Transient invagination of flagella by Tritrichomonas foetus.

We describe an experimental system for the study of rapid and reversible formation of pseudocysts, which are spherical forms that lack a true cyst wall, by Tritrichomonas foetus, a trichomonad parasite of the bovine genitourinary tract. It highlights the dynamics of the plasma membrane and cytoskeleton of this parasite, which is perpetually devoid of any sort of protective cell wall, and can reflect a responsive survival mechanism. We have found that cooling of axenic cultures of T. foetus from their normal 37 degrees C to below about 16 degrees C can trigger pseudocyst formation. The three anterior flagella and the single recurrent flagellum can be fully internalized within 1 3 min at 37 degrees C, with the axonemes and flagellar membranes remaining intact within the cell body. Electron microscopy confirms that the internalized flagella are surrounded by a separate membrane. At 37 degrees C the internalized flagella can resume beating movements and become externalized as quickly as within 10 min. We have begun to elucidate the mechanisms of this unusual phenomenon, characterizing its temperature dependence and exploring the effects of agents that interfere with various aspects of the cytoskeleton, phagocytosis, endocytosis, and exocytosis.

The ligand binding site of the formyl peptide receptor maps in the transmembrane region.

We propose that the N-formyl-Met-Leu-Phe binding site in the human neutrophil formyl peptide receptor (FPR) lies in the predicted transmembrane region. We examined the expression, binding, and G protein coupling of 28 mutated forms of FPR in stably transfected Chinese hamster ovary cells. The amino acids we mutated are: 1) predicted to be oriented toward the interhelical space; 2) analogous to those required for ligand binding in various other G protein-coupled receptors; 3) divergent from lipoxin A4 receptor, a low affinity receptor for formylated peptides; and 4) either highly conserved or divergent in other G protein-coupled receptors. Some mutations resulted in intracellular retention, suggesting that the receptors were misfolded. Most mutated receptors that were transported to the plasmalemma bound f-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with affinities similar to the wild-type receptor (Kd = 6 nM). However, mutations L78A (helix II), D106N, L109A (helix III), T157A (helix IV), R201A, I204Y, and R205A (helix V), W254A and Y257A (helix VI), and F291A (helix VII) resulted in reduced affinities (Kd = 30-128 nM). Of these mutations, D106N, R201A, and R205A also appeared to affect G protein coupling, suggesting that these residues may also be involved in signal transduction and/or are essential for proper folding of the molecule. Some of the FPR residues that appeared to be involved in binding of formylated peptides were located at sites analogous to those identified in ligand binding to certain other G protein-coupled receptors. It is thus possible that several G protein-coupled receptors have a common placement of ligand-binding amino acids.

Identification of a neutrophil chemotactic factor from Tritrichomonas foetus as superoxide dismutase.

Antibodies to a neutrophil chemotactic factor from Tritrichomonas foetus were used to screen a T. foetus cDNA expression library in lambda gt11. All positive clones were identified as homologs of iron-containing superoxide dismutase (SOD). Native gel electrophoresis showed that the antibodies indeed recognized T. foetus antigens with SOD activity. Two SOD genes were found in T. foetus, and cloned and sequenced as parts of larger genomic segments of 3844 and 4089 base pairs. Transcription initiated between the first and second methionine codons of each genomic open reading frame, generating mRNAs with 5' untranslated regions of 11-15 bases, and encoding proteins of 195 amino acids. The two SOD coding sequences lacked obvious introns. They were 79% identical at both the nucleotide and amino acid levels. Both SOD genes were inserted into a eukaryotic expression vector and stably expressed in mammalian cells; both proteins were recognized by the antibodies, and both assumed a cytosolic, extranuclear distribution in these cells. Histidine-tagged forms of both T. foetus SODs were expressed in E. coli and after purification, found to have neutrophil chemotactic activity similar to the non-recombinant factor purified from T. foetus. Identification of this neutrophil chemotactic factor as SOD provides additional insight into the host-parasite interaction.

Transmembrane domain mutations influence the cellular distribution of lysosomal membrane glycoprotein A.

The Igp/LAMP family of mammalian and avian lysosomal type I membrane glycoproteins features short, conserved, cytosolic tails that possess lysosomal targeting information. The sequences of the adjacent transmembrane domains are also highly conserved, with six amino acids identical in all sixteen known Igp variants. These six residues are found along one side of a hypothetical alpha-helix that may comprise this domain. We substituted or deleted some of the conserved transmembrane residues in mouse Igp-A and stably expressed the proteins in Chinese hamster ovary cells. We examined various properties and transport characteristics of the normal and modified proteins and concluded that the transmembrane domain serves as more than just a membrane anchor, as it subtly influences the cellular distribution of the protein as well.

Rapid internalization and degradation of surface-bound antibodies by Tritrichomonas foetus.

Tritrichomonas foetus is a protozoan parasite of cattle that can be cultured axenically. Three monoclonal antibodies specific for surface antigens of T. foetus were found to be rapidly internalized and degraded by these cells after binding. Degradation was not due to secreted or artificially liberated proteases but depended on targeting to internal degradative compartments. Radiolabeled catabolites of the antibodies were subsequently incorporated into the parasite's own proteins. Antibody degradation could be inhibited by certain protease inhibitors or lowered temperatures; a sharp reduction in degradation between 20 C and 15 C was similar to a well documented block in endocytic transport to degradative compartments of mammalian cells. Growth and proliferation of T.foetus in the continuous presence of the antibodies appeared unhindered, but there was a general shift toward expression of both more and less of each epitope among cells within each population. Subclones of these populations always exhibited striking variability in epitope expression levels, with patterns similar to the parent cultures. These findings may lead to a better understanding of how T. foetus resists host immune responses.

Cell surface accumulation of overexpressed hamster lysosomal membrane glycoproteins.

We cloned and sequenced cDNAs encoding two lysosomal membrane glycoproteins, lgp-A and lgp-B, from Chinese hamster ovary cells. The deduced amino acid sequences of these proteins are similar to those of the other known members of this conserved family (also known as "LAMP" proteins). We used the cDNAs to generate stable lines of hamster lgp-expressing mouse NIH-3T3 cells, rat NRK cells, and monkey CV-1 cells. We also generated hybridomas that secrete antibodies specific for hamster lgp-A and lgp-B, enabling us to distinguish foreign from endogenous lgps in a wider variety of transfected cell lines. One line of mouse NIH-3T3 cells that expresses hamster lgp-B was studied in detail. Whereas most of the hamster lgp-B appeared to be transported to lysosomes in these cells, butyrate-induced overexpression resulted in the accumulation of a significant proportion of the total on the plasma membrane. In addition, overexpression of this foreign lgp-B also resulted in the appearance of the endogenous mouse lgp-A and lgp-B on the plasma membrane. Characterization of this accumulation suggested that it resulted from competition for one or more limited components in the transport pathway(s) to lysosomes. Endocytosis from the plasma membrane appeared to be one step that was saturable.

Characterization and cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells.

lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines. Partial sequencing of mouse lgp110 allowed oligonucleotide probes to be constructed for the screening of several mouse cDNA libraries. A partial cDNA clone for mouse lgp110 was found and used for additional library screening, generating a cDNA clone covering all of the coding sequence of mature rat lgp110 as well as genomic clones covering most of the mouse gene. These new clones bring to seven the number of lysosomal membrane proteins whose amino acid sequences can be deduced, and two distinct but highly similar groups (designated lgp-A and lgp-B) can now be defined. Sequence comparisons suggest that differences within each group reflect species variations of the same protein and that lgp-A and lgp-B probably diverged from a common ancestor prior to the evolup4f1ary divergence of birds and mammals. Individual cells and individual lysosomes possess both lgp-A and lgp-B, suggesting that these two proteins have different functions. Mouse lgp110 is encoded by at least seven exons; intron positions suggest that the two homologous ectodomains of each lgp arose through gene duplication.

Derived protein sequence, oligosaccharides, and membrane insertion of the 120-kDa lysosomal membrane glycoprotein (lgp120): identification of a highly conserved family of lysosomal membrane glycoproteins.

The 120-kDa lysosomal membrane glycoprotein (lgp120) is an acidic, heavily glycosylated membrane protein enriched in the lysosomal membrane. To determine the basis for its selective transport to and stability in lysosomes, we have investigated the structure of lgp120. By using an oligonucleotide probe corresponding to the amino terminus of rat lgp120, we isolated and characterized cDNA clones containing the entire coding region. The deduced amino acid sequence demonstrates that lgp120 contains a putative signal peptide, 18 sites for N-linked glycosylation, a single membrane-spanning segment, and a short (11 amino acid) cytosolic tail. The sequence suggests a distinct domain organization, with two luminal glycosylated regions separated by a nonglycosylated proline-rich region. Proteolysis in detergent showed that the protein was not intrinsically resistant to exogenous or endogenous proteases. The N-linked oligosaccharides on lgp120, tetraantennary structures with two lactosamine repeats on one of the branches, were not different from those of glycoproteins on the plasma membrane. lgp120 was similar in its domain organization and portions of its amino acid sequence to the avian 100-kDa lysosomal membrane protein LEP100 [Fambrough, D. M., Takeyasu, K., Lippincott-Schwartz, J., Siegel, N. R. & Somerville, D. (1988) J. Cell Biol. 106, 61-67], and to a distinct 110-kDa lysosomal membrane protein (lgp110) that colocalizes with lgp120. The similarities between lysosomal membrane glycoproteins from diverse species, coupled with the fact that at least two distinct lysosomal membrane glycoproteins are expressed in a single species, indicate the existence of a conserved family of glycoproteins enriched in the lysosomal membrane.

Appearance of new variants of membrane skeletal protein 4.1 during terminal differentiation of avian erythroid and lenticular cells.

The erythrocyte plasma membrane is lined with a network of extrinsic proteins, mainly spectrin and actin, which constitute a reticulum tethered to the intrinsic anion transport protein of the lipid bilayer through a linker protein, ankyrin. Protein 4.1 forms a stable ternary complex with spectrin and actin, thereby strengthening the reticulum and anchoring it directly to the lipid bilayer or to another intrinsic protein, glycophorin. It has been found recently that spectrin, ankyrin and protein 4.1 are not erythrocyte-specific; this has elucidated further the mechanisms of plasma membrane assembly and modelling during the differentiation of diverse tissues. We have shown previously that protein 4.1 in chickens is most abundant in erythrocytes and lens cells, but is scarce or absent from other spectrin-rich cell types. In addition, it exists as a family of related polypeptides showing differential expression in these two tissues, suggesting variant-specific functions. Here we show that the pattern of protein 4.1 variants changes during the terminal differentiation of erythroid and lenticular cells, with novel variants appearing in postmitotic cells. The accumulation of these variants may lead to the final stabilization of the plasma membrane skeletons of these cells.

Expression of the intermediate-filament-associated protein synemin in chicken lens cells.

Synemin, a 230-kilodalton polypeptide component of avian muscle and erythrocyte intermediate filaments, is also found in association with the vimentin filaments of lens tissue. In chicken lens cells, synemin is bound to the core vimentin polymer with the same 180-nm periodicity that it exhibits in erythrocytes. Its solubility properties are characteristic of those of intermediate filaments in general and similar to those of synemin in muscle cells and erythrocytes. Synemin appears at an early stage of lens development and undergoes a dramatic accumulation as the epithelial cells elongate and differentiate into fiber cells. In contrast to synemin in cultured skeletal muscle, lens synemin is not confined to postmitotic, terminally differentiating cells but is present in proliferative cells as well. It is lost from the fibers near the center of the lens, as are many other cellular structures including intermediate filaments. These findings provide new information about the occurrence and expression of avian synemin and new insight regarding its presumptive role as a modulator of intermediate-filament function.

Membrane skeletal protein 4.1 of avian erythrocytes is composed of multiple variants that exhibit tissue-specific expression.

The avian analog of mammalian erythrocyte protein 4.1, a structural component of the membrane skeleton, has been identified. It is present at the plasma membranes of avian erythrocytes and lens cells, but has not been found elsewhere in comparable amounts. In chickens, it exists as six variants with molecular masses of 87, 100, 115, 150, 160, and 175 kd. The corresponding polypeptides in turkeys are each about 3 kd smaller, suggesting that all may be encoded by a single gene. These variants have similar solubility properties and nearly identical two-dimensional iodopeptide maps that are similar to those of mammalian protein 4.1, but they are differentially phosphorylated. The three smallest variants are the predominant forms in avian erythrocytes, while the two largest variants predominate in avian lens cells. In contrast, mammalian erythrocytes and lens cells exhibit patterns of variants that are more similar to each other. These results show that only a subset of spectrin-containing cells possess protein 4.1, and that these cells differentially express the variants of protein 4.1 in a manner that may reflect corresponding functional differences.

Avian lens spectrin: subunit composition compared with erythrocyte and brain spectrin.

Chicken lens spectrin is composed predominantly of equimolar amounts of two polypeptides with solubility properties similar, but not identical, to erythrocyte spectrin. The larger polypeptide, Mr 240,000 (lens alpha-spectrin), co-migrates with erythrocyte and brain alpha-spectrin on one- and two-dimensional SDS polyacrylamide gels and cross-reacts with antibodies specific for chicken erythrocyte alpha-spectrin; the smaller polypeptide, Mr 235,000 (lens gamma-spectrin), co-migrates with brain gamma-spectrin and does not cross-react with either the alpha-spectrin antibodies specific for chicken erythrocyte beta-spectrin. Minor amounts of polypeptides antigenically related to erythrocyte beta-spectrin with a greater electrophoretic mobility than lens gamma-spectrin are also detected in lens. The equimolar ratio of lens alpha- and gamma-spectrin is invariantly maintained during the extraction of lens plasma membranes under different conditions, or after immunoprecipitation of whole extracts of lens with erythrocyte alpha-spectrin antibodies. Two-dimensional peptide mapping reveals that whereas alpha-spectrins from chicken erythrocytes, brain, and lens are highly homologous, the gamma-spectrins, although related, have some cell-type-specific peptides and are substantially different from erythrocyte beta-spectrin. Thus, the expression of cell-type-specific gamma- and beta-spectrins may be the basis for the assembly of a spectrin-plasma membrane complex whose molecular composition is tailored to the functional requirements of the particular cell-type.

Expression of the major neurofilament subunit in chicken erythrocytes.

The 70,000-dalton core polypeptide of neurofilaments, thought to exist only in neurons, has been detected in chicken erythrocytes, where it coexists with vimentin and synemin as a component of the intermediate filament network. It is present in the circulating erythroid cells of embryos and young chickens but is nearly absent from the erythroid cells of adults. These findings are inconsistent with current models of intermediate filament expression, but provide another example of unexpected similarities between the nervous and hemopoietic systems.

Structural associations of synemin and vimentin filaments in avian erythrocytes revealed by immunoelectron microscopy.

Intermediate filament structure and distribution were studied by antibody decoration and low-angle shadowing of sonicated chicken erythrocytes and embryonic erythroid cells. Intermediate filaments containing vimentin and synemin form a three-dimensional network in these cells, interlinking the nucleus and plasma membrane. This filament network is spatially segregated from the marginal band of microtubules, indicating that these two systems do not interact directly in the development or maintenance of cell shape. Incubation of sonicated cells with an antiserum specific for vimentin results in uniform decoration of the intermediate filaments; incubation with antisynemin results in decoration of periodically spaced foci. Measurement of the synemin periodicity under a specified set of sample preparation conditions gives average values of 180 nm for adult erythrocytes and 230 nm for 10 day old embryonic erythroid cells, suggesting some fundamental change in the structure of the filaments during erythropoiesis. Registration of these foci in laterally associated filaments, and decoration of bridges between slightly separated filaments, suggest that synemin mediates crosslinking of intermediate filaments through self-interaction.

Widespread occurrence of avian spectrin in nonerythroid cells.

We have prepared an antibody against chicken erythrocyte alpha spectrin, using as immunogen protein purified by two-dimensional polyacrylamide gel electrophoresis. One- and two-dimensional immunoautoradiography show that this antiserum reacts only with alpha spectrin in chicken erythrocytes and crossreacts with alpha spectrin in erythrocytes from various mammals. Immunofluorescence reveals that this antiserum reacts with a plasma membrane component in erythrocytes as well as in most nonerythroid avian and mammalian cells. Intense staining is seen at or near the plasma membrane in neurons, lens cells, endothelial and epithelial cells of the gastrointestinal and respiratory tracts, skeletal and cardiac muscle, as well as skeletal myotubes grown in tissue culture. Immunoautoradiography indicates that the crossreactive antigen in these nonerythroid tissues has the same molecular weight and isoelectric point as the chicken erythrocyte antigen. Smooth muscle, tracheal cilia, myelin and mature sperm stain weakly or not at all. These results suggest that spectrin is more extensively distributed than previously recognized, and that the functions of spectrin elucidated for erythrocytes may apply to other cell types as well.

Synemin and vimentin are components of intermediate filaments in avian erythrocytes.

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.

Synemin: a new high molecular weight protein associated with desmin and vimentin filaments in muscle.

A 230,000 dalton polypeptide co-purifies through cycles of depolymerization and polymerization with the intermediate filament subunits, desmin and vimentin, from avian smooth muscle. This protein is also present in skeletal muscle and is distinct from myosin and filamin. Double immunofluorescence microscopy of cultured cells, using antisera shown to be specific by immunoautoradiography, has revealed that this protein has the same spatial distribution as desmin and vimentin. During skeletal myogenesis, all three antigens exist initially in multinucleate myotubes as wavy filaments throughout the cytoplasm. Within a week after myoblast fusion, they begin to coalesce at the peripheries of the myofibril Z discs, thereby attaining the distribution observed in mature muscle, a network of interlinked rings within the Z plane. Treatment of cultured myotubes with colcemid causes the filamentous forms of these three proteins to co-aggregate into cytoplasmic bundles, but has little effect on them when they are associated with the Z discs. Extraction of cells with nonionic detergent and high salt leaves cytoskeletons containing desmin, vimentin and the 230,000 dalton polypeptide with immunofluorescent patterns that are indistinguishable from one another. These data suggest that this high molecular weight protein is closely associated with desmin and vimentin filaments in muscle cells; to indicate this, we have named the protein synemin, from the Greek oa uv (with) and v eta mu alpha (filament).