Subtomogram analysis: The sum of a tomogram's particles reveals molecular structure in situ

Cryo-electron tomography is uniquely suited to provide insights into the molecular architecture of cells and tissue in the native state. While frozen hydrated specimens tolerate sufficient electron doses to distinguish different types of particles in a tomogram, the accumulating beam damage does not allow resolving their detailed molecular structure individually. Statistical methods for subtomogram averaging and classification that coherently enhance the signal of particles corresponding to copies of the same type of macromolecular allow obtaining much higher resolution insights into macromolecules. Here, I review the developments in subtomogram analysis at Wolfgang Baumeister's laboratory that make the dream of structural biology in the native cell become reality

Different Evolutionary Modifications as a Guide to Rewire Two-Component Systems

Two-component systems (TCS) are short signalling pathways generally occurring in prokaryotes. They frequently regulate prokaryotic stimulus responses and thus are also of interest for engineering in biotechnology and synthetic biology. The aim of this study is to better understand and describe rewiring of TCS while investigating different evolutionary scenarios. Based on large-scale screens of TCS in different organisms, this study gives detailed data, concrete alignments, and structure analysis on three general modification scenarios, where TCS were rewired for new responses and functions: (i) exchanges in the sequence within single TCS domains, (ii) exchange of whole TCS domains; (iii) addition of new components modulating TCS function. As a result, the replacement of stimulus and promotor cassettes to rewire TCS is well defined exploiting the alignments given here. The diverged TCS examples are non-trivial and the design is challenging. Designed connector proteins may also be useful to modify TCS in selected cases

Zu einigen Problemen der weltanschaulich-atheistischen Erziehung: Expertise

Der vorliegende Beitrag ist eine Expertise des Zentralinstituts für Jugendforschung der DDR zu Problemen der weltanschaulichen-atheistischen Erziehung in der DDR Anfang der siebziger Jahre. Die Studie stützt sich auf Datenmaterial von Erhebungen des Instituts sowie aus Arbeiten sowjetischer Autoren zur Thematik. Die Autoren legen einleitend die Notwendigkeit der weltanschaulichen Bildung und Erziehung der Jugend hinsichtlich der Grundfragen der Weltanschauung dar. Es folgen Thesen zu Einstellungen der DDR-Jugend zum wissenschaftlichen Atheismus seit 1945, wobei auf die Institutionen verwiesen wird, die die weltanschaulichen Positionen der Jugendlichen mitbeeinflussen. In diesem Zusammenhang sind die Verfasser der Auffassung, daß ein idealistischer oder neutraler weltanschaulicher Standpunkt sich negativ auf die gesamte politische Position der Persönlichkeit auswirkt. Im folgenden führen die Autoren wesentliche Bedingungen und Einflußfaktoren der Bildung weltanschaulicher Positionen bei Jugendlichen zur Stärkung einer atheistischen Grundhaltung an. Abschließend führen sie einen Katalog von Empfehlungen an, wie eine Verbesserung der weltanschaulichen Erziehung im Sinne eines sozialistischen Atheismus erreicht werden kann. (ICC

Self-assembly of magnetic iron oxide nanoparticles into cuboidal superstructures

This chapter describes the synthesis and some characteristics of magnetic iron oxide nanoparticles, mainly nanocubes, and focus on their self-assembly into crystalline cuboids in dispersion. The influence of external magnetic fields, the concentration of particles, and the temperature on the assembly process is experimentally investigated

Нові інтелектуальні технології та методи оптимізації для дослідження складних систем

Protein transport into the endoplasmic reticulum (ER) is essential for all eukaryotic cells and evolutionary related to protein transport into and across the cytoplasmic membrane of eubacteria and archaea. It is based on amino-terminal signal peptides in the precursor polypeptides plus various transport components in cytosol plus ER and can occur either cotranslationally or posttranslationally. The two mechanisms merge at the heterotrimeric Sec61 complex in the ER membrane, which forms an aqueous polypeptide-conducting channel. Since the mammalian ER is also the main intracellular calcium storage organelle, the Sec61 complex is tightly regulated in its dynamics between the open and closed conformations by various ligands, such as precursor polypeptides at the cytosolic face and the Hsp70-type molecular chaperone BiP at the ER lumenal face (Hsp, heat shock protein). Furthermore, BiP binding to the incoming precursor polypeptide contributes to unidirectionality and efficiency of transport. Recent insights into the structural dynamics of the Sec61 complex and related complexes in eubacteria and archaea have various mechanistic and functional implications

Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co- or post translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the pre cursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting vari ous peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients

Sucrose-mediated translational stalling involves a conserved ribosomal pocket

Within eukaryotes, 20-50% of the mRNAs contain short open reading frames (uORFs) located upstream of the main ORF. A significant fraction of these uORFs encode conserved peptides (CPuORFs) that regulate translation in response to specific metabolites. A well-studied example includes uORF2 of the plant growth inhibiting transcription factor bZIP11. Elevated intracellular sucrose levels lead to ribosome stalling at the stop codon of uORF2, thus reducing bZIP11 protein synthesis. Similar examples can be found in bacteria and animals, e.g. on the bacterial TnaC and human CDH1-NPN* ORFs that both induce stalling at the stop codon when in the presence of tryptophan and the drug-like molecule PF846, respectively. In this study, we affinity-purified in vitro translated sucrose-stalled wheat ribosomes translating bZIP11-uORF2 and determined the ribosomes’ structures using cryo-electron microscopy. This revealed density inside a pocket in the ribosomal exit tunnel of the plant Triticum aestivum, that colocalizes with the binding locations of tryptophan and PF846 in E. coli and humans, respectively. We suggest this density corresponds to sucrose. Tryptophan and PF846 mode-of-action was previously proposed to inhibit release factor binding or function. Mutation of the uORF2 stop codon shows that its presence is crucial for sucrose-induced stalling, suggesting that the stalling only manifests during termination and not elongation. Moreover, the structural similarities with tryptophan-induced stalled ribosomes near the peptidyl transferase center indicates that an analogous mechanism of inhibition of release factor function is likely. Our findings suggest a conserved mechanistic framework across different organisms, wherein specific molecules interact with the nascent peptide and ribosome to modulate protein synthesis

Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1- involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach