Osmoregulation and Contractile Vacuole Dynamics

作为原生动物的细胞器，收缩液泡（CVs）已经被发现了200多年，但它们的功能直到现在才被发现。该实验室最近对草履虫的研究表明，CVs含有一种渗透物的高渗溶液，其中K+和Cl-离子最为丰富。由于细胞质的高渗，多余的水可以通过渗透从细胞质进入CV。研究还表明，CV是一种独特的细胞器，它经历周期性的圆缩和松弛循环，显然没有收缩细胞骨架系统的帮助。CV的膜包含一个固有的定时机制，触发CV舍入，伴随着膜张力增加35到50倍。在此过程中，多余的CV膜在地形上转化为直径40纳米的管，与CV保持连续。本研究将尝试(1)开发一种微毛细管渗透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管腔以建立高渗梯度，(3)了解质子转运v - atp酶如何被用来为细胞器提供能量，(4)确定CV如何如此戏剧性地增加其膜张力。为了进行这些研究，将CV膜的生物化学特性与草履虫中的其他膜进行比较，以确定这种膜与那些不能圆缩和张力发展的膜有何不同。膜片钳电生理学将被用于表征这种技术可以检测到的CV膜中的离子通道、水通道和电致泵的类型。我们将继续研究原生动物和藻类的渗透调节细胞器——可收缩液泡的功能。渗透调节是在细胞和环境之间平衡水和盐的过程。膜片钳电生理学和生化研究将帮助我们了解淡水原生动物如何在水不断进入细胞质的环境中解决生命问题。了解“原始”细胞中的水分调节可能为了解更高级的生物体中的水分调节提供线索。

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.