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S M Wolniak

Publications and source records attributed to S M Wolniak.

At least 19 recordsLinked to original sources
PubMed2001

Expression of the mitogen-activated protein kinase kinase ZmMEK1 in the primary root of maize.

We have analyzed changes in the distribution and abundance of a mitogen-activated protein kinase kinase (MAPKK) enzyme (EC 2.7.1.37) known as ZmMEK1. in root apices of Zea mays L. under normal growth conditions, and after treatments that alter patterns of proliferation, as a means to assess the potential physiological role of the MAP kinase cascade in growth and development. The ZmMEK1 protein is most abundant within immature tissues such as the roots and leaves of seedlings, and is nearly undetectable in mature leaf tissue. Along the longitudinal axis of growing roots, ZmMEK1 mRNA and protein are abundant throughout the apical 12 mm. Two anti-ZmMEK1 antibody-reactive proteins can be resolved within the apical 4 mm of the root, spatially coinciding with the meristem and distal elongation zone. Phosphatase treatments suggest that both immunoreactive bands are forms of ZmMEK1 that differ in their state of phosphorylation. Expression of ZmMEK1, histone H4 and cyclin-dependent protein kinase (CDK) in roots after 7 days of exposure to low temperature and during a 48-hour recovery period was monitored during the coincident alterations in growth. Levels of ZmMEK1 mRNA and protein within these roots were indistinguishable from those of control roots. However, a slower-migrating form of ZmMEK1 temporally coincided with the observed increase in CDK levels during the transition into proliferative activity. We demonstrate that the ZmMEK1 MAPK activator is expressed and is differentially phosphorylated within the root meristem and distal elongation zone. We suggest that post-translational modifications of the protein regulate the function of ZmMEK1 within the root. Changes in ZmMEK1 phosphorylation state correlate with changes in proliferation in the root apex.

DNA-Binding Proteins↗
PubMed2001

Centrin is necessary for the formation of the motile apparatus in spermatids of Marsilea.

During spermiogenesis in the water fern, Marsilea vestita, basal bodies are synthesized de novo in cells that lack preexisting centrioles, in a particle known as a blepharoplast. We have focused on basal body assembly in this organism, asking what components are required for blepharoplast formation. Spermiogenesis is a rapid process that is activated by placing dry microspores into water. Dry microspores contain large quantities of stored protein and stored mRNA, and inhibitors reveal that certain proteins are translated from stored transcripts at specific times during development. Centrin translation accompanies blepharoplast appearance, while beta-tubulin translation occurs later, during axonemal formation. In asking whether centrin is an essential component of the blepharoplast, we used antisense, sense, and double-stranded RNA probes made from the Marsilea centrin cDNA, MvCen1, to block centrin translation. We employed a novel method to introduce these RNAs directly into the cells. Antisense and sense both arrest spermiogenesis when blepharoplasts should appear, and dsRNA made from the same cDNA is an effective inhibitor at concentrations at least 10 times lower than either of the single-stranded RNA used in these experiments. Blepharoplasts are undetectable and basal bodies fail to form. Antisense, sense, and dsRNA probes made from Marsilea beta-tubulin permitted normal development until axonemes form. In controls, antisense, sense, and dsRNA, made from a segment of HIV, had no effect on spermiogenesis. Immunoblots suggest that translational blocks induced by centrin-based RNA are gene specific and concentration dependent, since neither beta-tubulin- nor HIV-derived RNAs affects centrin translation. The disruption of centrin translation affects microtubule distributions in spermatids, since centrin appears to control formation of the cytoskeleton and motile apparatus. These results show that centrin plays an essential role in the formation of a motile apparatus during spermiogenesis of M. vestita.

Amanitins↗
PubMed2001

Cell cycle arrest allows centrin translation but not basal body formation during spermiogenesis in Marsilea.

Spermiogenesis in the water fern Marsilea vestita is a rapid process that requires the de novo formation of basal bodies in a cytoplasmic particle known as a blepharoplast. Spermiogenesis is activated by placing dry spores into water and is dependent upon the translation of new proteins from stored mRNAs with little, if any, new transcription. We looked at the necessity of cell division cycles in the gametophyte as a prerequisite for the activation of centrin translation and for the consequent formation of blepharoplasts. Cell cycle arrest was induced by treatments of gametophytes with hydroxyurea, with olomoucine, or after RNAi, employing dsRNA derived from Marsilea cyclin A or cyclin B. In all cases, centrin is translated from stored mRNA at the normal time, approximately 4 hours after imbibition, and it accumulates to maximal levels approximately 6 hours after imbibition. In spite of the fact that centrin is translated at essentially normal times and accumulates to nearly normal levels, no blepharoplasts form in the gametophytes where division cycles have been disrupted. These results provide a clear demonstration that the new translation of centrin, by itself, is insufficient for blepharoplast formation, the de novo formation of basal bodies, and the assembly of a motile apparatus.

Calcium-Binding Proteins↗
PubMed2000

The efficacy of RNAi in the study of the plant cytoskeleton.

Recent studies on a variety of organisms point to the ubiquity of RNA interference (RNAi) as a means to induce a gene-specific block to translation. RNAi has gained popularity in the last few years in the study of a number of problems in development. In this review, we highlight recent findings with RNAi using several different kinds of animals and fungi, and we show how these responses parallel cosuppression effects described in plants nearly a decade earlier. We then point to the efficacy of RNAi in studying minor and regulatory components of the plant cytoskeleton, and we highlight some recent studies using this approach with the water fern, Marsilea vestita.

Actins↗
PubMed2000

Control of development and motility in the spermatozoids of lower plants.

The spermatozoids of lower plants have long been recognized as remarkably complex motile gametes. Spermatozoids differ markedly from the other gametophyte cells that surround or give rise to them. Their differentiation process involves the synthesis and assembly of a complex cytoskeleton and a motile apparatus that can be simple or complex, having as few as two to as many as thousands of ciliary axonemes. An important aspect of spermiogenesis involves the de novo synthesis of basal bodies in a cytoplasmic particle known as the blepharoplast: that is, the cells that produce spermatocytes do not contain centrioles. Thus, these cells provide an ideal system in which to study the formation of basal bodies. The cytoskeletons of spermatozoids from different organisms display a common architecture, with a multilayered structure (MLS) at the anterior end of the cell and a dorsally situated planar ribbon of crosslinked microtubules extending the length of the elongated gamete. The function of the MLS is not known, but it could be involved in cell-body elongation during development and in the control of ciliary motility in the mature gamete, particularly during chemotaxis. The application of modern techniques on these cells can shed light on long-standing problems relating to spermiogenesis and motility.

Cell Movement↗
PubMed1999

Molecular cloning of a centrin homolog from Marsilea vestita and evidence for its translational control during spermiogenesis.

Spermiogenesis in the water fern Marsilea vestita is a process that reaches completion 11 h after dry microspores are immersed in an aqueous medium at 20 degrees C. Each microspore produces 32 spermatozoids and each spermatozoid has a coiled cell body and approximately 140 cilia. The spermatids make basal bodies de novo, from a structure known as a blepharoplast. From the onset of development, the spores contain a large quantity of protein and stored mRNA. We have found previously that centrin, a protein involved in the function of microtubule organizing centers and present in association with basal bodies in motile cells, is made in large quantity approximately 4 h after the microspores are placed into liquid medium. In this paper, we show that a centrin cDNA (MvCen1) we isolated from M. vestita closely resembles centrin cDNAs from other eukaryotic organisms. MvCen1, synthesized in Escherichia coli as a GST-fusion protein, reacted with anti-centrin monoclonal antibodies on immunoblots. Northern blot analysis demonstrates that centrin mRNA is present in the dry microspore at the time of imbibition, at levels that remain constant over 10 h of development and are unaffected by treatment of spores with alpha-amanitin. The centrin transcripts, stored in dry microspores, cannot be translated in vitro for at least 30 min after imbibition.

Amino Acid Sequence↗
PubMed1998

Spermiogenesis in Marsilea vestita: a temporal correlation between centrin expression and blepharoplast differentiation.

The motile male gamete of the water fern Marsilea vestita is a spirally shaped cell that possesses a complex cytoskeletal array of microtubules and approximately 140 cilia. Spermiogenesis in this organism is a rapid process that requires only approximately 11 h at 20 degrees C and involves the de novo synthesis of basal bodies from an organelle known as a blepharoplast. The developmental program that gives rise to the spermatozoids begins with nine mitotic divisions that occur in rapid succession during the first 5.5 h after imbibition of the dry microspore. During the next 5.5 h, the spermatids undergo a complicated differentiation process. We have asked what new proteins must be made for differentiation to proceed. Inhibitor treatments reveal that some translation is a necessary prerequisite for the differentiation and release of spermatozoids, but methionine-labeling studies demonstrate that relatively few types of proteins must be translated for this developmental program to reach completion. We have found that the dry microspores contain alpha-, beta-, and gamma-tubulin, at levels that may be sufficient for the entire developmental process. The abundance of the tubulins remains essentially constant until very late stages of spermiogenesis. In contrast to the tubulins, we show that centrin begins to increase in abundance at approximately 4 h after imbibition and that it reaches a peak at 6 h after imbibition. We also show that centrin mRNA is stored in the dry microspore, and that centrin protein abundance is regulated at the translational level. We believe that the translation of stored centrin transcripts serves as a rate-limiting step in the rapid differentiation process of spermiogenesis M. vestita. We suggest that centrin functions in the microtubule organizing centers that are required for the construction of the cytoskeleton and the motile apparatus in these structurally complicated cells.

Amanitins↗
PubMed1998

Molecular cloning and characterization of maize ZmMEK1, a protein kinase with a catalytic domain homologous to mitogen- and stress-activated protein kinase kinases.

Mitogen- or stress-activated protein kinase kinases (M/SAPKKs) are dual-specificity protein kinases that are components of highly conserved signal transduction pathways. A cDNA clone (ZmMEK1) was isolated from a Zea mays (L.) root tip library. ZmMEK1 contains a complete open reading frame, encoding a 355-amino-acid protein with an estimated molecular mass of 39,874 Da. The predicted protein contains the 11 catalytic sub-domains conserved in all protein kinases, and a version of the sub-domain VIII S/TxxxS/TxVGT motif that is characteristic of M/SAPKK proteins (EC 2.7.1.37). The catalytic domain of ZmMEK1 is most closely related (65% identity) to the tomato M/SAPKK homolog LeMEK1, but exhibits similar identity (39-60%) to M/SAPKKs from other plants, animals and fungi. Northern blotting revealed ZmMEK1 mRNA in maize seedling roots and coleoptiles; in mature plants transcripts were detected in stems and low levels in leaves. Transcription of ZmMEK1 mRNA was also affected by environmental stimuli. The catalytic domain of ZmMEK1 was expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Nanogram quantities of the purified fusion protein reacted with anti-M/SAPKK antibodies on immunoblots. In vitro, the GST-ZmMEK1 fusion protein undergoes autophosphorylation, and will phosphorylate myelin basic protein, but will not phosphorylate histone H1. ZmMEK1 encodes an enzyme that is structurally and functionally similar to other M/SAPKK proteins.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of a functional A-type cyclin from maize.

Cyclins are involved in the regulation of cell cycle progression in eukaryotes. We have isolated a cyclin cDNA clone, cycZm2w, from maize root tip cells, which fits best into group A2 of current plant cyclin gene classification schemes. The cDNA encodes a protein with a domain homologous to the cyclin box of mitotic cyclins. Complementation studies revealed that cycZm2w was able to rescue a budding yeast cyclin-deficient mutant (BF305-15d#21). As expected, cycZm2w is expressed in organs of the maize plant that possess meristematic activity, but is especially prominent in the proliferating regions of the root apex.

Cyclin A↗
PubMed1995

The timing of protein kinase activation events in the cascade that regulates mitotic progression in Tradescantia stamen hair cells.

Stamen hair cells of the spiderwort plant Tradescantia virginiana exhibit unusually predictable rates of progression through mitosis, particularly from the time of nuclear envelope breakdown (NEBD) through the initiation of cytokinesis. The predictable rate of progression through prometaphase and metaphase has made these cells a useful model system for the determination of the timing of regulatory events that trigger entry into anaphase. A number of studies suggest that the elevation of one or more protein kinase activities is a necessary prerequisite for entry into anaphase. The current experiments employ two strategies to test when these elevations in protein kinase activity actually occur during metaphase. In perfusions, we added the protein kinase inhibitors K-252a, staurosporine, or calphostin C to living stamen hair cells for 10-min intervals at known times during prometaphase or metaphase and monitored the subsequent rate of progression into anaphase. Metaphase transit times were altered as a function of the time of addition of K-252a or staurosporine to the cells; metaphase transit times were extended significantly by treatments initiated in prometaphase through early metaphase and again late in metaphase. Transit times were normal after treatments initiated in mid-metaphase, approximately 15 to 21 min after NEBD. Calphostin C had no significant effect on the metaphase transit times. In parallel, cells were microinjected with known quantities of a general-purpose protein kinase substrate peptide, VRKRTLRRL, at predefined time points during prometaphase and metaphase. At a cytosolic concentration of 100 nM to 1 microM, the peptide doubled or tripled the metaphase transit times when injected into the cytosol of mitotic cells within the first 4 min after NEBD, at any point from 7.5 to 9 min after NEBD, at any point from 14 to 16 min after NEBD, at 21 min after NEBD, or at 24 min after NEBD. At the concentration used and during these brief intervals, the peptide appeared to act as a competitive inhibitor to reveal inflection points when protein kinase activation was occurring or when endogenous substrate levels approached levels of the peptide. The timing of these inflection points coincides with the changes in protein kinase activities during prometaphase and metaphase, as indicated by our perfusions of cells with the broad spectrum kinase inhibitors. Collectively, our results suggest that the cascade that culminates in anaphase is complex and involves several successive protein kinase activation steps punctuated by the activation of one or more protein phosphatases in mid-metaphase.

Alkaloids↗
PubMed1992

Changes in the metaphase transit times and the pattern of sister chromatid separation in stamen hair cells of Tradescantia after treatment with protein phosphatase inhibitors.

Stamen hair cells from the spiderwort plant, Tradescantia virginiana, exhibit remarkably predictable metaphase transit times, making them uniquely suitable for temporal studies on mitotic regulation. In this study, we describe two kinds of experiments that test whether protein phosphatase activity is a necessary prerequisite for entry into anaphase in living, mitotic cells. We treated cells at specific points during prophase, prometaphase and metaphase with the broad-spectrum protein phosphatase inhibitor, alpha-naphthyl phosphate (administered by microinjection), or with the naturally occurring, potent phosphatase inhibitors okadaic acid, microcystin-LR or microcystin-RR (administered by perfusion), and we have observed changes in the metaphase transit time that are primarily dependent on the time of initial exposure to the inhibitor. Maximal extensions of the metaphase transit time result from alpha-naphthyl phosphate microinjections initiated in mid-metaphase, 10-20 min after nuclear envelope breakdown. Perfusions with okadaic acid started during a specific interval in mid-metaphase, 15-20 min after nuclear envelope breakdown, resulted in a statistically significant extension of the metaphase transit time. Perfusions with either microcystin-LR or microcystin-RR initiated 15-26 min after nuclear envelope breakdown extended the metaphase transit times significantly. Treatments of cells with okadaic acid or with either of the microcystins initiated outside this mid-metaphase interval either were without effect or, alternatively, resulted in a significant shortening of the metaphase transit time. In addition to their effects on the timing of anaphase onset, treatments with these protein phosphatase inhibitors also resulted in a remarkable change in the way in which these cells enter anaphase. Sister chromatid separation in stamen hair cells typically requires only 5 seconds, but after treatment with any of these inhibitors some, but not all, of the chromatids split apart at anaphase onset. Those that split begin to migrate toward the spindle pole regions, while those that fail to split remain at the metaphase plate. Later, more of the paired chromatids split apart and begin moving toward the spindle pole regions. Those that fail to separate remain at the metaphase plate. This process can be repeated several times before all of the chromatids have separated. Thus, entry into anaphase becomes extremely asynchronous, and as much as 30 min can transpire between the centromeric separation of the first and last chromosomes. Some of the chromosomes complete their anaphase movements before others have even split apart at the metaphase plate. Asynchronous separation did not result in a permanent segregation anomaly.(ABSTRACT TRUNCATED AT 400 WORDS)

Anaphase↗
PubMed1990

1,2-Dioctanoylglycerol accelerates or retards mitotic progression in Tradescantia stamen hair cells as a function of the time of its addition.

We have treated living, intact stamen hair cells from the spiderwort plant, Tradescantia virginiana, with 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol, a potent and permeant activator of protein kinase C, and have observed the rates of progression of mitosis from prophase through anaphase. We have found that in addition to the concentration used, the time of initial treatment with 1,2-dioctanoylglycerol defines the response by the cells. The cells rapidly undergo nuclear envelope breakdown when this diglyceride is added in very late prophase, 0 to approximately 8 min prior to the time of normal nuclear envelope breakdown. Anaphase onset occurs 28 min after nuclear envelope breakdown, rather than after the 33 min interval observed in untreated cells. Rapid progression through metaphase is also observed if cells are treated with 0.5 microgram/ml 1,2-dioctanoylglycerol during prometaphase, up to 15 min after nuclear envelope breakdown. The addition of 0.5 microgram/ml 1,2-dioctanoylglycerol in late metaphase, approximately 26 min after nuclear envelope breakdown, results in sister chromatid separation slightly ahead of its normal time, 33 min after nuclear envelope breakdown, and in precocious cell plate vesicle aggregation, 3-5 min earlier than that observed in untreated cells. Treatment of cells with 60 micrograms/ml of 1,2-dioctanoylglycerol at any point during the interval from 0 to approximately 5 min prior to nuclear envelope breakdown results in precocious entry into anaphase. If cells are treated with either 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol earlier than 20 min before nuclear envelope breakdown, they do not enter mitosis, but instead revert to interphase without dividing. When 1,2-dioctanoylglycerol is added at other times during mitosis, the rate of subsequent mitotic progression is dramatically slowed; the cells require greater than 55 min to progress from nuclear envelope breakdown to anaphase onset, though once in anaphase, the cells progress onward to cytokinesis at normal rates. Treatments o of cells with 1,3-dioctanoylglycerol at any point during prophase, prometaphase, or metaphase are without effect on the rate of subsequent mitotic progression. The shifts in response by cells treated at specific times with 1,2-dioctanoylglycerol during mid- and late metaphase may be indicative of the existence of one or more regulatory switch points (i.e., checkpoints) just prior to anaphase onset.

Anaphase↗
PubMed1990

Role of microtubules in stimulating cytokinesis in animal cells.

The initiation of furrow formation is disrupted when microtubule elongation to the cell surface is inhibited either by promoting microtubule disassembly with hydrostatic pressure or by stabilizing the mitotic astral microtubules with taxol. The pressure studies confirmed Rappaport's earlier observation that stimulation of furrow formation is produced by a pair of asters and does not require chromosomes or a central spindle in the large dividing cells of echinoderm embryos. The taxol studies showed that furrow formation occurs between two stable asters when the asters are within 20 microns of the lateral cell surface. Furrow formation at the lateral edge of the cell does not appear to require microtubule dynamics, microtubule elongation, or contact of microtubule ends with the lateral cell surface. Microtubules may function to increase the concentrations of the active forms of diffusible stimulatory factors that interact with receptors at the inner cell surface to initiate the formation of the furrow apparatus and activate contraction.

Alkaloids↗
PubMed1989

Quin2-induced metaphase arrest in stamen hair cells can be reversed by 1,2-dioctanoylglycerol but not by 1,3-dioctanoylglycerol.

Elicitor molecules of the polyphosphoinositide cycle, inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol (1,2-DAG) play roles in the entry of calcium into the cytosol and in the elevation of protein kinase C activity, respectively. We have treated stamen hair cells of the spiderwort plant, Tradescantia virginiana, with a solution of quin2 (50 microM) or its acetoxymethyl ester, quin2-AM (50 microM) and have retarded the normally predictable rate of progression through metaphase. Metaphase arrest persists for longer than 80 min after treatment with this Ca2+-chelator, and, shortly thereafter, the cells revert to interphase without dividing. Reversal of metaphase arrest results from treatments with calcium chloride (100 microM) after 5 to 8 min or with 1,2-DAG (i.e., 60 micrograms/ml 1,2-dioctanoylglycerol) after 7 to 11 min. In control experiments, metaphase arrest could not be reversed by treatment with either magnesium sulfate or 1,3-dioctanoylglycerol. Anaphase onset was observed in these control cells after post-treatment with calcium chloride (after 4-9 min) or with 1,2-dioctanoylglycerol (after 7-13 min). The treatment of stamen hair cells in very early prophase with H-7, (i.e., 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), a potent protein kinase C inhibitor, extends the duration of metaphase significantly. Neither H-8 nor HA-1004, less active protein kinase C inhibitors in this class of molecules, alter the duration of metaphase to a significant extent. These results suggest that in cells arrested in metaphyase by quin2, calcium translocation plays a role in the sequence of events which culminate in anaphase.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoquinolines↗
PubMed1988

The regulation of mitotic spindle function.

The process of mitosis includes a series of morphological changes in the cell in which the directional movements of chromosomes are the most prominent. The presence of a microtubular array, known as the spindle or mitotic apparatus, provides at least a scaffold upon which these movements take place. The precise mechanism for chromosome movement remains obscure, but new findings suggest that the kinetochore may play a key role in chromosome movement toward the spindle pole, and that sliding interactions between or among adjacent microtubules may provide the mechanochemical basis for spindle elongation. The physiological regulation of the anaphase motors and of spindle operation either before or after anaphase remains equally elusive. Elicitors that may serve as controlling elements in spindle function include shifts in cytosolic calcium activity and perhaps the activation or inactivation of protein kinases, which in turn produce changes in the state of phosphorylation of specific spindle components.

Animals↗
PubMed1987

Mitotic progression in stamen hair cells of Tradescantia is accelerated by treatment with ruthenium red and Bay K-8644.

The normally predictable duration of metaphase in stamen hair cells from the spiderwort, Tradescantia virginiana, is shortened significantly by treatment during prometaphase with either ruthenium red or Bay K-8644. Ruthenium red is an inhibitor of Ca2+ translocation and Bay K-8644 is a Ca2+-channel agonist. Their action on mitotic progression appears to involve a rise in the cytosolic Ca2+ level that in turn has a pronounced effect on the duration of metaphase. The timing of addition of ruthenium red for accelerated progression through metaphase is less critical than that for Bay K-8644 which will promote metaphase progression only if added 0 to 12 min after nuclear envelope breakdown. In contrast, ruthenium red can be added at any time from approximately 10 min prior to nuclear envelope breakdown up to 25 min afterward. A reduction of extracellular Ca2+ is sufficient by itself to prolong the duration of metaphase in stamen hair cells, but the duration of metaphase by ruthenium red or Bay K-8644 is significantly shortened in identical solutions with Ca2+ buffered at levels greater than 1 microM. Metaphase progression rates with either agent are independent of changes in extracellular Mg2+ levels. Correlated with the precocious entry into anaphase was rapid formation of the spindle and a marked reduction in spindle rotation during metaphase. Interestingly, we observed a modest increase in the rate of anaphase chromosome separation, but the appearance of cell plate vesicles at the site of incipient cell plate formation occurred normally approximately 19 min after anaphase onset. Similarly, the initial appearance of cell plate vesicles in Bay K-8644 was normal, approximately 19 min after the onset of anaphase. These results further implicate shifts in cytosolic Ca2+ in the regulation of mitotic events.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Lithium alters mitotic progression in stamen hair cells of Tradescantia in a time-dependent and reversible fashion.

Stamen hair cells of Tradescantia exhibit remarkable precision in the timing of their mitotic events. This precision is altered dramatically with treatment in 50 microM to 1 mM LiCl, an inhibitor of the polyphosphoinositide cycle. Mitotic progression is altered as a function of the time of treatment with LiCl. If cells are treated during late prophase, greater than 80% fail to enter metaphase. Most of the cells that undergo nuclear envelope breakdown become arrested in metaphase. Treatment with LiCl earlier in prophase also results in metaphase arrest. Metaphase arrest can be reversed by the addition of 10 microM myo-inositol or 100 microM CaCl2 to the extracellular medium. The timing of reversal by myo-inositol takes 10 to 14 min while CaCl2 promotes anaphase onset in 2 to 5 min. The difference in kinetics for reversal between these two treatments suggests that myo-inositol addition overrides a biochemical pathway while Ca2+ addition supplants a phosphoinositide-mediated rise in the cation that may be necessary for anaphase onset. Buffer without myo-inositol or CaCl2 is insufficient for reversal. If the cells are treated with LiCl in mid-late-metaphase, at least 5 min prior to the expected time of anaphase onset, sister chromatids split at the normal time, 33 +/- 4 min after nuclear envelope breakdown, but further chromosome separation is arrested. Anaphase chromosome movement can be restored by treatment with either 10 microM myo-inositol or 100 microM CaCl2 in the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaphase↗