Linking Integrin α6β4-based Cell Adhesion to the Intermediate Filament Cytoskeleton: Direct Interaction between the β4 Subunit and Plectin at Multiple Molecular Sites

Recent studies with patients suffering from epidermolysis bullosa simplex associated with muscular dystrophy and the targeted gene disruption in mice suggested that plectin, a versatile cytoskeletal linker and intermediate filament-binding protein, may play an essential role in hemidesmosome integrity and stabilization. To define plectin's interactions with hemidesmosomal proteins on the molecular level, we studied its interaction with the uniquely long cytoplasmic tail domain of the β₄ subunit of the basement membrane laminin receptor integrin α₆β₄ that has been implicated in connecting the transmembrane integrin complex with hemidesmosome-anchored cytokeratin filaments. In vitro binding and in vivo cotransfection assays, using recombinant mutant forms of both proteins, revealed their direct interaction via multiple molecular domains. Furthermore, we show in vitro self-interaction of integrin β₄ cytoplasmic domains, as well as disruption of intermediate filament network arrays and dislocation of hemidesmosome-associated endogenous plectin upon ectopic overexpression of this domain in PtK2 and/or 804G cells. The close association of plectin molecules with hemidesmosomal structures and their apparent random orientation was indicated by gold immunoelectron microscopy using domain-specific antibodies. Our data support a model in which plectin stabilizes hemidesmosomes, via directly interlinking integrin β₄ subunits and cytokeratin filaments.     

Short-range functional interaction between connexin35 and neighboring chemical synapses

Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication--chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre- and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35- and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.     

High-resolution proteomic mapping in the vertebrate central nervous system: Close proximity of connexin35 to NMDA glutamate receptor clusters and co-localization of connexin36 with immunoreactivity for zonula occludens protein-1 (ZO-1)

Combined confocal microscopy and freeze-fracture replica immunogold labeling (FRIL) were used to examine the connexin identity at electrical synapses in goldfish brain and rat retina, and to test for “co-localization” vs. “close proximity” of connexins to other functionally interacting proteins in synapses of goldfish and mouse brain and rat retina. In goldfish brain, confocal microscopy revealed immunofluorescence for connexin35 (Cx35) and NMDA-R1 (NR1) glutamate receptor protein in Mauthner Cell/Club Ending synapses. By FRIL double labeling, NR1 glutamate receptors were found in clusters of intramembrane particles in the postsynaptic membrane extraplasmic leaflets, and these distinctive postsynaptic densities were in close proximity (0.1–0.3 μm) to neuronal gap junctions labeled for Cx35, which is the fish ortholog of connexin36 (Cx36) found at neuronal gap junctions in mammals. Immunogold labeling for Cx36 in adult rat retina revealed abundant gap junctions, including several previously unrecognized morphological types. As in goldfish hindbrain, immunogold double labeling revealed NR1-containing postsynaptic densities localized near Cx36-labeled gap junction in rat inferior olive. Confocal immunofluorescence microscopy revealed widespread co-localization of Cx36 and ZO-1, particularly in the reticular thalamic nucleus and amygdala of mouse brain. By FRIL, ZO-1 immunoreactivity was co-localized with Cx36 at individual gap junction plaques in rat retinal neurons. As cytoplasmic accessory proteins, ZO-1 and possibly related members of the membrane-associated guanylate kinase (MAGUK) family represent scaffolding proteins that may bind to and regulate the activity of many neuronal gap junctions. These data document the power of combining immunofluorescence confocal microscopy with FRIL ultrastructural imaging and immunogold labeling to determine the relative proximities of proteins that are involved in short- vs. intermediate-range molecular interactions in the complex membrane appositions at synapses between neurons.     

Toll-like receptor 4 gene polymorphism influences dendritic cell in vitro function and clinical outcomes in vaccinated melanoma patients

Toll-like receptor 4 (TLR4) is expressed on dendritic cells (DCs), sensing environmental danger molecules that induce their activation and maturation. Recently, we reported a method for the production of therapeutic DCs against melanoma, called tumor antigen-presenting cells (TAPCells), using a heat-shocked allogeneic melanoma cell lysate (TRIMEL) as an activation factor and antigen provider. Since TRIMEL contains endogenous TLR4 ligands, we evaluated the role of TLR4 in TAPCells differentiation by antibody neutralization and the association of a Tlr4 polymorphism (896A/G) (Asp299Gly), determined by PCR–RFLP, with the in vitro activation capacity and the clinical outcome of TAPCells-vaccinated patients. Antibody blocking of monocyte TLR4 inhibited surface expression, determined by flow cytometry, of the major histocompatibility complex class I, CCR7, CD80, CD83 and CD86 on TAPCells, reduced interleukin (IL)-6 and tumor necrosis factor -α gene expression evaluated by qRT-PCR, and also inhibited the TAPCells-mediated interferon-γ (IFN-γ) secretion of melanoma-specific CD8⁺ T cells determined by ELISpot (p?<?0.01). Moreover, CD8⁺ T-cell activation capacity was significantly reduced in TAPCells bearing the TLR4 Asp299Gly receptor (p?<?0.05). Finally, TAPCells-vaccinated stage-IV melanoma patients bearing the Tlr4 896G allele showed a shortened post-therapy median survival rate compared with those carrying the Tlr4 896A allele (p?<?0.05; log-rank test). Our results indicate that TLR4 is a key receptor for the tumor lysate-mediated in vitro generation of clinically efficient antigen-presenting cells. Further analysis of patients included in different vaccine protocols is necessary for definitively establishing a role for TLR4 polymorphism in clinical responses.     

Structural and functional analysis of the actin binding domain of plectin suggests alternative mechanisms for binding to F-actin and integrin beta4

Plectin is a widely expressed cytoskeletal linker. Here we report the crystal structure of the actin binding domain of plectin and show that this region is sufficient for interaction with F-actin or the cytoplasmic region of integrin alpha6beta4. The structure is formed by two calponin homology domains arranged in a closed conformation. We show that binding to F-actin induces a conformational change in plectin that is inhibited by an engineered interdomain disulfide bridge. A two-step induced fit mechanism involving binding and subsequent domain rearrangement is proposed. In contrast, interaction with integrin alpha6beta4 occurs in a closed conformation. Competitive binding of plectin to F-actin and integrin alpha6beta4 may rely on the observed alternative binding mechanisms and involve both allosteric and steric factors.     

An index of signal mode complexity based on orthogonal transformation

Irregular and complex signals are ubiquitous in nature. The principal aim of this paper is to develop an index, quantifying the complexity of such signals, which is based on the distribution of the strengths of its orthogonal oscillatory modes estimated by singular value decomposition. The index is first tested with simulated chaotic and/or stochastic maps and flows. Among neural data analysis, the index is first applied to a cognitive EEG data set recorded from two groups, musicians and non-musicians, during listening to music and resting state. In the gamma band (30–50 Hz), musicians showed robust changes in complexity, consistent over various scalp regions, during listening to music from resting condition, whereas such changes were minimal for non-musicians. Then the index is used to separate healthy participants from epileptic and manic patients based on spontaneous EEG analysis. Finally, it is used to track a tonic-clonic seizure EEG signal, and a conspicuous change was found in the complexity profiles of delta band (1–3.5 Hz) oscillations at the onset of seizure. We conclude that this index would be useful for quantification of a wide range of time series including neural oscillations.     

Recherches dans le massif d’Ardines : nouvelles galeries ornées de la grotte de Tito Bustillo

During the year 2002 we have continued the works in the massive of Ardines at Ribadesella, Asturias, and especially in its fundamental cave, Tito Bustillo. Here the prospection has permitted us to find several new elements of great cultural and graphic value. A consistent deposit in four cutted contours in the form of head of hind and also two new galleries, called gallery of the Bisons and gallery of the Anthropomorphes, communicated with the Principal Gallery of the cave and mutually. In this last one exist remains of adaptation of the space, bony remains that we are yet digging and two figures of painted anthropomorphes with an exceptional interest. Finally, in the ensemble N XI, place where it is found the habitation deposit, and where we have found large masses of red colourings prepared for its use, exists a small final cave, where also it has been painted in its interior, and where the mouth is opened among the remains of a great deposit in surface that occupies all its northern part.     

Crystal structure of a human peptidyl-tRNA hydrolase reveals a new fold and suggests basis for a bifunctional activity

Peptidyl-tRNA hydrolase (Pth) activity releases tRNA from the premature translation termination product peptidyl-tRNA. Two different enzymes have been reported to encode such activity, Pth present in bacteria and eukaryotes and Pth2 present in archaea and eukaryotes. Here we report the crystallographic structure of the Homo sapiens Pth2 at a 2.0-A resolution as well as its catalytic properties. In contrast to the structure of Escherichia coli Pth, H. sapiens Pth2 has an alpha/beta fold with a four-stranded antiparallel beta-sheet in its core surrounded by two alpha-helices on each side. This arrangement of secondary structure elements generates a fold not previously reported. Its catalytic efficiency is comparable with that reported for the archaeal Sulfolobus solfataricus Pth2 and higher than that of the bacterial E. coli Pth. Several lines of evidence target the active site to two close loops with highly conserved residues. This active site architecture is unrelated to that of E. coli Pth. In addition, intermolecular contacts in the crystal asymmetric unit cell suggest a likely surface for protein-protein interactions related to the Pth2-mediated apoptosis.