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John Kloetzel

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University of Zagreb

Uppsala University

University of East London

Armando Marques-Guedes

UNL - New University of Lisbon

University of Leicester

Gwen Robbins Schug

University of North Carolina at Greensboro

Gabriel Gutierrez-Alonso

University of Salamanca

Macquarie University

Universidade Federal do Rio Grande do Sul

Swansea University

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Papers by John Kloetzel

A Simplified Method of Preparing Optically Clear Flat Embedments with Epoxy Resin

Stain technology, 1973

Nuclear Roles in the Post-Conjugant Development of the CiliateEuplotes aediculatusII. Experimentally Induced Regeneration of Old Macronuclear Fragments1

The Journal of protozoology, Feb 1, 1981

Cells of the ciliate Euplotes, following conjugation, carry out a 4-day sequence of nuclear and c... more Cells of the ciliate Euplotes, following conjugation, carry out a 4-day sequence of nuclear and cortical changes that is unique to this sexual phase of the life cycle. The relative roles of the old preconjugant macronucleus (fragments of which persist in the exconjugant cytoplasm) and of the new postzygotic macronucleus (the maturing anlage) in controlling this "developmental program" following conjugation are not known. Experiments were performed in which anlage formation was prevented (by treatment of conjugating pairs with hydroxyurea, HU) or inhibited over a wide range of exconjugant stages (using HU, or a uv-microbeam focused on the anlage). A detailed analysis of the developmental consequences of such treatments has revealed that the anlage releases information essential for normal exconjugant development at two distinct times. Absence of the first information, normally released within the first few hours following conjugant separation, led to an "early death syndrome": the old macronucleus fragmented as usual, but the anterior fragments never carried out their normal pycnosis and resorption; the cortical reorganization responsible for restoration of a functional feeding apparatus never occurred; the cells gradually swelled and lysed by Day 3-4 of development. Cell death was unrelated to starvation, correlating rather in some way with the failure of the old anterior fragments to become inactivated. The analage's second developmental role corresponds in time with the period of peak polyteny of the anlage's chromosomes (34-37 hr), and is related to cortical morphogenesis. Anlage irradiation prior to this time prevented this morphogenesis, although the cells survived 3 weeks; if anlagen were irradiated even shortly after this time, cortical reorganization occurred on schedule (60-72 hr) and the cells were able to resume feeding, even though the damaged anlagen remained stunted and rudimentary. There are thus at least two distinct "developmental hurdles" that can be crossed only with a functional anlage. 20

Developmental changes in fine structure associated with secretion in larval salivary glands of Chironomus

Experimental Cell Research, Jun 1, 1970

Salivary glands from fourth-instar larvae, prepupae, and early pupae of Chironomus thummi were ex... more Salivary glands from fourth-instar larvae, prepupae, and early pupae of Chironomus thummi were examined with light-and electron microscopes to determine whether developmental changes in the structure of the large polytenic gland cells might be correlated with the gland's secretory activity. It was found that the Golgi complexes, previously implicated in the production of dense "secretory" granules [13], are most extensively developed during mid-prepupal stages, when the glands are the largest, swollen with secretion and apparently at a peak of saliva production. In recently molted pupae, which appear inactive in the production of salivary secretion, the glands are smaller and display Golgi bodies that lack the dense secretory material found associated with Golgi complexes at earlier stages. Those dense secretory granules remaining in the cytoplasm of pupal glands are found no longer accumulated just beneath the secretory brush border, as in earlier stages, but rather aggregated in clusters within the endoplasm, apparently in preparation for autophagic digestion. Electron microscope radioautographs prepared from glands labeled with radioactive amino acids in culture have provided further evidence that the dense secretory granules are indeed composed, at least in part, of proteins synthesized de novo by ihe large gland cells.

Feeding in Ciliated Protozoa

Journal of Cell Science, Jul 1, 1974

The ciliate Euplotes is able to expend a very large amount of membrane in the formation of food v... more The ciliate Euplotes is able to expend a very large amount of membrane in the formation of food vacuoles. Calculations based on the rate of ingestion of the food organism Tetrahymena indicate that an amount of food vacuole membrane equivalent to approximately 50-150% of the total Euplotes cell surface area can be produced within 5-10 min. An aggregation of osmiophilic, membrane-limited 'pharyngeal disks' is found packed in the cytoplasm just beneath the cell surface membrane in the region of the cell mouth and cytopharynx. These disks, which can be seen also in living cells, have average dimensions of 2 /Mn diameter by 100 nm thickness, and contain tightly packed layers of a thin lamellar material. Electron micrographs have revealed the apparent fusion of the limiting membrane of disks with the cell's plasma membrane at the base of the gullet. The lamellar disk contents are thereby released to the exterior medium in the buccal cavity, where they form a loosely packed layer over the surface membrane. It is postulated that the pharyngeal disks represent a repository of preformed membrane for use in food vacuole formation. The disk contents may also play a role in food ingestion, although this is not well defined at present. The myeloid content of old food vacuoles is very similar to that of nearby disks in the cytoplasm, suggesting that the disks may form by pinching from shrinking food vacuoles during the digestive cycle. Thus a cycle of membrane flow is envisaged, with the pharyngeal disks (1) coalescing with the surface membrane during food vacuole formation, (2) reforming by pinching from these food vacuoles during digestion, and (3) migrating back to the oral region to serve as a membrane store for subsequent food vacuole formation.

A fine-structural analysis of larval salivary gland function in Chironomus thummi (diptera)

Journal of Ultrastructure Research, Oct 1, 1969

ABSTRACT Salivary glands of Chironomus thummi larvae were examined with the light and electron mi... more ABSTRACT Salivary glands of Chironomus thummi larvae were examined with the light and electron microscopes to obtain information on the structure and function of the gland cells. The large gland cells (those containing giant polytene chromosomes) display a cytoplasm rich in granular endoplasmic reticulum and Golgi complexes, and an apical brush border. Dense “secretory” granules and “coated” vesicles are concentrated in the apical cytoplasm. The fine structure of these cells suggests that they are active in the synthesis of secretory proteins. These same cells possess an elaborate system of basal membrane infoldings with numerous associated mitochondria; this surface specialization is implicated in the uptake of hemal materials for transport to the saliva. Multivesicular bodies and larger inclusion bodies are numerous; inclusions resembling autophagic vacuoles are especially prevalent in the basal cytoplasm of the “special” lobe (SZ) cells. Additional cells, which form thin layers on the two flattened faces of the gland, appear to contribute a distinctive type of secretory material to the saliva.

Sequence and Properties of Cagein, a Coiled‐Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus

Journal of Eukaryotic Microbiology, Apr 15, 2021

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly p... more In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross‐linked into ordered arrays by scaffold structures known as “basal‐body cages.” The major protein comprising Euplotes cages has been previously identified and termed “cagein.” Screening a E. aediculatus cDNA expression library with anti‐cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear “nanochromosome” of ca. 1.5 kb in Southern blots against whole‐cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled‐coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.

Scanning Electron Microscopy of Stomatogenesis Accompanying Conjugation inEuplotes aediculatus*

The Journal of protozoology, Aug 1, 1975

SYNOPSIS. The succession of morphologic changes in the feeding apparatus (peristome) accompanying... more SYNOPSIS. The succession of morphologic changes in the feeding apparatus (peristome) accompanying conjugation and postconjugant develppment in the hypotrich Euplotes aediculatus has been examined by scanning electron microscopy (SEM). The details of stomatogenesis inferred from earlier light-microscopic studies of silver-stained preparations have been confirmed and extended. The elaborate peristome is the dominant surface feature of vegetative Euplotes. I n conjugation, the ciliates are joined in their peristomial regions; as the conjugants separate, the old feeding apparatus is seen to be disrupted and partially resorbed. In its place is the crescent-shaped primordium of a new peristome, which develops as part of a general cortical reorganization. This primordium expands anteriorly, unfurling a new crown of ciliary membranelles that soon replaces the remaining preconjugant membranellar band. The resulting "exconjugant peristome" is characterized by a greatly reduced number of adoral membranelles and the absence of paroral membranelles, buccal cavity, and cytostome. Exconjugants thus cannot feed for 2-3 days, until the missing peristomial components are replaced. This occurs by means of a 2nd cortical reorganization, during which new membranelles, developing from another peristomial rudiment, are added directly to the abbreviated exconjugant set. A new buccal cavity is concurrently sculpted as the primordial depression enlarges, and the cells can resume feeding sometime during the 4th day after separation. The implications of this mode of stomatogenesis and the nonfeeding condition are discussed, as are the advantages of SEM for studies of ciliate morphogenesis.

Intracellular, periodic structures in the gliding bacterium Myxococcus xanthus

Journal of Bacteriology, Nov 1, 1977

Electron microscopic observations of thin sections of Mvxococcus xatuthus vegetative cells reveal... more Electron microscopic observations of thin sections of Mvxococcus xatuthus vegetative cells revealed the presence of cvtoplasmic bundles of 4to 5-nmdiameter filaments, running longitudinally below the cell membrane and terminating in association with the envelope near one pole. Part of each bundle demonstrated a herringbone-like periodicity (-12-nm spacing). This structure was observed in cells from shake cultures and in gliding cells fixed by several methods. It is proposed that the structure may be attached to the envelope near both poles in gliding cells and that the motive force for motility may be provided by its contraction and relaxation. In one of four nongliding mutants examined, the periodicity was indistinct or lacking. In this mutant another structure, comprised of linearly arrayed beads, was observed in association with the filamentous bundle. Another structure, characterized by major, transverse bands (-34 nm apart), occurred in patches that may traverse the diameter of the wild-type cells in which the structure was observed.

Nuclear roles in the postconjugant development of the ciliated protozoan Euplotes aediculatus

Developmental Biology, Apr 1, 1981

Cells of the ciliate Euplotes, following conjugation, carry out a 4-day sequence of nuclear and c... more Cells of the ciliate Euplotes, following conjugation, carry out a 4-day sequence of nuclear and cortical changes that is unique to this sexual phase of the life cycle. The relative roles of the old preconjugant macronucleus (fragments of which persist in the exconjugant cytoplasm) and of the new postzygotic macronucleus (the maturing anlage) in controlling this "developmental program" following conjugation are not known. Experiments were performed in which anlage formation was prevented (by treatment of conjugating pairs with hydroxyurea, HU) or inhibited over a wide range of exconjugant stages (using HU, or a uv-microbeam focused on the anlage). A detailed analysis of the developmental consequences of such treatments has revealed that the anlage releases information essential for normal exconjugant development at two distinct times. Absence of the first information, normally released within the first few hours following conjugant separation, led to an "early death syndrome": the old macronucleus fragmented as usual, but the anterior fragments never carried out their normal pycnosis and resorption; the cortical reorganization responsible for restoration of a functional feeding apparatus never occurred; the cells gradually swelled and lysed by Day 3-4 of development. Cell death was unrelated to starvation, correlating rather in some way with the failure of the old anterior fragments to become inactivated. The analage's second developmental role corresponds in time with the period of peak polyteny of the anlage's chromosomes (34-37 hr), and is related to cortical morphogenesis. Anlage irradiation prior to this time prevented this morphogenesis, although the cells survived 3 weeks; if anlagen were irradiated even shortly after this time, cortical reorganization occurred on schedule (60-72 hr) and the cells were able to resume feeding, even though the damaged anlagen remained stunted and rudimentary. There are thus at least two distinct "developmental hurdles" that can be crossed only with a functional anlage. 20

Structure and Protein Composition of a Basal-Body Scaffold (“Cage”) in the Hypotrich CiliateEuplotes

Journal of Eukaryotic Microbiology, Nov 1, 2012

Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or... more Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or compound ciliary organelles, such as cirri or oral membranelles, used in locomotion and prey capture. A single polykinetid may contain more than 150 individual cilia; these emerge from basal bodies held in a closely spaced array within a scaffold or framework structure that has been referred to as a basal-body "cage". Cage structures were isolated free of cilia and basal bodies; the predominant component of such cages was found on polyacrylamide gels to be a 45-kDa polypeptide. Antisera were raised against this protein band and used for immunolocalizations at the light and electron microscope levels. Indirect immunofluorescence revealed the 45-kDa polypeptide to be localized exclusively to the bases of the ventral polykinetids. Immunogold staining of thin sections of intact cells further localized this reactivity to filaments of a double-layered dense lattice that appears to link adjoining basal bodies into ordered arrays within each polykinetid. Scanning electron microscopy of isolated cages reveals the lower or "basal" cage layer to be a fine lacey meshwork supporting the basal bodies at their proximal ends; adjoining basal bodies are held at their characteristic spacing by filaments of an upper or "medial" cage layer. The isolated cage thus resembles a miniature test-tube rack, able to accommodate varying arrangements of basal-body rows, depending on the particular type of polykinetid. Because of its clear and specific localization to the basal-body cages in Euplotes, we have termed this novel 45-kDa protein "cagein".

Nuclear Roles in the Post-Conjugant Development of the CiliateEuplotes aediculatus. III. Roles of Old Macronuclear Fragments in Nuclear and Cortical Development1

The Journal of protozoology, Aug 1, 1985

Characterization of a protein correlated with the production of knob-like protrusions on membrane... more Characterization of a protein correlated with the production of knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum. Proc. Natl. Acad. Sci. USA, 76: 8.-1980. Homology between a histidine-rich protein from Plasmodium lophurae and a protein associated with the knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum.

Programmed autophagocytosis accompanying conjugation in the ciliate Stylonychia mytilus

Developmental Biology, 1975

Conjugation and postconjligant development in Stylonychia is accompanied by a period of approxima... more Conjugation and postconjligant 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.

Cytoskeletal proteins with N-terminal signal peptides: plateins in the ciliateEuplotesdefine a new family of articulins

Journal of Cell Science, Apr 1, 2003

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Cellular dynamics of conjugation in the ciliateeuplotes aediculatus. I. Cytoskeletal elements

Journal of Morphology, Apr 1, 1987

The fate and possible roles of the cytoskeleton in the process of conjugation in the hypotrich ci... more The fate and possible roles of the cytoskeleton in the process of conjugation in the hypotrich ciliate Euplotes aediculatus were investigated. Following the coalescence of the plasma membranes of the conjugant cells, a fusion zone or bridge of cytoplasm contributed by both partners is constructed. The sub-alveolar microtubule layers of the vegetative cell cortex remain in place to define the fusion zone boundaries after cell union. The initial fusion zone consists primarily of featureless ground cytoplasm; soon the ground plasm becomes crowded with microtubules and anastomosing smooth endoplasmic reticulum, which become displaced only late in conjugation as the migratory pronuclei are exchanged between partners. Fusion zone microtubules, functioning in some undetermined way, may be involved in the nuclear migration. Resorption of the posterior portion of each partner's buccal apparatus results in the degradation of the component cilia within acid phosphatase-positive autophagic bodies. Silver staining for light microscopy shows that the late fusion zone contracts forward from the posterior border, then constricts to separate the conjugants. In some separating pairs remnants of a microfilamentous assembly are seen at the posterior edge of the fusion zone; the full extent of this system may be masked by partial degradation due to osmium tetroxide fixation. Treatment of conjugants for 6 hours with cytochalasin B prevents separation, possibly through inhibition of the actin-like microfilament assembly in the fusion zone. The observations and experiments favor a model of cell separation following conjugation in which the fusion zone is resorbed by motile or contractile processes occurring within or around the fusion bridge itself.

Cellular dynamics of conjugation in the ciliateeuplotes aediculatus. II. Cellular membranes

Journal of Morphology, Apr 1, 1987

The fate and possible roles of the cytoskeleton in the process of conjugation in the hypotrich ci... more The fate and possible roles of the cytoskeleton in the process of conjugation in the hypotrich ciliate Euplotes aediculatus were investigated. Following the coalescence of the plasma membranes of the conjugant cells, a fusion zone or bridge of cytoplasm contributed by both partners is constructed. The sub-alveolar microtubule layers of the vegetative cell cortex remain in place to define the fusion zone boundaries after cell union. The initial fusion zone consists primarily of featureless ground cytoplasm; soon the ground plasm becomes crowded with microtubules and anastomosing smooth endoplasmic reticulum, which become displaced only late in conjugation as the migratory pronuclei are exchanged between partners. Fusion zone microtubules, functioning in some undetermined way, may be involved in the nuclear migration. Resorption of the posterior portion of each partner's buccal apparatus results in the degradation of the component cilia within acid phosphatase-positive autophagic bodies. Silver staining for light microscopy shows that the late fusion zone contracts forward from the posterior border, then constricts to separate the conjugants. In some separating pairs remnants of a microfilamentous assembly are seen at the posterior edge of the fusion zone; the full extent of this system may be masked by partial degradation due to osmium tetroxide fixation. Treatment of conjugants for 6 hours with cytochalasin B prevents separation, possibly through inhibition of the actin-like microfilament assembly in the fusion zone. The observations and experiments favor a model of cell separation following conjugation in which the fusion zone is resorbed by motile or contractile processes occurring within or around the fusion bridge itself.

Plateins: A Novel Family of Signal Peptide-Containing Articulins in Euplotid Ciliates1

Journal of Eukaryotic Microbiology, 2003

In euplotid ciliates, the cortex is reinforced by alveolar plates-proteinaceous scales located wi... more In euplotid ciliates, the cortex is reinforced by alveolar plates-proteinaceous scales located within the membranous alveolar sacs, forming a monolayer just below the plasma membrane. This system appears to play a cytoskeletal role analogous to that provided by the fibrous epiplasm found beneath the cortical alveoli in other ciliates. In Euplotes aediculatus, the major alveolar plate proteins (termed a-, p-, and y-plateins) have been identified. Using anti-platein antibodies, an expression library of Euplotes genes was screened, and a platein gene identified, cloned, and completely sequenced. Comparison of its derived amino acid sequence with microsequences obtained directly from purified plateins identified this gene as encoding one of the closely related (3-or y-plateins. The derived protein, of 644 amino acids (74.9 kDa), is very acidic (PI = 4.88). Microsequences from authentic a-platein were then used to design oligonucleotide primers, which yielded, via a PCR-based approach, the sequences of two a-platein genes from E. aediculatus. Even more acidic proteins, the derived al-and a2-plateins contain 536 and 501 residues, respectively. Analyses of their amino acid sequences revealed the plateins to be members of the articulin superfamily of cytoskeletal proteins, first described in Euglena and now identified in the ciliate Pseudomicrothorax and in Plasmodium. The hallmark articulin repetitive motifs (based on degenerate valineand proline-rich 12-mers) are present in all three plateins. In Ply-platein this primary motif domain (27 repeats) is central in the molecule, whereas the primary repeats in the a-plateins lie near their C-termini. A cluster of proline-rich pentameric secondary repeats is found in the C-terminus of Ply-platein, but near the N-terminus of a-plateins. All three plateins contain canonical N-terminal signal sequences, unique among known cytoskeletal proteins. The presence of start-transfer sequences correlates well with the final intra-alveolar location of these proteins. This feature, and significant differences from known articulins in amino acid usage and arrangement within the repeat domains, lead us to propose that the plateins comprise a new family of articulin-related proteins. Efforts to follow microscopically the assembly of plateins into new alveolar plates during pre-fission morphogenesis are underway.

Intracellular, periodic structures in the gliding bacterium Myxococcus xanthus

Journal of Bacteriology, 1977

Electron microscopic observations of thin sections of Myxococcus xanthus vegetative cells reveale... more Electron microscopic observations of thin sections of Myxococcus xanthus vegetative cells revealed the presence of cytoplasmic bundles of 4- to 5-nm-diameter filaments running longtitudinally below the cell membrane and terminating in association with the envelope near one pole. Part of each bundle demonstrated a herringbone-like periodicity (approximately 12-nm spacing). This structure was observed in cells from shake cultures and in gliding cells fixed by several methods. It is proposed that the structure may be attached to the envelope near both poles in gliding cells and that the motive force for motility may be provided by its contraction and relaxation. In one of four nongliding mutants examined, the periodicity was indistinct or lacking. In this mutant another structure, comprised of linearly arrayed beads, was observed in association with the filamentous bundle. Another structure, characterized by major, transverse bands (approximately 34 nm apart), occurred in patches that ...

Compartmentalization of the Developing Macronucleus Following Conjugation in Stylonychia and Euplotes

The development of the macronucleus following conjugation in the hypotrichous ciliates Euplotes a... more The development of the macronucleus following conjugation in the hypotrichous ciliates Euplotes and Stylonychia has been examined with the electron microscope. Banded polytene chromosomes can be seen in thin sections of the macronuclear anlagen during the early periods of exconjugant development. As the chromosomes reach their maximum state of polyteny, sheets of fibrous material appear between the chromosomes and transect the chromosomes in the interband regions. Individual bands of the polytene chromosomes thus appear to be isolated in separate compartments. Subsequently, during the stage when the bulk of the polytenic DNA is degraded (1), these compartments swell, resulting in a nucleus packed with thousands of separate spherical chambers. Individual chromosomes are no longer discernible. The anlagen retain this compartmentalized condition for several hours, at the end of which time aggregates of dense material form within many of the compartments. The partitioning layers dispers...

Sequence and Properties of Cagein, a Coiled‐Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly p... more In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross‐linked into ordered arrays by scaffold structures known as “basal‐body cages.” The major protein comprising Euplotes cages has been previously identified and termed “cagein.” Screening a E. aediculatus cDNA expression library with anti‐cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear “nanochromosome” of ca. 1.5 kb in Southern blots against whole‐cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explain...

Feeding in ciliated protozoa. I. Pharyngeal disks in Euplotes: a source of membrane for food vacuole formation?

Journal of Cell Science, Jul 1, 1974

The ciliate Euplotes is able to expend a very large amount of membrane in the formation of food v... more The ciliate Euplotes is able to expend a very large amount of membrane in the formation of food vacuoles. Calculations based on the rate of ingestion of the food organism Tetrahymena indicate that an amount of food vacuole membrane equivalent to approximately 50-150% of the total Euplotes cell surface area can be produced within 5-10 min. An aggregation of osmiophilic, membrane-limited 'pharyngeal disks' is found packed in the cytoplasm just beneath the cell surface membrane in the region of the cell mouth and cytopharynx. These disks, which can be seen also in living cells, have average dimensions of 2 /Mn diameter by 100 nm thickness, and contain tightly packed layers of a thin lamellar material. Electron micrographs have revealed the apparent fusion of the limiting membrane of disks with the cell's plasma membrane at the base of the gullet. The lamellar disk contents are thereby released to the exterior medium in the buccal cavity, where they form a loosely packed layer over the surface membrane. It is postulated that the pharyngeal disks represent a repository of preformed membrane for use in food vacuole formation. The disk contents may also play a role in food ingestion, although this is not well defined at present. The myeloid content of old food vacuoles is very similar to that of nearby disks in the cytoplasm, suggesting that the disks may form by pinching from shrinking food vacuoles during the digestive cycle. Thus a cycle of membrane flow is envisaged, with the pharyngeal disks (1) coalescing with the surface membrane during food vacuole formation, (2) reforming by pinching from these food vacuoles during digestion, and (3) migrating back to the oral region to serve as a membrane store for subsequent food vacuole formation.

A Simplified Method of Preparing Optically Clear Flat Embedments with Epoxy Resin

Stain technology, 1973

Nuclear Roles in the Post-Conjugant Development of the CiliateEuplotes aediculatusII. Experimentally Induced Regeneration of Old Macronuclear Fragments1

The Journal of protozoology, Feb 1, 1981

Cells of the ciliate Euplotes, following conjugation, carry out a 4-day sequence of nuclear and c... more Cells of the ciliate Euplotes, following conjugation, carry out a 4-day sequence of nuclear and cortical changes that is unique to this sexual phase of the life cycle. The relative roles of the old preconjugant macronucleus (fragments of which persist in the exconjugant cytoplasm) and of the new postzygotic macronucleus (the maturing anlage) in controlling this "developmental program" following conjugation are not known. Experiments were performed in which anlage formation was prevented (by treatment of conjugating pairs with hydroxyurea, HU) or inhibited over a wide range of exconjugant stages (using HU, or a uv-microbeam focused on the anlage). A detailed analysis of the developmental consequences of such treatments has revealed that the anlage releases information essential for normal exconjugant development at two distinct times. Absence of the first information, normally released within the first few hours following conjugant separation, led to an "early death syndrome": the old macronucleus fragmented as usual, but the anterior fragments never carried out their normal pycnosis and resorption; the cortical reorganization responsible for restoration of a functional feeding apparatus never occurred; the cells gradually swelled and lysed by Day 3-4 of development. Cell death was unrelated to starvation, correlating rather in some way with the failure of the old anterior fragments to become inactivated. The analage's second developmental role corresponds in time with the period of peak polyteny of the anlage's chromosomes (34-37 hr), and is related to cortical morphogenesis. Anlage irradiation prior to this time prevented this morphogenesis, although the cells survived 3 weeks; if anlagen were irradiated even shortly after this time, cortical reorganization occurred on schedule (60-72 hr) and the cells were able to resume feeding, even though the damaged anlagen remained stunted and rudimentary. There are thus at least two distinct "developmental hurdles" that can be crossed only with a functional anlage. 20

Developmental changes in fine structure associated with secretion in larval salivary glands of Chironomus

Experimental Cell Research, Jun 1, 1970

Salivary glands from fourth-instar larvae, prepupae, and early pupae of Chironomus thummi were ex... more Salivary glands from fourth-instar larvae, prepupae, and early pupae of Chironomus thummi were examined with light-and electron microscopes to determine whether developmental changes in the structure of the large polytenic gland cells might be correlated with the gland's secretory activity. It was found that the Golgi complexes, previously implicated in the production of dense "secretory" granules [13], are most extensively developed during mid-prepupal stages, when the glands are the largest, swollen with secretion and apparently at a peak of saliva production. In recently molted pupae, which appear inactive in the production of salivary secretion, the glands are smaller and display Golgi bodies that lack the dense secretory material found associated with Golgi complexes at earlier stages. Those dense secretory granules remaining in the cytoplasm of pupal glands are found no longer accumulated just beneath the secretory brush border, as in earlier stages, but rather aggregated in clusters within the endoplasm, apparently in preparation for autophagic digestion. Electron microscope radioautographs prepared from glands labeled with radioactive amino acids in culture have provided further evidence that the dense secretory granules are indeed composed, at least in part, of proteins synthesized de novo by ihe large gland cells.

Feeding in Ciliated Protozoa

Journal of Cell Science, Jul 1, 1974

The ciliate Euplotes is able to expend a very large amount of membrane in the formation of food v... more The ciliate Euplotes is able to expend a very large amount of membrane in the formation of food vacuoles. Calculations based on the rate of ingestion of the food organism Tetrahymena indicate that an amount of food vacuole membrane equivalent to approximately 50-150% of the total Euplotes cell surface area can be produced within 5-10 min. An aggregation of osmiophilic, membrane-limited 'pharyngeal disks' is found packed in the cytoplasm just beneath the cell surface membrane in the region of the cell mouth and cytopharynx. These disks, which can be seen also in living cells, have average dimensions of 2 /Mn diameter by 100 nm thickness, and contain tightly packed layers of a thin lamellar material. Electron micrographs have revealed the apparent fusion of the limiting membrane of disks with the cell's plasma membrane at the base of the gullet. The lamellar disk contents are thereby released to the exterior medium in the buccal cavity, where they form a loosely packed layer over the surface membrane. It is postulated that the pharyngeal disks represent a repository of preformed membrane for use in food vacuole formation. The disk contents may also play a role in food ingestion, although this is not well defined at present. The myeloid content of old food vacuoles is very similar to that of nearby disks in the cytoplasm, suggesting that the disks may form by pinching from shrinking food vacuoles during the digestive cycle. Thus a cycle of membrane flow is envisaged, with the pharyngeal disks (1) coalescing with the surface membrane during food vacuole formation, (2) reforming by pinching from these food vacuoles during digestion, and (3) migrating back to the oral region to serve as a membrane store for subsequent food vacuole formation.

A fine-structural analysis of larval salivary gland function in Chironomus thummi (diptera)

Journal of Ultrastructure Research, Oct 1, 1969

ABSTRACT Salivary glands of Chironomus thummi larvae were examined with the light and electron mi... more ABSTRACT Salivary glands of Chironomus thummi larvae were examined with the light and electron microscopes to obtain information on the structure and function of the gland cells. The large gland cells (those containing giant polytene chromosomes) display a cytoplasm rich in granular endoplasmic reticulum and Golgi complexes, and an apical brush border. Dense “secretory” granules and “coated” vesicles are concentrated in the apical cytoplasm. The fine structure of these cells suggests that they are active in the synthesis of secretory proteins. These same cells possess an elaborate system of basal membrane infoldings with numerous associated mitochondria; this surface specialization is implicated in the uptake of hemal materials for transport to the saliva. Multivesicular bodies and larger inclusion bodies are numerous; inclusions resembling autophagic vacuoles are especially prevalent in the basal cytoplasm of the “special” lobe (SZ) cells. Additional cells, which form thin layers on the two flattened faces of the gland, appear to contribute a distinctive type of secretory material to the saliva.

Sequence and Properties of Cagein, a Coiled‐Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus

Journal of Eukaryotic Microbiology, Apr 15, 2021

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly p... more In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross‐linked into ordered arrays by scaffold structures known as “basal‐body cages.” The major protein comprising Euplotes cages has been previously identified and termed “cagein.” Screening a E. aediculatus cDNA expression library with anti‐cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear “nanochromosome” of ca. 1.5 kb in Southern blots against whole‐cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled‐coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.

Scanning Electron Microscopy of Stomatogenesis Accompanying Conjugation inEuplotes aediculatus*

The Journal of protozoology, Aug 1, 1975

SYNOPSIS. The succession of morphologic changes in the feeding apparatus (peristome) accompanying... more SYNOPSIS. The succession of morphologic changes in the feeding apparatus (peristome) accompanying conjugation and postconjugant develppment in the hypotrich Euplotes aediculatus has been examined by scanning electron microscopy (SEM). The details of stomatogenesis inferred from earlier light-microscopic studies of silver-stained preparations have been confirmed and extended. The elaborate peristome is the dominant surface feature of vegetative Euplotes. I n conjugation, the ciliates are joined in their peristomial regions; as the conjugants separate, the old feeding apparatus is seen to be disrupted and partially resorbed. In its place is the crescent-shaped primordium of a new peristome, which develops as part of a general cortical reorganization. This primordium expands anteriorly, unfurling a new crown of ciliary membranelles that soon replaces the remaining preconjugant membranellar band. The resulting "exconjugant peristome" is characterized by a greatly reduced number of adoral membranelles and the absence of paroral membranelles, buccal cavity, and cytostome. Exconjugants thus cannot feed for 2-3 days, until the missing peristomial components are replaced. This occurs by means of a 2nd cortical reorganization, during which new membranelles, developing from another peristomial rudiment, are added directly to the abbreviated exconjugant set. A new buccal cavity is concurrently sculpted as the primordial depression enlarges, and the cells can resume feeding sometime during the 4th day after separation. The implications of this mode of stomatogenesis and the nonfeeding condition are discussed, as are the advantages of SEM for studies of ciliate morphogenesis.

Intracellular, periodic structures in the gliding bacterium Myxococcus xanthus

Journal of Bacteriology, Nov 1, 1977

Electron microscopic observations of thin sections of Mvxococcus xatuthus vegetative cells reveal... more Electron microscopic observations of thin sections of Mvxococcus xatuthus vegetative cells revealed the presence of cvtoplasmic bundles of 4to 5-nmdiameter filaments, running longitudinally below the cell membrane and terminating in association with the envelope near one pole. Part of each bundle demonstrated a herringbone-like periodicity (-12-nm spacing). This structure was observed in cells from shake cultures and in gliding cells fixed by several methods. It is proposed that the structure may be attached to the envelope near both poles in gliding cells and that the motive force for motility may be provided by its contraction and relaxation. In one of four nongliding mutants examined, the periodicity was indistinct or lacking. In this mutant another structure, comprised of linearly arrayed beads, was observed in association with the filamentous bundle. Another structure, characterized by major, transverse bands (-34 nm apart), occurred in patches that may traverse the diameter of the wild-type cells in which the structure was observed.

Nuclear roles in the postconjugant development of the ciliated protozoan Euplotes aediculatus

Developmental Biology, Apr 1, 1981

Cells of the ciliate Euplotes, following conjugation, carry out a 4-day sequence of nuclear and c... more Cells of the ciliate Euplotes, following conjugation, carry out a 4-day sequence of nuclear and cortical changes that is unique to this sexual phase of the life cycle. The relative roles of the old preconjugant macronucleus (fragments of which persist in the exconjugant cytoplasm) and of the new postzygotic macronucleus (the maturing anlage) in controlling this "developmental program" following conjugation are not known. Experiments were performed in which anlage formation was prevented (by treatment of conjugating pairs with hydroxyurea, HU) or inhibited over a wide range of exconjugant stages (using HU, or a uv-microbeam focused on the anlage). A detailed analysis of the developmental consequences of such treatments has revealed that the anlage releases information essential for normal exconjugant development at two distinct times. Absence of the first information, normally released within the first few hours following conjugant separation, led to an "early death syndrome": the old macronucleus fragmented as usual, but the anterior fragments never carried out their normal pycnosis and resorption; the cortical reorganization responsible for restoration of a functional feeding apparatus never occurred; the cells gradually swelled and lysed by Day 3-4 of development. Cell death was unrelated to starvation, correlating rather in some way with the failure of the old anterior fragments to become inactivated. The analage's second developmental role corresponds in time with the period of peak polyteny of the anlage's chromosomes (34-37 hr), and is related to cortical morphogenesis. Anlage irradiation prior to this time prevented this morphogenesis, although the cells survived 3 weeks; if anlagen were irradiated even shortly after this time, cortical reorganization occurred on schedule (60-72 hr) and the cells were able to resume feeding, even though the damaged anlagen remained stunted and rudimentary. There are thus at least two distinct "developmental hurdles" that can be crossed only with a functional anlage. 20

Structure and Protein Composition of a Basal-Body Scaffold (“Cage”) in the Hypotrich CiliateEuplotes

Journal of Eukaryotic Microbiology, Nov 1, 2012

Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or... more Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or compound ciliary organelles, such as cirri or oral membranelles, used in locomotion and prey capture. A single polykinetid may contain more than 150 individual cilia; these emerge from basal bodies held in a closely spaced array within a scaffold or framework structure that has been referred to as a basal-body "cage". Cage structures were isolated free of cilia and basal bodies; the predominant component of such cages was found on polyacrylamide gels to be a 45-kDa polypeptide. Antisera were raised against this protein band and used for immunolocalizations at the light and electron microscope levels. Indirect immunofluorescence revealed the 45-kDa polypeptide to be localized exclusively to the bases of the ventral polykinetids. Immunogold staining of thin sections of intact cells further localized this reactivity to filaments of a double-layered dense lattice that appears to link adjoining basal bodies into ordered arrays within each polykinetid. Scanning electron microscopy of isolated cages reveals the lower or "basal" cage layer to be a fine lacey meshwork supporting the basal bodies at their proximal ends; adjoining basal bodies are held at their characteristic spacing by filaments of an upper or "medial" cage layer. The isolated cage thus resembles a miniature test-tube rack, able to accommodate varying arrangements of basal-body rows, depending on the particular type of polykinetid. Because of its clear and specific localization to the basal-body cages in Euplotes, we have termed this novel 45-kDa protein "cagein".

Nuclear Roles in the Post-Conjugant Development of the CiliateEuplotes aediculatus. III. Roles of Old Macronuclear Fragments in Nuclear and Cortical Development1

The Journal of protozoology, Aug 1, 1985

Characterization of a protein correlated with the production of knob-like protrusions on membrane... more Characterization of a protein correlated with the production of knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum. Proc. Natl. Acad. Sci. USA, 76: 8.-1980. Homology between a histidine-rich protein from Plasmodium lophurae and a protein associated with the knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum.

Programmed autophagocytosis accompanying conjugation in the ciliate Stylonychia mytilus

Developmental Biology, 1975

Conjugation and postconjligant development in Stylonychia is accompanied by a period of approxima... more Conjugation and postconjligant 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.

Cytoskeletal proteins with N-terminal signal peptides: plateins in the ciliateEuplotesdefine a new family of articulins

Journal of Cell Science, Apr 1, 2003

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Cellular dynamics of conjugation in the ciliateeuplotes aediculatus. I. Cytoskeletal elements

Journal of Morphology, Apr 1, 1987

The fate and possible roles of the cytoskeleton in the process of conjugation in the hypotrich ci... more The fate and possible roles of the cytoskeleton in the process of conjugation in the hypotrich ciliate Euplotes aediculatus were investigated. Following the coalescence of the plasma membranes of the conjugant cells, a fusion zone or bridge of cytoplasm contributed by both partners is constructed. The sub-alveolar microtubule layers of the vegetative cell cortex remain in place to define the fusion zone boundaries after cell union. The initial fusion zone consists primarily of featureless ground cytoplasm; soon the ground plasm becomes crowded with microtubules and anastomosing smooth endoplasmic reticulum, which become displaced only late in conjugation as the migratory pronuclei are exchanged between partners. Fusion zone microtubules, functioning in some undetermined way, may be involved in the nuclear migration. Resorption of the posterior portion of each partner's buccal apparatus results in the degradation of the component cilia within acid phosphatase-positive autophagic bodies. Silver staining for light microscopy shows that the late fusion zone contracts forward from the posterior border, then constricts to separate the conjugants. In some separating pairs remnants of a microfilamentous assembly are seen at the posterior edge of the fusion zone; the full extent of this system may be masked by partial degradation due to osmium tetroxide fixation. Treatment of conjugants for 6 hours with cytochalasin B prevents separation, possibly through inhibition of the actin-like microfilament assembly in the fusion zone. The observations and experiments favor a model of cell separation following conjugation in which the fusion zone is resorbed by motile or contractile processes occurring within or around the fusion bridge itself.

Cellular dynamics of conjugation in the ciliateeuplotes aediculatus. II. Cellular membranes

Journal of Morphology, Apr 1, 1987

The fate and possible roles of the cytoskeleton in the process of conjugation in the hypotrich ci... more The fate and possible roles of the cytoskeleton in the process of conjugation in the hypotrich ciliate Euplotes aediculatus were investigated. Following the coalescence of the plasma membranes of the conjugant cells, a fusion zone or bridge of cytoplasm contributed by both partners is constructed. The sub-alveolar microtubule layers of the vegetative cell cortex remain in place to define the fusion zone boundaries after cell union. The initial fusion zone consists primarily of featureless ground cytoplasm; soon the ground plasm becomes crowded with microtubules and anastomosing smooth endoplasmic reticulum, which become displaced only late in conjugation as the migratory pronuclei are exchanged between partners. Fusion zone microtubules, functioning in some undetermined way, may be involved in the nuclear migration. Resorption of the posterior portion of each partner's buccal apparatus results in the degradation of the component cilia within acid phosphatase-positive autophagic bodies. Silver staining for light microscopy shows that the late fusion zone contracts forward from the posterior border, then constricts to separate the conjugants. In some separating pairs remnants of a microfilamentous assembly are seen at the posterior edge of the fusion zone; the full extent of this system may be masked by partial degradation due to osmium tetroxide fixation. Treatment of conjugants for 6 hours with cytochalasin B prevents separation, possibly through inhibition of the actin-like microfilament assembly in the fusion zone. The observations and experiments favor a model of cell separation following conjugation in which the fusion zone is resorbed by motile or contractile processes occurring within or around the fusion bridge itself.

Plateins: A Novel Family of Signal Peptide-Containing Articulins in Euplotid Ciliates1

Journal of Eukaryotic Microbiology, 2003

In euplotid ciliates, the cortex is reinforced by alveolar plates-proteinaceous scales located wi... more In euplotid ciliates, the cortex is reinforced by alveolar plates-proteinaceous scales located within the membranous alveolar sacs, forming a monolayer just below the plasma membrane. This system appears to play a cytoskeletal role analogous to that provided by the fibrous epiplasm found beneath the cortical alveoli in other ciliates. In Euplotes aediculatus, the major alveolar plate proteins (termed a-, p-, and y-plateins) have been identified. Using anti-platein antibodies, an expression library of Euplotes genes was screened, and a platein gene identified, cloned, and completely sequenced. Comparison of its derived amino acid sequence with microsequences obtained directly from purified plateins identified this gene as encoding one of the closely related (3-or y-plateins. The derived protein, of 644 amino acids (74.9 kDa), is very acidic (PI = 4.88). Microsequences from authentic a-platein were then used to design oligonucleotide primers, which yielded, via a PCR-based approach, the sequences of two a-platein genes from E. aediculatus. Even more acidic proteins, the derived al-and a2-plateins contain 536 and 501 residues, respectively. Analyses of their amino acid sequences revealed the plateins to be members of the articulin superfamily of cytoskeletal proteins, first described in Euglena and now identified in the ciliate Pseudomicrothorax and in Plasmodium. The hallmark articulin repetitive motifs (based on degenerate valineand proline-rich 12-mers) are present in all three plateins. In Ply-platein this primary motif domain (27 repeats) is central in the molecule, whereas the primary repeats in the a-plateins lie near their C-termini. A cluster of proline-rich pentameric secondary repeats is found in the C-terminus of Ply-platein, but near the N-terminus of a-plateins. All three plateins contain canonical N-terminal signal sequences, unique among known cytoskeletal proteins. The presence of start-transfer sequences correlates well with the final intra-alveolar location of these proteins. This feature, and significant differences from known articulins in amino acid usage and arrangement within the repeat domains, lead us to propose that the plateins comprise a new family of articulin-related proteins. Efforts to follow microscopically the assembly of plateins into new alveolar plates during pre-fission morphogenesis are underway.

Intracellular, periodic structures in the gliding bacterium Myxococcus xanthus

Journal of Bacteriology, 1977

Electron microscopic observations of thin sections of Myxococcus xanthus vegetative cells reveale... more Electron microscopic observations of thin sections of Myxococcus xanthus vegetative cells revealed the presence of cytoplasmic bundles of 4- to 5-nm-diameter filaments running longtitudinally below the cell membrane and terminating in association with the envelope near one pole. Part of each bundle demonstrated a herringbone-like periodicity (approximately 12-nm spacing). This structure was observed in cells from shake cultures and in gliding cells fixed by several methods. It is proposed that the structure may be attached to the envelope near both poles in gliding cells and that the motive force for motility may be provided by its contraction and relaxation. In one of four nongliding mutants examined, the periodicity was indistinct or lacking. In this mutant another structure, comprised of linearly arrayed beads, was observed in association with the filamentous bundle. Another structure, characterized by major, transverse bands (approximately 34 nm apart), occurred in patches that ...

Compartmentalization of the Developing Macronucleus Following Conjugation in Stylonychia and Euplotes

The development of the macronucleus following conjugation in the hypotrichous ciliates Euplotes a... more The development of the macronucleus following conjugation in the hypotrichous ciliates Euplotes and Stylonychia has been examined with the electron microscope. Banded polytene chromosomes can be seen in thin sections of the macronuclear anlagen during the early periods of exconjugant development. As the chromosomes reach their maximum state of polyteny, sheets of fibrous material appear between the chromosomes and transect the chromosomes in the interband regions. Individual bands of the polytene chromosomes thus appear to be isolated in separate compartments. Subsequently, during the stage when the bulk of the polytenic DNA is degraded (1), these compartments swell, resulting in a nucleus packed with thousands of separate spherical chambers. Individual chromosomes are no longer discernible. The anlagen retain this compartmentalized condition for several hours, at the end of which time aggregates of dense material form within many of the compartments. The partitioning layers dispers...

Sequence and Properties of Cagein, a Coiled‐Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly p... more In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross‐linked into ordered arrays by scaffold structures known as “basal‐body cages.” The major protein comprising Euplotes cages has been previously identified and termed “cagein.” Screening a E. aediculatus cDNA expression library with anti‐cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear “nanochromosome” of ca. 1.5 kb in Southern blots against whole‐cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explain...

Feeding in ciliated protozoa. I. Pharyngeal disks in Euplotes: a source of membrane for food vacuole formation?

Journal of Cell Science, Jul 1, 1974

The ciliate Euplotes is able to expend a very large amount of membrane in the formation of food v... more The ciliate Euplotes is able to expend a very large amount of membrane in the formation of food vacuoles. Calculations based on the rate of ingestion of the food organism Tetrahymena indicate that an amount of food vacuole membrane equivalent to approximately 50-150% of the total Euplotes cell surface area can be produced within 5-10 min. An aggregation of osmiophilic, membrane-limited 'pharyngeal disks' is found packed in the cytoplasm just beneath the cell surface membrane in the region of the cell mouth and cytopharynx. These disks, which can be seen also in living cells, have average dimensions of 2 /Mn diameter by 100 nm thickness, and contain tightly packed layers of a thin lamellar material. Electron micrographs have revealed the apparent fusion of the limiting membrane of disks with the cell's plasma membrane at the base of the gullet. The lamellar disk contents are thereby released to the exterior medium in the buccal cavity, where they form a loosely packed layer over the surface membrane. It is postulated that the pharyngeal disks represent a repository of preformed membrane for use in food vacuole formation. The disk contents may also play a role in food ingestion, although this is not well defined at present. The myeloid content of old food vacuoles is very similar to that of nearby disks in the cytoplasm, suggesting that the disks may form by pinching from shrinking food vacuoles during the digestive cycle. Thus a cycle of membrane flow is envisaged, with the pharyngeal disks (1) coalescing with the surface membrane during food vacuole formation, (2) reforming by pinching from these food vacuoles during digestion, and (3) migrating back to the oral region to serve as a membrane store for subsequent food vacuole formation.