Inter-strain Variability of Structural Proteins in Tetrahymena*

SYNOPSIS The high molecular weight proteins found in isolated pellicles of Tetrahymena have been compared in several individual strains within the genus using SDS polyacrylamide gel electrophoresis. Three variants of the B-protein of epiplasm (MW 174,000; 155,000; 145,000) and 2 of the C-protein (MW 140,000; 122,000) were found among the strains examined. No variation was observed in the major kinetodesmal fiber protein (MW 250,000). The variation found between strains in the proteins of a structure which is (as far as we know) the same in all strains indicates a disjunction between evolutionary change at the 2 levels of organization. The taxonomic implications of the observed variation in structural proteins in Tetrahymena are discussed.     

Protein Similarities in the Genus Tetrahymena and a Description of Tetrahymena leucophrys n. sp.1

The potential utility of protein arrays visualized by SDS-PAGE in taxonomic studies of protozoa has been investigated within the genus Tetrahymena. Matching coefficients have been obtained from one-dimensional separations of cytoskeletal proteins in a study involving 40 strains of Tetrahymena. In separate studies, the method was estimated to be accurate to within 10%, and the limit of intraspecific variation within the genus was estimated at ? 30%. Accordingly, it is suggested that strains showing matching coefficients 0.6 in these preparations probably represent different species. Further tests using strains identified to species by breeding criteria have shown that the 0.6 rule is asymmetric, i.e. although matching coefficients lower than this indicate separate species, values higher than this do not prove identity. Protein comparisons included in the present study indicate that strain Tur, previously identified as a strain of T. vorax, is distinct from all other macrostome-forming species. It is here designated Tetrahymena leucophrys n. sp.     

Growth Factors and Steroid Mediated Regulation of Cytoskeletal Protein Expression in Serum-Deprived Primary Astrocyte Cultures

In this research we aimed to investigate the interactions between growth factors (GFs) and dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression under different experimental conditions. Condition I: 24 h pretreatment with bFGF, subsequent 72 h switching in serum-free medium (SFM) and final addition of GFs, alone or by two in the last 24 h, after a prolonged (60 h) DEX treatment. Condition II: 36 h pretreatment with DEX (with bFGF in the last 24 h), followed by SFM for 60 h and final addition for 24 h with growth factors alone or two of them togheter. Western blot analysis data showed a marked GFAP expression in cultures submitted to Condition I comparing results to untreated or treated controls. VIM expression was instead significantly reduced after GFs addition in the last 24 h of 60 h DEX treatment, respect to control DEX-pretreated ones. Referring data to untreated controls, VIM expression was significantly enhanced after GFs addition. GFAP showed also a significant increase in astrocytes submitted to Condition II, respect to untreated or treated control cultures. VIM expression was up and down regulated under Condition II. Collectively, our findings evidence an interactive dialogue between GFs and DEX in astroglial cultures, co-pretreated with DEX and bFGF, regulating cytoskeletal network under stressfull conditions. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

The epiplasm gene EPC1 influences cell shape and cortical pattern in Tetrahymena thermophila

The cortical protein Epc1p is the most abundant protein in the membrane skeleton, or epiplasm, of Tetrahymena thermophila. A partial sequence of the EPC1 gene was obtained and used to obtain a knockout construct that was successful in transforming Tetrahymena thermophila cells. The results support the conclusion that Epc1p influences cell shape and the fidelity of cortical development. It was further observed that this protein is transferred from plus to minus cells during conjugation, and that the imported protein is assembled into the epiplasm of the recipient cell in a discreet series of steps.     

ARL1 Plays a Role in the Binding of the GRIP Domain of a Peripheral Matrix Protein to the Golgi Apparatus in Plant Cells

ARF GTPases play a central role in regulating membrane dynamics and protein transport in eukaryotic cells. ARF-like (ARL) proteins are close relatives of the ARF regulators of vesicular transport, but their function in plant cells is poorly characterized. Here, by means of live cell imaging and site-directed mutagenesis, we have investigated the cellular function of the plant GTPase ARL1. We provide direct evidence for a role of this ARL family member in the association of a plant golgin with the plant Golgi apparatus. Our data reveal the existence of key residues within the conserved GRIP-domain of the golgin and within the GTPase ARL1 that are central to ARL1–GRIP interaction. Mutations of these residues abolish the interaction of GRIP with the GTP-bound ARL1 and induce a redistribution of GRIP into the cytosol. This indicates that the localization of GRIP to the Golgi apparatus is strongly influenced by the interaction of GRIP with Golgi-localized ARL1. Our results assign a cellular role to a member of the Arabidopsis ARL family in the plant secretory pathway and propose mechanisms for localization of peripheral golgins to the plant Golgi apparatus. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Comparative Study of Seed Protein Profiles in the Genus Pisum

Seed protein profiles of 24 wild and cultivated taxa of Pisum have been compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No consistent differences were detected either among wild taxa or between wild and cultivated taxa. This shows that Pisum forms a single-species complex on the basis of seed protein profiles.     

In vivo secretion and association of clusterin (SGP-2) in luminal fluid with spermatozoa in the rat testis and epididymis.

Clusterin (sulfated glycoprotein-2) is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells. An antigenically similar form is synthesized and secreted by the epididymis. The goal of this study was to define the epididymal regions in which clusterin is present and the regions in which clusterin is secreted and interacts with developing spermatozoa. Seminiferous tubule (STF), caput, corpus, and cauda fluids were collected by micropuncture and/or microperfusion and two-dimensional Western blot analysis was performed with a polyclonal antibody directed against Sertoli cell clusterin. Clusterin was found in both STF and epididymal fluid. STF contained predominantly the clusterin heavy chain (45 kd); however, a 70 Kd heterodimer was present under nonreducing conditions. Two subunits of clusterin with lower molecular weights (41 kd, heavy chain; 32 kd, light chain) and higher isoelectric points were present in the luminal fluid of all epididymal regions. The intraluminal levels of the heavy and light chains decreased from caput to cauda. Analysis by two-dimensional gel electrophoresis of proteins secreted directly into the epididymal luminal fluid revealed that clusterin was secreted by caput epithelium and not by the corpus and cauda epithelium. Western blots of membrane extracts from testicular, caput, and cauda spermatozoa revealed that testicular clusterin was associated with testicular sperm and epididymal clusterin with predominantly caput sperm. Our findings suggest that clusterin is secreted into the caput epididymal lumen, where it binds to sperm and then dissociates from sperm to be endocytosed by cells of the distal epididymal epithelium.     

Novel cytoskeletal proteins in the cortex of Tetrahymena

An important unsolved problem lies in the mechanisms that determine overall size, shape, and the localization of subcellular structures in eukaryotic cells. The membrane skeleton must play a central role in these processes in many cell types, and the ciliate membrane skeleton, or epiplasm, offers favorable opportunities for exploring the molecular determinants of cortical organization. Among the ciliates, Tetrahymena is well suited for the application of a wide range of molecular and cellular approaches. Progress has been made in the identification and sequencing of genes and proteins that encode epiplasmic and cortical proteins. The amino acid sequences of these proteins suggest that they define new classes of cytoskeletal proteins, distinct from the articulin and epiplasmin proteins. We will also discuss recent in vivo and in vitro studies of the regulation of assembly of these cortical proteins. This will include information regarding the down-regulation of epiplasmic proteins during cleavage, their topographic regulation in the cell cycle, and the results of in vitro assembly and binding studies of the epiplasmic C protein.     

Glial Fibrillary Acidic Protein Increases in the Spinal Cord of Lewis Rats with Acute Experimental Autoimmune Encephalomyelitis

Glial fibrillary acidic protein (GFAP) in the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) was quantitated by densitometry of both stained gels and immunoblots of electrophoretically separated cytoskeletal proteins. The experimental period ranged from 7 to 65 days postinoculation (dpi). Greater than 92% of the total spinal cord GFAP was recovered in the Triton-insoluble cytoskeletal pellet; less than 2% was truly soluble. GFAP increased gradually and significantly with time, reaching a level one-and-a-half to two times greater than that of controls by 35 dpi and remaining elevated at 65 dpi. In EAE animals, GFAP was 33% of the total Triton-insoluble protein (excluding histones and other small basic proteins) at 7 dpi, rising to 48% at 65 dpi. Increases in vimentin were also noted, following a time course similar to that of GFAP. An increase in immunocytochemical staining of GFAP was noticeable at 10 dpi and became marked at 14 dpi, a time before GFAP levels had increased significantly. Thus, enhanced staining at the peak of the disease cannot be explained simply by an increase in antigen protein. Other possible explanations, such as an increase in soluble GFAP content, proteolytic degradation, or modifications in the immunochemical properties of GFAP in EAE animals, were ruled out. Both the biochemical and immunocytochemical increases in GFAP persisted through 65 dpi, even though the animals recovered from clinical signs at approximately 18 dpi.     

Membrane-Associated Cytoskeletal Proteins in Squid Giant Axons

Abstract: Cytoskeletal proteins (e.g., tubulin, actin, and neurofilament proteins) in the squid giant axon are separable into KF-soluble and -insoluble forms. The KF-insoluble cytoskeletal components appear to constitute the major proteins in the subaxolemmal fibrous network on the inner surface of the axon. These cytoskeletal proteins and the subaxolemmal network are both highly soluble in KI solutions. Whereas giant axons tolerate prolonged perfusions in KF solutions with no loss of excitable properties, a relatively short perfusion with KI solution completely eliminates the excitability of the axon. The loss of this excitability correlates with the simultaneous dissolution of the subaxolemmal network of cytoskeletal proteins and the release of its proteins into the perfusate. These data support the hypothesis that cytoskeletal proteins associated with the inner surface of the axolemma are involved in the regulation of axonal excitability.     

The identification of thymic nurse cells in vivo and the role of cytoskeletal proteins in thymocyte internalization

Much debate has been generated about the existence of thymic nurse cells within the thymus. Until now, the authenticity of an epithelial cell capable of internalizing developing thymocytes within the thymic cortex has been in question. Here, we use the thymic nurse cell-specific monoclonal antibody, ph91, to define the in vivo location of thymic nurse cells. For the first time, thymic nurse cells enclosing several thymocytes were detected in the subcapsular region of the thymic cortex in a “honeycomb-like” configuration. In vitro studies show the internalization process using digitalized time-lapse microscopy. Internalized thymocytes have also been reported to interact with macrophages within the TNC complex. The cytoplasmic interaction between thymocytes and macrophages was detected using time-lapse microscopy. Using fluorescence microscopy, we show polymerization of actin within macrophages at the contact point with thymocytes, which is indicative of an immunological synapse. Microfilaments and microtubules within TNCs were shown to be associated with thymocyte binding and internalization, but neither interacted with macrophages. Also, we provide data to show that thymocytes are actively involved in the internalization process. These experiments show for the first time the existence of thymic nurse cells within the thymic microenvironment. They provide a visual documentation of thymocyte uptake by thymic nurse cells, and define an interaction between thymocytes and macrophages within the TNC complex.     

Platelet activating factor induces cytoskeletal reorganization through Rho family pathway in THP-1 macrophages

In the process of atherosclerosis, platelet activating factor (PAF) promotes the infiltration of inflammatory cells into atherosclerotic plaque by modulating their cytoskeleton. Here, we examined whether Rho family proteins are involved in PAF-induced cytoskeletal reorganization in THP-1 macrophages. PAF stimulation rapidly induced cell elongation, accompanied by filopodia formation. The inhibition of Rho family proteins by the overexpression of Rho-GDI attenuated the PAF-mediated morphological changes. Both RhoA and Cdc42 were activated in response to PAF. Inhibition of RhoA or Cdc42 by dominant negative mutants abrogated morphological changes induced by PAF. Collectively, PAF regulates cytoarchitecture through Rho family proteins in macrophages.     

Protein binding to expanded telomere repeats in Tetrahymena thermophila

The ends of eukaryotic chromosomes are protected by DNA-protein structures called telomeres. Telomeric DNA is highly conserved, usually consisting of long tracts of a repeating G-rich sequence. Tetrahymena thermophila telomeric DNA consists of alternating blocks of GGGG and TT sequences (i.e. a G4T2 repeat sequence). We examined the relative importance of the guanine and thymine elements of the repeat sequence in promoting in vitro binding by T. thermophila proteins. We identified single- and, for the first time, double-stranded telomere binding activities from a crude T. thermophila protein extract and tested the binding of these activities to altered telomere repeat sequences. All deletions or substitutions made to the guanine element virtually abolished binding, indicating that four G's are essential for recognition by the binding activity. However, G's alone are not sufficient for efficient binding, as elimination of the thymine element dramatically reduced binding. By contrast, substantial expansion of the thymine element was well tolerated, even though one such change, G4T4, is lethal in vivo. We tested up to a four-fold expansion of the thymine element and found that highly efficient binding was still achieved. These results suggest a minimal recognition sequence for T. thermophila proteins, with the T element providing an important spacer between essential G elements.     

Cytoskeletal Alterations in Human Fetal Astrocytes Induced by Interleukin-1β

Abstract: Previous studies in this and other laboratories have shown that interleukin-1β (IL-1β) is a selective and potent activator of human astrocytes with respect to induction of cytokines and hematopoietic growth factors. To study the effect of recombinant human IL-1β (rhIL-1β) on astrocyte morphology, glial fibrillary acidic protein (GFAP) and vimentin expression, and actin organization, we conducted a systematic survey using dissociated human fetal astrocyte cultures. Within hours of stimulation with IL-1β, the majority of astrocytes converted from flat, polygonal cells to small, contracted, highly branched cells. This change in morphology was more striking when serum was eliminated from the medium. Complete dissolution of filamentous actin occurred simultaneously with the change in cell shape, as demonstrated by fluorescein-phalloidin binding. These “activated” astrocytes displayed intense GFAP and vimentin immunoreactivity in the small perikarya and processes. In contrast, the large, flat astrocytes in control cultures showed diffuse pale immunoreactivity for GFAP and vimentin. To quantify the changes in GFAP and vimentin content with IL-1β stimulation, densitometric analyses of northern and western blots were performed. Northern blot analysis of IL-1β-stimulated astrocytes revealed a transient, marked decrease in steady-state levels of mRNA for GFAP, vimentin, and microtubule-associated protein 4. The decrease in mRNA levels was evident by 4–8 h and fell to the lowest level at 16–24 h (80–98% decrease by densitometry) with partial recovery by 72 h. By immunoblotting, a significant decrease in both GFAP and vimentin protein content was observed after IL-1β stimulation. Furthermore, metabolic labeling studies revealed an almost total loss of GFAP synthesis following stimulation with IL-1β for 16 h. These observations are consistent with the idea that increases in immunoreactivity were related to factors such as redistribution of epitope, rather than increases in total protein content. We hypothesize that in IL-1β-stimulated astrocytes, synthesis of other proteins, e.g., inflammatory cytokines, occurs at the expense of structural proteins and that the decrease in content of cytoskeletal proteins may reflect an “activated” state of astrocytes.     

Ca2+-mediated phosphorylation and proteolysis activity associated with the cytoskeletal fraction from cerebral cortex of rats

We describe a Triton-insoluble cytoskeletal fraction extracted from cerebral cortex of young rats retaining an endogenous Ca²⁺-mediated mechanism acting in vitro on Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII) activity and on phosphorylation and proteolysis of the 150 kDa neurofilament subunit (NF-M), α and β tubulin. Exogenous Ca²⁺ induced a 70% decrease in the in vitro phosphorylation of the NF-M and tubulins and a 30–50% decrease in the total amount of these proteins. However, when calpastatin was added basal phosphorylation and NF-M and tubulin content were recovered. Furthermore, exogenous Ca²⁺/calmodulin induced increased in vitro phosphorylation of the cytoskeletal proteins and CaM-KII activity only in the presence of calpastatin, suggesting the presence of Ca²⁺-induced calpain-mediated proteolysis. This fraction could be an interesting model to further studies concerning the in vitro effects of Ca²⁺-mediated protein kinases and proteases associated with the cytoskeletal fraction.     

Contractile proteins in podocytes: immunocytochemical localization of actin and alpha-actinin in normal and nephrotic rat kidneys

Actin and alpha-actinin immunoreactive sites have been localized at the electron microscope level by the protein A-gold immunocytochemical technique in podocytes of normal and nephrotic rat renal tissues. In normal renal glomeruli, fibrillar networks located in the core of foot processes or bundles of micro filaments interconnecting them were found to be labelled for these two cytoskeletal proteins. On the other hand, in nephrotic renal glomeruli, concomitant with the loss of podocytic foot processes a reorganization of the podocytic cytoskeleton and a concentration of some of its elements into thick uniform bands was observed. Actin and alpha-actinin were revealed in these bands. Control experiments confirmed the specificity of the labelling obtained. Our results suggest that normal podocytes contain an actin-based contractile system that might contribute to the maintenance of the particular cell shape of these cells and that the rearrangement of the podocytic cyto-skeleton occurring in the nephrotic syndrome might account for the changes in the foot processes and contribute to the alteration in glomerular function. This work was supported by grants from the Medical Research Council of Canada