Calcium buffer injections delay cleavage in Xenopus laevis blastomeres

Abstract

Abstract. Microinjection of calcium buffers into the two-cell Xenopus laevis embryo delays cell division in a dose-dependent manner. Four calcium buffers in the BAPTA series with different affinities for calcium were used to distinguish between a localized calcium gradient regulating cleavage and the global calcium concentration regulating this event. DibromoBAPTA (Kd = 1.5 #M) was found to delay cleavage at the lowest intracellular concentration (1.3 mM) of the four buffers tested. The effectiveness of the calcium buffers was dependent upon the buffer dissociation constant but not in a linear fashion. The concentration of buffer required to delay cleavage increased as the buffer's dissociation constant shifted above or below that of the optimum buffer, dibromoBAPTA. This relationship between a calcium buffer's effectiveness at delaying cleavage and its calcium affinity provides support for the hypothesis that a calcium concentration gradient is required for normal cell cycle progression (Speksnijder, J. E., A. L. Miller, M. H. Weisenseel, T.-H.Chert, and L. F. Jaffe. 1989. Proc. Natl. Acad. Sci. USA. 86:6607-6611). DibromoBAPTA was also injected with two different mounts of coinjected calcium to test the possibility that the free calcium concentration of the buffer solution is the important parameter for delaying cleavage. However, we found that changes in buffer concentration have a much stronger effect than changes in the free calcium concentration. This observation supports the hypothesis that BAPTA-type buffers exert their effect by shuttling calcium from regions of high concentration to those of lower concentration, reducing any calcium concentration gradients present in the Xenopus embryo.ansient increase in cytoplasmic [Ca2+]i during the events of mitosis and cell cleavage is considered to be an important regulatory step in both plant (11,12,36) and animal cells (15,16,22,23,25,26,28,30 The calcium gradient model proposed by Lionel Jaffe (27) could account for many of the discrepancies encountered during the investigations of calcium as a regulator of mitotic events. In particular, this could explain the lack of consistency in measured [Ca2+]i changes. This model asserts that calcium increases only in localized areas of the cell, generating highly localized calcium gradients that might not

An endogenous calcium oscillator may control early embryonic division

“…They also showed that the addition of 1 mM CaCl 2 has no effect on cyclin destruction (observation 10). Furthermore, the introduction of the calcium buffer BAPTA in dividing embryos slows but does not kill division cycles (8 Fig. 1 and are mathematically modeled in a manner similar to that used by Dupont and Goldbeter (23).…”

“…One possibility is that these transients are essential, and that the mechanism that controls variations in [Ca 2ϩ ] i is a fundamental component of the master clock. The following observations (among others) suggest this scenario: (i) [Ca 2ϩ ] i transients* accompany early embryonic cell cycles (2-7); (ii) the injection of calcium buffers into intact Xenopus blastomeres delays or blocks division (8); and (iii) in the absence of division, [Ca 2ϩ ] i transients appear with the same frequency as that of division (3,5,6).…”

“…Each of these injections partially or completely arrested the cell cycle. Moreover, to various degrees each type of BAPTA buffer delays cleavage (observation 7) and at higher concentrations blocks cleavage for at least 4-6 hr (7-10 cleavage cycles) (observation 8) (8).…”

An increase in [Ca2+]i is sufficient but not necessary for driving mitosis in early mouse embryos

“…This unambiguous role for Ca 2+ in cell-cycle progression at fertilization led to the discovery that Ca 2+ plays a role in cell-cycle progression in several different cell types. Evidence supporting a role for Ca 2+ in mitosis includes the fact that Ca 2+ transients have been recorded at key mitotic transitions including, nuclear-envelope breakdown (NEBD) (Poenie et al, 1985;Kao et al, 1990;Tombes et al, 1990), the metaphase-anaphase transition (Poenie et al, 1986;Ratan et al, 1988;Tombes and Borisy, 1989;Groigno and Whitaker, 1998) and cytokinesis (Fluck et al, 1991;Snow and Nuccitelli, 1993;Chang and Meng, 1995;Muto et al, 1996). These cell-cycle Ca 2+ transients are stimulated by cell-cycle-associated changes in the level of the Ca 2+ -mobilizing second messenger inositol-(1,4,5)-trisphosphate [Ins(1,4,5)P 3 ] (Ciapa et al, 1994).…”

Advances in Cytokinesis Research. The Molecular Mechanism of Targeted Vesicle Transport in Cytokinesis.

“…Several studies have suggested that Ca 2+ binding proteins, such as synaptotagmin or calmodulin, function in the membrane fusion (Südhof and Rizo, 1996;Chamberlain et al, 1995). During cytokinesis, microinjections of calcium buffers result in inhibition of furrowing in Xenopus eggs (Miller et al, 1993;Snow and Nuccitelli, 1993). This may result from the inhibition of vesicle fusion as well as the inhibition of assembly and/or contraction of an actomyosin ring.…”