CHROMOSOMA

Chromosoma (Berl) (1984) 90:50-56

~, Springer-Verlag 1984

The ultrastructure of an intraspindle membrane system in meiosis of spider spermatocytes Dwayne Wise Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762, USA

Abstract. An extensive system of membranes was found in the spindles of spermatocytes of two wolf spider species, Lycosa georgieola and L. rabida. Serial section reconstructions of this membrane system revealed that each meiotic bivalent is encased in a tube of membrane, which encloses both kinetochore microtubule bundles and approaches to within a few microns of the centriolar complex. The membrane tube is open at the polar ends. The membrane composing the tube is doubled and resembles smooth endoplasmic reticulum (ER). No evidence of nuclear pore complexes has been found in the intraspindle membrane system, but typical pores are present on the nuclear envelope of prophase cells. The membrane tubes are fenestrated and microtubules sometimes penetrate these fenestrae. Besides its possible function in the regulation of chromosome movement, the intraspindle membrane system may participate in the nonrandom segregation of the sex chromosomes at meiosis in these spiders.

Introduction

It is becoming more and more apparent that the mitotic apparatus of eukaryotic cells contains more than the chromosomes, microtubules, and microtubule organizing centers (e.g., kinetochores and centrioles), which have received so much attention from cell biologists for decades. It is still not clear that these structures are sufficient for both the generation and regulation of the force that moves chromosomes in the eukaryotic spindle (Nieklas 1971; Inou6 1981 ; Pickett-Heaps 1982; Bajer and Mol~-Bajer 1981 ; Paweletz and Finze 1981). The work of Harris (1975) and Hepler (1980) called attention to the presence of an extensive system of membranes in certain kinds of both plant and animal cells. Membranous elements of various kinds have been found associated with the mitotic apparatus of an increasing number of organisms (Hepler 1980; Hepler et al. 1981; Jackson and Doyle 1982; Kubai 1982). The finding that dividing cells have the ability to sequester microinjected calcium (Kiehart 1981), lends credence to the possibility that spindle-associated membrane systems regulate (at the least) force production by modulating the intracellular concentration of this divalent cation (Harris 1981; Sisken 1980). This possibility is further strengthened by the known Ca 2 § activity in isolated mitotic apparatus (Petzelt 1979), the localization of Ca 2. in the spindle of

fixed (Forer et al. 1980) and living cells (Wolniak and Hepler 1981; Wolniak et al. 1983), and the isolation of Ca 2 +-sequestering vesicles from sea urchin embryo spindles (Silver et al. 1980). In this paper, I report on the ultrastructure of an extensive intraspindle membrane system in spermatocytes of two spider species. This membrane system differs from others in that each meiotic bivalent, along with its attendant kinetochore microtubule bundles, appears to be completely enclosed at metaphase by these membranes. The opportunity for local Ca 2 + regulation by this membrane system seems greater than in any yet reported because every part of the spindle is pervaded by membrane. Materials and methods

The spermatocytes studied were from two species of wolf spider, Lycosa rabida and L. georgicola. Cells for analysis were prepared in either of two ways. (1)Testes were dissected from animals immobilized with CO 2 into a PIPES buffered saline solution that was approximately isotonic with the spermatocytes (Nicklas et al. 1979). These testes were then fixed in an aqueous solution of 4% glutaraldehyde, 100 mM PIPES, and 0.3% NaC1, and prepared according to standard procedures for electron microscopy (Kubai and Wise 1981). Serial sections were examined in a Siemens 101 electron microscope operating at 80 kV. (2) Single cell fixations were made using a microinjection technique (Nicklas et al. 1979). Briefly, this entailed making a preparation of living spermatocytes in a well-slide, injecting fixative into the immediate environment of a single cell, embedding, and serial sectioning. In this way, a single cell whose immediate history was known could be reconstructed from serial sections. "Two-dimensional" reconstructions were made by tracing structures from photos of adjacent sections onto acetate sheets, then laying the acetate sheets over each other (Kubai and Wise 1981 ; Nicklas et al. 1982). This produced a reconstruction that was distorted in the third dimension, but that gave a good idea of the arrangement of some structures. Two cells prepared by this method were partially reconstructed. One cell was near metaphase; the other was judged to be in mid-prometaphase. Cells from whole-testis fixations were reconstructed in the same way. Several prometaphase cells and one cell at early anaphase were partially reconstructed. In each case, the outlines of chromosomes and of cross-sectional profiles of the membranes were traced. This produced an underesti-

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Fig. 1. Electron micrograph of a section through a spermatocyte of L),cosa rabida near metaphase fixed by the microinjection technique. Bivalents (B) are arranged at the equator of the cell. One kinetochore (K) was included in the plane of this section. Each bivalent and kinetochore microtubule bundle is encased in a double membrane tube (arrows). These tubes extend to within a few microns of the centriole pair (C). Bar represents 1 jam

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Fig. 2. An enlargement of part of the cell in Figure 1 showing one kinetochore (K). Note that the membrane composing the tube is doubled and that most of the mierotubules (arrowheads) in this section occur within the membrane tube. Fenestrae in the membrane tube are shown by closed arrows. An adjacent bivalent is completely enclosed by membrane (open arrow). Bar represents 1 gm mate of the amount of membrane present because membranes were difficult to recognize in tangential section.

Results In late prometaphase each bivalent, along with its kinetochore microtubule bundles, appears to be completely encased in a tube of double-membrane envelope (Fig. 1). Figure 2 shows a higher magnification of the kinetochore microtubule bundle and attendant membrane seen in Figure 1. Note that the membrane tube is fenestrated, and microtubules can sometimes be seen penetrating these fenestrae (arrows in Fig. 2). An adjacent bivalent whose kinetochore is not in the plane of this section is closely surrounded by a cross-sectional membrane profile. A bivalent completely enwrapped by membrane would yield this kind of image when sectioned in any plane except the one occupied by the kinetochore. Note that the membrane profile extends the length of the kinetochore microtubule bundle to within 1.5 gm of the pericentriolar material (examine the centriole shown in Fig. 1). Few microtubules were seen outside the membrane tubes (Fig. 2). A partial two-dimensional reconstruction of the bivalent (marked by B) shown in Figure 1 revealed that the kinetochore fiber is completely encased by the membrane tube, which appears to close when the kinetochore is not in the plane of sectioning.

Figure 3 shows another cell in late prometaphase fixed by the microinjection technique. Here again bivalents are enclosed by cross-sectional profiles of doubled membranes. The membrane system in this cell is less well organized than that shown in Figure 1, however, vestiges of the membrane tubes can be seen in the half-spindle and near the pole. Note that the sex chromosomes (S) are not enclosed by membrane. Two-dimensional reconstructions of the sex chromosomes (there are two: Xt, X2) indicate that these chromosomes occasionally lie near a membrane tube but are not encased in membrane tubes. In early prometaphase cells the intraspindle membrane is less well organized than in late prometaphase cells. Partial two-dimensional reconstructions of spermatocytes in early prometaphase showed each bivalent enclosed in membrane, but the membrane did not continue along the kinetochore microtubule bundle. Typically in early prometaphase cells arrays of membrane were located at the periphery of the spindle, and a dense cluster of osmiophilic vesicles was present. The distribution of the membrane system at anaphase is shown in Figure 4. Very few cells in anaphase were available for study, however, a few were encountered in the whole-testis preparations. The cell shown in Figure 4 was judged to be in early anaphase because two-dimensional reconstruction of serial sections clearly showed separating half-bivalents. The preliminary indication from this cell is

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Fig. 3. Electron micrograph of a spermatocyte in late prometaphase. Bivalents (B) are aligned near the equator and the membrane system is present (arrows). The sex chromosomes (S) lie close to one spindle pole and are not surrounded by membrane. Bar represents 1 pm that each chromosome is invested with membrane, but kinetochore fibers are free of organized membrane tubes. Also, a considerable amount of membrane is present in the spindle periphery. This pattern of membrane distribution is similar to that of early prometaphase cells. A more precise determination of the anaphase distribution of the membrane requires the reconstruction of several more such cells. The intraspindle membrane system is indistinguishable from endoplasmic reticulum (ER)-like membrane found in these spider spermatocytes at meiotic prophase (Fig. 5). Whether the ER system contributes to the intraspindle membrane system present in dividing cells cannot be determined from the data presented here. Nuclear envelope of prophase cells has typical nuclear pore complexes; no evidence of these was seen in the intraspindle membranes. Discussion

It has been shown that a highly organized, ER-like system of membranes permeates the spindle of dividing lycosid spermatocytes. An intraspindle membrane system of this extent and of this high degree of organization has not been reported in cells of any higher eukaryote. The only similar case has been reported in the sporozoan, So'locephalus longicollus, where each chromosome is invested with membrane at mitosis (Heath 1980). At metaphase in the spider cells, the membrane corn-

pletely encloses each bivalent along with its two kinetochore microtubule bundles. At early prometaphase bivalents but not kinetochore microtubule bundles are encased and arrays of ER-like membrane are present at the periphery of the spindle. The data argue that the membrane system becomes more organized as prometaphase progresses. This might explain the unusual way in which bivalents in these cells congress (Revell 1947; Wise, unpublished observations of living cells). Bivalents congress to the equator slowly, one at a time, and some bivalents remain near the spindle poles for nearly the duration of prometaphase. Typically in these cells one or two bivalents are located at the equator, with all the other chromosomes clustered around the spindie poles. It is possible that the organization of a kinetochore fiber "membrane tube" is necessary for congressional movements to occur. One of the most intriguing questions about the intraspindle membrane system in these cells is its origin. One possibility is that it is merely rearranged nuclear envelope. Two pieces of evidence argue against this hypothesis: (1) The nuclear envelope of meiotic prophase cells contains typical nuclear pore complexes; these are not present on the intraspindle membranes. However, it is possible that the nuclear pore complexes could be lost between diakinesis and prometaphase. (2) The surface area of membrane in the nuclear envelope at diakinesis is insufficient to enclose thirteen bivalents

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Fig. 4. Electron micrograph of a section through a spermatocyte in early anaphase. Half-bivalents (H) were in the process of moving toward the poles, one of which, along with the centriole (C) is included in this section. Two kinetochores (K) are in the plane of sectioning. Note membranes (arrows) in the spindle periphery and around the chromosomes, but not enclosing the kinetochore fibers. Bar represents 1 gm

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Fig. 5a. Electron micrograph of a prophase spermatocyte from a whole-testis fixation. This is a section through the centriole pair but does not include the nucleus. Note the cloud of osmiophilic vesicles and ER-like membrane (arrows) surrounding the centrioles and radiating into the cytoplasm, b Enlargement of the area shown in the box in a. Note vesicles and membrane (arrowheads)surrounding the centrioles (C). Bars represent I /am (for these lycosids, N = I 3 + X 1 , X 2 ) and their kinetochore microtubule bundles. A t diakinesis, the nucleus in these ceils has a radius (r) o f a b o u t 10 pro. Therefore, the surface area (A) of the diakinesis nucleus is 1,256 p m z (A = 4 nr2). To calculate the surface area o f the bivalents and their kinetochore fibers, we assumed each bivalent to be a cylinder o f radius 3 lain and height (h) 5 pm, and each kinetochore fiber to be a cylinder o f radius 2 ~tm and height 7 jam. The total surface area o f all bivalents and their kinetochore fibers is 3,510.5 i.tm 2 ( A = 2 nrh). It seems clear, therefore, that the intraspindle m e m b r a n e system cannot be derived from rearrangement o f the nuclear envelope because roughly three times as much m e m b r a n e is necessary than is present in the diakinesis nuclear envelope. Either new membrane is synthesized, or other cellular membranes are recruited (or both o f these occur).

A n attractive hypothesis a b o u t the function o f the intraspindle m e m b r a n e system is that it controls the polymerization-depolymerization cycle o f microtubules by releasing or sequestering calcium ions. This would provide a means whereby events such as synchronous separation of chromatids, beginning o f c h r o m o s o m e movement, and the accompanying b r e a k d o w n o f spindle microtubules could be triggered (Wolniak et al. 1983; Harris 1981, Izant 1983). The possibility that the intraspindle m e m b r a n e system modulates local calcium concentration can be tested in these cells in a number o f ways. In living cells o f Haemanthus, the use o f permeant, nontoxic, calcium-sensitive fluorescent probes has shown that the spindles o f these cells are rich in membrane-associated Ca 2 ~ and that there is a reduction in the a m o u n t o f membrane-associated Ca z+ just before the onset o f anaphase (Wolniak and Hepler 1981; W o l n i a k

56 et al. 1980, 1983). Preliminary results indicate that the intraspindle m e m b r a n e system o f spider spermatocytes rep o r t e d here also is rich in membrane-associated calcium (Wise and W o l n i a k 1983). The hypothesis can be tested further by treatment o f these cells with c o m p o u n d s which produce electron-opaque calcium precipitates (Wick and Hepler 1980; Slocum and R o u x 1982; Caswell 1979; Zechmeister 1979). If, for example, the m e m b r a n e system sequesters Ca 2 before or during spindle formation, then one might expect to find significant amounts of Ca 2 + precipitate in diakinesis or early p r o m e t a p h a s e membranes. Likewise, if Ca 2+ is released just before onset of anaphase, this should be reflected in the a m o u n t of Ca 2 + precipitate present in the membranes at anaphase. In a cell with such a highly organized intraspindle m e m b r a n e system, definitive questions about the role o f such membranes in mitotic calcium regulation might be answered. A n alternative hypothesis a b o u t the role o f these particular intraspindle membranes involves their possible particip a t i o n in the unusual segregation behavior of the sex chromosomes. It has been known for some time that lycosids (and m a n y other spider groups) have the sex c h r o m o s o m e constitution X1X 2 for males and X I X 1 X , X 2 for females (White 1973; Wise 1983). The two n o n h o m o l o g o u s X chromosomes, which a p p e a r to be physically unconnected, orient and segregate to the same spindle pole at meiosis I. R a n d o m segregation would dictate that the X chromosomes move to opposite poles 50% of the time. Therefore, the segregational behavior o f these chromosomes is nonrandora. E x a m i n a t i o n o f several spermatocytes in early and late p r o m e t a p h a s e shows that the two X chromosomes are not enclosed in the intraspindle m e m b r a n e s ; as a matter of fact, they a p p e a r to be completely free of m e m b r a n e (see Fig. 3). It is therefore possible that the n o n r a n d o m behavior o f these chromosomes is brought a b o u t somehow because they are not encased in membrane. Perhaps chromosomes without m e m b r a n e cannot reorient or move. If the two X chromosomes are already attached to one spindle pole at diakinesis, the membranes may prevent their orientation to opposite poles. A n attractive compromise is that the intraspindle membrane system in these cells functions both to regulate X c h r o m o s o m e segregation and to modulate the intracellular levels o f calcium.

Acknowledgements. I am grateful to Donna Kubai for providing serial sections of two of the cells studied. I thank Suzanne Olah for expert technical assistance and Allen Brady for help in identifying the spiders. This research was supported by grant GM 28660 from the National Institutes of Health. References Bajer AS, Mol&Bajer J (1981) Mitosis: studies of living cells a revision of basic concepts. In: Zimmerman and Forer (eds) Mitosis/cytokinesis. Academic Press, NY, pp 277 296 Caswell AH (1979) Methods for measuring intracellular calcium. Int Rev Cytol 56:145-181 Forer A, Gupta BL, Hall TA (1980) Electron probe X-ray microanalysis of calcium and other elements in meiotic spindles, in frozen sections of spermatocytes from crane fly testes. Exp Cell Res 126:217-226 Harris P (1975) The role of membranes in the organization of the mitotic apparatus. Exp Cell Res 94:409425 Harris P (1981) Calcium regulation of cell cycle events. In: Zimmerman and Forer (eds) Mitosis/cytokinesis. Academic Press, NY, pp 29-52

Heath IB (1980) Variant mitoses in lower eukaryotes: indicators of the evolution of mitosis? Int Rev Cytol 64:1-80 Hepler PK (1980) Membrane in the mitotis apparatus of barley cells. J Cell Biol 86:490-499 Hepler PK, Wick SM, Wolniak SM (1981) The structure and role of membranes in the mitotic apparatus. In: HG Schweiger (ed) International cell biology, Springer-Verlag, Heidelberg, pp 673-686 Inou6 S (1981) Cell division and the mitotic spindle. J Cell Biol 91 : 131 s-147s Izant JG (1983) The role of calcium ions during mitosis. Calcium participates in the anaphase trigger. Chromosoma 88 : 1-10 Jackson WT, Doyle BG (1982) Membrane distribution in dividing endosperm cells of Haemanthus. J Cell Biol 94:637-643 Kiehart DP (1981) Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system. J Cell Biol 88:604-617 Kubai DF (1982) Meiosis in Sciara coprophila: structure of the spindle and chromosome behavior during the first meiotic division. J Cell Biol 93 : 655-669 Kubai DF, Wise DA (1981) Nonrandom chromosome segregation in Neocurtilla (Gryllotalpa) hexadactyla: an ultrastructural study. J Cell Biol 88:281-293 Nicklas RB (1971) Mitosis. Adv Cell Biol 2:225-297 Nicklas RB, Brinkley BR, Pepper DA, Kubai DF, Rickards GK (1979) Electron microscopy of spermatocytes previously studied in life: Methods and some observations on micromanipulated chromosomes. J Cell Sci 35:87- 104 Nicklas RB, Kubai DF, tlays TS (1982) Spindle microtubules and their mechanical associations after micromanipulation in anaphase. J Cell Biol 95:91-104 Paweletz N, Finze E-M (1981) Membranes and microtubules of the mitotic apparatus of mammalian cells. J Ultrastruet Res 76:123-133 Petzelt C (1979) Biochemistry of the mitotic spindle. Int Rev Cytol 60: 53-92 Pickett-Heaps JD (1982) Rethinking mitosis. Cell 29:792- 744 Revell SH (1947) Controlled X-segregation in Tegenaria. Heredity t : 337-347 Silver RB, Cole RD, Cande WZ (1980) Isolation of mitotic apparatus containing vesicles with calcium sequestration activity. Cell 19:505-516 Sisken JE (1980) The significance and regulation of calcium during mitotic events. In: Nuclear-cytoplasmic interactions in the cell cycle. Academic Press, NY, pp 271-292 Slocum RD, Roux SJ (1982) An improved method for the subcellular localization of calcium using a modification of the antimonate precipitation technique. J Histochem Cytochem 30:617-629 White MJD (1973) Animal cytology and evolution. 3rd ed. Cambridge University Press, London, pp 669-672 Wick SM, Hepler PK (1980) Localization of Ca * +-containing antimonate precipitates during mitosis. J Cell Biol 86:500-513 Wise DA (1983) An electron microscope study of the karyotypes of two wolf spiders. Can J Genet Cytol 25:161-168 Wise DA, Wolniak SM (1983) The intraspindle membrane system of spider spermatocytes is rich in associated calcium. J Cell Biol 97:41a Wolniak SM, Hepler PK (1981) The coincident distribution of calcium-rich membranes and kinetochore fibers at metaphase in living endosperm cells of Haemanthus. Eur J Cell Biol 25 : 171-174 Wolniak SM, Hepler PK, Jackson WT (1980) Detection of the membrane-calcium distribution during mitosis in Haemanthus endosperm with chlorotetracycline. J Cell Biol 87:23- 32 Wolniak SM, Hepler PK, Jackson WT (1983) Ionic changes in the mitotic apparatus at the metaphase/anaphase transition. J Cell Biol 96 : 598-605 Zechmeister A (1979) A new selective ultrahistochemical method for the demonstration of calcium using N,N-naphthaloylhydroxylamine. Histochemistry 61 : 223-232 Received December 19, 1983 / in revised form March 14, 1984 Accepted by J.tt. Taylor