The force of gravity can induce a variety of developmental events and growth responses in many different kinds of cells. How a single cell senses gravity and transduces this stimulus into a signal for a polarized response remains a mystery. Here we report first-time evidence that gravity can polarize the flow of the intracellular signaling ion, calcium, into and out of a single cell. Ceratopteris is an aquatic fern that generates large, single celled spores. After the germination of these spores has been initiated by light, there is a limited period of time, usually 24 hours, during which gravity can fix their developmental polarity. The results of polarity fixation are that the cell nucleus migrates downward, the plane of the first cell division is positioned asymmetrically and perpendicular to the vector of gravity, and the two cell types produced grow in opposite directions parallel to the vector of gravity. To investigate whether calcium is involved in this polarization event, we used a self-referencing calcium selective electrode (Kuhtreiber and Jaffe, 1990) and recorded the net movement of calcium acrosss the cell membrane at the top, side, and bottom of the spore after germination was initiated. A strong efflux of calcium was seen at the top of the spore, which increased sharply at approximately 6 hours after germination was initiated. There was also a calcium efflux from the sides of the spore, but it was 20 folds smaller than the top efflux. An influx of calcium was seen at the bottom of the spore peaking at 2 hours after the peak at the top. The movement of calcium was seen only in the first 24 hours after germination initiation, after which it declined at all three points to low steady state levels. Thus the period of maximal polarization of the calcium current coincides with the period during which the developmental polarity of the spores was fixed by gravity. To verify that this polarized movement of calcium was indeed attributable to gravity and not to the intrinsic polarity of the cell, spores were sown in a wire mesh to keep them locked in a fixed orientation. Measurements taken at the top of the spore again revealed a large efflux of calcium. The wire mesh was then """"""""flipped"""""""" 1800, and within the 5 minutes it took to establish a new stable recording, the same calcium polarity was reestablished, showing the same high level of efflux at the newly positioned top of the cell. This response was unaffected by shining unidirectional white light at various positions onto the spores thereby eliminating the possibility of light response. To test the specificity of this response for calcium, we utilized an H+ probe and examined the relative flux of H+ ions during the same period of time. The results demonstrate that during the first day after the cells are induced to germinate, the movement of calcium out of the cell is polarized and is strongest in a direction that opposes the vector of gravity. This suggests that while gravity is fixing the polarity of the cells it is also activating calcium pumps along the top and sides of the cell and inducing calcium channels to open along the bottom, resulting in a calcium current that moves from the bottom to the top of the cell. Because the efflux from the top and sides is much greater than the influx from the bottom, some release of internal calcium stores probably participates in sustaining the current. Because this polarized current develops so rapidly after reorientation it is probably one of the earliest cell-level responses induced by gravity and could play an important role in guiding subsequent polar events, such as the downward migration of the nucleus. The fact that the magnitude of the current drops off dramatically after the developmental polarity of the cell has been fixed suggests a probable role for this current in establishing that polarity.
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