CALCIUM INVOLVEMENT IN DEVELOPMENT OF CELLULAR POLARITY

地心引力可以引发各种发育事件。 在许多不同种类的细胞中的生长反应。怎么一首单曲 细胞感知重力并将这种刺激转换为信号 两极分化的反应仍然是一个谜。在这里我们是第一次报道 重力可以使细胞内的流动极化的证据 信号离子，钙，进入和离开单个细胞。金龟属 是一种水生蕨类植物，能产生大的单细胞孢子。之后 这些孢子的萌发是由光引发的，有 一段有限的时间，通常是24小时，在此期间，重力可以 修复他们的发育两极。极性固定的结果 是细胞核向下迁移，第一个平面 细胞分裂的位置不对称，并垂直于 重力矢量，产生的两种细胞类型以相反的方式生长 方向与重力向量平行。调查是否 钙参与了这一极化事件，我们使用了 自参照钙选择电极(Kuhtreiber和Jaffe， 1990)，并记录了细胞内钙的净移动 萌发后孢子顶端、侧端和底端的膜 被启动了。在牙槽顶部可见一股强烈的钙外流。 孢子，在大约6小时后急剧增加 萌发开始了。此外，还有一种钙外流。 孢子的侧面，但比顶端的排泄物小20倍。 在孢子底部可见钙离子流入，在2时达到峰值。 在顶峰几个小时后。可以看到钙的移动。 仅在萌发开始后的前24小时内，之后 这三个点都下降到了较低的稳态水平。因此， 钙电流的最大极化周期与 分生孢子发育的极化期 靠重力固定的。来验证钙离子的这种极化运动 确实归因于重力而不是内在的极性 在细胞中，孢子被播种在铁丝网中，以保持它们被锁在 固定方向。再次在孢子顶端进行测量 显示出大量的钙外流。然后，铁丝网被“翻转”。 1800年，在5分钟内建立了一个新的马厩 记录，重新建立相同的钙极性，显示 在新放置的单元格顶部有同样高水平的外流。 这种反应不受单向白光照射的影响 在孢子上的各种位置，从而消除了 对光的反应。以测试此响应的特异性 钙，我们使用了H探针并检测了H的相对通量 离子在同一时间段内。结果表明， 在细胞被诱导萌发后的第一天， 钙离子从细胞外的运动是极化的，在 与重力矢量相反的方向。这表明 当重力固定细胞的极性时，它也 激活细胞顶部和两侧的钙泵，并 诱导钙通道沿着底部打开，导致 从细胞底部向顶部移动的钙电流。 因为从顶部和侧面流出的流量远远大于 从底部涌入，一些内部的钙库释放出来 可能参与了维持海流的过程。因为这件事 极化电流在重新定向后发展得如此之快 可能是重力引起的最早的细胞水平反应之一 并可能在指导随后的极地事件方面发挥重要作用， 比如原子核的下移。事实是， 电流的大小在 细胞的发育极性已经确定，这表明 这股电流在建立这种两极中的作用。

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.