Nuclear envelope breakdown and mitosis in sand dollar embryos is inhibited by microinjection of calcium buffers in a calcium-reversible fashion, and by antagonists of intracellular Ca2+ channels.
Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)
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Laboratory of Molecular Biology, University of Wisconsin, Madison 53706.
D000818:Animals; D001667:Binding, Competitive; D001757:Blastomeres / Q000187:drug effects* / Q000648:ultrastructure; D002021:Buffers; D002118:Calcium / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002122:Calcium Chloride / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007202:Indicators and Reagents; D008845:Microinjections; D008938:Mitosis / Q000187:drug effects*; D009282:Naphthalenesulfonates / Q000494:pharmacology; D009685:Nuclear Envelope / Q000187:drug effects* / Q000502:physiology; D012433:Ryanodine / Q000494:pharmacology; D012617:Sea Urchins / Q000196:embryology*; D008941:Spindle Apparatus / Q000187:drug effects
D002021:Buffers; D002121:Calcium Channel Blockers; D007202:Indicators and Reagents; D009282:Naphthalenesulfonates; D012433:Ryanodine; D004533:Egtazic Acid; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; C016238:antipyrylazo III; D002122:Calcium Chloride; D002118:Calcium