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Study of calcium signaling in cell cleavage using confocal microscopy.

Intracellular calcium is an important second messenger that is involved in many important cellular functions. Several recent studies have implicated Caf+ signaling in cell cleavage, particularly cytokinesis ( l-4). Cytokinesis is an important part of the cell cycle; it also plays a key role in development. The spatial location and the timing of cytokinesis are both precisely regulated (2), but the regulatory mechanisms of cytokinesis are not well understood at present. The evidence implicating intracellular Cat’ in cytokinesis has, thus far, been indirect. The only relevant calcium images available, from medaka eggs, were obtained by the use of aequorin (3). Because of the limitations of spatial and temporal resolution, the changes in Ca++ level cannot yet be correlated with the onset of cytokinesis. To shed some light on this problem, we decided to combine a number of recently developed imaging techniques to examine the variation of intracellular free Caf+ in zebrafish embryos during early cell cleavage. The major goal was to improve greatly the spatial and temporal resolution so that a more definitive Ca++ signal in association with cytokinesis could be characterized. Our approach was to micro-inject a Ca++ indicator probe, Calcium Green-l conjugated to 10 kD dextran (from Molecular Probes, final concentration = 2-5 FM), into the embryo and then to use a laser-scanning confocal microscope (Bio-Rad) to measure the fluorescent signal; such signals can be converted into Ca++ concentrations with a standard calibration curve. This method has several advantages. First, because of the conjugation with dextran, our probe is not sequestered and is distributed only in the cytosol. Thus, the observed fluorescent signals reflect more faithfully the cytosolic free Ca++ concentration ([Ca”]i). Second, a relatively large dose of Calcium Green-l can be injected into the zebrafish embryo without affecting its normal development. Thus, a high signal-to-noise ratio can be obtained. Also, the measurement can be done at a shorter time, thus providing better temporal resolution. Third, using the confocal microscope, we can measure the variation of [Ca”]i within a specific optical section; thus the spatial resolution can be greatly improved. Using this method, we have examined the spatial and temporal variation of [Ca++li in the zebrafish embryo during the second and third cell cleavages. Our findings, based on 24 experiments, can be summarized as follows: