Sergej Masich

The Human Skin Barrier Is Organized as Stacked Bilayers of Fully Extended Ceramides with Cholesterol Molecules Associated with the Ceramide Sphingoid Moiety

The skin barrier is fundamental to terrestrial life and its evolution; it upholds homeostasis and protects against the environment. Skin barrier capacity is controlled by lipids that fill the extracellular space of the skins surface layer--the stratum corneum. Here we report on the determination of the molecular organization of the skins lipid matrix in situ, in its near-native state, using a methodological approach combining very high magnification cryo-electron microscopy (EM) of vitreous skin section defocus series, molecular modeling, and EM simulation. The lipids are organized in an arrangement not previously described in a biological system-stacked bilayers of fully extended ceramides (CERs) with cholesterol molecules associated with the CER sphingoid moiety. This arrangement rationalizes the skins low permeability toward water and toward hydrophilic and lipophilic substances, as well as the skin barriers robustness toward hydration and dehydration, environmental temperature and pressure changes, stretching, compression, bending, and shearing.

In situ transcription and splicing in the Balbiani ring 3 gene

The Balbiani ring 3 (BR3) gene contains 38 introns, and more than half of them are co‐transcriptionally excised. We have determined the in situ structure of the active BR3 gene by electron tomography. Each of the 20–25 nascent transcripts on the gene is present together with splicing factors and the RNA polymerase II in a nascent transcript and splicing complex, here called the NTS complex. The results indicate that extensive changes in overall shape, substructure and molecular mass take place repeatedly within an NTS complex as it moves along the gene. The volume and calculated mass of the NTS complexes show that, maximally, one complete spliceosome is assembled on the multi‐intron transcript at any given time point. The structural data show that the spliceosome is not a structurally well‐defined unit in situ and that the C‐terminal domain of the elongating RNA polymerase II cannot carry spliceosomal components for all introns in the BR3 transcript. Our data indicate that spliceosomal factors are continuously added to and released from the NTS complexes during transcription elongation.

Nuclear poly(A)-binding protein PABPN1 is associated with RNA polymerase II during transcription and accompanies the released transcript to the nuclear pore.

The nuclear poly(A)-binding protein, PABPN1, has been previously shown to regulate mRNA poly(A) tail length and to interact with selected proteins involved in mRNA synthesis and trafficking. To further understand the role of PABPN1 in mRNA metabolism, we used cryo-immunoelectron microscopy to determine the fate of PABPN1 at various stages in the assembly and transport of the Chironomus tentans salivary gland Balbiani ring (BR) mRNA ribonucleoprotein (mRNP) complex. PABPN1 is found on BR mRNPs within the nucleoplasm as well as on mRNPs docked at the nuclear pore. Very little PABPN1 is detected on the cytoplasmic side of the nuclear envelope, suggesting that PABPN1 is displaced from mRNPs during or shortly after passage through the nuclear pore. Surprisingly, we also find PABPN1 associated with RNA polymerase II along the chromatin axis of the BR gene. Our results suggest that PABPN1 binds to the polymerase before, at, or shortly after the start of transcription, and that the assembly of PABPN1 onto the poly(A) tail may be coupled to transcription. Furthermore, PABPN1 remains associated with the released BR mRNP until the mRNP is translocated from the nucleus to the cytoplasm.

Two pools of vesicles associated with the presynaptic cytosolic projection in Drosophila neuromuscular junctions

Synapses that sustain neurotransmitter release at high rates often contain special presynaptic cytosolic projections (PCPs) that are believed to facilitate synaptic vesicle (SV) movements to the sites of fusion. The genetically modifiable Drosophila neuromuscular junction (NMJ) serves as one of the model systems to investigate the functions of these structures. Using electron microscope tomography we determined the three-dimensional organization of the Drosophila PCP immobilized by high-pressure freezing, followed by cryo-substitution. We show that it is composed of three structural components: (1) the central core, (2) legs, organized in a regular grid at the bottom of the central core, and (3) cytoplasmic extensions. The extensions are comprised of thin filaments emerging from the central core. SVs connected to the extensions are either linked to the vesicles accumulated around the PCP or to the presynaptic membrane. This suggests that SVs associated with the PCP loose their connections with other vesicles in the cluster during translocation to the site of fusion.

Structural organization of the presynaptic density at identified synapses in the locust central nervous system.

In a synaptic active zone, vesicles aggregate around a densely staining structure called the presynaptic density. We focus on its three‐dimensional architecture and a major molecular component in the locust. We used electron tomography to study the presynaptic density in synapses made in the brain by identified second‐order neuron of the ocelli. Here, vesicles close to the active zone are organized in two rows on either side of the presynaptic density, a level of organization not previously reported in insect central synapses. The row of vesicles that is closest to the densitys base includes vesicles docked with the presynaptic membrane and thus presumably ready for release, whereas the outer row of vesicles does not include any that are docked. We show that a locust ortholog of the Drosophila protein Bruchpilot is localized to the presynaptic density, both in the ocellar pathway and compound eye visual neurons. An antibody recognizing the C‐terminus of the Bruchpilot ortholog selectively labels filamentous extensions of the presynaptic density that reach out toward vesicles. Previous studies on Bruchpilot have focused on its role in neuromuscular junctions in Drosophila, and our study shows it is also a major functional component of presynaptic densities in the central nervous system of an evolutionarily distant insect. Our study thus reveals Bruchpilot executes similar functions in synapses that can sustain transmission of small graded potentials as well as those relaying large, spike‐evoked signals. J. Comp. Neurol. 520:384–400, 2012.

The central element of the synaptonemal complex in mice is organized as a bilayered junction structure

ABSTRACT The synaptonemal complex transiently stabilizes pairing interactions between homologous chromosomes during meiosis. Assembly of the synaptonemal complex is mediated through integration of opposing transverse filaments into a central element, a process that is poorly understood. We have, here, analyzed the localization of the transverse filament protein SYCP1 and the central element proteins SYCE1, SYCE2 and SYCE3 within the central region of the synaptonemal complex in mouse spermatocytes using immunoelectron microscopy. Distribution of immuno-gold particles in a lateral view of the synaptonemal complex, supported by protein interaction data, suggest that the N-terminal region of SYCP1 and SYCE3 form a joint bilayered central structure, and that SYCE1 and SYCE2 localize in between the two layers. We find that disruption of SYCE2 and TEX12 (a fourth central element protein) localization to the central element abolishes central alignment of the N-terminal region of SYCP1. Thus, our results show that all four central element proteins, in an interdependent manner, contribute to stabilization of opposing N-terminal regions of SYCP1, forming a bilayered transverse-filament–central-element junction structure that promotes synaptonemal complex formation and synapsis. Summary: We describe a bilayered junction structure essential for organization of the synaptonemal complex. This structure is crucial for synapsis of homologous chromosomes and for chromosome segregation.

Skin Lamellar Bodies are not Discrete Vesicles but Part of a Tubuloreticular Network.

Improved knowledge of the topology of lamellar bodies is a prerequisite for a molecular-level understanding of skin barrier formation, which in turn may provide clues as to the underlying causes of barrier-deficient skin disease. The aim of this study was to examine the key question of continuity vs. discreteness of the lamellar body system using 3 highly specialized and complementary 3-dimensional (3D) electron microscopy methodologies; tomography of vitreous sections (TOVIS), freeze-substitution serial section electron tomography (FS-SET), and focused ion beam scanning electron microscopy (FIB-SEM) tomography. We present here direct evidence that lamellar bodies are not discrete vesicles, but are part of a tubuloreticular membrane network filling out the cytoplasm and being continuous with the plasma membrane of stratum granulosum cells. This implies that skin barrier formation could be regarded as a membrane folding/unfolding process, but not as a lamellar body fusion process.

Two splicing isoforms of the Y‐box protein ctYB‐1 appear on the same mRNA molecule

Y‐box proteins constitute an evolutionarily conserved family of DNA‐ and RNA‐binding proteins involved in the regulation of transcription and translation. In the dipteran Chironomus tentans, a homologue to the vertebrate Y‐box protein YB‐1 was recently characterized and designated ctYB‐1. It is transferred from nucleus to cytoplasm bound to mRNA and is likely to affect translation. It appears in two size variants, p40 and p50. We further analysed the two size variants and their interaction with mRNA. Southern blot analysis, in situ hybridization and RT‐PCR analysis suggested that there is just one YB‐1 gene, and that the two size variants represent splicing isoforms. In a C. tentans epithelial cell line, only p40 is present, whereas both variants appear together in eight tissues from fourth‐instar larvae, although in somewhat different proportions. Furthermore, the appearance of the two isoforms was studied in relation to a specific 35–40 kb mRNA transcript in the salivary glands, the Balbiani ring mRNA. Because of their exceptional size, Balbiani ring messenger ribonucleoprotein particles in nucleoplasm and Balbiani ring polysomes in cytoplasm could be identified and selectively studied. We were able to establish that both isoforms are associated with both nuclear and cytoplasmic Balbiani ring mRNA. In addition, a p50‐specific antibody coimmunoprecipitated p40 from Balbiani ring polysomes, suggesting that the two splicing isoforms are located along the same Balbiani ring mRNA molecule. The functional significance of the two isoforms is being discussed.

The Viral Protein Corona Directs Viral Pathogenesis and Amyloid Aggregation

Nanoparticles accumulate a layer of host factors on their surface (protein corona) in biological fluids, which influences the nanoparticle activity. We hypothesized that viruses also constitute nanoparticles in this respect and we provide evidence for the existence of a viral protein corona that has implications for viral infectivity, immune cell activation and catalysis of amyloid aggregation. We demonstrate that respiratory syncytial virus (RSV), a major cause of respiratory tract infections, accumulates a rich and distinctive protein corona in different biological fluids including human plasma, human bronchoalveolar lavage fluid, non-human primate plasma and fetal bovine serum. Additionally, corona pre-coating differentially affects viral infectivity and its ability to activate human monocyte-derived dendritic cells (moDCs) depending on the biological fluid. Furthermore, we demonstrate that RSV can bind and catalyze the amyloid aggregation of an amyloidogenic peptide derived from the islet amyloid polypeptide (IAPP) via surface-assisted nucleation. Similarly, we show that herpes simplex virus 1 (HSV-1) possesses a protein corona and catalyzes the amyloid aggregation of the amyloid-beta (Aβ42) peptide which is the major constituent of amyloid plaques in Alzheimer’s disease (AD). We also show that HSV-1 infection accelerates Aβ42 aggregation in the hippocampi of a mouse model of AD. Our results provide a proof-of-concept for the presence of a viral protein corona layer that is dependent on the microenvironment and influences viral-host interactions. Additionally, the demonstration of corona-driven amyloid catalysis illustrates convergence between viral and amyloid pathologies in the extracellular environment suggesting a novel mechanistic link that warrants further investigation.

ProViz: a tool for explorative 3-D visualization and template matching in electron tomograms

Visual understanding is a key aspect when studying electron tomography data-sets, aside quantitative assessments such as registration of high-resolution structures. We here present the free software tool ProViz (Protein Visualization) for visualisation and template matching in electron tomograms of biological samples. The ProViz software contains methods and tools which we have developed, adapted and computationally optimised for easy and intuitive visualisation and analysis of electron tomograms with low signal-to-noise ratio. ProViz complements existing software in the application field and serves as an easy and convenient tool for a first assessment and screening of the tomograms. It provides enhancements in three areas: (1) improved visualisation that makes connections as well as intensity differences between and within objects or structures easier to see and interpret, (2) interactive transfer function editing with direct visual result feedback using both piecewise linear functions and Gaussian function elements, (3) computationally optimised template matching and tools to visually assess and interactively explore the correlation results. The visualisation capabilities and features of ProViz are demonstrated on various biological volume data-sets: bacterial filament structures in vitro, a desmosome and the transmembrane cadherin connections therein in situ, and liposomes filled with doxorubicin in solution. The explorative template matching is demonstrated on a synthetic IgG data-set.