ABSTRACT
The formation of skeletal muscle fibers is an intricate process controlled by a multitude of signaling pathways, including Wnt, Shh, and FGF. However, the role of the Hippo pathway during vertebrate myofiber formation has conflicting reports, which we decided to address in chick muscle cultures. We found that the transcriptional regulator Yes-associated protein (YAP) was highly concentrated within the nuclei of myoblasts. As cells differentiate into myotubes, YAP localization shifted to the cell cytoplasm in more mature myotubes. Treatment of cultures with XMU-MP-1 (XMU), a MST1/2 inhibitor, stimulated the nuclear localization of YAP in myoblasts and in myotubes, upregulated myogenin, and promoted myoblast fusion, ultimately resulting in the formation of large and fully striated multinucleated myotubes. The XMU-induced phenotype was blocked by the protein kinase C (PKC) inhibitor calphostin, which raises the possibility that the Hippo pathway controls the growth of skeletal muscle fibers through a PKC-dependent mechanism.
Subject(s)
Muscle Development , Muscle Fibers, Skeletal , Cell Differentiation , Muscle Fibers, Skeletal/metabolism , Myoblasts/metabolism , Signal Transduction/geneticsABSTRACT
ß-Adrenergic signaling regulates many physiological processes in skeletal muscles. A wealth of evidence has shown that ß-agonists can increase skeletal muscle mass in vertebrates. Nevertheless, to date, the specific role of ß-adrenergic receptors in different cell phenotypes (myoblasts, fibroblasts, and myotubes) and during the different steps of embryonic skeletal muscle differentiation has not been studied. Therefore, here we address this question through the analysis of embryonic chick primary cultures of skeletal muscle cells during the formation of multinucleated myotubes. We used isoproterenol (ISO), a ß-adrenergic receptor agonist, to activate the ß-adrenergic signaling and quantified several aspects of muscle differentiation. ISO induced an increase in myoblast proliferation, in the percentage of Pax7-positive myoblasts and in the size of skeletal muscle fibers, suggesting that ISO activates a hyperplasic and hypertrophic muscle response. Interestingly, treatment with ISO did not alter the number of fibroblast cells, suggesting that ISO effects are specific to muscle cells in the case of chick myogenic cell culture. We also show that rapamycin, an inhibitor of the mammalian target of rapamycin signaling pathway, did not prevent the effects of ISO on chick muscle fiber size. The collection of these results provides new insights into the role of ß-adrenergic signaling during skeletal muscle proliferation and differentiation and specifically in the regulation of skeletal muscle hyperplasia and hypertrophy.
ABSTRACT
The Sonic Hedgehog signaling (Shh) pathway has been implicated in both proliferation of myoblast cells and terminal differentiation of muscle fibers, and contradictory results of these effects have been described. To clarify the role of Shh during myogenesis, we decided to study the effects of recombinant Shh and the distribution of Gli-1 during in vitro and in situ embryonic chick skeletal muscle differentiation at later stages of development. Gli-1 was found in small aggregates near the nucleus in mononucleated myoblasts and in multinucleated myotubes both in vitro and in situ chick muscle cells. Some Gli-1 aggregates colocalized with gamma-tubulin positive-centrosomes. Gli-1 was also found in striations and at the subsarcolemmal membrane in muscle fibers in situ. Recombinant Shh added to in vitro grown muscle cells induced the nuclear translocation of Gli-1, as well as an increase in the number of myoblasts and in the number of nuclei within myotubes. We suggest that Gli-1 aggregates observed in chick muscle cells near the nuclei of myoblasts and myotubes could be a storage site for the rapid cellular redistribution of Gli-1 upon specific signals during muscle differentiation.
Subject(s)
Hedgehog Proteins/metabolism , Muscle Development , Zinc Finger Protein GLI1/metabolism , Animals , Cell Nucleus/metabolism , Cell Proliferation , Centrosome/metabolism , Chick Embryo , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Myoblasts/metabolism , Protein Aggregates , Protein Transport , Sarcolemma/metabolismABSTRACT
BACKGROUND INFORMATION: The costa is a prominent striated fibre that is found in protozoa of the Trichomonadidae family that present an undulating membrane. It is composed primarily of proteins that have not yet been explored. In this study, we used cell fractionation to obtain a highly enriched costa fraction whose structure and composition was further analysed by electron microscopy and mass spectrometry. RESULTS: Electron microscopy of negatively stained samples revealed that the costa, which is a periodic structure with alternating electron-dense and electron-lucent bands, displays three distinct regions, named the head, neck and body. Fourier transform analysis showed that the electron-lucent bands present sub-bands with a regular pattern. An analysis of the costa fraction via one- and two-dimensional electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) allowed the identification of 54 hypothetical proteins. Fourteen of those proteins were considered to be major components of the fraction. CONCLUSIONS: The costa of T. foetus is a complex and organised cytoskeleton structure made of a large number of proteins which is assembled into filamentous structures. Some of these proteins exhibit uncharacterised domains and no function related according to gene ontology, suggesting that the costa structure may be formed by a new class of proteins that differ from those previously described in other organisms. Seven of these proteins contain prefoldin domains displaying coiled-coil regions. This propriety is shared with proteins of the striated fibres of other protozoan as well as in intermediate filaments. SIGNIFICANCE: Our observations suggest the presence of a new class of the cytoskeleton filaments in T. foetus. We believe that our data could auxiliate in determining the specific locations of these proteins in the distinct regions that compose the costa, as well as to define the functional roles of each component. Therefore, our study will help in the better understanding of the organisation and function of this structure in unicellular organisms.
Subject(s)
Cytoskeleton/chemistry , Protozoan Proteins/chemistry , Trichomonadida/metabolism , Cell Fractionation , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Microscopy, Electron , Protozoan Proteins/metabolism , Protozoan Proteins/ultrastructure , Tandem Mass Spectrometry , Trichomonadida/chemistry , Trichomonadida/ultrastructureABSTRACT
Tritrichomonas foetus is a protist that causes bovine trichomoniasis and presents a well-developed Golgi. There are very few studies concerning the Golgi in trichomonads. In this work, monoclonal antibodies were raised against Golgi of T. foetus and used as a tool on morphologic and biochemical studies of this organelle. Among the antibodies produced, one was named mAb anti-Golgi 20.3, which recognized specifically the Golgi complex by fluorescence and electron microscopy. By immunoblotting this antibody recognized two proteins with 60 and 66 kDa that were identified as putative beta-tubulin and adenosine triphosphatase, respectively. The mAb 20.3 also recognized the Golgi complex of the Trichomonas vaginalis, a human parasite. In addition, the nucleotide coding sequences of these proteins were identified and included in the T. foetus database, and the 3D structure of the proteins was predicted. In conclusion, this study indicated: (1) adenosine triphosphatase is present in the Golgi, (2) ATPase is conserved between T. foetus and T. vaginalis, (3) there is new information concerning the nucleic acid sequences and protein structures of adenosine triphosphatase and beta-tubulin from T. foetus and (4) the mAb anti-Golgi 20.3 is a good Golgi marker and can be used in future studies.
Subject(s)
Adenosine Triphosphatases/metabolism , Antibodies, Protozoan/immunology , Antigens, Protozoan/immunology , Golgi Apparatus/ultrastructure , Protozoan Infections, Animal/parasitology , Tritrichomonas foetus/ultrastructure , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Base Sequence , Cattle , Female , Golgi Apparatus/chemistry , Golgi Apparatus/metabolism , Mice , Mice, Inbred BALB C , Microscopy, Electron, Transmission/veterinary , Microscopy, Fluorescence/veterinary , Models, Molecular , Protozoan Proteins/chemistry , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Sequence Alignment/veterinary , Sequence Analysis, DNA/veterinary , Trichomonas vaginalis/enzymology , Trichomonas vaginalis/immunology , Tritrichomonas foetus/enzymology , Tritrichomonas foetus/genetics , Tritrichomonas foetus/immunologyABSTRACT
Trichomonas vaginalis causes trichomoniasis in humans, a sexually transmitted disease commonly treated with metronidazole (MTZ), a drug that presents some toxicity, causing undesirable side effects. In addition, an increase in metronidazole-resistant parasites has been reported. Thus, the development of alternative treatment is recommended. To date, the search for antiparasitic drugs has been based on different approaches: identification of active natural products, identification of parasite targets, and the use of available compounds active against other pathogenic microorganisms. Here, we analyzed the in vitro antiproliferative and ultrastructural effects on T. vaginalis of BPQ-OH, a hydroxiquinuclidine derivative that inhibits squalene synthase and is active against several protozoa and fungi. We also compared the effects of BPQ-OH on T. vaginalis and mammalian cells with those of MTZ. We found that BPQ-OH inhibits in vitro proliferation of T. vaginalis, with an IC50 of 46 µM after 24 h. Although this IC50 is 16 times higher than that of MTZ (1.8 µM), BPQ-OH is less toxic for human cell lines than MTZ, with LC50 values of 2,300 and 70 µM, and selective indexes of 50 and 39, respectively. Ultrastructural analyses demonstrated that BPQ-OH induced alterations in T. vaginalis, such as rounded and wrinkled cells, membrane blebbing and intense vacuolization, leading to cell death, whereas MTZ also caused significant changes, including a decrease in hydrogenosomes size and endoflagellar forms. Our observations identify BPQ-OH as a promising leading compound for the development of novel anti-T. vaginalis drugs and highlight the need for further testing this molecule using experimentally infected animals.
Subject(s)
Antiprotozoal Agents/pharmacology , Metronidazole/pharmacology , Quinuclidines/pharmacology , Trichomonas vaginalis/drug effects , Animals , Caco-2 Cells , Cell Proliferation/drug effects , Farnesyl-Diphosphate Farnesyltransferase/antagonists & inhibitors , HeLa Cells , Humans , Organelles/drug effects , Trichomonas vaginalis/cytology , Trichomonas vaginalis/ultrastructureABSTRACT
Trichomonas vaginalis causes trichomoniasis in humans, a sexually transmitted disease commonly treated with metronidazole (MTZ). MTZ is known to cause undesirable side effects, and MTZ-resistant parasites have been reported. Thus, the development of an alternative treatment is desirable. Miltefosine (MLT) is an alkylphosphocholine synthetic lipid analogue that displays antiparasitic activity against Leishmania, Trypanosoma cruzi, Entamoeba histolytica, Acanthamoeba spp., Giardia lamblia, T. vaginalis and some fungi. Moreover, it has been used for oral treatment of visceral leishmaniosis in several countries. Here, we analysed the MLT-induced antiproliferative effect on T. vaginalis as well its effect on the fine structure and viability of the parasite. We observed a dose-dependent effect with an IC50 of 14.5 and 20 µM after 24 and 48 h, respectively. Furthermore, reversibility assays demonstrated that new incubations were necessary in order to maintain the antiproliferative effect. Ultrastructural analyses demonstrated that MLT induced several alterations, including the appearance of wrinkled and rounded cells, membrane blebbing, intense vacuolization and nuclear condensation, all indicative of cell death by apoptosis. In addition, the quantitative analyses of the viability assays using combined markers of live and dead cells demonstrated that treatment with the IC50 concentration of MLT significantly reduced the number of viable parasites compared with untreated cells. Taken together, these observations suggest that MLT is a promising compound for the treatment of trichomoniasis.
Subject(s)
Antiprotozoal Agents/pharmacology , Phosphorylcholine/analogs & derivatives , Trichomonas vaginalis/drug effects , Dose-Response Relationship, Drug , Inhibitory Concentration 50 , Phosphorylcholine/pharmacology , Trichomonas vaginalis/ultrastructureABSTRACT
Tritrichomonas foetus is a pathogenic protozoan that causes bovine trichomoniasis. In addition to its importance in veterinary medicine, this parasite is also a good representative of one the earliest eukaryotic cells available for study. T. foetus contains organelles that are common to all eukaryotic cells as well as uncommon cell structures such as hydrogenosomes and a complex and elaborate cytoskeleton that constitutes the mastigont system. The mastigont system is mainly formed by several proteinaceous structures that are associated with basal bodies, the pelta-axostylar complex and the costa. Although the structural organization of trichomonad cytoskeletons has been analyzed using several techniques, observation using a new generation of scanning electron microscopes with a resolution of 0.8nm has allowed more detailed visualization of the three-dimensional organization of the mastigont system. Moreover, this study revealed the presence of new structures, such as the costa accessory filament, and the presence of two groups of microtubules that form the pelta-axostylar system.
Subject(s)
Microtubules/ultrastructure , Tritrichomonas foetus/ultrastructure , Animals , Cattle , Cytoskeleton/ultrastructure , Male , Microscopy, Electron, Scanning , Protozoan Proteins/ultrastructureABSTRACT
Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.
Subject(s)
Actin Cytoskeleton/metabolism , Nuclear Envelope/metabolism , Actin Cytoskeleton/genetics , Actin Cytoskeleton/ultrastructure , Animals , Cells, Cultured , Chick Embryo , Intrinsically Disordered Proteins/metabolism , Lysosomes/metabolism , Lysosomes/ultrastructure , Microscopy, Electron, Transmission/methods , Mitochondria/metabolism , Mitochondria/ultrastructure , Nuclear Envelope/ultrastructure , Proteome/genetics , Proteome/metabolism , ZebrafishABSTRACT
Epithelial and mesenchymal cell types are basic for animal multicellularity and they have complementary functions coordinated by cellular interactions. Sponges are especially important model organisms to address the evolutionary basis of morphogenetic programs for epithelial and mesenchymal organization in animals. Evolutionary studies in sponges can contribute to the understanding of the mechanisms that control tissue maintenance and tumor progression in humans. In the present study, sponge mesenchymal and epithelial cells were isolated from the demosponge Hymeniacidon heliophila, and aggregate formation was observed by video microscopy. Epithelial-mesenchymal interaction, epithelial transition, and cell migration led to sponge cell aggregation after drastic stress. Based on their different morphologies, adhesion specificities, and motilities, we suggest a role for different sponge cell types as well as complementary functions in cell aggregation. Micromanipulation under the microscope and cell tracking were also used to promote specific grafting-host interaction, to further test the effects of cell type interaction. The loss of cell polarity and flattened shape during the epithelial to mesenchymal cell transition generated small immobile aggregates of round/amoeboid cells. The motility of these transited epithelial-cell aggregates was observed by cell tracking using fluorescent dye, but only after interaction with streams of migratory mesenchymal cells. Cell motility occurred independently of morphological changes, indicating a progressive step in the transition toward a migratory mesenchymal state. Our data suggest a two-step signaling process: (a) the lack of interaction between mesenchymal and epithelial cells triggers morphological changes; and (b) migratory mesenchymal cells instruct epithelial cells for directional cell motility. These results could have an impact on the understanding of evolutionary aspects of metastatic cancer cells. HIGHLIGHTS: Morphogenetic movements observed in modern sponges could have a common evolutionary origin with collective cell migration of human metastatic cells. A sponge regenerative model was used here to characterize epithelial and mesenchymal cells, and for the promotion of grafting/host interactions with subsequent cell tracking. The transition from epithelial to mesenchymal cell type can be observed in sponges in two steps: (a) withdrawal of epithelial/mesenchymal cell interactions to trigger morphological changes; (b) migratory mesenchymal cells to induce epithelial cells to a collective migratory state.
Subject(s)
Cell Movement , Cell Shape , Epithelial Cells/cytology , Epithelial-Mesenchymal Transition , Mesoderm/cytology , Porifera/cytology , Animals , Cell Aggregation , Epithelial Cells/ultrastructure , Mesoderm/ultrastructure , Porifera/ultrastructureABSTRACT
CAPN3 is a muscle-specific and an intrinsically disordered protein. Thus, as a scaffolding protein CAPN3 could play a role during early stages of myogenesis. To test this hypothesis, we studied the distribution and function of CAPN3 during myogenesis using embryonic chick muscle cells grown in vitro. Super-resolution microscopy showed CAPN3 distribution in (i) amorphous patches in myoblasts, (ii) a region near the nuclei of myotubes; (iii) adhesion plaques in myotubes, (iv) stress fiber-like structures in myotubes, and (v) filaments in fibroblasts. Downregulation of CAPN3 induced a decrease in the number of muscle cells and in the size of myotubes formed. These data show a diverse intracellular distribution of CAPN3, compatible with a scaffolding protein, and suggest a multitude of different interactions of CAPN3 with other partners during muscle formation.
Subject(s)
Calpain/metabolism , Muscle Fibers, Skeletal/metabolism , Myoblasts/metabolism , Animals , Chick Embryo , Fibroblasts/metabolism , Intracellular Space/metabolism , Models, Biological , Muscle DevelopmentABSTRACT
The protist Tritrichomonas foetus (Excavata, Parabasalia) is a parasite that causes bovine and feline trichomonosis. Bovine trichomonosis is a venereal disease that leads to abortion and reproductive problems in herds. Feline trichomonosis affects domestic cats. Here, we report the genome sequence of the T. foetus K strain, isolated in Brazil.
ABSTRACT
Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. It grows in axenic media as a trophozoite with a pear-shaped body, three anterior flagella, and one recurrent flagellum. However, under some well-controlled experimental conditions in vitro, as well as in vivo in infected bulls, the parasite acquires a spherical or elliptical shape, and the flagella are internalized but the cells do not display a cyst wall. This form, known as the endoflagellar or pseudocystic form, is viable, and can be transformed back to trophozoites with pear-shaped body. We used confocal laser scanning microscopy, and high resolution scanning electron microscopy to examine the changes that take place in the protozoan cytoskeleton during trophozoite-pseudocyst transformation. Results confirmed previous studies and added new structural information to the organization of cytoskeletal structures during the transformation process. We observed that changes take place in the pseudocysts' axostyle and costa, which acquired a curved shape. In addition, the costa of multinucleated/polymastigont pseudocysts took variable conformations while curved. The costa accessory structure, as well as a network of filaments connecting this structure to the region where the recurrent flagellum associates to the protozoan body, was not seen in pseudocysts. In addition, the axostyle was fragmented during trophozoite-pseudocyst transformation.
Subject(s)
Cytoskeleton/ultrastructure , Tritrichomonas foetus/growth & development , Tritrichomonas foetus/ultrastructure , Animals , Cattle , Flagella/ultrastructure , Microscopy, Electron, Scanning/methods , Microtubules/ultrastructure , Tritrichomonas foetus/cytology , Trophozoites/ultrastructureABSTRACT
The mosquito Aedes aegypti can spread the dengue, chikungunya and yellow fever viruses. Thus, the search for key molecules involved in the mosquito survival represents today a promising vector control strategy. High Mobility Group Box (HMGB) proteins are essential nuclear factors that maintain the high-order structure of chromatin, keeping eukaryotic cells viable. Outside the nucleus, secreted HMGB proteins could alert the innate immune system to foreign antigens and trigger the initiation of host defenses. In this work, we cloned and functionally characterized the HMGB1 protein from Aedes aegypti (AaHMGB1). The AaHMGB1 protein typically consists of two HMG-box DNA binding domains and an acidic C-terminus. Interestingly, AaHMGB1 contains a unique alanine/glutamine-rich (AQ-rich) C-terminal region that seems to be exclusive of dipteran HMGB proteins. AaHMGB1 is localized to the cell nucleus, mainly associated with heterochromatin. Circular dichroism analyses of AaHMGB1 or the C-terminal truncated proteins revealed α-helical structures. We showed that AaHMGB1 can effectively bind and change the topology of DNA, and that the AQ-rich and the C-terminal acidic regions can modulate its ability to promote DNA supercoiling, as well as its preference to bind supercoiled DNA. AaHMGB1 is phosphorylated by PKA and PKC, but not by CK2. Importantly, phosphorylation of AaHMGB1 by PKA or PKC completely abolishes its DNA bending activity. Thus, our study shows that a functional HMGB1 protein occurs in Aedes aegypt and we provide the first description of a HMGB1 protein containing an AQ-rich regulatory C-terminus.
Subject(s)
HMGB1 Protein/chemistry , Insect Proteins/chemistry , Aedes , Amino Acid Sequence , Animals , Cell Nucleus/metabolism , Cloning, Molecular , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA, Superhelical/metabolism , DNA-Binding Proteins/metabolism , HMGB1 Protein/isolation & purification , Molecular Sequence Data , Phosphorylation , Protein Kinase C/metabolismABSTRACT
Tritrichomonas foetus and Trichomonas vaginalis are extracellular parasites of the urogenital tract of cattle and humans, respectively. They cause infertility and abortion, but there is no documented information on the susceptibility of bovine sperm cells to this cattle parasite. The aim of this present work was to study the effects provoked by T. foetus and T. vaginalis when in interaction with bovine and human sperm cells. The bovine and human spermatozoa were obtained from uninfected bulls and men, respectively, and were exposed to living trichomonads over different periods of time. Light microscopy, video microscopy, scanning, and transmission electron microscopy first revealed a tropism, then a close proximity followed by a tight adhesion between these two different cells. A decrease in the spermatozoa motility was observed as well intense semen agglutination. The adhesion between trichomonads to the sperm cell occurred either by the flagella or sperm head. Motile parasites were observed during the next 12 h, whereas sperm cells in contact with the parasites rapidly became immotile. The parasites were able to maintain the sperm cells attached to their cell surface, followed by phagocytosis. This process began with a tight membrane-membrane adhesion and the incorporation of the sperm cell within an intracellular vacuole. Afterwards, the sperm cell was gradually digested in lysosomes. Many trichomonads were injured and/or died on making contact with the spermatozoa possibly due to necrosis. Results from this study demonstrated that both T. foetus and T. vaginalis interact with sperm cells provoking damage and death of these reproductive cells. Differences in the behavior of both trichomonads were evident, showing that T. vaginalis was much more virulent than T. foetus. The possible role of trichomonads in reproductive failure is discussed.
Subject(s)
Cell Adhesion , Host-Parasite Interactions , Phagocytosis , Spermatozoa/parasitology , Trichomonas vaginalis/physiology , Tritrichomonas foetus/physiology , Animals , Cattle , Humans , Male , Microscopy, Electron , Spermatozoa/physiology , Spermatozoa/ultrastructure , Trichomonas Infections/parasitology , Trichomonas vaginalis/ultrastructure , Tritrichomonas foetus/ultrastructureABSTRACT
Hydrogenosomes are found in organisms that lack typical mitochondria. Cardiolipin is a phospholipid located exclusively in bacterial membranes and the inner membrane of mitochondria. Here we show, by cell fractionation, thin-layer chromatography, high-pressure liquid chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry that hydrogenosomes of Tritrichomonas foetus, a cattle vaginal parasite, contain cardiolipin, which is strong evidence for its endosymbiotic origin.