Controls for centrosome reproduction in animal cells: issues and recent observations.
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Biomedical subjects
G Sluder
Publications and source records attributed to G Sluder.
Nuclear envelope breakdown is under nuclear not cytoplasmic control in sea urchin zygotes.
Nuclear envelope breakdown (NEB) and entry into mitosis are though to be driven by the activation of the p34cdc2-cyclin B kinase complex or mitosis promoting factor (MPF). Checkpoint control mechanisms that monitor essential preparatory events for mitosis, such as DNA replication, are thought to prevent entry into mitosis by downregulating MPF activation until these events are completed. Thus, we were surprised to find that when pronuclear fusion in sea urchin zygotes is blocked with Colcemid, the female pronucleus consistently breaks down before the male pronucleus. This is not due to regional differences in the time of MPF activation, because pronuclei touching each other break down asynchronously to the same extent. To test whether NEB is controlled at the nuclear or cytoplasmic level, we activated the checkpoint for the completion of DNA synthesis separately in female and male pronuclei by treating either eggs or sperm before fertilization with psoralen to covalently cross-link base-paired strands of DNA. When only the maternal DNA is cross-linked, the male pronucleus breaks down first. When the sperm DNA is cross-linked, male pronuclear breakdown is substantially delayed relative to female pronuclear breakdown and sometimes does not occur. Inactivation of the Colcemid after female NEB in such zygotes with touching pronuclei yields a functional spindle composed of maternal chromosomes and paternal centrosomes. The intact male pronucleus remains located at one aster throughout mitosis. In other experiments, when psoralen-treated sperm nuclei, over 90% of the zygote nuclei do not break down for at least 2 h after the controls even though H1 histone kinase activity gradually rises close to, or higher than, control mitotic levels. The same is true for normal zygotes treated with aphidicolin to block DNA synthesis. From these results, we conclude that NEB in sea urchin zygotes is controlled at the nuclear, not cytoplasmic, level, and that mitotic levels of cytoplasmic MPF activity are not sufficient to drive NEB for a nucleus that is under checkpoint control. Our results also demonstrate that the checkpoint for the completion of DNA synthesis inhibits NEB by acting primarily within the nucleus, not by downregulating the activity of cytoplasmic MPF.
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The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores.
During mitosis in Ptk1 cells anaphase is not initiated until, on average, 23 +/- 1 min after the last monooriented chromosome acquires a bipolar attachment to the spindle--an event that may require 3 h (Rieder, C. L., A. Schultz, R. W. Cole, and G. Sluder. 1994. J. Cell Biol. 127:1301-1310). To determine the nature of this cell-cycle checkpoint signal, and its site of production, we followed PtK1 cells by video microscopy prior to and after destroying specific chromosomal regions by laser irradiation. The checkpoint was relieved, and cells entered anaphase, 17 +/- 1 min after the centromere (and both of its associated sister kinetochores) was destroyed on the last monooriented chromosome. Thus, the checkpoint mechanism monitors an inhibitor of anaphase produced in the centromere of monooriented chromosomes. Next, in the presence of one monooriented chromosome, we destroyed one kinetochore on a bioriented chromosome to create a second monooriented chromosome lacking an unattached kinetochore. Under this condition anaphase began in the presence of the experimentally created monooriented chromosome 24 +/- 1.5 min after the nonirradiated monooriented chromosome bioriented. This result reveals that the checkpoint signal is not generated by the attached kinetochore of a monooriented chromosome or throughout the centromere volume. Finally, we selectively destroyed the unattached kinetochore on the last monooriented chromosome. Under this condition cells entered anaphase 20 +/- 2.5 min after the operation, without congressing the irradiated chromosome. Correlative light microscopy/elctron microscopy of these cells in anaphase confirmed the absence of a kinetochore on the unattached chromatid. Together, our data reveal that molecules in or near the unattached kinetochore of a monooriented PtK1 chromosome inhibit the metaphase-anaphase transition.
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Feedback control of the metaphase-anaphase transition in sea urchin zygotes: role of maloriented chromosomes.
To help ensure the fidelity of chromosome transmission during mitosis, sea urchin zygotes have feedback control mechanisms for the metaphase-anaphase transition that monitor the assembly of spindle microtubules and the complete absence of proper chromosome attachment to the spindle. The way in which these feedback controls work has not been known. In this study we directly test the proposal that these controls operate by maloriented chromosomes producing a diffusible inhibitor of the metaphase-anaphase transition. We show that zygotes having 50% of their chromosomes (approximately 20) unattached or monoriented initiate anaphase at the same time as the controls, a time that is well within the maximum period these zygotes will spend in mitosis. In vivo observations of the unattached maternal chromosomes indicate that they are functionally within the sphere of influence of the molecular events that cause chromosome disjunction in the spindle. Although the unattached chromosomes disjoin (anaphase onset without chromosome movement) several minutes after spindle anaphase onset, their disjunction is correlated with the time of spindle anaphase onset, not the time their nucleus breaks down. This suggests that the molecular events that trigger chromosome disjunction originate in the central spindle and propagate outward. Our results show that the mechanisms for the feedback control of the metaphase-anaphase transition in sea urchin zygotes do not involve a diffusible inhibitor produced by maloriented chromosomes. Even though the feedback controls for the metaphase-anaphase transition may detect the complete absence of properly attached chromosomes, they are insensitive to unattached or mono-oriented chromosomes as long as some chromosomes are properly attached to the spindle.
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Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle.
To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment--well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.
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Centrosome inheritance in starfish zygotes. II: Selective suppression of the maternal centrosome during meiosis.
Although both gametes may contribute a centrosome to the zygote at fertilization, only one of these centrosomes is used in development. Thus, specific mechanisms must exist to control centrosome inheritance in all sexually reproducing organisms. We use starfish as a model system to characterize these control mechanisms because the eggs complete meiosis I and meiosis II after fertilization; this allows us to directly follow the fate of all parental centrosomes in vivo. Only the paternal centrosome is used in starfish development. Although the microtubule organizing center activity of the maternal centrosome persists, the functional loss of this centrosome involves the suppression of its ability to double, or reproduce, at successive mitoses (Sluder et al., 1989. Dev. Biol. 131, 567-579). To determine when the reproductive capacity of the maternal centrosome is degraded, we transfer meiosis I and meiosis II spindles from just fertilized eggs into other zygotes that are in prophase of first mitosis. Meiosis I spindles are stable during first mitosis and are disassembled in first telophase in concert with the host spindle. In 61% of the cases a variable number of formerly meiotic centrosomes are active at second mitosis and reproduce in a normal fashion between subsequent mitoses. However, when meiosis II spindles are transferred in the same manner, in only 26% of the cases do any of the centrosomes persist past first mitosis or reproduce in a normal fashion thereafter. In the remainder of the cases the remnants of the maternal centrosomes organize a single monaster that does not double between mitoses. Control transfers of first mitosis spindles indicate that these results are not due to nonspecific damage to the meiotic spindles or to the recipient zygotes. These observations indicate that the reproductive capacity of maternal centrosomes is degraded during meiosis I, not during oogenesis. Our results also show that the cytoplasmic conditions which eliminate this reproductive capacity are no longer active once the zygote has entered the first mitotic cell cycle.
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Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.
Immunofluorescence microscopy is widely used to characterize the cellular distribution of both soluble and structural proteins. Control experiments generally address only the specificity of the antibodies used. The permeabilization/fixation conditions used to prepare cells for antibody application are assumed to preserve faithfully the in vivo distributions of the protein(s) being examined. We systematically tested the extent to which soluble proteins are redistributed into inappropriate locations and are differentially extracted from native locations during the permeabilization and fixation of the cells before antibody application. We separately introduce six soluble FITC-conjugated proteins of different net charges and sizes into living cultured cells. The labeled proteins do not adhere to the external surfaces of living cells and are evenly distributed throughout the cytoplasm with the larger proteins being excluded from the nucleus. The cells are then prepared as if for immunofluorescence using several conditions that encompass many of the methods commonly used for this purpose. Cells permeabilized with 0.1-0.2% Triton X-100 before fixation with 3.7% paraformaldehyde show a striking localization of all but one of the test proteins to the nucleus and/or nucleoli of 60-80% of labeled cells. Punctate cytoplasmic labeling and cytoskeletal-like arrays of labeled protein are also observed. Extraction with 1% detergent prior to fixation removes most but not always all of the exogenous proteins from the cell remnants. Permeabilization of cells with 0.1% detergent after paraformaldehyde fixation leaves a reticular, uneven cytoplasmic distribution of the labeled proteins, and some of the larger proteins are redistributed to the nuclei. Direct fixation/permeabilization with -20 degrees C methanol largely preserves the in vivo distributions of fluorescent proteins with some preferential localization of these proteins to nuclei, nucleoli and the perinuclear region. These results show that misleading apparent localizations of soluble proteins can result from their redistribution and/or differential extraction during the preparation of cells for primary antibody application.
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Functional properties of kinetochores in animal cells.
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Protein synthesis and the cell cycle: centrosome reproduction in sea urchin eggs is not under translational control.
The reproduction, or duplication, of the centrosome is an important event in a cell's preparation for mitosis. We sought to determine if centrosome reproduction is regulated by the synthesis and accumulation of cyclin proteins and/or the synthesis of centrosome-specific proteins at each cell cycle. We continuously treat sea urchin eggs, starting before fertilization, with a combination of emetine and anisomycin, drugs that have separate targets in the protein synthetic pathway. These drugs inhibit the postfertilization incorporation of [35S]methionine into precipitable material by 97.3-100%. Autoradiography of SDS-PAGE gels of drug-treated zygotes reveals that [35S]methionine incorporates exclusively into material that does not enter the gel and material that runs at the dye front; no other labeled bands are detected. Fertilization events and syngamy are normal in drug-treated zygotes, but the cell cycle arrests before first mitosis. The sperm aster doubles once in all zygotes to yield two asters. In a variable but significant percentage of zygotes, the asters continue to double. This continued doubling is slower than normal, asynchronous between zygotes, and sometimes asynchronous within individual zygotes. High voltage electron microscopy of serial semithick sections from drug-treated zygotes reveals that 90% of the daughter centrosomes contain two centrioles of normal appearance. From these results, we conclude that centrosome reproduction in sea urchin zygotes is not controlled by the accumulation of cyclin proteins or the synthesis of centrosome-specific proteins at each cell cycle. New centrosomes are assembled from preexisting pools of ready-to-use subunits. Furthermore, our results indicate that centrosomal and nuclear events are regulated by separate pathways.
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Reproductive capacity of sea urchin centrosomes without centrioles.
For animal cells, the relative roles of the centrioles and the pericentriolar material (the centrosomal microtubule organizing center) in controlling the precise doubling of the centrosome before mitosis have not been well defined. To this end we devised an experimental system that allowed us to characterize the capacity of the centrosomal microtubule organizing center to double regularly in the absence of centrioles. Sea urchin eggs were fertilized, stripped of their fertilization envelopes, and fragmented before syngamy. Those activated egg fragments containing just the female pronucleus assembled a monaster at first mitosis. A serial section ultrastructural analysis of such monasters revealed that the radially arrayed microtubules were organized by a hollow fenestrated sphere of electron-dense material, of the same appearance as pericentriolar material, that was devoid of centrioles. We followed individual fragments with only a female pronucleus through at least three cell cycles and found that the monasters did not double between mitoses. The observation that fragments with only a male pronucleus repeatedly divided in a normal fashion indicates that the assembly and behavior of monasters were not artifacts of egg fragmentation. Our results demonstrate that the activity that controls the precise doubling of the centrosome before mitosis is distinct and experimentally separable from the centrosomal microtubule organizing center. Our observations also extend the correlation between the reproductive capacity of a centrosome and the number of centrioles it contains (G Sluder and CL Rieder, 1985a: J. Cell Biol. 100:887-896). For a cell that normally has centrioles, we show that a centrosome without centrioles does not reproduce between mitoses.
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Centrosome inheritance in starfish zygotes: selective loss of the maternal centrosome after fertilization.
The mature egg inherits a centrosome from the second meiotic spindle, and the sperm introduces a second centrosome at fertilization. Since only one of these centrosomes survives to be used in development, specific mechanisms must exist to control centrosome inheritance. To investigate how centrosome inheritance is controlled we used starfish eggs as a model system, because they undergo meiosis after fertilization. As a result, the fate of the maternal and paternal centrosomes can be followed by light microscopy and experimentally manipulated in vivo. We show initially that only the paternal centrosome is used in starfish zygote development; the maternal centrosome retained from meiosis II is functionally lost before first mitosis. We then tested a number of possible ways in which the zygote could exert this differential control over the stability of centrosomes initially residing in the same cytoplasm. The results of these experiments can be summarized as follows: (1) Although the microtubule organizing center activity of the maternal centrosome is not degraded after meiosis, the ability of this centrosome to double at successive mitoses is lost. (2) The sperm centrosome is not "masked" from cytoplasmic conditions which could destabilize all centrosomes during or after the meiotic sequence. (3) The functional loss of the maternal centrosome is not due to its cortical location. (4) The loss of this doubling capacity is determined by the egg, not by putative inhibitory factors from the fertilizing sperm. (5) The destabilization of the maternal centrosome is not due to the complete loss of its centrioles. Together, these results demonstrate that all maternal centrosomes are equivalent and that they are intrinsically different from the paternal centrosome. This intrinsic difference, in concert with a change in cytoplasmic conditions after meiosis, determines the selective loss of the maternal centrosome inherited from the meiosis II spindle.
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Centrosomes and the cell cycle.
Centrosomes are the ensemble of organelles that form the poles of the mitotic spindle. We have examined the properties of the mechanisms that control the precise doubling, or reproduction, of centrosomes during the cell cycle. A functional analysis of this event in sea urchin eggs indicates that it is limited by the reproduction of determinants that we call polar organizers, around which the centrosome is organized. Each centrosome contains two polar organizers whose splitting, physical separation, and duplication control the reproduction of the centrosome. The splitting and duplication events are distinct processes that can be experimentally put out of phase with each other for several cell cycles. A serial section ultrastructural analysis of centrosomes with altered reproduction shows that the reproductive capacity of a centrosome is correlated with the number of centrioles it contains. In other experiments we show that centrosome reproduction is cytoplasmically controlled; centrosomes repeatedly double in a normal fashion in physically enucleated sea urchin eggs and eggs in which DNA synthesis has been inhibited by aphidicolin. In addition, we show that centrosomes, through the spindle microtubules they nucleate, play an important role in the mechanisms that control the timing of mitotic events and the overall duration of the cell cycle. Taken together, our observations in concert with those of others suggest that the cell cycle is a cytoplasmic phenomenon that does not require nuclear activities for cells that are not growth-limited.
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Relationship between nuclear DNA synthesis and centrosome reproduction in sea urchin eggs.
The importance of nuclear DNA synthesis for the doubling, or reproduction, of centrosomes in cells that are not growth-limited, such as sea urchin eggs, has not been clearly defined. Studies of enucleated, fertilized eggs show that nuclear activities are not required at each cell cycle for the normal reproduction of the complete centrosome. However, other studies report that the inhibition of nuclear DNA synthesis in intact eggs by the drug aphidicolin prevents centrosome reproduction and entry into mitosis as seen by nuclear envelope breakdown. To resolve this paradox, we systematically characterized the effect of aphidicolin on cell division in eggs from three species of sea urchins. Eggs were continuously treated with 5 or 10 micrograms/ml aphidicolin starting 5 min after fertilization. This blocked total incorporation of 3H-thymidine into DNA by at least 90%, as previously reported. We found that the sperm aster always doubles prior to first mitosis. Over a period of several hours, the centrosomes reproduce in the normal 2-4-8-16 fashion, with a period that is longer and more variable than normal. In every culture, a variable percentage of the eggs undergoes nuclear envelope breakdown. Once broken down, the nuclear envelope never visibly reforms even though centrosomes continue to double. Fluorescent labeling of DNA revealed that the chromatin does not condense into discrete chromosomes. Whether or not the nuclear envelope breaks down, the chromatin appears as an amorphous mass of fibers stretched between first two and then four asters. Later, the nuclear envelope/chromatin loses its association with some or all centrosomes. Our results were the same for all eggs at both drug concentrations. Thus, nuclear DNA synthesis is not required for centrosome reproduction in sea urchin eggs.
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The role of spindle microtubules in the timing of the cell cycle in echinoderm eggs.
Spindle microtubules play an important role in the mechanisms that control the timing of cell cycle events in the eggs of the sea urchins L. variegatus and L. pictus. However, recent work which used colchicine to block microtubule assembly in the eggs of two other echinoderms, S. purpuratus and D. excentricus, has raised serious questions about the generality of this role for spindle microtubules. Thus, we have systematically examined the role of spindle microtubules in the timing of the cell cycle in the fertilized eggs of these latter species. We treated eggs of both species with 5-10 microM Colcemid for several minutes starting 30 min after fertilization to completely prevent spindle microtubule assembly for several h. We used Colcemid, instead of colchicine, because it is effective at lower doses and, at these doses, shows no detectable toxic side effects. We compared for control and treated eggs the time course of nuclear envelope breakdown/reformation and DNA synthesis. We found for both species that the eggs continue to cycle without spindle microtubules; mitosis is up to twice the normal duration while interphase remains essentially unaffected. To test for the possible toxic side effects of the 1-2 mM colchicine used earlier on S. purpuratus and D. excentricus, we treated eggs of these two species, and also those of L. variegatus, with 1 mM lumi-colchicine. This photo-inactivated form of colchicine, which does not bind to tubulin, substantially prolongs mitosis and, to a lesser extent, interphase. Thus, the results of the earlier work are most easily explained by the combination of specific and nonspecific effects of the 1-2 mM colchicine used. Our present results indicate that the importance of spindle microtubules in the mechanisms that control the timing of the mitosis portion of the cell cycle is a general phenomenon.
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The reproduction of centrosomes: nuclear versus cytoplasmic controls.
The tight coordination normally found between nuclear events and the doubling of centrosomes at each cell cycle suggests that nuclear activities may be part of the mechanism that controls the reproduction of centrosomes. To determine if this is the case, we used a micropipette to completely remove the nucleus from eggs of the sea urchin Lytechinus variegatus at prophase of the first mitosis, leaving only one centrosome in the cell. The subsequent behavior of this centrosome was then followed in vivo with the polarization microscope. In all cases the centrosome reproduced in a precise 1:2:4:8 fashion with a periodicity that was slightly slower than the centrosome cycle of control eggs. The cell cycle-related changes in centrosome morphology were identical to those of control eggs in that: (a) the astral birefringence varied cyclically to a normal extent, (b) the astral focus enlarged and then flattened during the telophase equivalent, (c) cleavage furrows were initiated as the astral birefringence faded, and (d) daughter centrosomes separated before the increase in astral birefringence at the onset of each mitosis. To determine if centrioles also reproduced normally, enucleate eggs were followed in vivo until they contained eight centrosomes. They were then individually removed from the preparations, fixed, and embedded. Each egg was serially 0.25-micron sectioned for observation with the high voltage electron microscope. We completely reconstructed 23 centrosomes in four eggs; all centrosomes contained two centrioles apiece. These results demonstrate that the subunits for complete centrosome assembly can be stockpiled ahead of time and that the properly controlled use of these subunits for centrosome reproduction does not require nuclear transcription or nuclear DNA synthesis at each cell cycle.
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Centrosomes are required for the assembly of a bipolar spindle in animal cells.
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Centriole number and the reproductive capacity of spindle poles.
The reproduction of spindle poles is a key event in the cell's preparation for mitosis. To gain further insight into how this process is controlled, we systematically characterized the ultrastructure of spindle poles whose reproductive capacity had been experimentally altered. In particular, we wanted to determine if the ability of a pole to reproduce before the next division is related to the number of centrioles it contains. We used mercaptoethanol to indirectly induce the formation of monopolar spindles in sea urchin eggs. We followed individually treated eggs in vivo with a polarizing microscope during the induction and development of monopolar spindles. We then fixed each egg at one of three predetermined key stages and serially semithick sectioned it for observation in a high-voltage electron microscope. We thus know the history of each egg before fixation and, from earlier studies, what that cell would have done had it not been fixed. We found that spindle poles that would have given rise to monopolar spindles at the next mitosis have only one centriole whereas spindle poles that would have formed bipolar spindles at the next division have two centrioles. By serially sectioning each egg, we were able to count all centrioles present. In the twelve cells examined, we found no cases of acentriolar spindle poles or centriole reduplication. Thus, the reproductive capacity of a spindle pole is linked to the number of centrioles it contains. Our experimental results also show, contrary to existing reports, that the daughter centriole of a centrosome can acquire pericentriolar material without first becoming a parent. Furthermore, our results demonstrate that the splitting apart of mother and daughter centrioles is an event that is distinct from, and not dependent on, centriole duplication.
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