Fine Structure of Colpoda steinii During Encystment and Excystment

SYNOPSIS. The encystment and excystment of Colpoda steinii was examined by electron microscopy. Cellular organelles including cilia are retained in the cyst without any fundamental alteration in structure. During encystment, the cell becomes surrounded by 2 coats, the inner of which is the more substanial and regular and is about 1600 A or more thick. It is probably formed in the main from material contained in bodies which have no obvious structure and which may be seen in the cell during cyst formation. Discharging vacuoles containing sheet-like material, probably derived from ingested bacteria, are particularly visible during encystment but probably play no direct role in the formation of the main cyst coat. During excystment, this coat is eroded away and, when it becomes thin enough, the motile cell bursts its way out.     

Protein phosphorylation in encystment-induced Colpoda cucullus: localization and identification of phosphoproteins

In Colpoda cucullus, the morphogenetic transformation was preceded by an enhancement of the in vivo protein phosphorylation level. Immunofluorescence microscopy using antiphosphoserine antibody showed that these phosphorylated proteins were localized in the macronucleus and other cytoplasmic regions. Biotinylated Phos-tag/ECL assays of isolated macronuclei showed that a 33-kDa protein (p33) was localized within them. The p33 obtained from isolated macronuclei was tentatively identified as ribosomal P0 protein by LC-MS/MS analysis. In addition, among the encystment-specific phosphoproteins obtained by phosphate-affinity chromatography, the 29-, 31-, and 33-kDa proteins (p29, p31, and p33) were tentatively identified as ribosomal P0 protein, whereas the 24-kDa phosphoprotein (p24) was tentatively identified as ribosomal S5 protein.     

Signaling in temperature-induced resting cyst formation in the ciliated protozoan Colpoda cucullus

The terrestrial ciliated protozoan Colpoda cucullus inhabits soil. When the habitat conditions become unfavorable, the vegetative cells of C. cucullus quickly transform into resting cysts. C. cucullus culture is established in our laboratory, and encystment is routinely induced by the addition of Ca²⁺ to overpopulated vegetative cells. However, an increase in Ca²⁺ concentration and overpopulation of vegetative cells do not always occur in natural. We investigated the effect of temperature and found that cyst formation was induced by a rapid increase of 5 °C within 2 min but not by a decrease. Moreover, an increase in intracellular Ca²⁺ concentrations is essential, but Ca²⁺ inflow does not necessarily occur during encystment. Ca²⁺ image analysis showed that Ca²⁺ is stored in vesicular structures and released into the cytoplasm within 60 s after temperature stimulation. Multiple signaling pathways are activated after the release of Ca²⁺ from vesicles, and cAMP is a candidate second messenger with a crucial role in the process of temperature-induced encystment. Further studies are needed to clarify the mechanism underlying the sensing of temperature and release of Ca²⁺ from vesicles.     

Identification of Differentially Expressed Water‐insoluble Proteins in the Encystment Process of Colpoda cucullus by Two‐dimensional Electrophoresis and LC‐MS/MS Analysis

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h–1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water‐insoluble proteins by two‐dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween‐80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60‐kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55‐kDa protein (p55; actin) and a 49‐kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50‐kDa protein (p50d; α‐tubulin), a 25‐kDa protein (p25; α‐tubulin) and a 52‐kDa protein (p52c; β‐tubulin) was enhanced.     

Culture Age, Intracellular Ca2+ Concentration, and Protein Phosphorylation in Encystment-Induced Colpoda cucullus

In Colpoda cucullus, intracellular Ca²⁺ mediates the encystment induction and protein phosphorylation that occur just prior to morphogenetic transformation into the resting form. When rapidly growing cells were stimulated to encyst, encystment was not readily induced, and the protein phosphorylation level was lower. On the other hand, in post-growing cells stimulated to encyst, the encystment rate and protein phosphorylation level were elevated. These results suggest that protein phosphorylation is closely linked to encystment induction. Why, then, are the protein phosphorylation level and encystment rate difficult to elevate in the rapidly growing cells? Fura 2 ratiometry showed that the intracellular Ca²⁺ concentration (F₃₄₀/F₃₈₀ ratio) was raised in rapidly growing cells as well as in post-growing cells when the cells were stimulated to encyst. It is presumed that the Ca²⁺-mediated signal transduction pathways for protein phosphorylation and encystment may be triggered in rapidly growing cells, but downstream certain steps may be suppressed by certain intracellular components.     

Chromatin extrusion in resting encystment of Colpoda cucullus: a possible involvement of apoptosis-like nuclear death

The extrusion of macronuclear chromatin is a remarkable characteristic during encystment in Colpoda, but the biological significance of this phenomenon has not been fully elucidated. Here we demonstrate that chromatin extrusion occurs with high frequency when encystment was induced by increasing Ca(2+) in growing cells in various stages of the cell cycle. The Feulgen-DNA reaction revealed that vegetatively growing cells have more macronuclear DNA than cells in the stationary phase, suggesting an association of macronuclear DNA content with the execution of chromatin extrusion. Using 4',6-diamidino-2-phenylindole (DAPI), we found that the size of the macronuclear extrusion body was reduced with time and eventually disappeared approximately 24h after encystment induction. In addition, oligonucleosome-sized DNA cleavage was confirmed to occur concomitant with the size reduction, suggesting that the extrusion body is selectively degraded, while the normal macronucleus remains alive. Combined use of acridine orange and Hoechst 33342 demonstrated that the extruded body was increasingly acidified before final resorption. These features are reminiscent of the nuclear degradation process in conjugating Tetrahymena, and therefore we conclude that chromatin extrusion in Colpoda might occur to adjust the macronuclear DNA content prior to encystment. In this way, it is similar to the apoptotic-like nuclear death that occurs during the conjugation of other ciliates.     

Cyst Wall Protein Synthesis and Some Enzyme Changes on Starvation and Encystment in Colpoda steinii

SYNOPSIS. During starvation-induced encystment, Colpoda steinii loses some 30% of its nitrogen before synthesizing a glutamic acid-rich protein coat, which after 24 hr accounts for 18% of the cyst protein. Settling cells contain 29 ± 2 pg/cell of glutamic acid (free acid plus that released on hydrolysis) whilst encysted cells contain 51 ± 3 pg/cell, the coat glutamic acid being adequate to account for the increase. Thus substantial glutamic acid and protein biosynthesis occur during starvation. Assayed in homogenates, some relevant enzymes appeared to decrease rather than increase in activity as encystment proceeded. Intra-cellular proteolytic activity showed little alteration but ribonuclease, acid phosphatase, L-alanine: 2-oxoglutarate aminotransferase (E.C.2.6.1.2) and L-glutamate:NAPD oxidoreductase (E.C.1.4.1.4) were considerably reduced. The total carbohydrate content of the cell also increased during starvation.     

Identification of Precystic Stages and Encystment Kinetics Analysis of Colpoda inflata by Using an Immunofluorescent Method

ABSTRACT. The application of an immunocytochemical method to identify precystic stages and to analyse the encystment kinetics, by using a polyclonal antiserum against isolated cyst walls from the ciliate Colpoda inflata , is reported for the first time. Three different precystic phases were chosen on the basis of morphological changes and degree of cyst wall formation. By using this procedure a better identification of mature resting cysts with regard to precystic cells or young cysts is provided. An average consensus encystment kinetics of C. inflata , by using an accumulated class frequency analysis, is reported.     

Ca(2+)-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca²⁺ and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca²⁺ or without the addition of Ca²⁺. Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca²⁺/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca²⁺ inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca²⁺-triggered signaling pathways, may be involved in encystment induction.     

Ultrastructural Changes During Encystment of Schizopyrenus russelli*

Ultrastructural changes associated with the encystment of Schizopyrenus russelli have been studied by electron microscopy. Before encystment small “black bodies” appear in the cytoplasm and later migrate toward the periphery. The outer cyst wall is secreted at this stage as a thin discontinuous layer which thickens and subsequently becomes continuous. Concomitant with this, the endoplasmic reticulum surrounds the mitochondria. The inner cyst wall later appears as a multilayered structure which presumably is cast off from the plasma membrane. Between the inner and outer layers of the cyst wall, there is a middle, less electron-dense layer wherein extruded cytoplasmic material is found embedded at certain places.     

Effect of Tricarboxylic Acid Cycle Intermediates and Related Compounds on the Respiratory Rate of Urostyla cristata and Colpoda cucullus*

SYNOPSIS. The respiratory rates of vegetative forms of Urostyla cristata and vegetative and encysted Colpoda cucullus were measured in balanced salt solution and after addition of Na salts of various organic acids, including Krebs- and glyoxylic-cycle intermediates. The results point to some peculiarities in Urostyla metabolism; it was poisoned by succinate—an effect partly abolished by malonate; respiration was stimulated by malonate. Respiration of vegetative Colpoda was accelerated by Krebs- and glyoxylic-acid cycle intermediates. Most of these intermediates inhibited respiration of Urostyla. In experiments of short duration respiration of encysted Colpoda was not stimulated by these intermediates.     

Differentiation During Encystment and Excystment in Oxytricha fallax*

During encystment of Oxytricha fallax, a wall composed of 4 distinct layers, each derived from a different kind of endoplasmic vesicle, is formed between the 2 unit membranes that cover the vegetative cell. Numerous autophagic vacuoles arise in the endoplasm and later (during excystment undergo internal changes comparable to those characteristic of food vacuoles. Mitochondria aggregate into a band. The 2 macronuclei fuse and their nucleoli become homogeneous. Except for the 2 cell membranes, all visible cortical structures, including cilia, kinetosomes, and microtubules, disappear. Despite the absence of visible ciliature in the mature cyst, the various primordia of the normal vegetative ciliature arise during excystment in the same positional relations to one another as is characteristic of developments during cell division.     

Sequential Metabolic Events During Encystment of Azobacter vinelandii

Cells of Azotobacter vinelandii are specifically induced to encyst by beta-hydroxybutyrate (BHB). The process of differentiation, which occurs over a period of 36 h, was characterized by an ordered sequence of biochemical events. Upon initiation of encystment, nitrogen fixation and glucose-6-phosphate dehydrogenase activities decreased immediately to very low levels. This was followed by an increase in the specific activities of BHB dehydrogenase, isocitrate dehydrogenase, isocitrate lyase, and malate synthase first at 3 h and then again at 21 h. The peak activity of fructose 1,6-diphosphate aldolase occurred at 6 h, and the enzyme activity then decreased gradually. Fructose 1,6-diphosphatase had peak activities at 9 and 27 h. Deoxyribonucleic acid synthesis ceased just prior to the final cell division at 4 to 6 h, but ribonucleic acid synthesis continued until the 12th h. From labeling studies and the appearance of new enzyme activities, it appeared that protein synthesis continued throughout encystment.     

Morphology and Life History of Colpoda maupasi, Bensonhurst Strain

Strains of Colpoda maupasi previously reported were found to produce only octogenic reproductive cysts and monogenic wrinkled resting cysts. The present form of C. maupasi (Bensonhurst strain), isolated by the senior author in 1949, was found to produce, in addition to the above, quadrigenic reproductive cysts, digenic corrugated (wrinkled) resting cysts, and smooth thick-walled monogenic, digenic and quadrigenic resting cysts. Some of the factors leading to the development of these cysts in the Bensonhurst strain are believed to be related to nutrition, age and size of the trophic forms. The cytological changes in encystment and excystment were followed with particular attention to aging monogenic resting cysts. The latter were observed for over 4 years.