Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins

The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. We produced rabbit antisera against isolated ciliary membranes and against partially purified immobilization antigens derived from three serotypes (A, B, and H), and used these antisera as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross- reactivities of the antisera were examined. We identified the antigens recognized by these sera using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), approximately 250,000 mol wt. Several secondary antigens, including a family of polypeptides of 42,000-45,000 mol wt, are common to the membranes of serotypes A, B, and H, and antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for our studies on the effects of the antibodies on electrophysiological properties of cells and electron microscopic localization studies, which are reported in the accompanying paper. We have also used these antibodies to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i- antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. We conclude that antigen-antibody interaction per se is not sufficient for immobilization, and that antibody bivalency, which allows antigen cross-linking, is essential.     

Endosymbionts in paramecium

Paramecium species are extremely valuable organisms to enable experiments for the reestablishment of endosymbiosis. This is investigated in two different systems, the first with Paramecium caudatum and the endonuclear symbiotic bacterium Holospora species. Although most endosymbiotic bacteria cannot grow outside the host cell as a result of their reduced genome size, Holospora species can maintain their infectivity for a limited time. We found that an 89-kDa periplasmic protein has an important function for Holospora's invasion into the target nucleus, and that Holospora alters the host gene expression; the host thereby acquires resistance against various stresses. The second system is the symbiosis between P. bursaria and symbiotic Chlorella. Alga-free P. bursaria and the algae retain the ability to grow without a partner. Consequently, endosymbiosis between the aposymbiotic host cells and the symbiotic algae can be reestablished easily by mixing them. We now found four checkpoints for the reestablishment of the endosymbiosis between P. bursaria and the algae. The findings in the two systems provide excellent opportunities for us to elucidate not only infection processes but also to assess the associations leading to eukaryotic cell evolution. This paper summarizes recent progresses on reestablishment of the primary and the secondary endosymbiosis in Paramecium.     

Ionic regulation of cyclic AMP levels in Paramecium tetraurelia in vivo

cAMP levels in Paramecium increased dose dependently after a step increase of [Ca] or [Sr] in the incubation, provided K was present. Two mM Ca or Sr tripled cAMP concentrations within 3 s and induced an increase in forward swimming speed. The increase in cAMP formation was strictly dependent on the Donnan ratio [K]: square root [Ca]. Na, Li, or tetraethylammonium could not replace K. The data provide evidence for regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the K: Ca ratio.     

Control of exocytotic processes: cytological and physiological studies of trichocyst mutants in Paramecium tetraurelia

The trichocysts of Paramecium tetraurelia constitute a favorable system for studying secretory process because of the numerous available mutations that block, at various stages, the development of these secretory vesicles, their migration towards and interaction with the cell surface, and their exocytosis. Previous studies of several mutants provided information (a) on the assembly and function of the intramembranous particles arrays in the plasma membrane at trichocyst attachment sites, (b) on the autonomous motility of trichocysts, required for attachment to the cortex, and (c) on a diffusible cytoplasmic factor whose interaction with both trichocyst and plasma membrane is required for exocytosis to take place. We describe here the properties of four more mutants deficient in exocytosis ability, nd6, nd7, tam38, and tam6, which were analyzed by freeze-fracture, microinjection of trichocysts, and assay for repair of the mutational defect through cell-cell interaction during conjugation with wild-type cells. As well as providing confirmation of previous conclusions, our observations show that the mutations nd6 and tam6 (which display striking abnormalities in their plasma membrane particle arrays and are reparable through cell-cell contact but not by microinjection of cytoplasm) affect two distinct properties of the plasma membrane, whereas the other two mutations affect different properties of the trichocysts. Altogether, the mutants so far analyzed now provide a rather comprehensive view of the steps and functions involved in secretory processes in Paramecium and demonstrate that two steps of these processes, trichocyst attachment to the plasma membrane and exocytosis, depend upon specific properties of both the secretory vesicle and the plasma membrane.     

Ionic regulation of adenylate cyclase from the cilia of Paramecium tetraurelia.

The kinetics of the ionic regulation of an adenylate cyclase associated with the excitable ciliary membrane from Paramecium tetraurelia was examined. Glycerol (30%, v/v) stabilized the enzyme, and activated by an increase in Vmax. (3-fold) and a decrease in the apparent Km for MgATP (6-fold). Kinetic analysis of Mg2+ effects showed a stimulation via a single metal-binding site separate from the substrate site, with a dissociation constant, Ks, of 0.27 mM. Analysis of Ca2+ effects showed (i) an uncompetitive inhibition with respect to substrate MgATP, and (ii) dependence of the extent of inhibition on the free Mg2+ concentration. Ki values ranged from 4 to 130 microM-Ca2+ in the presence of 0.55-2 mM-Mg2+ respectively. This indicates competition between Mg2+ and Ca2+ at the metal-binding site. The Ca2+ effect was specific; Sr2+ and Ba2+ were almost without effect, and 100 microM-Ba2+ did not interfere with the Ca2+ inhibition. The actions of Ca2+ were readily reversible after addition of EGTA. K+ activated the adenylate cyclase at concentrations around 20 mM. The stimulatory potency of K+ was dependent on the free Mg2+ concentration. At 1 mM free Mg2+, 20 mM-K+ doubled the adenylate cyclase activity. The inhibitory Ca2+ and stimulatory K+ inputs were independent of each other.     

草履虫的冬季培养

草履虫作为原生动物纤毛纲的代表 ,在动物学教学和实验中是必不可少的 ,在遗传学、细胞生物学、生物化学以及生理学等领域内也被广泛采用 ,且需要量大 ,纯度要求高。关于草履虫的培养方法 ,已有诸多报道 ,这里介绍一种冬季在人工控温条件下用熟鸡蛋黄培养草履虫的方法。1　 草履虫的简易纯化与培养取普通多孔水浴锅一只 ,在锅内安装一只暖棒 (恒温加热器 ,温度范围 16 - 32℃ ) ,加水、通电、调温至 2 5℃ ;同时安放 5 0ｍｌ、 10 0 0ｍｌ烧杯各一只 ,内盛自来水 2 / 3左右。将野外采集的草履虫液在水浴锅中放置一周左右 ,取上层含有草履虫…     

植物矮化突变体的激素调控

作者主要从赤霉素（GA）、油菜素内酯（BR）和生长素（IAA）三方面介绍了植物矮化突变体的激素水平变化及其调控,其对其他与激素调控有关的植物筹化体研究进展作了介绍。     

The role of mutants in metabolic regulation

Many Indian students of biochemistry and molecular biology do not appreciate the role of mutants in elucidation of metabolic pathways in general and metabolic regulation in particular. One reason is that a majority of biochemistry and enzymology textbooks used in India do not give adequate emphasis and importance to the role of biochemical genetics in unravelling metabolic pathways and their regulation. Teachers and students depend heavily on these textbooks. Many teachers pay little attention to the experimental strategies employed for the elucidation of metabolic/enzyme regulation. Asking specific questions about metabolic regulation, and requiring the students to read some important and key review articles for the answers could effectively rectify this malady.     

Motor activity of paramecium

A systemic study of the mechanisms of motor activity of paramecium was carried out. The movements of paramecium responding to various influences were photographed. The analysis of the data revealed the time dependences of the rate of movement, rate of rotation, and the radius and the pitch of the helix trajectory. Mathematical models of the membrane and a unit that transforms the calcium signal to programs of regulating the effectors were constructed. A system of equations for constructing the trajectory of movement was proposed. It is concluded that the biomolecular system that involves calmodulin, calmodulin-dependent ionic channels, adenylate cyclase, guanylate cyclase, phosphodiesterases, Ca(2+)-calmodulin, cAMP, cGMP-dependent protein kinases, and phosphoprotein phosphatases is capable of regulating motor reactions necessary for complex maneuvering of paramecium under various conditions.     

Restoration of nucleo-mitochondrial compatibility in paramecium

In Paramecium, as previously described, the nuclear mutation cl(1) is incompatible with wild-type mitochondria (M(+)); however, all cl(1)/cl(1)M(+) cells eventually overcome this incompatibility (Sainsard, Claisse and Balmefrezol 1974). We have studied the kinetics and genetic basis of the spontaneous restoration of harmony between nucleus and mitochondria. We also studied the modification of these kinetics following microinjection of compatible mutant mitochondria into cl(1)/cl(1)M(+) cells. We demonstrate that nucleo-mitochondrial readjustment is always achieved by mitochondrial changes that fall into two classes. The first class corresponds to spontaneous mitochondrial mutations affecting the amount of cytochrome aa(3) and is similar to the previously described M(cl) and M(su) mutations (Sainsard-Chanet 1978; Sainsard 1975). The nature of the second class of modification is not yet understood; it may correspond either to a mitochondrial "adaptation" or to an unusual type of mutation arising and reverting at high frequency.     

Ion channels in the regulation of autophagy

Autophagy is a cellular process in which the cell degrades and recycles its own constituents. Given the crucial role of autophagy in physiology, deregulation of autophagic machinery is associated with various diseases. Hence, a thorough understanding of autophagy regulatory mechanisms is crucially important for the elaboration of efficient treatments for different diseases. Recently, ion channels, mediating ion fluxes across cellular membranes, have emerged as important regulators of both basal and induced autophagy. However, the mechanisms by which specific ion channels regulate autophagy are still poorly understood, thus underscoring the need for further research in this field. Here we discuss the involvement of major types of ion channels in autophagy regulation.     

Molecular genetics of regulated secretion in paramecium

Paramecium is a unicell in which cellular processes are amenable to genetic dissection. Regulated secretion, which designates a secretory pathway where secretory products are first stored in intracellular granules and then released by exocytotic membrane fusion upon external trigger, is an important function in Paramecium, involved in defensive response through the release of organelles called trichocysts. In this review, we focus on recent advances in the molecular genetics of two major aspects of the regulated pathway in Paramecium, the biogenesis of the secretory organelles and their exocytosis.