Osmoregulation and Contractile Vacuole Dynamics

收缩泡（Contractile vacuoles，CV）作为原生动物的细胞器，已被发现200年，但其功能直到现在才被发现。 本实验室最近对草履虫的研究表明，CV含有渗透剂的高渗溶液，其中K+和Cl-离子是最丰富的。 由于对细胞质具有高渗性，过量的水可以通过渗透作用从细胞质进入CV。 它还表明，CV是一个独特的细胞器，经历周期性的圆形和松弛周期，显然没有一个收缩的细胞骨架系统的帮助。 CV的膜包含一种先天的计时机制，该机制触发CV变圆，伴随着膜张力增加35至50倍。 在该圆化过程中，过量CV膜在形貌上转化为与CV保持连续的40 nm直径的管。本研究将尝试（1）开发一种微毛细管渗透压计来测量细胞内和细胞器内的总渗透压，（2）确定K+和Cl-离子如何穿过CV膜进入CV腔以建立这种高渗梯度，（3）观察质子移位的V-ATP酶如何用于为该细胞器供能，以及（4）确定CV如何如此显著地增加其膜张力。 为了进行这些研究，CV膜的生物化学将与草履虫中的其他膜进行比较，以确定该膜与那些不能变圆和张力发展的膜有何不同。 膜片钳电生理学将用于表征CV膜中存在的可用该技术检测的离子通道、水通道和生电泵的类型。 对原生动物和藻类的调节细胞器--收缩泡功能的研究将继续进行。 渗透调节是平衡细胞与其环境之间的水和盐的过程。 膜片钳电生理学和生物化学研究将帮助我们了解淡水原生动物如何解决水不断进入细胞质的环境中的生命问题。 了解“原始”细胞中的水调节可能会为更高级生物的水调节提供线索。

Contractile vacuoles (CVs), as cellular organelles of protozoa, have been known for 200 years but how they function is only now being discovered. Recent work on Paramecium from this laboratory shows that the CVs contain a hypertonic solution of osmolytes of which K+ and Cl- ions are the most abundant. Being hypertonic to the cytosol, excess water can enter the CV by osmosis from the cytoplasm. It has also been shown that the CV is a unique organelle that undergoes periodic rounding and relaxing cycles, apparently without the aid of a contractile cytoskeletal system. The membrane of the CV contains an innate timing mechanism that triggers CV rounding that is accompanied by a 35- to 50-fold increase in membrane tension. During this rounding excess CV membrane is topographically transformed into 40-nm diameter tubes that remain continuous with the CV. This research will attempt to (1) develop a microcapillary osmometer to measure the total intracellular and intraorganellar osmolarities, (2) determine how K+ and Cl- ions cross the CV membrane into the CV lumen to set up this hypertonic gradient, (3) see how the proton-translocating V-ATPases are used to energize this organelle and (4) determine how the CV can increase its membrane tension so dramatically. To carry on these studies the biochemistry of the CV membrane will be compared with other membranes in Paramecium to determine how this membrane differs from those membranes that are incapable of rounding and tension development. Patch-clamp electrophysiology will than be used to characterize the types of ion channels, water channels and electrogenic pumps present in the CV membranes that can be detected with this technique. A study of the function of contractile vacuoles, osmoregulatory organelles of protozoa and algae, will be continued. Osmoregulation is the process of balancing water and salts between cells and their environments. Patch clamp electrophysiology and biochemical studies will help us understand how fresh water protozoa have solved the problem of life in an environment where water continuously enters the cell's cytoplasm. Understanding water regulation in "primitive" cells is likely to provide clues on water regulation in much more advanced organisms.