Actin and myosin inhibitors block elongation of kinetochore fibre stubs in metaphase crane-fly spermatocytes.

We used an ultraviolet microbeam to cut individual kinetochore spindle fibres in metaphase crane-fly spermatocytes. We then followed the growth of the "kinetochore stubs", the remnants of kinetochore fibres that remain attached to kinetochores. Kinetochore stubs elongate with constant velocity by adding tubulin subunits at the kinetochore, and thus elongation is related to tubulin flux in the kinetochore microtubules. Stub elongation was blocked by cytochalasin D and latrunculin A, actin inhibitors, and by butanedione monoxime, a myosin inhibitor. We conclude that actin and myosin are involved in generating elongation and thus in producing tubulin flux in kinetochore microtubules. We suggest that actin and myosin act in concert with a spindle matrix to propel kinetochore fibres poleward, thereby causing stub elongation and generating anaphase chromosome movement in nonirradiated cells.

Pac-Man does not resolve the enduring problem of anaphase chromosome movement.

The Pac-Man hypothesis suggests that poleward movement of chromosomes during anaphase A is brought about by: disassembly of kinetochore microtubules (MTs) at the kinetochore; generation of the poleward force exclusively at or very close to the kinetochore; and the required energy coming from coupled disassembly of these MTs. This model has become widely accepted and cited as the sole or major mechanism of anaphase A. Rarely acknowledged are several significant phenomena that refute some or all of these postulates. We summarise these anomalies as follows: poleward movement of chromosomes occurring without insertion of any MTs at the kinetochore; "anaphase" shortening of kinetochore fibres in spindles entirely devoid of chromosomes and, presumably, kinetochores; continued movement of chromosomes while their severed kinetochore stub elongated poleward after treatment with UV microbeams; and fluxing of tubulin subunits through kinetochore MTs during anaphase A, indicating that during anaphase, kinetochore MTs disassemble partly or solely at the poles.

Effects of anti-myosin drugs on anaphase chromosome movement and cytokinesis in crane-fly primary spermatocytes.

To investigate whether myosin is involved in crane-fly primary spermatocyte division, we studied the effects of myosin inhibitors on chromosome movement and on cytokinesis. With respect to chromosome movement, the myosin ATPase inhibitor 2,3-butanedione 2-monoxime (BDM) added during autosomal anaphase reversibly perturbed the movements of all autosomes: autosomes stopped, slowed, or moved backwards during treatment. BDM added before anaphase onset altered chromosome movement less than when BDM was added during anaphase: chromosome movements only rarely were stopped. They often were normal initially and then, if altered at all, were slowed. To confirm that the effects of BDM were due to myosin inhibition, we treated cells with ML-7, a drug that inhibits myosin light chain kinase (MLCK), an enzyme necessary to activate myosin. ML-7 affected anaphase movement only when added in early prometaphase: this treatment prevented chromosome attachment to the spindle. We treated cells with H-7 as a control for possible non-myosin effects of ML-7. H-7, which has a lower affinity than ML-7 for MLCK but a higher affinity than ML-7 for other potential targets, had no effect. These data confirm that the BDM effect is on myosin and indicate that the myosin used for chromosome movement is activated near the start of prometaphase. With respect to cytokinesis, BDM did not block furrow initiation but did block subsequent contraction of the contractile ring. When BDM was added after initiation of the furrow, the contractile ring either stalled or relaxed. ML-7 blocked contractile ring contraction when added at all stages after autosomal anaphase onset, including when added during cytokinesis. H-7 had no effect. These results confirm that the effects of BDM are on myosin and indicate that the myosin used for cytokinesis is activated starting from autosomal anaphase and continuing throughout cytokinesis.

Evidence that kinetochore fibre microtubules shorten predominantly at the pole in anaphase flea-beetle spermatocytes.

Kinetochore spindle fibres in flea-beetle metaphase primary spermatocytes have two regions with distinct morphologies. As seen after staining with antibodies against tubulin, the kinetochore microtubules are tightly bundled in the 5 microm closest to the kinetochore but they splay out in the region closest to the pole. This morphology persists throughout anaphase. This distinct morphology allows one to deduce the site where kinetochore microtubules depolymerise during anaphase. During poleward movement of the autosomes in anaphase, the bundled region shortens by about 0.25 microm for each 1 microm the chromosome moves poleward; this suggests that, during anaphase, 75% of the kinetochore microtubule shortening occurs at the pole end. Sex chromosomes in metaphase cells are separated from the autosomes and do not move poleward at the same time as the autosomes: they are reported to move poleward when the autosomes are in metaphase, to stop part way to the poles, and to move poleward again as the autosomes do (Virkki 1970). Kinetochore microtubules of the sex chromosomes also have bundled and splayed regions; measurements of those regions suggest that these chromosomes may move poleward before the autosomes enter anaphase, but not afterwards.

Evidence that actin and myosin are involved in the poleward flux of tubulin in metaphase kinetochore microtubules of crane-fly spermatocytes.

We studied the effects of various drugs on the poleward flux of tubulin in kinetochore microtubules in metaphase-I crane-fly spermatocytes. We used as a measure of tubulin flux a 'gap' in acetylation of kinetochore microtubules immediately poleward from the kinetochore; the 'gap' is caused by a time lag between incorporation of new tubulin subunits at the kinetochore and subsequent acetylation of those subunits as they flux to the pole. We confirmed that the 'gap' is due to flux by showing that the 'gap' disappeared when cells were treated briefly with the anti-tubulin drug nocodazole, which decreases microtubule dynamics. The 'gap' disappeared when cells were treated for 10 minutes with anti-actin drugs (cytochalasin D, latrunculin B, swinholide A), or with the anti-myosin drug 2,3-butanedione 2-monoxime. The 'gap' did not disappear when cells were treated with the actin stabilizing drug jasplakinolide. We studied whether these drugs altered spindle actin. We used fluorescent phalloidin to visualize spermatocyte F-actin, which was associated with kinetochore spindle fibers as well as the cell cortex, the contractile ring and finger-like protrusions at the poles. Spindle F-actin was no longer seen after cells were treated with cytochalasin D, swinholide A or a high concentration of latrunculin B, whereas a low concentration of latrunculin B, which did not completely remove the 'gap', caused reduced staining of spindle actin. Neither 2,3-butanedione 2-monoxime nor jasplakinolide altered spindle actin. These data suggest that an actomyosin mechanism drives the metaphase poleward tubulin flux.

From megakaryocytes to platelets: platelet morphogenesis takes place in the bloodstream.

We studied megakaryocyte processes formed in rat bone marrow and spleen, using both the transmission and scanning electron microscopes. Some processes were bulky, others slender and beaded. The bulky megakaryocyte processes developed a specialized arrangement of organelles at the site at which they entered the lumen: filaments present around the outside of the process seemed to support a central cylinder in which organelles flowed along microtubules. Megakaryocyte processes were present in platelet-rich plasma from both human and rat blood. When followed in living preparations, bulky processes developed pointed tips, elongated, and became slender and beaded. Fusiform proplatelets also were present in the platelet rich plasma, with pointed tips at both ends of what appeared to be single "beads"; we assume that the long, beaded megakaryocyte processes would have fragmented were we to have had proper culture conditions. The straight, shorter fusiform proplatelets in living preparations underwent characteristic curving and bending motions, eventually forming disk-shaped cells which sometimes had appendages. This behaviour suggests that the entire process of platelet morphogenesis takes place in plasma: megakaryocyte processes first elongate, then bead and fragment, and then curve and fuse to form disk-shaped platelets. This interpretation is strengthened by finding in freshly isolated plasma many of the shapes seen in the transformations studied in living cell preparations. The megakaryocyte processes and the proplatelets seemed to appear in plasma with a periodicity related to light and dark cycles--that is, with a circadian rhythm. In particular, megakaryocyte processes appear in human blood within a few hours after sunrise; we argue that this might be related to similar peak periods for heart attacks.

Cytochalasin D and latrunculin affect chromosome behaviour during meiosis in crane-fly spermatocytes.

Living crane-fly spermatocytes were treated with 10-20 microg/ml cytochalasin D (CD) or 0.3 microg/ml latrunculin (LAT) at various stages of meiosis I. The drugs had the same effects on chromosome behaviour, but CD effects were reversible and LAT effects generally were not. When applied in mid-prometaphase to metaphase, both drugs altered subsequent anaphase poleward movements: half-bivalents either moved more slowly than normal, or moved more slowly after a brief period of movement at normal rate or stalled for 10 min or more immediately after disjunction. CD effects were reversible: within 1 min after washing out the CD, stopped chromosomes started moving and slowed chromosomes sped up. When applied in anaphase, both drugs stopped or slowed poleward chromosome movements, usually reversibly. When applied near the end of prophase, both drugs often prevented one or more bivalents in the cell from attaching to the spindle. Attached bivalents behaved as in cells treated with drugs at later stages, as described above. Unattached bivalents in the same cells moved to poles or cytoplasm in early prometaphase, where they remained motionless; at anaphase they sometimes did not disjoin, but when they did disjoin the half-bivalents did not move, either in the continued presence of the drug or when CD was washed out, confirming that they were not atttached. When CD or LAT prevented all bivalents in the cell from attaching, spindles kept in the drug were invaded by granules at about the time of normal anaphase. Conversely, when CD was washed out during late prometaphase, chromosomes often attached to spindle fibres and later entered anaphase. As CD and LAT are different antiactin drugs, but have the same effect on chromosome behaviour, the results implicate actin in early interactions of chromosomes with spindle fibres and in anaphase chromosome movements.

Effects of ultraviolet-microbeam irradiation of kinetochores in crane-fly spermatocytes.

Ultraviolet (UV)-microbeam irradiation of a single kinetochore during anaphase generally causes all 6 of the half-bivalents in the cell to stop poleward motion within 1 min after the irradiation. The half-bivalents regain movement after remaining stopped for an average of 8.7 min, through different pairs in the same cell can resume at different times. Once movement resumes they usually continue movement until they reach the poles. As controls, to see if the effect is due to alteration of the kinetochore, we irradiated spindle fibers and chromosome arms using the same doses and wavelengths as for kinetochore irradiation. After spindle fiber irradiation, only the half-bivalent associated with the irradiated spindle fiber and its partner stop moving poleward while the other half-bivalents in the same cell are not affected. After irradiation of a chromosome arm, the movement of the two partner half-bivalents associated with irradiated arm either slowed or moved with unchanged velocity; no other half-bivalents in the cell were affected. Therefore, only irradiation of a kinetochore stops the movement of all the half-bivalents in the same cell. We suggest that the irradiated kinetochore sends a "stop" signal to the other kinetochores in the same cell.

Ultraviolet microbeam irradiations of epithelial and spermatocyte spindles suggest that forces act on the kinetochore fibre and are not generated by its disassembly.

Ultraviolet (UV) microbeam irradiations of crane-fly spermatocyte and newt epithelial spindles severed kinetochore fibres (KT-fibres), creating areas of reduced birefringence (ARBs): the remnant KT-fibre consists of two "stubs," a pole-stub attached to the pole and a KT-stub attached to the kinetochore. KT-stubs remained visible but pole-stubs soon became undetectable [Forer et al., 1996]. At metaphase, in both cell types the KT-stub often changed orientation immediately after irradiation and its tip steadily moved poleward. In spermatocytes, the chromosome attached to the KT-stub remained at the equator as the KT-stub elongated. In epithelial cells, the KT-stub sometimes elongated as the associated chromosome remained at the equator; other times the associated chromosome moved poleward together with the KT-stub, albeit only a short distance toward the pole. When an ARB was generated at anaphase, chromosome(s) with a KT-stub often continued to move poleward. In spermatocytes, this movement was accompanied by steady elongation of the KT-stub. In epithelial cells, chromosomes accelerated polewards after irradiation until the KT-stubs reached the pole, after which chromosome movement returned to normal speeds. In some epithelial cells fine birefringent fibres by chance were present along one edge of ARBs; these remnant fibres buckled and broke as the KT-stub and chromosome moved polewards. Similarly, KT-stubs that moved into pole stubs (or astral fibres) caused the pole stubs (or astral fibres) to bend sharply from the point of impact. Our results contradict models of chromosome movement that postulate that force is generated by the kinetochore disassembling the KT-fibre. Instead, these results suggest that poleward directed forces act on the KT-fibre and the KT-stub and suggest that continuity of microtubules between kinetochore and pole is not obligatory for achieving anaphase motion to the pole.

Traction fibre: toward a "tensegral" model of the spindle.

Most current hypotheses of mitotic mechanisms are based on the "PAC-MAN" paradigm in which chromosome movement is generated and powered by disassembly of kinetochore microtubules (k-MTs) by the kinetochore. Recent experiments demonstrate that this model cannot explain force generation for anaphase chromosome movement [Pickett-Heaps et al., 1996: Protoplasma 192:1-10]. Another such experiment is described here: a UV-microbeam cut several kinetochore fibres (k-fibres) in newt epithelial cells at metaphase and the half-spindle immediately shortened: in several cells, the remaining intact spindle fibres bowed outwards as they came under increased compression. Thus, severing of k-MTs can lead to increased tension between chromosomes and poles. This observation cannot be explained by models in which force is produced by motor molecules at the kinetochore actively disassembling k-MTs. Rather, we argue that tensile forces act along the whole k-fibre, which, therefore, can be considered as a classic "traction fibre." We suggest that anaphase polewards force is generated by MTs interacting with the spindle matrix and when k-MTs are severed, polewards force continues to act on the remaining kMT-stub; spindle MTs act as rigid struts concurrently resisting and being controlled by these forces. We suggest that the principles of "cellular tensegrity" [Ingber, 1993: J. Cell Sci. 104:613-627] derived from the behaviour and organization of the interphase cell apply to the spindle. In an evolutionary context, this argument further suggests that the spindle might originally have evolved as the mechanism by which a single tensegral unit (cytoplast) is divided into two cytoplasts; use of the spindle for segregating chromosomes might represent a secondary, more recent development of this primary function. If valid, this concept has implications for the way the spindle functions and for the spindle's relationship to cytokinesis.

Effects of nanomolar taxol on crane-fly spermatocyte spindles indicate that acetylation of kinetochore microtubules can be used as a marker of poleward tubulin flux.

Kinetochore microtubules (kMTs) in meiosis-I crane-fly spermatocytes label strongly with antibodies to acetylated alpha-tubulin, except near the kinetochore, where there is a "gap" in labelling [Wilson and Forer, 1989: Cell Motil. Cytoskeleton 14:237-250]. Previously we measured the length of gaps in metaphase and anaphase cells, and from these data deduced that during anaphase kMTs disassemble primarily at the pole [Wilson et al., 1994: J. Cell Sci. 107:3015-3027]. However, the study rested on our assumption that the gap is due to a time lag between polymerisation at the kinetochore and acetylation of the polymerised MTs: the subunits enter kMTs at the kinetochore and do not become acetylated until they have moved poleward. In the present study we tested our interpretation of the gap by treating spermatocytes with paclitaxel (taxol) to reduce microtubule dynamics [e.g. Jordan et al., 1993: Proc. Natl. Acad. Sci. U.S.A. 90:9552-9556]. We expected that if our assumptions were correct, taxol would slow tubulin addition at the kinetochore but acetylation would continue, and the gap in acetylation would get smaller. We found that 5 to 50 nM taxol results in increased acetylation of kMTs at the kinetochore, supporting our interpretation of the gap. Nanomolar taxol also increases the level of acetylation in other microtubule populations and causes changes in spindle morphology.

Coordinated movements between autosomal half-bivalents in crane-fly spermatocytes: evidence that 'stop' signals are sent between partner half-bivalents.

During anaphase-I in crane-fly spermatocytes, sister half-bivalents separate and move to opposite poles. When we irradiate a kinetochore spindle fibre with an ultraviolet microbeam, the associated half-bivalent temporarily stops moving and so does the partner half-bivalent with which it was paired during metaphase. To test whether a 'signal' is transmitted between partner half-bivalents we irradiated the spindle twice, once in the interzone (the region between separating partner half-bivalents) and once in a kinetochore fibre. For both irradiations we used light of wavelength 290 microns and a dose that, after irradiating a spindle fibre only, altered movement in 63% of irradiations (12/19); in 11 of the 12 cells both partner half-bivalents stopped moving after the irradiation. In control experiments we irradiated the interzone only: these irradiations generally did not stop chromosomal poleward motion but sometimes (14/29) caused poleward movement to each pole to be abruptly reduced to about half the velocity prior to irradiation. In double irradiation experiments we varied the order of the irradiations. In some double irradiation experiments we irradiated the interzonal region first and the spindle fibre second; in 75% (9/12) of the cells the half-bivalent associated with the irradiated fibre stopped moving while the partner half-bivalent moved normally, i.e. in 9/12 cells the interzonal irradiations uncoupled the movements of the partner half-bivalents. In other double irradiation experiments we irradiated the spindle fibre first and the interzone second: in 80% (4/5) of the cells the half-bivalents not associated with the irradiated spindle fibre resumed movement immediately after the irradiation while the other half-bivalent remained stopped. Interzonal irradiations therefore uncouple the poleward movements of sister half-bivalents and the uncoupling does not depend on the order of the irradiation. Our experiments suggest therefore that the irradiation of a spindle fibre causes negative ('stop') signals to be transmitted across the interzone and that irradiation of the interzone blocks the transmission of the stop signal.

Evidence for polewards forces on chromosome arms during anaphase.

We have identified new mitotic forces in crane-fly spermatocytes, separate from forces on the kinetochore, that propel chromosome arms in anaphase towards the spindle pole. In normal spermatocytes, the chromosome arms in anaphase generally trail the kinetochore to the pole. After ultraviolet-microbeam irradiation of a kinetochore spindle fibre, however, chromosome arms moved closer to the pole than the kinetochore. This poleward arm-movement occurred regardless of whether the irradiation stopped the movement of the associated chromosomes, and occurred both in chromosomes associated with the irradiated fibre and in chromosomes not associated with the irradiated fibre. Arms that moved ahead of the kinetochore continued to lead the kinetochore to the pole for the duration of anaphase. Ultraviolet-microbeam-irradiation-induced movement of arms ahead of the kinetochore is specific for irradiation of spindle fibres: irradiations of the cytoplasm outside the spindle had no effect, and irradiations of the region between spindle and mitochondrial sheath (that outlines the spindle) and irradiations of the interzonal region are much less effective than irradiations of spindle fibres in causing arms to move. We argue that in crane-fly spermatocytes forces propelling chromosome arms toward the pole are part of normal anaphase.

Molecular cloning of an intracellular P-type ATPase from Dictyostelium that is up-regulated in calcium-adapted cells.

Results from a number of laboratories suggest that intracellular Ca2+ is involved in the regulation of Dictyostelium discoideum growth and development. To learn more about the regulation and function of intracellular Ca2+ in this organism, we have cloned and sequenced cDNAs that encode a putative P-type Ca2+ ATPase designated patA. The deduced protein product of this gene (PAT1) has a calculated molecular mass of 120,718 daltons. It exhibits about 46% amino acid identity with Ca2+ ATPases of the plasma membrane Ca2+ ATPase family and lower identity with sarco(endo)plasmic reticulum Ca2+ ATPase family members and monovalent cation pumps. However, PAT1 lacks the highly conserved calmodulin-binding domain present in the C-terminal region of most plasma membrane Ca2+ ATPase-type enzymes. When Dictyostelium amoebae are adapted to grow in the presence of 80 mM CaCl2, both the patA message and protein product are up-regulated substantially. These cells also exhibit an increase in the rate and magnitude of intracellular P-type Ca2+ uptake activity. Immunofluorescence analysis indicates that PAT1 colocalizes with bound calmodulin to intracellular membranes, probably components of the contractile vacuole complex. The presence of PAT1 on the contractile vacuole suggests that in Dictyostelium this organelle might function in Ca2+ homeostasis as well as in water regulation.

Evidence that kinetochore microtubules in crane-fly spermatocytes disassemble during anaphase primarily at the poleward end.

Anaphase chromosome motion involves the disassembly of kinetochore microtubules. We wished to determine the site of kinetochore microtubule disassembly during anaphase in crane-fly spermatocytes. In crane-fly spermatocyte spindles, monoclonal antibody 6-11B-1 to acetylated alpha-tubulin labels kinetochore microtubules almost exclusively, with an area immediately adjacent to the kinetochore being weakly or not labelled. This 'gap' in acetylation at the kinetochore serves as a natural marker of kinetochore microtubules in the kinetochore fibre. We measured the length of the gap on kinetochore fibres in metaphase and anaphase in order to deduce the fate of the gap during anaphase; we used this information to determine where kinetochore microtubules disassemble in anaphase. Gap lengths were measured from confocal microscope images of fixed spermatocytes dual labelled with 6-11B-1 to acetylated alpha-tubulin and YL1/2 to tyrosinated alpha-tubulin, the latter being used to determine the positions of kinetochores. In metaphase the average gap length was 1.7 microns. In anaphase, the gap appeared to decrease in length abruptly by about 0.4 microns, after which it decreased in length by about 0.2 microns for every 1 microns that the chromosome moved poleward. PacMan models of chromosome movement predict that this 'gap' in staining should disappear in anaphase at a rate equal to that of chromosome movement. Thus, our results do not support theories of chromosome motion that require disassembly solely at the kinetochore; rather, in crane-fly spermatocytes kinetochore microtubule disassembly in anaphase seems to take place primarily at the poles.

A homologue of the human regulator of mitotic spindle assembly protein (RMSA-1) is present in crane fly and is associated with meiotic chromosomes.

In a previous study, we have shown that a newly identified chromosomal protein, RMSA-1 (Regulator of Mitotic Spindle Assembly-1), identified and cloned using a human autoimmune, serum, is essential for mitotic spindle assembly; we proposed that RMSA-1 was a previously unknown physiological substrate for cdc 2 kinase. In the present study, we show that this protein is present in crane fly and is associated with the chromosomes of spermatocytes. A 31 kDa molecule in extracts from crane-fly nuclei, isolated from larvae, pupae and adults, reacts with affinity-purified anti-RMSA-1 autoantibody, shown by immunoblotting. The autoantibody reacts, as shown by immunofluorescence, with crane-fly spermatocyte chromosomes in prophase through anaphase of both meiosis-1 and meiosis-II but does not react with preprophase or telophase nuclei or with spermatid nuclei. In all meiotic stages, the crane-fly sex chromosomes stain more intensely than the autosomes. We conclude that, since RMSA-1 is present in insect and mammalian cells, it is conserved across a variety of animal species. Further, since RMSA-1 binds to chromosomes in meiotic cells, it also may be essential for assembly of the meiotic spindle.

Blood platelet heterogeneity: evidence for two classes of platelets in man and rat.

The platelet population of man and rat can be divided into two classes of about equal size on the basis of presence/absence of an acid phosphatase which acts on para-nitrophenylphosphate (a PNPase), at pH 5. The cytochemical reaction product is in the platelet cytoplasmic matrix, without apparent association with organelles or membrane systems. We could not relate differences in staining to differences in function: all cells responded the same to activation by thrombin, ADP, or collagen, in fibrinogen binding to activated platelets, by endocytosis of fluid-phase tracers, and in internalization of latex particles. With respect to possible physiological substrates for the PNP-ase, there was no reaction product from beta-glycerophosphate, AMP, ADP, ATP, GTP, CMP, IMP, cAMP, creatine phosphate, and inositol phosphates, and the enzyme was not inhibited by 40 mM lithium. There was reaction product from tyrosine phosphate suggesting that the physiological substrate for PNP-ase is tyrosine phosphate. In rat bone marrow, megakaryocytes also were of two classes, PNPase positive and PNPase negative, suggesting that different classes of platelets arise from different classes of megakaryocytes.

Ultraviolet microbeam irradiation of chromosomal spindle fibres in Haemanthus katherinae endosperm. I. Behaviour of the irradiated region.

We used an ultraviolet microbeam to irradiate chromosomal spindle fibres in metaphase Haemanthus endosperm cells. An area of reduced birefringence (ARB) was formed at the position of the focussed ultraviolet light with all wavelengths we used (260, 270, 280, and 290 nm). The chromosomal spindle fibre regions (kinetochore microtubules) poleward from the ARBs were unstable: they shortened (from the ARB to the pole) either too fast for us to measure or at rates of about 40 microns per minute. The chromosomal spindle fibre regions (kinetochore microtubules) kinetochore-ward from the ARBs were stable: they did not change length for about 80 seconds, and then they increased in length at rates of about 0.7 microns per minute. The lengthening chromosomal spindle fibres sometimes grew in a direction different from that of the original chromosomal spindle fibre. The chromosome associated with the irradiated spindle fibre sometimes moved off the equator a few micrometers, towards the non-irradiated half-spindle. We discuss our results in relation to other results in the literature and conclude that kinetochores and poles influence the behaviour of kinetochore microtubules.

Microinjection into crane-fly spermatocytes.

We were successful in microinjecting fluorescently labelled material into crane-fly spermatocytes. In our experiments, we obtained four results. (i) In most attempts, the membrane stretched around the micropipette and prevented entry of fluorescent material, even when the micropipette appeared to be pushed completely through the cell. This confirms suppositions from earlier micromanipulation experiments that the elastic membrane prevents the micropipette needle from entering the cell. (ii) In some attempts, cells lysed upon contact with the micropipette. (iii) In other attempts, we successfully injected fluorescent material into cells. (iv) Fluorescent material left the cells after injection, often passing into adjacent cells. Although our success rate is low, microinjection into crane-fly spermatocytes is indeed possible.

Rhodamine-labelled phalloidin stains components in the chromosomal spindle fibres of crane-fly spermatocytes and Haemanthus endosperm cells.

In crane-fly spermatocytes and Haemanthus endosperm, all metaphase and anaphase chromosomal spindle fibres were stained with rhodamine-labelled phalloidin. In crane-fly spermatocytes, each kinetochore was stained with rhodamine-labelled phalloidin at diakinesis of prophase and after colcemid caused metaphase spindles to depolymerize. Since phalloidin stains actin filaments, the distributions of rhodamine-labelled phalloidin-stained material in crane-fly spermatocytes and Haemanthus endosperm suggest that actin filaments might interact with microtubules to produce forces that move chromosomes during cell division, either directly or via an intermediate motor molecule.