The Skin's Barrier: A Cryo-EM Based Overview of its Architecture and Stepwise Formation.

A major role of the skin is to serve as a barrier toward the environment. The skin's permeability barrier consists of a lipid structure positioned in the stratum corneum. Recent progress in high-resolution cryo-electron microscopy (cryo-EM) has allowed for elucidation of the architecture of the skin's barrier and its stepwise formation process representing the final stage of epidermal differentiation. In this review, we present an overview of the skin's barrier structure and its formation process, as evidenced by cryo-EM.

Molecular Reorganization during the Formation of the Human Skin Barrier Studied In Situ.

In vertebrates, skin upholds homeostasis by preventing body water loss. The skin's permeability barrier is located intercellularly in the stratum corneum and consists of stacked lipid lamellae composed of ceramides, cholesterol, and free fatty acids. We have combined cryo-electron microscopy with molecular dynamics modeling and electron microscopy simulation in our analysis of the lamellae's formation, a maturation process beginning in stratum granulosum and ending in stratum corneum. Previously, we have revealed the lipid lamellae's initial- and end-stage molecular organizations. In this study, we reveal two cryo-electron microscopy patterns representing intermediate stages in the lamellae's maturation process: a single-band pattern with 2.0‒2.5 nm periodicity and a two-band pattern with 5.5‒6.0 nm periodicity, which may be derived from lamellar lipid structures with 4.0‒5.0 nm and 5.5‒6.0 nm periodicity, respectively. On the basis of the analysis of the data now available on the four maturation stages identified, we can present a tentative molecular model for the complete skin barrier formation process.

Human skin barrier structure and function analyzed by cryo-EM and molecular dynamics simulation.

In the present study we have analyzed the molecular structure and function of the human skin's permeability barrier using molecular dynamics simulation validated against cryo-electron microscopy data from near native skin. The skin's barrier capacity is located to an intercellular lipid structure embedding the cells of the superficial most layer of skin - the stratum corneum. According to the splayed bilayer model (Iwai et al., 2012) the lipid structure is organized as stacked bilayers of ceramides in a splayed chain conformation with cholesterol associated with the ceramide sphingoid moiety and free fatty acids associated with the ceramide fatty acid moiety. However, knowledge about the lipid structure's detailed molecular organization, and the roles of its different lipid constituents, remains circumstantial. Starting from a molecular dynamics model based on the splayed bilayer model, we have, by stepwise structural and compositional modifications, arrived at a thermodynamically stable molecular dynamics model expressing simulated electron microscopy patterns matching original cryo-electron microscopy patterns from skin extremely closely. Strikingly, the closer the individual molecular dynamics models' lipid composition was to that reported in human stratum corneum, the better was the match between the models' simulated electron microscopy patterns and the original cryo-electron microscopy patterns. Moreover, the closest-matching model's calculated water permeability and thermotropic behaviour were found compatible with that of human skin. The new model may facilitate more advanced physics-based skin permeability predictions of drugs and toxicants. The proposed procedure for molecular dynamics based analysis of cellular cryo-electron microscopy data might be applied to other biomolecular systems.

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

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.

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.

Exclusion of mRNPs and ribosomal particles from a thin zone beneath the nuclear envelope revealed upon inhibition of transport.

We have studied the nucleocytoplasmic transport of a specific messenger RNP (mRNP) particle, named Balbiani ring (BR) granule, and ribosomal RNP (rRNP) particles in the salivary glands of the dipteran Chironomus tentans. The passage of the RNPs through the nuclear pore complex (NPC) was inhibited with the nucleoporin-binding wheat germ agglutinin, and the effects were examined by electron microscopy. BR mRNPs bound to the nuclear basket increased in number, while BR mRNPs translocating through the central channel decreased, suggesting that the initiation of translocation proper had been inhibited. The rRNPs accumulated heavily in nucleoplasm, while no or very few rRNPs were recorded within nuclear baskets. Thus, the transport of rRNPs had been blocked prior to the entry into the baskets. Remarkably, the rRNPs had been excluded both from baskets and the space in between the baskets. We propose that normally basket fibrils move freely and repel RNPs from the exclusion zone unless the particles have affinity for and bind to nucleoporins within the baskets.

Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin.

Messenger ribonucleoprotein particles (mRNPs) move randomly within nucleoplasm before they exit from the nucleus. To further understand mRNP trafficking, we have studied the intranuclear movement of a specific mRNP, the BR2 mRNP, in salivary gland cells in Chironomus tentans. Their polytene nuclei harbor giant chromosomes separated by vast regions of nucleoplasm, which allows us to study mRNP mobility without interference of chromatin. The particles were fluorescently labeled with microinjected oligonucleotides (DNA or RNA) complementary to BR2 mRNA or with the RNA-binding protein hrp36, the C. tentans homologue of hnRNP A1. Using high-speed laser microscopy, we followed the intranuclear trajectories of single mRNPs and characterized their motion within the nucleoplasm. The Balbiani ring (BR) mRNPs moved randomly, but unexpectedly, in a discontinuous manner. When mobile, they diffused with a diffusion coefficient corresponding to their size. Between mobile phases, the mRNPs were slowed down 10- to 250-fold but were never completely immobile. Earlier electron microscopy work has indicated that BR particles can attach to thin nonchromatin fibers, which are sometimes connected to discrete fibrogranular clusters. We propose that the observed discontinuous movement reflects transient interactions between freely diffusing BR particles and these submicroscopic structures.

Close coupling between transcription and exit of mRNP from the cell nucleus.

Transcription is intimately coupled to co-transcriptional formation of mRNP particles and their preparation for export. In the dipteran Chironomus tentans we have now investigated whether on-going transcription is closely linked also to the ensuing transfer of the mRNPs from genes to cytoplasm. The assembly and nucleocytoplasmic transport of a specific mRNP particle, the Balbiani ring (BR) RNP granule, were visualized in larval salivary glands by electron microscopy. When transcription was inhibited with DRB or actinomycin D (AMD), the growing BR mRNPs disappeared from the genes. The two inhibitors affected the distribution of BR mRNPs in the nucleoplasm and in the nuclear pores in essentially the same way. At the nuclear pore complexes (NPCs) the basket-associated and translocating mRNPs were substantially reduced in number, the translocating RNPs being essentially absent after 90 min treatment. Remarkably, the amount of BR mRNPs in the nucleoplasm did not change. We conclude that on-going transcription is required for the mRNPs to exit from the cell nucleus. Interruption of transcription seems to primarily affect the intranuclear movement of BR mRNPs and/or prevent the binding of mRNPs to the NPCs rather than to directly interfere with translocation per se.

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.

A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography.

We describe a novel approach for the accurate alignment of images in electron tomography of vitreous cryo-sections. Quantum dots, suspended in organic solvents at cryo-temperatures, are applied directly onto the sections and are subsequently used as fiducial markers to align the tilt series. Data collection can be performed from different regions of the vitreous sections, even when the sections touch the grid only at a few places. We present high-resolution tomograms of some organelles in cryo-sections of human skin cells using this method. The average error in image alignment was about 1nm and the resolution was estimated to be 5-7nm. Thus, the use of section-attached quantum dots as fiducial markers in electron tomography of vitreous cryo-sections facilitates high-resolution in situ 3D imaging of organelles and macromolecular complexes in their native hydrated state.

A specific SR protein binds preferentially to the secretory protein gene transcripts in salivary glands of Chironomus tentans.

The members of the serine-arginine (SR) family of proteins play multiple roles in posttranscriptional gene expression. Initially considered as essential splicing factors confined to the nucleus and regulating constitutive and alternative splicing, SR proteins are now known to shuttle between the nucleus and the cytoplasm and to be involved in mRNA biogenesis, transport, and translation. In Chironomus tentans, hrp45 is an SR protein structurally similar to the Drosophila SRp55/B52 SR protein. We have studied how hrp45, hrp36 [a heterogenous nuclear ribonucleoprotein (hnRNP) protein], and small nuclear RNP (snRNP) proteins are distributed in the transcriptionally active loci of polytene chromosomes in C. tentans. Immunofluorescence visualization of the proteins in double-labeling experiments revealed that hrp45 preferentially associates with a small number of puffs. On the other hand, hrp36 and snRNP proteins were found distributed in a large number of loci with little quantitative difference. Remarkably, hrp45-labeled loci coincide with the sites of transcription of premessenger RNPs of secretory protein (sp) genes. Because the labeling was found sensitive to RNase A treatment, we conclude that the SR protein hrp45 preferentially binds to sp gene transcripts in salivary gland cells. The preferential association of a specific SR protein with a particular type of gene transcripts reflects substrate-specific function(s) of an SR protein, in vivo. The possible roles that hrp45 might be playing in speedy and efficient processing of sp gene transcripts are discussed.

A novel Ded1-like RNA helicase interacts with the Y-box protein ctYB-1 in nuclear mRNP particles and in polysomes.

We have characterized a novel mRNA-binding protein, designated hrp84, in the dipteran Chironomus tentans and identified it as a DEAD-box RNA helicase. The protein contains the typical helicase core domain, a glycine-rich C-terminal part and a putative nuclear export signal in the N terminus. The protein belongs to the Ded1 subgroup of DEAD-box helicases, which is highly conserved from yeast (Ded1p) to mammals (DDX3). In tissue culture cells, hrp84 is present both in the nucleus and cytoplasm and, as shown by in vivo UV cross-linking, is bound to mRNA in both compartments. Immunoprecipitation experiments revealed that hpr84 is associated with the C. tentans homologue (ctYB-1) of the vertebrate Y-box protein YB-1 both in the nucleus and cytoplasm, and the two proteins also appear together in polysomes. The interaction is likely to be direct as shown by in vitro binding of purified components. We conclude that the mRNA-bound hrp84.ctYB-1 complex is formed in the nucleus and is translocated with mRNA into the cytoplasm and further into polysomes. As both Ded1 and YB-1 are known to regulate the initiation of translation, we propose that the RNA helicase-Y-box protein complex affects the efficiency of mRNA translation, presumably by modulating the conformation of the mRNP template.

Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export.

A specific messenger ribonucleoprotein (RNP) particle, Balbiani ring (BR) granules in the dipteran Chironomus tentans, can be visualized during passage through the nuclear pore complex (NPC). We have now examined the transport through the nuclear basket preceding the actual translocation through the NPC. The basket consists of eight fibrils anchored to the NPC core by nucleoprotein Nup153. On nuclear injection of anti-Nup153, the transport of BR granules is blocked. Many granules are retained on top of the nuclear basket, whereas no granules are seen in transit through NPC. Interestingly, the effect of Nup153 seems distant from the antibody-binding site at the base of the basket. We conclude that the entry into the basket is a two-step process: an mRMP first binds to the tip of the basket fibrils and only then is it transferred into the basket by a Nup153-dependent process. It is indicated that ribosomal subunits follow a similar pathway.

Conspicuous accumulation of transcription elongation repressor hrp130/CA150 on the intron-rich Balbiani ring 3 gene.

Chromosomal puffs on the polytene chromosomes in the dipteran Chironomus tentans offer the possibility of comparing the appearance of RNA-binding proteins at different transcription sites. We raised a monoclonal antibody that recognized a 130 kDa protein, designated hrp130. Immunocytological analysis of isolated chromosomes showed that hrp130 is heavily accumulated in a specific puff, called Balbiani ring 3; only occasionally is hrp130 abundant in one or two additional puffs on other chromosomes. The immunolabeling was sensitive to RNase treatment, suggesting that hrp130 is associated with nascent ribonucleoproteins. As shown by immunoelectron microscopy hrp130 is distributed along the active BR3 genes. The full sequence of hrp130 was determined by cDNA cloning. The protein comprises 1028 amino acids and contains three WW domains in the N-terminal half and six FF domains in the C-terminal half of the molecule. The protein is conserved from Caenorhabditis elegans to mammals; the human homolog is known as the transcription elongation repressor CA150. We propose that the abundance of hrp130/CA150 in BR3 is connected with the exceptionally high level of splicing in this locus and that hrp130/CA150 adjusts the transcription rate to the numerous splicing events taking place along the gene to ensure proper splicing.

An actin-ribonucleoprotein interaction is involved in transcription by RNA polymerase II.

To determine the function of actin in the cell nucleus, we sought to identify nuclear actin-binding proteins in the dipteran Chironomus tentans using DNase I-affinity chromatography. We identified the RNA-binding protein hrp65 as an actin-binding protein and showed that the C-terminal sequence of the hrp65-2 isoform is able to interact directly with actin in vitro. In vivo crosslinking and coimmunoprecipitation experiments indicated that hrp65 and actin are also associated in the living cell. Moreover, in vivo administration of a competing peptide corresponding to the C-terminal sequence of hrp65-2 disrupted the actin-hrp65-2 interaction and caused a specific and drastic reduction of transcription as judged by puff regression and diminished bromo-UTP incorporation. Our results indicate that an actin-based mechanism is implicated in the transcription of most if not all RNA polymerase II genes and suggest that an actin-hrp65-2 interaction is required to maintain the normal transcriptional activity of the cell. Furthermore, immunoelectron microscopy experiments and nuclear run-on assays suggest that the actin-hrp65-2 complex plays a role in 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.

A p50-like Y-box protein with a putative translational role becomes associated with pre-mRNA concomitant with transcription.

In vertebrates free messenger ribonucleoprotein (RNP) particles and polysomes contain an abundant Y-box protein called p50 (YB-1), which regulates translation, presumably by affecting the packaging of the RNA. Here, we have identified a p50-like protein in the dipteran Chironomus tentans and studied its relation with the biogenesis of mRNA in larval salivary glands. The salivary gland cells contain polytene chromosomes with the transcriptionally active regions blown up as puffs. A few giant puffs, called Balbiani rings (BRs), generate a transcription product, a large RNP particle, which can be visualised (with the electron microscope) during its assembly on the gene and during its transport to and through the nuclear pores. The p50-like protein studied, designated Ct-p40/50 (or p40/50 for short), was shown to contain a central cold-shock domain, an alanine- and proline-rich N-terminal domain, and a C-terminal domain with alternating acidic and basic regions, an organisation that is characteristic of p50 (YB-1). The p40/50 protein appears in two isoforms, p40 and p50, which contain 264 and 317 amino acids, respectively. The two isoforms share the first 258 amino acids and thus differ in amino-acid sequence only in the region close to the C-terminus. When a polyclonal antibody was raised against p40/50, western blot analysis and immunocytology showed that p40/50 is not only abundant in the cytoplasm but is also present in the nucleus. Immunolabelling of isolated polytene chromosomes showed that p40/50 appears in transcriptionally active regions, including the BRs. Using immunoelectron microscopy we revealed that p40/50 is added along the nascent transcripts and is also present in the released BR RNP particles in the nucleoplasm. Finally, by UV crosslinking in vivo we showed that p40/50 is bound to both nuclear and cytoplasmic poly(A) RNA. We conclude that p40/50 is being added cotranscriptionally along the growing BR pre-mRNA, is released with the processed mRNA into the nucleoplasm and probably remains associated with the mRNA both during nucleocytoplasmic transport and protein synthesis. Given that the p40/p50 protein, presumably with a role in translation, is loaded onto the primary transcript concomitant with transcription, an early programming of the cytoplasmic fate of mRNA is indicated.

A novel protein localized to the fibrillar compartment of the nucleolus and to the brush border of a secretory cell.

We report the identification and molecular characterization of a novel abundant nucleolar protein of the dipteran Chironomus tentans. As shown by Western blot analysis, this protein is present in nuclear extracts in a phosphorylated form with a mobility corresponding to 100 kDa. Therefore, the protein has been termed Chironomus tentans p100, or p100 for short. Analysis of the cDNA-derived primary structure of p100 indicates a protein that contains a combination of structural domains which could be involved in interactions with proteins and nucleic acids: twelve alternating acidic and basic repeats, a glycine-arginine-rich domain and a region with two zinc fingers of the C4-type. Acidic and basic repeats are typical for a group of nonribosomal nucleolar proteins. The best-studied representatives of this group are Nopp140 and nucleolin, proteins with structural and regulatory functions in rDNA transcription. Immunocytology and immunoelectron microscopy of Chironomus tentans salivary gland cells have shown that the p100 protein is located in the fibrillar compartment of the nucleolus, while it is almost absent from the granular compartment and from the nucleoplasm. The p100 protein remains in the nucleolus after removal of RNA and DNA by digestion with nucleases. This indicates that p100 might be a constituent of the nucleolar proteinaceous framework. Remarkably, p100 is also localized in the brush border in the apical part of the salivary gland cell. The presence of p100 both in the nucleolus and at the apical plasma membrane suggests that it could be involved in coordination of the level of protein production and export from the cell through regulation of the level of rRNA production in the nucleolus.

Nuclear actin is associated with a specific subset of hnRNP A/B-type proteins.

Pre-mRNP complexes were isolated from rat liver nuclei as 40S hnRNP particles, and actin-binding proteins were collected by DNase I affinity chromatography. The bound proteins were analyzed by 2D gel electrophoresis, and the following five hnRNP A/B-type proteins were identified by tandem mass spectrometry: DBP40/CBF-A (CArG binding factor A), a minor hnRNP A2 variant and three minor hnRNP A3 (mBx) variants. DBP40 was chosen for further analysis of the association of actin with the pre-mRNP complex. It was shown in vitro that purified actin binds to recombinant DBP40 suggesting that the interaction between actin and DBP40 is direct in the pre-mRNP particles. The association of actin with DBP40 was further explored in vivo. It was shown in a transfection study that DBP40 appears both in the nucleus and cytoplasm. Microinjection experiments revealed that DBP40 is exported from the nucleus to the cytoplasm. Finally, RNA-protein and protein-protein cross-linking experiments showed that DBP40 interacts with poly(A)(+) RNA as well as actin, both in the nucleus and cytoplasm. We propose that actin associated with DBP40, and perhaps with additional hnRNP A/B-type proteins, is transferred from nucleus to cytoplasm bound to mRNA.

The mRNA export factor Dbp5 is associated with Balbiani ring mRNP from gene to cytoplasm.

The DEAD box RNA helicase Dbp5 is essential for nucleocytoplasmic transport of mRNA-protein (mRNP) complexes. Dbp5 is present mainly in the cytoplasm and is enriched at the cytoplasmic side of nuclear pore complexes (NPCs), suggesting that it acts in the late part of mRNP export. Here, we visualize the assembly and transport of a specific mRNP particle, the Balbiani ring mRNP in the dipteran Chironomus tentans, and show that a Dbp5 homologue in C.tentans, Ct-Dbp5, binds to pre-mRNP co-transcriptionally and accompanies the mRNP to and through the nuclear pores and into the cytoplasm. We also demonstrate that Ct-Dbp5 accumulates in the nucleus and partly disappears from the NPC when nuclear export of mRNA is inhibited. The fact that Ct-Dbp5 is present along the exiting mRNP fibril extending from the nuclear pore into the cytoplasm supports the view that Ct-Dbp5 is involved in restructuring the mRNP prior to translation. Finally, the addition of the export factor Dbp5 to the growing transcript highlights the importance of the co-transcriptional loading process in determining the fate of mRNA.