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Autophagy is an evolutionarily conserved catabolic process by which cells degrade their own components through the lysosomal machinery. In physiological conditions, the mechanism is tightly regulated and contributes to maintain a balance between synthesis and degradation in cells undergoing intense metabolic activities. Autophagy is associated with major tissue remodeling processes occurring through the embryonic, fetal and early postnatal periods of vertebrates. Here we survey current information implicating autophagy in cellular death, proliferation or differentiation in developing vertebrates. In developing systems, activation of the autophagic machinery could promote different outcomes depending on the cellular context. Autophagy is thus an extraordinary tool for the developing organs and tissues.
The capability of osmoadaptation is a prerequisite of organisms that live in an environment with changing salinities. Halobacillus halophilus is a moderately halophilic bacterium that grows between 0.4 and 3 M NaCl by accumulating both chloride and compatible solutes as osmolytes. Chloride is absolutely essential for growth and, moreover, was shown to modulate gene expression and activity of enzymes involved in osmoadaptation. The synthesis of different compatible solutes is strictly salinity- and growth phase-dependent. This unique hybrid strategy of H. halophilus will be reviewed here taking into account the recently published genome sequence. Based on identified genes we will speculate about possible scenarios of the synthesis of compatible solutes and the uptake of potassium ion which would complete our knowledge of the fine-tuned osmoregulation and intracellular osmolyte balance in H. halophilus.
Introduction: The involvement of platelets in various diseases has been increasingly recognized in the recent decades. This contribution is believed to involve platelet secretion and formation of reactive microparticles. Platelets contain two functionally important forms of vesicles, alpha and dense granules, which are secreted upon activation of platelets. Alpha granules incorporate larger molecules such as adhesive proteins, e.g. P-selectin, vWF and fibrinogen; chemokines like PF4 and RANTES and growth hormones like VEGF and PDGF are among the most important proteins attributed to the involvement of platelets in pathological conditions. In contrast, dense granules contain small molecules like ADP, ATP, serotonin and histamine, and they are more rapidly and completely secreted than alpha granules. Like in all secreting cells, regulated exocytosis in platelets is mediated by “zippering” of three different classes of SNARE proteins. The subtypes of these proteins found to be involved in platelet secretion are SNAP-23, syntaxin-2 and -4 and VAMP-3 and -8. Apart from SNARE proteins, other conserved proteins influencing exocytosis by e.g. acting on SNARE proteins have been described, one of the most important ones being Munc13. Platelets contribute to the progression of atherosclerosis by local deposition of inflammatory mediators like PF4, RANTES and CD40L, which leads to enhanced leukocyte recruitment and plaque formation. In 1865, Armand Trousseau first described the correlation between cancer and thrombotic events. Since the 1960s, an increasing number of studies have found an involvement of platelets also in the progression of cancer, especially in the formation of metastases. Platelets bind to circulating tumor cells and may shield them from NK cell attacks and shear stress. Platelets may also facilitate the interaction of tumor cells with other cell types and the vessel wall. Lastly, they may secrete molecules that influence the tumor cell phenotype and invasiveness.
Aims of this study: We sought to generate and describe genetically modified mouse lines with defective platelet secretion and to employ these mouse lines in murine models of atherosclerosis and tumor progression to study the role of platelet secretion under pathological in vivo conditions.
Results: Clostridial toxins cleave members of the SNARE protein family and can thus completely block exocytosis of neuronal and other cells. We generated three transgenic mouse lines expressing tetanus, botulinum-E or -C light chains and two transgenic mouse lines with dominant-negative mutations of SNAP-23 under the control of the platelet-specific PF4 promotor. None of these constructs was able to interfere with platelet secretion despite expression of the transgene. A functional null mutant of the only Munc13 isoform expressed in platelets, Munc13-4, showed complete lack of dense granule secretion, measured by ATP release, while alpha granule release as determined by PF4 and vWF secretion, was unaltered. Morphology, composition and adhesion of these platelets were also normal. Aggregation in response to U46619 and collagen and formation of large aggregates in flow chamber assays was attenuated. Munc13-4-deficient mice showed a severe defect in bleeding time and no formation of stable aggregates in FeCl3 thrombosis model. In response to B16 melanoma and LLC1 carcinoma cells, Munc13-4 KO platelets also showed complete abrogation of dense granule secretion, whereas alpha granule secretion and binding of platelets to tumor cells was unchanged. Interestingly, wild-type platelets, but not Munc13-4 KO platelets, enhanced transmigration of B16 and LLC1 cells through an endothelial cell layer. Exogenous ATP was able to mimic the effect of wild-type platelets and the ATP-degrading enzyme apyrase blocked platelet-mediated tumor cell transmigration. Platelets incubated with tumor cells secreted large amounts of ATP. Murine endothelial cells showed perturbed adherens junctions identified by irregular VE-cadherin staining and gap formation when incubated with supernatants from tumor cell-activated platelets as well as increased permeability under the same conditions. Addition of apyrase preserved normal endothelial morphology and function. In vivo, primary tumor growth and weight was comparable in wild-type and Munc13-4 KO mice upon B16 or LLC1 flank injection but formation of lung metastases was strongly reduced. Number, but not size of metastases was also reduced upon i.v. injection of B16 and LLC1 cells. We found P2Y2 and P2X4 receptors to be the most abundantly expressed endothelial metabotropic and ionotropic ATP receptors, respectively. Neither knock-down nor inhibition of P2X4 in endothelial cells influenced platelet-mediated transendothelial migration of B16 cells, but knock-down of P2Y2, for which no specific antagonist is available, strongly reduced plateletdependent tumor cell transmigration. When B16 melanoma cells were injected i.v. shortly after FITC-dextran (70 kDa) into wild-type mice, prominent leakage of FITC-dextran was observed three hours post-injection at extraluminal sites in the lung. In contrast, leakage into the lung parenchyma was at basal levels in Munc13-4 KO and P2Y2 KO mice after B16 cell injection. Marginal vascular leakage in Munc13-4 KO mice lacking platelet ATP secretion and in P2Y2 KO mice lacking the main endothelial ATP receptor correlated with strongly reduced extravasation of CFSE-labeled B16 melanoma cells 6 hours post-injection in these mice. Consistently, P2Y2 KO mice showed strongly reduced formation of metastases in the lung after i.v. injection of B16 or LLC1 tumor cells. Bone marrow-transplanted LDLR KO mice reconstituted with Munc13-4-deficient or wildtype bone marrow and subjected to 16 weeks of high fat diet showed no significant difference in atherosclerotic plaque formation in the aorta.
Discussion: We hereby provide a thorough analysis of a mouse line with an exclusive defect in platelet dense granule secretion, thus representing a unique genetic tool to study the role of dense granule secretion in various contexts without interfering with other platelet functions. We also provide evidence how extravasation of circulating tumor cells is facilitated by tumor cell-induced ATP release from platelets. This ATP release destabilizes endothelial barriers and facilitates tumor cell extravasation and formation of metastases in the target organ. Since metastasis is the leading cause of cancer death, pharmacological interference with endothelial P2Y2 receptor function may represent a promising therapeutic strategy.
Iron-rich structures have been described in the beak of homing pigeons, chickens and several species of migratory birds and interpreted as magnetoreceptors. Here, we will briefly review findings associated with these receptors that throw light on their nature, their function and their role in avian navigation. Electrophysiological recordings from the ophthalmic nerve, behavioral studies and a ZENK-study indicate that the trigeminal system, the nerves innervating the beak, mediate information on magnetic changes, with the electrophysiological study suggesting that these are changes in intensity. Behavioral studies support the involvement of magnetite and the trigeminal system in magnetoreception, but clearly show that the inclination compass normally used by birds represents a separate system. However, if this compass is disrupted by certain light conditions, migrating birds show 'fixed direction' responses to the magnetic field, which originate in the receptors in the beak. Together, these findings point out that there are magnetite-based magnetoreceptors located in the upper beak close to the skin. Their natural function appears to be recording magnetic intensity and thus providing one component of the multi-factorial 'navigational map' of birds.
The 25S rRNA of yeast contains several base modifications in the functionally important regions. The enzymes responsible for most of these base modifications remained unknown. Recently, we identified Rrp8 as a methyltransferase involved in m1A645 modification of 25S rRNA. Here, we discovered a previously uncharacterized gene YBR141C to be responsible for second m1A2142 modification of helix 65 of 25S rRNA. The gene was identified by reversed phase–HPLC screening of all deletion mutants of putative RNA methyltransferase and was confirmed by gene complementation and phenotypic characterization. Because of the function of its encoded protein, YBR141C was named BMT2 (base methyltransferase of 25S RNA). Helix 65 belongs to domain IV, which accounts for most of the intersubunit surface of the large subunit. The 3D structure prediction of Bmt2 supported it to be an Ado Met methyltransferase belonging to Rossmann fold superfamily. In addition, we demonstrated that the substitution of G180R in the S-adenosyl-l-methionine–binding motif drastically reduces the catalytic function of the protein in vivo. Furthermore, we analysed the significance of m1A2142 modification in ribosome synthesis and translation. Intriguingly, the loss of m1A2142 modification confers anisomycin and peroxide sensitivity to the cells. Our results underline the importance of RNA modifications in cellular physiology.
DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.
Introduction: Gastropoda are guided by several sensory organs in the head region, referred to as cephalic sensory organs (CSOs). These CSOs are innervated by distinct nerves. This study proposes a unified terminology for the cerebral nerves and the categories of CSOs and then investigates the neuroanatomy and cellular innervation patterns of these cerebral nerves, in order to homologise them. The homologisation of the cerebral nerves in conjunction with other data, e.g. ontogenetic development or functional morphology, may then provide insights into the homology of the CSOs themselves.
Results: Nickel-lysine axonal tracing (“backfilling”) was used to stain the somata projecting into specific nerves in representatives of opisthobranch Gastropoda. Tracing patterns revealed the occurrence, size and relative position of somata and their axons and enabled these somata to be mapped to specific cell clusters. Assignment of cells to clusters followed a conservative approach based primarily on relative location of the cells. Each of the four investigated cerebral nerves could be uniquely identified due to a characteristic set of soma clusters projecting into the respective nerves via their axonal pathways.
Conclusions: As the described tracing patterns are highly conserved morphological characters, they can be used to homologise nerves within the investigated group of gastropods. The combination of adequate number of replicates and a comparative approach allows us to provide preliminary hypotheses on homologies for the cerebral nerves. Based on the hypotheses regarding cerebral nerve homology together with further data on ultrastructure and immunohistochemistry of CSOs published elsewhere, we can propose preliminary hypotheses regarding homology for the CSOs of the Opisthobranchia themselves.
Background: Protein translocation across membranes is a central process in all cells. In the past decades the molecular composition of the translocation systems in the membranes of the endoplasmic reticulum, peroxisomes, mitochondria and chloroplasts have been established based on the analysis of model organisms. Today, these results have to be transferred to other plant species. We bioinformatically determined the inventory of putative translocation factors in tomato (Solanum lycopersicum) by orthologue search and domain architecture analyses. In addition, we investigated the diversity of such systems by comparing our findings to the model organisms Saccharomyces cerevisiae, Arabidopsis thaliana and 12 other plant species.
Results: The literature search end up in a total of 130 translocation components in yeast and A. thaliana, which are either experimentally confirmed or homologous to experimentally confirmed factors. From our bioinformatic analysis (PGAP and OrthoMCL), we identified (co-)orthologues in plants, which in combination yielded 148 and 143 orthologues in A. thaliana and S. lycopersicum, respectively. Interestingly, we traced 82% overlap in findings from both approaches though we did not find any orthologues for 27% of the factors by either procedure. In turn, 29% of the factors displayed the presence of more than one (co-)orthologue in tomato. Moreover, our analysis revealed that the genomic composition of the translocation machineries in the bryophyte Physcomitrella patens resemble more to higher plants than to single celled green algae. The monocots (Z. mays and O. sativa) follow more or less a similar conservation pattern for encoding the translocon components. In contrast, a diverse pattern was observed in different eudicots.
Conclusions: The orthologue search shows in most cases a clear conservation of components of the translocation pathways/machineries. Only the Get-dependent integration of tail-anchored proteins seems to be distinct. Further, the complexity of the translocation pathway in terms of existing orthologues seems to vary among plant species. This might be the consequence of palaeoploidisation during evolution in plants; lineage specific whole genome duplications in Arabidopsis thaliana and triplications in Solanum lycopersicum.
Ribosome biogenesis is best understood in the yeast Saccharomyces cerevisiae. In human or mammalian ribosome biogenesis, it has been shown that basic principles are conserved to yeast, but additional features have been reported. Our understanding about the interplay between proteins and RNA in human ribosome biogenesis is far from complete.
The present study focused on the analysis of the human ribosome biogenesis co-factors PWP2, EMG1 and Exportin 5 (XPO5) to understand the degree of conservation of ribosome biogenesis. The proteins were characterized in respect to their localization and interaction partners. For the early 90S co-factor, PWP2, it was possible to pull down and identify the human UTP-B complex with MALDI mass spectrometry. Besides the orthologues of the members of this complex known in yeast (TBL3, WDR3, WDR36, UTP6, UTP18), the human UTP-B complex is not only conserved from yeast to humans, but contains also additional components, like the DEAD-box RNA helicase DDX21, which lacks a yeast orthologue. DDX21 was localized to the nucleus, assembled to the native UTP-B complex and co-precipitated also with other UTP-B complex members, presumably extending the functions of this complex in ribosome biogenesis.
This phenomenon was also observed for the 90S co-factor EMG1, an RNA methyltransferase, whose mutant form causes the Bowen-Conradi syndrome, if aspartic acid is mutated to glycine at position 86. This study revealed that the mutant, EMG1-D86G, clearly lost its nucleolar localization and co-precipitated to histones for unknown reasons.
A participation of the nuclear export receptor XPO5 in human ribosome biogenesis was shown in this study. Pulldown analysis, sucrose density gradients and UV crosslinking and analysis of cDNAs of XPO5 revealed the involvement of XPO5 in pre-60S subunit maturation. Moreover, besides the known pre-miRNAs and tRNAs as substrates for nuclear export, XPO5 crosslinked to snoRNAs. XPO5 was further demonstrated to interact with the miRNA Let-7a, which has an important regulatory function for MYC, a transcription factor required for ribosome biogenesis.
All results support a role of these proteins in human ribosome biogenesis and therefore it seems that the biogenesis of ribosomes in human cells requires additional components, like DDX21 and XPO5.
In the first part of this work, the development of a novel two-dimensional native gel electrophoretic system (2-D BN/hrCNE) is described. This new system simplifies proteomics and biochemical analysis of mega protein complexes that are dissociated into the constituent complexes during 2-D electrophoresis, thereby reducing the complexity of the system considerably. This technique is exceptionally well suited for the in-gel detection of fluorescence-labeled proteins and the identification of individual enzymes and protein complexes by specific in-gel assays on native gels.
In the second part, a new technique for the native immunoblotting of blue native gels (NIBN) was developed. This new technique allows for the identification of conformation-specific antibodies and the discrimination of antibodies recognizing linear epitopes of denatured proteins. Identification of conformation-specific antibodies is becoming increasingly important not only for the electron microscopic identification of native proteins but also for structural investigations in general. For this purpose, a commonly used protocol for Western blotting of blue native gels was modified in such a way that the native state of proteins and protein complexes was retained throughout the complete protocol. Instead of using the denaturing methanol in Western blotting protocols, mild detergents such as Tween 20, digitonin and Brij 35 were used for the obligatory removal of protein bound Coomassie-dye.
The detection of respiratory complex I by activity staining on the blot membrane demonstrated that all three non-ionic detergents preserved the native state of complex I. The native state of the enzyme on the blot membrane was also monitored and confirmed with the help of a set of conformation-specific antibodies. NIBN can be used as a simple alternative method to the demanding native ELISA to screen for conformation-specific antibodies for structural studies. Unlike the time consuming native ELISA, NIBN does not require introduction of appropriate affinity tags and purification of the target protein by chromatography. Thus, the NIBN technique is especially useful for microscale projects and for proteins not easily accessible to genetic manipulation.
The third part aimed at identification of the immediate protein interaction partners of Cox26, a hydrophobic protein that has been identified by our group as a novel component of yeast respiratory supercomplex. Multi-dimensional electrophoretic techniques were applied to identify non-covalent and covalent protein-protein interactions of Cox26. Three-dimensional electrophoresis (BNE/BNE/SDS-PAGE) gave both qualitative and quantitative information on covalent and non-covalent interactions of Cox26 and subunits of cytochrome c oxidase (complex IV), and showed that most of the Cox26 protein was non-covalently bound to the complex IV moiety of the respirasomes. Four-dimensional electrophoresis (BNE/BNE/SDS/SDS-PAGE) applying reducing and non-reducing conditions revealed that a minor fraction of Cox26 used a single cysteine residue in the center of a predicted transmembrane helix to form a disulfide bond with the Cox2 subunit of complex IV. A structural role of Cox26 protein in the assembly/stability of respiratory strings or patches has been suggested.
The last part of this work focused on the isolation and characterization of native and morphologically intact nucleoids from bovine heart mitochondria, since only a few studies on nucleoid organization and composition have been carried out on mammalian tissues. The nucleoids appeared as distinct bands (apparent mass around 30-36 MDa) in blue native-PAGE on large pore gels. The moderate variation in particle size seems to reflect variations in the binding of loosely nucleoid-associated components like respiratory chain complexes. The estimated 30-36 MDa mass of nucleoids on native gels suggested that each nucleoid contains one mtDNA molecule provided that nucleoids contains equal amounts of DNA, protein and RNA (Miyakawa et al., 1987).
Electron microscopic analysis of native nucleoids, which was performed by Dr. Karen Davies from the Max-Planck-Institute of Biophysics, Department of Structural Biology, Frankfurt, showed homogenous pool of particles with dimensions in 85x100 nm (in negative stain) and 100x150 nm (in cryo-tomography). Some of the nucleoids showed dumbbell-shape indicating dimerization of nucleoids. Recent EM and high-resolution light microscopy analysis of mammalian nucleoids have reported that nucleoids have a size of 70 nm in average. We also observed the same size of 70 nm in cryo-tomogramms when we applied harsher treatment of the native nucleoid particles with dimensions 100x150 nm. This observation is in agreement with published nucleoid sizes from both EM and high-resolution light microscopy, if we assume that native nucleoids have been dissociated under harsher treatment.
The protein composition of bovine heart mt-nucleoids was analyzed by a number of complementary approaches to identify low and highly abundant, easily dissociating and tightly bound proteins, and to rank the 90 most abundant mt-nucleoid proteins. Native and denaturing gel electrophoresis techniques were coupled to LC-MS/MS to achieve a comprehensive protein component analysis. Qualitative MS analysis of highly purified nucleoids identified more than 400 proteins, including well known nucleoid proteins such as mitochondrial transcription factor and mtDNA-binding protein (TFAM), mitochondrial single-stranded DNA-binding protein (mtSSB), mitochondrial DNA polymerase subunit gamma-2 (POLG2) and mitochondrial helicase C26H10ORF2 protein (Twinkle). These proteins were ranked according to Mascot scores, and sorted according to presumed functional properties. A large group of proteins involved in protein synthesis comprised an almost complete set of subunits of mitochondrial ribosomes suggesting that the nucleoids contained significant amounts of mitochondrial ribosomes. Identification of sixty six proteins from the oxidative phosphorylation (OXPHOS) system comprising around 100 proteins in total suggested that OXPHOS proteins are also associated with mt-nucleoids.
Interestingly, TFAM, described as a main mtDNA packaging factor in human and other mammalian cells, was not confirmed here as a major nucleoid component from bovine heart mitochondria. Fluorescence staining of protein spots on 2-D IEF/SDS gels clearly identified TFAM, but according to the stain intensity, this protein did not rank in the list of the 90 most abundant nucleoid proteins. Western blot analysis of sucrose gradient fractions revealed an enrichment of putative TFAM isoform in nucleoid fractions. Unexpectedly, the uncharacterized mitochondrial protein Es1 was identified as the most abundant nucleoid protein in bovine heart nucleoids instead. This implicates that nucleoid organization may differ between species and tissues. A functional characterization of Es1 is required to clarify its role in mammalian nucleoids.