter (Baird Atomic, Inc., Bed- ford, Mass .) . Photography and Data Analysis Photographic records were made with a Nikon Microflex AFM camera system, using a Zeiss IOx ocular and Kodak Plux X 35-mm film . The films were developed in Kodak Microdol X Developer at 75°F . Brief exposure times were required to record changes in BR b and kinetochore position after temperature shifts, because changes
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