CALCIUM INVOLVEMENT IN DEVELOPMENT OF CELLULAR POLARITY

重力可以诱发多种发育事件 以及许多不同种类细胞的生长反应。 一个单一的 细胞感受重力，并将这种刺激转化为信号， 两极化反应仍然是个谜。 在这里我们第一时间报道 重力可以阻碍细胞内 信号离子，钙离子，进出单个细胞。 水蕨 是一种水生蕨类植物，能产生大的单细胞孢子。 后 这些孢子的萌发是由光引发的， 一段有限的时间，通常是24小时，在此期间，重力可以 固定他们的发展极性。 极性固定的结果 细胞核向下移动， 细胞分裂的位置是不对称的，并垂直于 重力矢量，产生的两种细胞类型以相反的方向生长 与重力矢量平行的方向。 调查是否 钙参与了这一极化事件，我们使用了 自参比钙选择性电极（Kuhtreiber和Jaffe， 1990），并记录了细胞内钙的净运动 萌发后孢子顶部、侧面和底部的膜 被发起。 在细胞的顶部可以看到强烈的钙外流。 孢子，其在约6小时后急剧增加， 开始发芽。 还有一个钙外流从 孢子的两侧，但它是20倍小于顶部流出。 在孢子底部观察到钙离子的流入，在2 几个小时后到达顶峰。 可见钙离子运动 仅在萌发开始后的前24小时内，之后 它在所有三个点都下降到较低的稳态水平。 因此 钙电流的最大极化周期与 在此期间，孢子的发育极性是 由重力固定。 为了证实钙离子的极化运动 确实归因于引力而不是内在极性 孢子被种在铁丝网里，以使它们被锁在一个 固定方向。 再次在孢子顶部进行测量 显示有大量钙离子流出 铁丝网随后被“翻转” 1800年，在5分钟内，它花了建立一个新的稳定 记录，重新建立了相同的钙极性，显示了 在新定位的细胞顶部具有相同的高水平流出。 这种反应不受单向白色光照射的影响， 在孢子上的不同位置，从而消除了 光的反应。 为了测试这种反应的特异性 钙，我们利用了H+探针，并检查了H+的相对通量 离子在同一时间内。 结果表明 在诱导细胞萌发后的第一天， 细胞外钙离子的运动是极化的，在细胞内最强。 与重力矢量相反的方向。 这表明 当重力固定细胞的极性时， 沿着细胞的顶部和侧面激活钙泵沿着， 诱导钙通道沿底部沿着开放，导致 从细胞底部流向顶部的钙电流。 因为从顶部和侧面流出的流量比 从底部涌入，一些内部钙储存的释放 可能参与维持电流。 因为这 极化电流在重定向后发展得如此之快， 可能是最早的细胞水平反应之一 并可能在指导随后的极地事件中发挥重要作用， 例如核的向下迁移。 的事实 电流的大小在 细胞的发育极性已被固定，这表明 这股电流在建立极性中的作用。

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.