Osmoregulation and Contractile Vacuole Function

渗透调节是所有细胞建立水盐平衡的过程，使细胞的细胞质对外部环境保持轻微的高渗或等渗。在淡水原生动物中，通常被认为负责渗透调节的细胞器是可收缩液泡复合体（CVC），它被认为分泌低渗溶液。然而，由于细胞膜对水是可渗透的，这些细胞或任何细胞如何能够沿着水的浓度梯度向上积聚、储存和排出水或低渗溶液仍然是一个悖论。本项目采用电生理、免疫细胞化学和细胞生物学等方法对CVC细胞器系统进行研究。所选择的模式生物是草履虫，它是一种纤毛虫，具有以下优点：(1)具有可识别且独立的CVC区室，用于液体摄取、液体运输和液体储存/排出；(2)便于测定细胞总液体输出。宿主实验室对该细胞及其CVC有丰富的经验，详细研究了其CVC的超微结构。从初步工作中获得了针对该纤毛虫CVC不同部分的单克隆抗体。了解渗透调节是如何在“原始”单细胞真核生物中实现的，将建立多细胞生物在进化过程中形成自己的渗透调节系统的基本原理。(1)离子选择微电极用于测定体内CV液的离子含量。(2)测量了CV膜的电势、电容和离子电导，并与质膜进行了比较。膜离子通道和电致泵活性在电生理学和药理学上有区别，使用特定的离子通道和电致泵阻滞剂。在分离的CV膜上使用贴片夹紧来确定单个通道或泵ps的性质。(3)使用针对泵的特定多肽的单克隆抗体确认15 nm的“peg”结构代表v型H+ atp酶。利用分子生物学技术对含有该泵的小管进行了分离和表征。(4)提出了流体分离机理。研究了渗透胁迫对CVC的影响，以确定这些影响是否与机制一致。这些研究的结果有助于理解这种单细胞淡水真核生物渗透调节的基本机制，并为理解CVC调节提供了一个开始，并为最终理解高等动物渗透调节的进化发展途径提供了一步。即使细胞生活在恶劣的环境中，细胞也必须将盐和水的含量维持在有利于生命的浓度。如果这些浓度偏离正常水平，细胞将无法继续其代谢功能，并将死亡。这个项目是对生活在淡水中的单细胞如何能够分泌水来维持水和盐的适当平衡的探索。原生动物有一个可收缩的液泡系统（CVC），大概就是它在做这个工作。然而，尚不清楚的是水是如何在CVC中积累的，因为包括CVC周围的所有膜都是可渗透的。解决这个矛盾的一种方法是，细胞将盐泵入CVC，以保持CVC内的溶液与细胞质的盐浓度相等。这样，水就会进入CVC，水就会回流到c细胞质中。然后，水和盐都会从细胞中分泌出来。然而，细胞能够不断地排泄什么盐仍然是未知的。因此，进行调查以确定淡水原生动物草履虫中CVC的含量，从而确定细胞通过其CVC排泄的物质。(1)将离子选择微电极插入活的CVC中，测量不同离子的浓度。(2)微电极还用于测定CVC膜的电势、电阻和电容。关于离子通道和离子泵类型的线索，将通过使用阻断特定通道或泵的药物进行实验。单个离子载体的生物化学可以通过分析孤立膜片（膜片夹紧）的电学性质来确定。(3)使用针对泵组分的单克隆抗体，可以直观地验证泵的身份。(4)提出并验证了CVC在这种原生动物中的作用机制。这项研究为我们提供了关于渗透调节（细胞中盐和水的平衡）在自然界中是如何实现的线索，以及这个过程是如何从“简单”的生命形式进化到高等生物目前的状态的。

9505910 Allen Osmoregulation is the process by which all cells establish their water and salt balance keeping the cell's cytosol slightly hypertonic or isotonic to its external environment. In fresh water protozoa the organelle generally regarded to be responsible for osmoregulation is the contractile vacuole complex (CVC) which is thought to secrete a hypotonic solution. However, as cellular membranes are permeable to water, how these or any cells can accumulate, store and expel water or hypotonic solution uphill against a concentration gradient of water remains a paradox. This project studies the poorly understood CVC organelle system with electrophysiological, immunocytochemical and cell biological methods. The model organism selected is Paramecium, a ciliate which offers the advantages (1) of having recognizable and separate CVC compartments for fluid uptake, fluid transport and fluid storage/expulsion and (2) of allowing an easy assay for determining total cellular fluid output. The host laboratory has extensive experience with this cell and its CVC, having studied in detail its CVC ultrastructure. Monoclonal antibodies to different parts of the CVC of this ciliate are available from preliminary work. An understanding of how osmoregulation is achieved in "primitive" single-celled eukaryotes will establish the fundamental principles on which multicellular organisms may have fashioned their own osmoregulatory systems as they evolved. (1) Ion-selective microelectrodes are used to determine the ionic contents of the in vivo CV fluid. (2) The electrical potential, capacitance, and ionic conductance of the CV membrane is measured and compared with the values found for plasma membrane. Membrane ion channels and electrogenic pump activity are discriminated electrophysiologically and pharmacologically, usine specific ion channel and pump blockers. Patch-clamping is used on isolated CV membrane to determine the properties of individual channels or pum ps. (3) Confirmation that 15 nm "peg" structures represent the V-type H+ ATPase is made using monoclonal antibodies to specific polypeptides of the pump. The tubules containing the pump are isolated and characterized using the techniques of molecular biology. (4) A fluid segregation mechanism is proposed. The effects of osmotic stress on the CVC is studied to see if these effects are consistent with the mechanism. The outcome of these studies contribute to an understanding of the basic mechanism of osmoregulation in this single-celled fresh- water eukaryote as well as give a start on understanding CVC regulation and provides a step in the eventual understanding of the path of evolutionary development of osmoregulation in higher animals. %%% Cells must maintain their content of salts and water at a concentration which is favorable for life even though the cells live in a harsh environment. If these concentrations waver from the norm, the cell will not be able to carry on its metabolic functions and will die. This project is an exploration of how a single cell living in fresh water is capable of secreting water to maintain the proper balance of water and salts. Protozoa have a contractile vacuole system (CVC) which presumably does this job. However, what is not understood is how the water is accumulated within this CVC since all membranes including those surrounding the CVC are permeable to water. One way around the paradox would be for the cell to pump salts into the CVC to maintain a solution within the CVC equal in salt concentration to the cell's cytoplasm. In this way water would enter the CVC and there would be net backflow of water into the c cytoplasm. Both water and salts would then be secreted from the cell. However, what salts the cell is capable of continually excreting remains unknown. Thus the investigation is undertaken to determine what the contents of the CVC are in the fresh water protozoan Paramecium, thereby determining what the ce ll is excreting through its CVC. (1) Ion- selective microelectrodes are inserted into a living CVC to measure the concentration of different ions. (2) Microelectrodes are also used to determine the electrical potential, resistance, and capacitance of the CVC membrane. Clues about the types of ion channels and ion pumps driven by the electrical potential across the membrane will be followed-up with experiments using drugs which block specific channels or pumps. The biochemistry of individual ion carriers can be determined using by assaying the electrical properties in an isolated patch of membrane (patch clamping). (3) Using monoclonal antibodies against pump components, the identity of the pump can be verified visually. (4) A probable mechanism of how the CVC works in this protozoa is suggested and tested. This study gives us clues about how osmoregulation (the balance of salt and water in cells) is made possible in nature and how this process may have evolved from "simpler" forms of life to its present state in higher organisms.