Asynchronous entry into anaphase induced by okadaic acid: spindle microtubule organization and microtubule/kinetochore attachments

Larsen, Paul M.; Wolniak, Stephen M.

doi:10.1007/bf01403399

Cited by 13 publications

(8 citation statements)

References 22 publications

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“…Protein kinase elevations early in prometaphaselearly metaphase and again late in metaphase are separated by an interval of elevated protein phosphatase activity in mid-metaphase. The elevation of protein phosphatase activity is necessary for the synchronous separation of sister chromatids at anaphase onset in both plant and animal cells (Wolniak and Larsen, 1992;Larsen and Wolniak, 1993) but does not appear to be involved in gross aspects of spindle organization or in the attachment of spindle microtubules to the kinetochores (Larsen and Wolniak, 1993). …”

Section: Discussionmentioning

confidence: 99%

“…It is important to provide a sense of the magnitude of this injection. In stamen hair cells, we estimate conservatively (using both unpublished and published immunofluorescence data that we have generated; Larsen and Wolniak, 1993) that each half spindle comprises -1000 microtubules and has a length of 4 0 to 15 pm. Based on these numbers and accounting for nonuniformity of microtubule length in the spindle, there are -10 to 15 million GTP binding sites on the tubulin molecules in the spindle alone.…”

Section: Microinjection Protocolsmentioning

confidence: 99%

“…Protein phosphatase activation also appears necessary for the synchronous separation of sister chromatids (Wolniak and Larsen, 1992), but asynchronous entry into anaphase does not appear to involve either major changes in spindle organization or the disruption of attachments between kinetochores and spindle microtubules (Larsen and Wolniak, 1993). The transition from elevated kinase to elevated phosphatase activity occurs 4 5 min after NEBD.…”

Section: Introductionmentioning

confidence: 99%

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

Wolniak

Larsen

1995

Plant Cell

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Microinjection Protocolsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Wolniak

Larsen

1995

Plant Cell

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“…OA is known to stim u late pre ma ture mi to sis and to in crease phosphorylation of mi to sis-spe cific proteins (Larsen andWolniak 1993, Ajiro et al 1996). This ac cel er a tion of the G2/M tran si tion is at tributed to MPF ac ti va tion throught a PP2A-de pend ent cdc25 phosphatase path way.…”

Section: A B E L I N G I N D E X [%] M I T O T I C I N D E X [%] T mentioning

confidence: 97%

Effect of OA-inhibitor of protein phosphatases PP1 and PP2A — on initiation of DNA replication and mitosis in Vicia faba root meristems

Polit

Maszewski

2005

Acta Physiol Plant

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Key words: car bo hy drate star va tion, con trol points of the cell cy cle, okadaic acid, pro tein phosphatases, Vicia faba var. mi nor Ab stractAc cord ing to the prin ci pal con trol point (PCP) hy poth e sis, exper i ments with ex cised, car bo hy drate-starved sta tion ary root meristems of Vicia faba var. mi nor have dem on strated that cells which pre vi ously di vided asynchronously were pref er entially blocked in G 1 (PCP1) and G 2 (PCP2) phases. When station ary phase meristems are sup plied with ex og e nous car bohy drate (2 % su crose), the G1-and G2-ar rested cells start out DNA rep li ca tion and mi totic di vi sions, re spec tively. The resump tion of DNA syn the sis and mi to sis is not im me di ate and the de lays of G 1 -and G 2 -ar rested cells are found dif fer ent. Using this model, we ex am ined the ef fects of 4 pulse in cu ba tions with okadaic acid (OA), a spe cific in hib i tor of PP1 and PP2A, on the du ra tion of in ter vals elaps ing be tween the pro vi sion of su crose and the first ap pear ance of S-and M-phase cells. We have found that de pend ing on the pe riod dur ing which OA had been ap plied, the re lease from G1 and G2 phase ar rest-points be comes pro longed, show ing dif fer ent time-course mod i fi cations. The ob tained data pro vide ev i dence that ac ti va tion of PP1 and PP2A is re quired to al low the cells for both PCP1→S and PCP2→M tran si tions in root meristems of V. faba.List of ab bre vi a tions: CDKs -cyclin-de pend ent kin ases, OA -okadaic acid, PCP: PCP1, PCP2 -prin ci pal con trol point: 1, 2, PPases: PP1, PP2A, PP2B, PP2C -serine/threonine pro tein phosphatases: 1, 2A, 2B, 2C, pRb -ret i no blas toma pro tein, PTPases -pro tein ty ro sine phosphatases In tro duc tion Pro gres sion through the cell di vi sion cy cle inEukaryotes is achieved by the op er a tion of a se ries of check points, each of them en sur ing the com pletion of one step of the cy cle prior to ini ti a tion of the next. The ex is tence of check point con trol sys tem in plants was first rec og nized 30 years ago by Van't Hof and Kovacs (1972). Their ex per i ments with ex cised, car bo hy drate-starved sta tion ary plant root meristems dem on strated, that cells which pre viously di vided asynchronously, be came pref er entially blocked in G1 and G2 phase. The stop page of the cell cy cle was re vers ible and the pro vi sion of ex cised roots with su crose stim u lated cell cy cle. Cells ar rested in G1 started out the S phase, while cells ar rested in G2 started out M phase. The reproducibility of this phe nom e non was the ba sis for the prin ci pal con trol point (PCP) hy poth e sis in plant cells. The first prin ci pal con trol point in G1 phase was termed PCP1, while the sec ond prin ci pal con trol point in G2 phase was termed PCP2 (reviewed by Van't Hof 1985). Pro tein phosphorylation and dephosphorylation -cat a lyzed by pro tein kin ases and phosphatases, re spec tively -are the key 303 ACTA PHYSIOLOGIAE PLANTARUM

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“…Standard procedures were used as if the cells were being prepared for antibody labeling including fixation with paraformaldehyde and glutaraldehyde (Wick and Duniec 1986), and treatment with wall-digestive enzymes and/or detergents (Larsen and Wolniak 1993), The results show that the distribution of the injected CaM and dextran changed dramatically during and after fixation (Fig. 6), with three quarters of the cells showing abnormal distributions, including leakage of CaM and dextran into the vacuole or neighboring cells, and movement of CaM and dextran onto the chromosomes, nucleoli, phragmoplast, and cell plate (Fig.…”

Section: Redistribution and Differential Extraction Of Cam And Dextramentioning

confidence: 99%

Calmodulin is uniformly distributed during cell division in living stamen hair cells ofTradescantia virginiana

Vos

Hepler

1998

Protoplasma

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Summary.Because the activity of calmodulin (CAM) may be dependent upon its structural distribution, we have examined its spatial localization in living cells. We have focused on ceil division and ceil plate formation, where conventional immunofluorescence studies report that CaM is specifically associated with microtubules (MTs) of the spindle and the phragmoplast. In dividing stamen hair cells of Tradescantia virginiana that were injected with fluorescently labeled CaM and examined by confocal laser scanning microscopy (CLSM), we found that the labeled protein is uniformly distributed throughout the cell and is not localized with the phragmoplast MTs or any other obvious structure. To explore why these images from live cells differ from those prepared by immunolabeling, we investigated the fate of CaM during fixation and compared it with the localization of fixable dextran and tubulin. The results show that fixation causes severe changes in cell morphology and in the distribution of CaM and dextran in three quarters of the cells. Conversely, injected rhodamine-tubulin did not show redistribution after fixation. We conclude that in the live cell, CaM is largely uniformly distributed throughout the cytoplasm, and secondly that conventional chemical fixation does not preserve CaM, and probably many other soluble proteins, in its in vivo distribution. The role postulated for CaM in mitosis, solely based on indirect immunofluorescence microscopy, has to be re-evaluated.

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Asynchronous entry into anaphase induced by okadaic acid: spindle microtubule organization and microtubule/kinetochore attachments

Cited by 13 publications

References 22 publications

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

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

Effect of OA-inhibitor of protein phosphatases PP1 and PP2A — on initiation of DNA replication and mitosis in Vicia faba root meristems

Calmodulin is uniformly distributed during cell division in living stamen hair cells ofTradescantia virginiana

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