Subcellular Localization of the TFF Peptides xP1 and xP4 in the Xenopus laevis Gastric/Esophageal Mucosa: Different Secretion Modes Reflecting Diverse Protective Functions.

The TFF peptides xP1 and xP4 from Xenopus laevis are orthologs of TFF1 and TFF2, respectively. xP1 is secreted as a monomer from gastric surface mucous cells and is generally not associated with mucins, whereas xP4 is a typical secretory peptide from esophageal goblet cells, and gastric mucous neck and antral gland cells tightly associated as a lectin with the ortholog of mucin MUC6. Both TFF peptides have diverse protective functions, xP1 as a scavenger for reactive oxygen species preventing oxidative damage and xP4 as a constituent of the water-insoluble adherent inner mucus barrier. Here, we present localization studies using immunofluorescence and immunoelectron microscopy. xP1 is concentrated in dense cores of secretory granules of surface mucous cells, whereas xP4 mixes with MUC6 in esophageal goblet cells. Of note, we observe two different types of goblet cells, which differ in their xP4 synthesis, and this is even visible morphologically at the electron microscopic level. xP4-negative granules are recognized by their halo, which is probably the result of shrinkage during the processing of samples for electron microscopy. Probably, the tight lectin binding of xP4 and MUC6 creates a crosslinked mucous network forming a stabile granule matrix, which prevents shrinkage.

Comparison of residual alpha- and beta-structures between two intrinsically disordered proteins by using NMR.

Intrinsically disordered proteins contain some residual structures, which may fold further upon binding to the partner protein for function. The residual structures observed in two intrinsically disordered proteins, including the C-terminal segment of peripherin-2 (63 residues) and measles virus nucleocapsid protein Ntail (125 residues), were compared using NMR. Differences in the chemical shifts of alpha-, beta- and carbonyl carbons between the observed structure and calculated random coil revealed the existence of a helix and some possible beta-structures in both proteins. The intensity of signals in the C-terminal segment of peripherin-2 in NMR spectra was informative and locally low, particularly in the middle and N-terminal parts: this suggested the broadening of the signals caused by the formation of residual structures in those areas. Furthermore, the protection of exchange of amide protons was significantly observed at the N-terminus. Conversely, the intensities of signals for Ntail were random beyond the overall areas of protein, and indicated no characteristic pattern. Only a faint protection of amide-proton exchange in Ntail was observed in the C-terminus. It was concluded that Ntail was more intrinsically disordered than the C-terminal segment of peripherin-2. The combination of chemical shifts with the amide-proton exchanges and signal intensities was useful for the analyses of the remaining secondary structures. The beta-structure might be more detectable by the protection of amide-proton exchange than the helical structure, although the changes in chemical shifts were sensitive for the detection of elements of both secondary structures.

Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution.

Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.

Structural origin of the drastic modification of second harmonic generation intensity pattern occurring in tail muscles of climax stages xenopus tadpoles.

Second harmonic generation (SHG) microscopy is a powerful tool for studying submicron architecture of muscles tissues. Using this technique, we show that the canonical single frequency sarcomeric SHG intensity pattern (SHG-IP) of premetamorphic xenopus tadpole tail muscles is converted to double frequency (2f) sarcomeric SHG-IP in metamorphic climax stages due to massive physiological muscle proteolysis. This conversion was found to rise from 7% in premetamorphic muscles to about 97% in fragmented muscular apoptotic bodies. Moreover a 66% conversion was also found in non-fragmented metamorphic tail muscles. Also, a strong correlation between predominant 2f sarcomeric SHG-IPs and myofibrillar misalignment is established with electron microscopy. Experimental and theoretical results demonstrate the higher sensitivity and the supra resolution power of SHG microscopy over TPEF to reveal 3D myofibrillar misalignment. From this study, we suggest that 2f sarcomeric SHG-IP could be used as signature of triad defect and disruption of excitation-contraction coupling. As the mechanism of muscle proteolysis is similar to that found in mdx mouse muscles, we further suggest that xenopus tadpole tail resorption at climax stages could be used as an alternative or complementary model of Duchene muscular dystrophy.

Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution.

One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41 ± 7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.

The Cryo-EM structure of pannexin 1 reveals unique motifs for ion selection and inhibition.

Pannexins are large-pore forming channels responsible for ATP release under a variety of physiological and pathological conditions. Although predicted to share similar membrane topology with other large-pore forming proteins such as connexins, innexins, and LRRC8, pannexins have minimal sequence similarity to these protein families. Here, we present the cryo-EM structure of a frog pannexin 1 (Panx1) channel at 3.0 Å. We find that Panx1 protomers harbor four transmembrane helices similar in arrangement to other large-pore forming proteins but assemble as a heptameric channel with a unique constriction formed by Trp74 in the first extracellular loop. Mutating Trp74 or the nearby Arg75 disrupt ion selectivity, whereas altering residues in the hydrophobic groove formed by the two extracellular loops abrogates channel inhibition by carbenoxolone. Our structural and functional study establishes the extracellular loops as important structural motifs for ion selectivity and channel inhibition in Panx1.

The allosteric role of the Ca2+ switch in adhesion and elasticity of C-cadherin.

Modular proteins such as titin, fibronectin, and cadherin are ubiquitous components of living cells. Often involved in signaling and mechanical processes, their architecture is characterized by domains containing a variable number of heterogeneous "repeats" arranged in series, with either flexible or rigid linker regions that determine their elasticity. Cadherin repeats arranged in series are unique in that linker regions also feature calcium-binding motifs. While it is well known that the extracellular repeats of cadherin proteins mediate cell-cell adhesion in a calcium-dependent manner, the molecular mechanisms behind the influence of calcium in adhesion dynamics and cadherin's mechanical response are not well understood. Here we show, using molecular dynamics simulations, how calcium ions control the structural integrity of cadherin's linker regions, thereby affecting cadherin's equilibrium dynamics, the availability of key residues involved in cell-cell adhesion, and cadherin's mechanical response. The all-atom, multi-nanosecond molecular dynamics simulations involved the entire C-cadherin extracellular domain solvated in water (a 345,000 atom system). Equilibrium simulations show that the extracellular domain maintains its crystal conformation (elongated and slightly curved) when calcium ions are present. In the absence of calcium ions, however, it assumes a disordered conformation. The conserved residue Trp(2), which is thought to insert itself into a hydrophobic pocket of another cadherin molecule (thereby providing the basis for cell-cell adhesion), switches conformation from exposed to intermittently buried upon removal of calcium ions. Furthermore, the overall mechanical response of C-cadherin's extracellular domain is characterized at low force by changes in shape (tertiary structure elasticity), and at high force by unraveling of secondary structure elements (secondary structure elasticity). This mechanical response is modulated by calcium ions at both low and high force, switching from a stiff, rod-like to a soft, spring-like behavior upon removal of ions. The simulations provide an unprecedented molecular view of calcium-mediated allostery in cadherins, also illustrating the general principles of linker-mediated elasticity of modular proteins relevant not only for cell-cell adhesion and sound transduction, but also muscle elasticity.

A novel karyoskeletal protein: characterization of protein NO145, the major component of nucleolar cortical skeleton in Xenopus oocytes.

The nucleolus is a ubiquitous, mostly spheroidal nuclear structure of all protein-synthesizing cells, with a well-defined functional compartmentalization. Although a number of nonribosomal proteins involved in ribosome formation have been identified, the elements responsible for the shape and internal architecture of nucleoli are still largely unknown. Here, we report the molecular characterization of a novel protein, NO145, which is a major and specific component of a nucleolar cortical skeleton resistant to high salt buffers. The amino acid sequence of this polypeptide with a SDS-PAGE mobility corresponding to M(r) 145,000 has been deduced from a cDNA clone isolated from a Xenopus laevis ovary expression library and defines a polypeptide of 977 amino acids with a calculated mass of 111 kDa, with partial sequence homology to a synaptonemal complex protein, SCP2. Antibodies specific for this protein have allowed its recognition in immunoblots of karyoskeleton-containing fractions of oocytes from different Xenopus species and have revealed its presence in all stages of oogenesis, followed by a specific and rapid degradation during egg formation. Immunolocalization studies at the light and electron microscopic level have shown that protein NO145 is exclusively located in a cage-like cortical structure around the entire nucleolus, consisting of a meshwork of patches and filaments that dissociates upon reduction of divalent cations. We propose that protein NO145 contributes to the assembly of a karyoskeletal structure specific for the nucleolar cortex of the extrachromosomal nucleoli of Xenopus oocytes, and we discuss the possibility that a similar structure is present in other cells and species.

Cryo-EM structures of the triheteromeric NMDA receptor and its allosteric modulation.

N-methyl-d-aspartate receptors (NMDARs) are heterotetrameric ion channels assembled as diheteromeric or triheteromeric complexes. Here, we report structures of the triheteromeric GluN1/GluN2A/GluN2B receptor in the absence or presence of the GluN2B-specific allosteric modulator Ro 25-6981 (Ro), determined by cryogenic electron microscopy (cryo-EM). In the absence of Ro, the GluN2A and GluN2B amino-terminal domains (ATDs) adopt "closed" and "open" clefts, respectively. Upon binding Ro, the GluN2B ATD clamshell transitions from an open to a closed conformation. Consistent with a predominance of the GluN2A subunit in ion channel gating, the GluN2A subunit interacts more extensively with GluN1 subunits throughout the receptor, in comparison with the GluN2B subunit. Differences in the conformation of the pseudo-2-fold-related GluN1 subunits further reflect receptor asymmetry. The triheteromeric NMDAR structures provide the first view of the most common NMDA receptor assembly and show how incorporation of two different GluN2 subunits modifies receptor symmetry and subunit interactions, allowing each subunit to uniquely influence receptor structure and function, thus increasing receptor complexity.

Use of intact Xenopus oocytes in nucleocytoplasmic transport studies.

Because of its large nucleus, the Xenopus laevis oocyte offers an excellent system to study nucleocytoplasmic transport. This system, in combination with electron microscopy, has provided much of our insight into the mechanisms of nuclear import and export. In a typical experiment, the nuclear transport substrate is first labeled with colloidal gold, and the resulting complex is injected into the cytoplasm (to study nuclear import) or the nucleus (to study nuclear export) of Xenopus oocytes. The oocytes are then fixed, dehydrated, infiltrated, and embedded into an epoxy resin. Following resin polymerization, thin sections of oocyte nuclei are obtained and examined under an electron microscope. Subsequent evaluation of the position and distribution of the gold-labeled substrate reveals whether the substrate has undergone nuclear import (or export) and the position of rate-limiting events. This chapter describes in detail the protocols for performing electron microscopy import assays with Xenopus oocytes and presents some data illustrating the types of experiments possible using this system.

Crystal structure of the metazoan Nup62•Nup58•Nup54 nucleoporin complex.

Nuclear pore complexes (NPCs) conduct nucleocytoplasmic transport and gain transport selectivity through nucleoporin FG domains. Here, we report a structural analysis of the FG Nup62•58•54 complex, which is a crucial component of the transport system. It comprises a ≈13 nanometer-long trimerization interface with an unusual 2W3F coil, a canonical heterotrimeric coiled coil, and a kink that enforces a compact six-helix bundle. Nup54 also contains a ferredoxin-like domain. We further identified a heterotrimeric Nup93-binding module for NPC anchorage. The quaternary structure alternations in the Nup62 complex, which were previously proposed to trigger a general gating of the NPC, are incompatible with the trimer structure. We suggest that the highly elongated Nup62 complex projects barrier-forming FG repeats far into the central NPC channel, supporting a barrier that guards the entire cross section.

Expression of vomeronasal receptor genes in Xenopus laevis.

In the course of evolution, the vomeronasal organ (VNO) first appeared in amphibians. To understand the relationship between the VNO and the vomeronasal receptors, we isolated and analyzed the expression of the vomeronasal receptor genes of Xenopus laevis. We identified genes of the Xenopus V2R receptor family, which are predominantly expressed throughout the sensory epithelium of the VNO. The G-protein Go, which is coexpressed with V2Rs in the rodent VNO, was also extensively expressed throughout the vomeronasal sensory epithelium. These results strongly suggest that the V2Rs and Go are coexpressed in the vomeronasal receptor cells. The predominant expression of the Xenopus V2R families and the coexpression of the V2Rs and Go imply that V2Rs play important roles in the sensory transduction of Xenopus VNO. We found that these receptors were expressed not only in the VNO, but also in the posterolateral epithelial area of the principal cavity (PLPC). Electron microscopic study revealed that the epithelium of the PLPC is more like that of the VNO than that of the principal and the middle cavity. These results suggest that in adult Xenopus the V2Rs analyzed so far are predominantly expressed in the vomeronasal and vomeronasal-like epithelium. The analysis of V2R expression in Xenopus larvae demonstrates that V2Rs are predominantly expressed in the VNO even before metamorphosis.

Dissection of the unusual structural and functional properties of the variant H2A.Bbd nucleosome.

The histone variant H2A.Bbd appeared to be associated with active chromatin, but how it functions is unknown. We have dissected the properties of nucleosome containing H2A.Bbd. Atomic force microscopy (AFM) and electron cryo-microscopy (cryo-EM) showed that the H2A.Bbd histone octamer organizes only approximately 130 bp of DNA, suggesting that 10 bp of each end of nucleosomal DNA are released from the octamer. In agreement with this, the entry/exit angle of the nucleosomal DNA ends formed an angle close to 180 degrees and the physico-chemical analysis pointed to a lower stability of the variant particle. Reconstitution of nucleosomes with swapped-tail mutants demonstrated that the N-terminus of H2A.Bbd has no impact on the nucleosome properties. AFM, cryo-EM and chromatin remodeling experiments showed that the overall structure and stability of the particle, but not its property to interfere with the SWI/SNF induced remodeling, were determined to a considerable extent by the H2A.Bbd docking domain. These data show that the whole H2A.Bbd histone fold domain is responsible for the unusual properties of the H2A.Bbd nucleosome.

XMAP215 is a long thin molecule that does not increase microtubule stiffness.

XMAP215 is a microtubule associated protein that speeds microtubule plus end growth by seven- to tenfold and protects these ends from destabilization by the Kin I kinesin, XKCM1. To understand the mechanisms responsible for these activities, it is necessary to know the structure of XMAP215. By unidirectional shadowing and electron microscopy, XMAP215 appeared as an elongate molecule of 60+/-18 nm, suggesting that XMAP215 could span up to seven to eight tubulin dimers along a protofilament. Most XMAP215 molecules were straight but a subset were bent suggesting that XMAP215 is flexible. Antibodies to the C terminus labeled one end of XMAP215 with no evidence for XMAP215 dimerization. Incubation of XMAP215 and tubulin at 4 degrees C resulted in assembly of curved protofilaments, which appeared to be incomplete tubulin rings. Measurements from rotary shadowed samples showed that tubulin/XMAP215 partial rings had an average width of 8.8+/-1.8 nm compared with 5.6+/-1.1 nm for rings assembled from tubulin dimers alone, suggesting that XMAP215 adds a width of approximately 3.2 nm to the curved tubulin protofilament. XMAP215 did not change the radius of curvature of these partial tubulin rings. Measurements of microtubule flexural rigidity by thermal fluctuations showed that XMAP215 did not change microtubule rigidity. Finally, sequence analysis shows that the N-terminal half of XMAP215 contains four repeats, each composed of multiple HEAT repeats.

Visualization of the reconstituted FRGY2-mRNA complexes by electron microscopy.

Xenopus oocytes store large quantities of translationally dormant mRNA in the cytoplasm as storage messenger ribonucleoprotein particles (mRNPs). The Y-box proteins, mRNP3 and FRGY2/mRNP4, are major RNA binding components of maternal storage mRNPs in oocytes. In this study, we show that the FRGY2 proteins form complexes with mRNA, which leads to mRNA stabilization and translational repression. Visualization of the FRGY2-mRNA complexes by electron microscopy reveals that FRGY2 packages mRNA into a compact RNP. Our results are consistent with a model that the Y-box proteins function in packaging of mRNAs to store them stably for a long time in the oocyte cytoplasm.