Cellular dynamics of conjugation in the ciliate euplotes aediculatus. II. Cellular membranes

The formation and subsequent dissolution of a common bridge of cytoplasm between conjugating ciliated protozoan cells provides an excellent opportunity to follow the dynamics of the cellular membrane systems involved in this process. In particular, separation of conjugant partners offers the chance to observe, at a fixed site on the cell surface, how the ciliate surface complex of plasma and alveolar membranes (collectively termed the “pellicle”) is constructed. Consequently, cortical and cellular membranes of Euplotes aediculatus were studied by light and electron microscopy through the conjugation sequence. A conjugant fusion zone of shared cytoplasm elaborates between the partner cells within their respective oral fields (peristomes) to include microtubules, cytosol, and a concentrated endoplasmic reticulum (heavily stained by osmium impregnation techniques) that may also be continuous with cortical ER of each cell. Cortical membranes displacd by fusion are autolyzed in acid phosphatase-positive lysosomes in the fusion zone. As conjugants separate, expansion of the plasma membrane may occur through the fusion of vesicles with the plasma membrane, presumably at bare membrane, presumably at bare membrane patches near the fusion zone. The underlying cortical alveolar membranes and their plate-like contents are reconstructed beneath the plasma membrane, apparently by multiple fusions of dense-cored alveolar precursor vesicles (APVs). These precursor vesicles themselves appear to condense directly from the smooth ER present in the fusion zone. No Golgi apparatus was visible in the fusion zone cytoplasm, and no step of APV maturation that might involve the Golgi complex was noted.     

Cytoskeletal elements in bacteria

It has become clear recently that bacteria contain all of the cytoskeletal elements that are found in eukaryotic cells, demonstrating that the cytoskeleton has not been a eukaryotic invention, but evolved early in evolution. Several proteins that are involved in cell division, cell structure and DNA partitioning have been found to form highly dynamic ring structures or helical filaments underneath the cell membrane or throughout the length of the cell. These exciting findings indicate that several highly dynamic processes occur within prokaryotic cells, during growth or differentiation, that are vital for a wide range of cellular tasks.     

Sequence of the small subunit ribosomal RNA gene from the hypotrichous ciliate Euplotes aediculatus

We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene of the hypotrichous ciliate Euplotes aediculatus. It is 1882 nucleotides long and contains several inserts not present in the small subunit rRNA genes of the hypotrichs Oxytricha nova and Stylonychia pustulata. A comparison of the sequences suggests that E. aediculatus is much less closely related to these other two hypotrichs than they are to each other. Although the gene sequence of E. aediculatus is drifting more rapidly than those of these other two species, its faster evolutionary clock is not enough to account for the degree of difference between them.     

Programmed autophagocytosis accompanying conjugation in the ciliate Stylonychia mytilus

Conjugation and postconjugant development in Stylonychia is accompanied by a period of approximately 80 hr during which the cells are unable to ingest food. This period is one of considerable synthetic activity, encompassing important portions of the development of the new macronucleus. Light- and electron-microscopic observations of conjugating pairs and exconjugant cells reveal a process of autophagocytosis that may provide the supplies of energy (and precursors) necessary for postconjugant developmental events. Small autophagosomes (AP) form in conjugants; degenerating mitochondria are prominent among the inclusions observed in these bodies. Soon after separation of pairs, large dense AP appear, apparently by coalescence and condensation of the smaller AP. These “mature” AP give only a slight acid phosphatase reaction. The number of AP slowly declines during postconjugant development; about 60 hr after separation all the AP have been digested. Several observations suggest that this autophagocytosis is part of the developmental program initiated soon after mating begins: (1) The first indications of AP formation occur while conjugating pairs are still able to feed, and thus cannot be attributed to the stress of starvation; (2) formation of large numbers of AP is rather abrupt, whereas their dissolution is very gradual, covering most of the nonfeeding period; and (3) the pattern of AP formation and dissolution is similar in cells whose new macronuclear development has been prevented by brief hydroxyurea treatment.     

Ca2+-dependent contractility of isolated and demembranated macronuclei in the hypotrichous ciliate Euplotes aediculatus

The hypotrichous ciliated protozoan Euplotes aediculatus possesses a characteristic C-shaped somatic nucleus (macronucleus) within the cytoplasm, which shows dynamic shape change during the cell cycle. It is shown that isolated macronuclei possess Ca(2+)-dependent contractility. Macronuclei were isolated, stuck fast on the glass surface, and subjected to different concentrations of Ca(2+) in a Ca(2+)-EGTA buffer. The nuclei became expanded at [Ca(2+)]<10(-7)M, and they contracted on subsequent addition of higher concentrations of Ca(2+). Cycles of expansion and contraction of the nucleus could be repeated many times by alternate addition of EGTA and Ca(2+), indicating that the size of isolated nuclei can be regulated by [Ca(2+)] alone. The nuclear contraction was observed in all phases of the cell cycle, but contractility was less evident around replication bands in the S phase. In addition to the hypotrichous ciliate Euplotes, similar Ca(2+)-dependent nuclear contractility was found to exist in other cell types, including protozoans of different taxa (a heliozoon Actinophrys sol and a peniculine ciliate Paramecium bursaria), and also mammalian culture cells (HeLa cells). Our findings suggest a possibility that Ca(2+)-dependent nuclear contractility may be shared among diverse eukaryotic organisms.     

Cytoskeletal elements in arthropod sensilla and mammalian photoreceptors.

Ciliary receptor cells, typified by cilia or modified cilia, are very common in the animal kingdom. In addition to the cytoskeleton of their ciliary processes these receptors possess other specific prominent cytoskeletal elements. Two representative systems are presented: i) scolopidia, mechanosensitive sensilla of various arthropod species; and ii) photoreceptor cells of the retina of the bovine eye. Two cytoskeletal structures are characteristic for arthropod scolopidia: a scolopale typifies the innermost auxiliary cell, and long ciliary rootlets are extending well into the sensory cells. The latter element is also characteristic for the inner segment of the photoreceptor cells in bovine. The scolopale of scolopidia is mainly composed of actin filaments. In the absence of myosin, the uniform polarity of the actin filaments and their association with tropomyosin all indicate a stabilizing role of the filament bundles within the scolopale. This function and a certain elasticity of actin filament bundles may be important during stimulation of the sensilla. The ciliary rootlets of both systems originate at the basal bodies at the ciliary base of the sensory cells and project proximally. These rootlets are composed of longitudinally oriented, fine filaments forming a characteristic regular cross-striation. An alpha-actinin immunoreactivity was detected within the ciliary rootlets of scolopidia. In addition, antibodies to centrin react with the rootlets of both types of receptors. Since centrin is largely responsible for the contraction of the flagellar rootlets in green algae, contraction may also occur in the ciliary rootlets of insect sensilla and vertebrate photoreceptors. In both systems, contraction or relaxation of the ciliary rootlets could serve in sensory transduction or adaptation.     

Cytoskeletal dynamics in rabbit synovial fibroblasts: I. Effects of acrylamide on intermediate filaments and microfilaments

Rabbit synovial fibroblasts respond to changes in cell shape and cytoskeletal architecture by altering specific gene expression. We have tested the ability of acrylamide, a neurotoxin that alters the distribution of intermediate filaments in cultured PtK1 cells, to induce metalloprotease expression in synovial fibroblasts. Cells treated with 2-20 mM acrylamide for 5 to 24 h underwent shape changes similar to cells treated with the tumor promoter phorbol myristate acetate. Intermediate filaments visualized with anti-vimentin antibodies did not collapse into a perinuclear cap in these rounded cells, but were still present in the extended cell processes. Unexpectedly, when actin was visualized in acrylamide-treated cells, extensive dissociation and clumping of microfilaments was observed. Concentrations of acrylamide greater than 10 mM were cytotoxic, but cells recovered completely after 24 h incubation with 5 mM acrylamide. Like other agents that alter cell shape and actin distribution in synovial fibroblasts, acrylamide also induced expression of the secreted metalloprotease collagenase. Although some recent evidence suggests that acrylamide may be able to exert its collagenase-inducing effects extracellularly, perhaps through transmembrane matrix receptors, our observation that this neurotoxin dramatically alters protein synthesis in synovial fibroblasts suggests that direct effects on cell metabolism may also play a role in acute acrylamide intoxication.     

The cyclic AMP control system in the development of Dictyostelium discoideum. I. Cellular dynamics.

A model for the synthesis and release of cyclic AMP in aggregating cells of Dictyostelium discoideum is developed. The model shows transitions from low level steady release of cAMP to excitable pulsatile release and then to autonomous periodic pulsatile release of cAMP as starvation proceeds. Finally, there is a transition to high level continuous release of cAMP. A detailed correspondence is drawn between these transitions and the phenomena that are observed to appear sequentially during the aggregation phase, specifically: cloud formation, relaying competence, autonomous competence, and tip activity. The only assumptions necessary to the model are that there is a autocatalytic mechanism for cAMP synthesis, a negative feedback regulation of cAMP through another variable C, and a source term for C that declines with starvation. By analogy with other systems across the phylogenetic scale, in which cAMP activates catabolic pathways and catabolites depress cAMP levels, C is tentatively identified as some measure of the level of energy-yielding catabolites in the cell and the source term for C, as a measure of the cells stored reserves. Starvation for C induces catabolism of stored reserves S through a rise in cAMP. As S, the source term for C declines, the feedback regulation through C can no longer maintain homeostosis and the control loop may be destabilised by small perturbations, i.e. it becomes excitable. A further decline in S can produce limit cycle oscillations in the catabolite-cAMP feedback loop. As S declines even further, continuous steady release of cAMP may ensue.In addition to incorporating the four developmental transitions observed during the aggregation phase as direct consequences of starvation, the model features a super-exponential emergence of relaying competence, phase shifts and acceleration of development by cAMP pulses, and a decreasing refractory period that becomes less than the period of an autonomous cell. All these features closely parallel experimental findings. Finally, the model suggests further experiments critical to an understanding of the dynamics underlying aggregation.     

Preparatory changes and the development of the conjugation junction in a ciliate,Dileptus

Summary The present observations concern changes in the cortical structure of the ciliateDileptus prior to and during conjugation. The results can be summarized as follows: (1) Mating cells join each other in heteropolar configuration, i.e., the distal part of the oral apparatus of one partner faces the proximal part of the oral apparatus of another partner. (2) The structural changes prior to conjugation occur in the area of the oral apparatus. (3) The bonding area is situated within the oral apparatus, while the oral ciliature that encircles the oral apparatus remains outside the bonding area. (4) The fusion area is formed within the bonding area as a newly formed zone, without cortical organelles typically associated with the cell membrane. Two aspects of the formation of cell-to-cell union inDileptus are discussed: firstly, the heteropolar configuration of mating cells and its possible bearing upon an ability to form double cells when partners separation is inhibited. Secondly, the bonding area ofDileptus is compared to bonding areas of peniculine and hymenostome ciliates. A striking likeness in structural changes was found within the bonding areas of all three ciliates, in spite of the fact that the changes in question are localized within the oral apparatus ofDileptus, while inParamecium andTetrahymena the changed territory is situated between the oral apparatus and the anterior pole of the cell.     

Cytoskeletal dynamics ofApedinella radians (Pedinellophyceae) I. Pre-division development and the formation of spine-scales and body scales

Summary Specific events preceding cell division in the unicellular phytoflagellateApedinella radians (Pedinellophyceae) were investigated ultrastructurally and by immunofluorescence microscopy. These events include the sequential formation and secretion of a duplicate set of elongate spine-scales, the duplication and development of basal bodies/flagella, the generation of two new dictyosomes at the future sites of spindle poles, and the constriction (not division) of chloroplasts. Actin and microtubules are involved in spine-scale formation, while actin alone appears to play a role during body scale formation. Both scale types are formed intracellularly within specialized vesicles located adjacent to the posterior cell membrane. The length of spine-scales is not limited by cell size, as they form within cell extensions. Following secretion, spine-scales remain attached below the equator of the cell, and are not deployed to their interphase positions until after the completion of cell division.Abbreviations 08/28 polyclonal anti-centrin antibody - 17E10 monoclonal anti-centrin antibody - 26/14-1 polyclonal anti-centrin antibody - BSA bovine serum albumin - BSFV body scale forming vesicle - FITC fluorescein isothiocyanate - Ssc polyclonal anti-centrin antibody - SSFV spine-scale forming vesicle     

Cytoskeletal dynamics: A view from the membrane

ConclusionsAs more mechanistic connections emerge between the membrane and the cytoskeleton, it is becoming clear that a new generation of tools is needed. In particular, being able to track the dynamics and localization of specific lipid species, as well as physical methods to measure membrane rigidity in living cells, is critical. Additionally, most studies have been performed in individual cells, but not in the context of developing tissues or varied extracellular environments. Thus, how mechanical strains on the membrane translate into cytoskeletal reorganization ultimately effecting cell physiology and development constitutes the next generation of questions in cytoskeletal dynamics.     

Cytoskeletal elements are required for the formation and maturation of autophagic vacuoles.

We evaluated the role of cytoskeletal elements in the degradation of endogenous proteins via autophagy using biochemical and morphological techniques. In the absence of exogenous amino acids, degradation of endogenous proteins was enhanced in cultured normal rat kidney cells. This enhanced degradative state was accompanied by a 4-fold increase in the occurrence of autophagic vacuoles. In the presence of drugs that induce the depolymerization of microfilaments (cytochalasins B and D) or microtubules (nocodazole), protein degradation was not enhanced in nutrient-deprived cells. Although these drugs had similar inhibitory effects on the protein degradation, their effect on autophagy differed. Cytochalasins B and D interfered with the formation of the autophagosome. In cells treated with these drugs, the fractional volume represented by autophagic vacuoles was not substantially increased despite nutrient depletion. On the contrary, nocodazole appeared to have no effect on the formation of autophagosomes. Instead, this drug suppressed the delivery of hydrolytic enzymes, thereby resulting in an accumulation of acidic autophagic vacuoles containing undegraded cellular components.     

Cellular responses of the ciliate, Tetrahymena thermophila, to far infrared irradiation.

Infrared rays from sunlight permeate the earth's atmosphere, yet little is known about their interactions with living organisms. To learn whether they affect cell structure and function, we tested the ciliated protozoan, Tetrahymena thermophila. These unicellular eukaryotes aggregate in swarms near the surface of freshwater habitats, where direct and diffuse solar radiation impinge upon the water-air interface. We report that populations irradiated in laboratory cultures grew and mated normally, but major changes occurred in cell physiology during the stationary phase. Early on, there were significant reductions in chromatin body size and the antibody reactivity of methyl groups on lysine residues 4 and 9 in histone H3. Later, when cells began to starve, messenger RNAs for key proteins related to chromatin structure, intermediary metabolism and cellular motility increased from two- to nearly nine-fold. Metabolic activity, swimming speed and linearity of motion also increased, and spindle shaped cells with a caudal cilium appeared. Our findings suggest that infrared radiation enhances differentiation towards a dispersal cell-like phenotype in saturated populations of Tetrahymena thermophila.     

Cytoskeletal interactions of synapsin I in non-neuronal cells

Synapsin I is a neuronal phosphoprotein involved in the localization and stabilization of synaptic vesicles. Recently, synapsin I has been detected in several non-neuronal cell lines, but its function in these cells is unclear. To determine the localization of synapsin I in non-neuronal cells, it was transiently expressed in HeLa and NIH/3T3 cells as an enhanced green fluorescent protein fusion protein. Synapsin I-enhanced green fluorescent protein colocalized with F-actin in both cell lines, particularly with microspikes and membrane ruffles. It did not colocalize with microtubules or vimentin and it did not cause major alterations in cytoskeletal organization. Synapsin Ia-enhanced green fluorescent protein colocalized with microtubule bundles in taxol-treated HeLa cells and with F-actin spots at the plasma membrane in cells treated with cytochalasin B. It did not noticeably affect F-actin reassembly following drug removal. Synapsin Ia-enhanced green fluorescent protein remained colocalized with F-actin in cells treated with nocodazole, and it did not affect reassembly of microtubules following drug removal. These results demonstrate that synapsin I interacts with F-actin in non-neuronal cells and suggest that synapsin I may have a role in regions where actin is highly dynamic.     

Fine structure of division in ciliate protozoa. I. Micronuclear mitosis in Blepharisma

The mitotic, micronuclear division of the heterotrichous genus Blepharisma has been studied by electron microscopy. Dividing ciliates were selected from clone-derived mass cultures and fixed for electron microscopy by exposure to the vapor of 2% osmium tetroxide; individual Blepharisma were encapsulated and sectioned. Distinctive features of the mitosis are the presence of an intact nuclear envelope during the entire process and the absence of centrioles at the polar ends of the micronuclear figures. Spindle microtubules (SMT) first appear in advance of chromosome alignment, become more numerous and precisely aligned by metaphase, lengthen greatly in anaphase, and persist through telophase. Distinct chromosomal and continuous SMT are present. At telophase, daughter nuclei are separated by a spindle elongation of more than 40 µ, and a new nuclear envelope is formed in close apposition to the chromatin mass of each daughter nucleus and excludes the great amount of spindle material formed during division. The original nuclear envelope which has remained structurally intact then becomes discontinuous and releases the newly formed nucleus into the cytoplasm. The micronuclear envelope seems to lack the conspicuous pores that are typical of nuclear envelopes. The morphology, size, formation, and function of SMT and the nature of micronuclear division are discussed.