Hidden Multivalency in Phosphatase Recruitment by a Disordered AKAP Scaffold.

Disordered scaffold proteins provide multivalent landing pads that, via a series of embedded Short Linear Motifs (SLiMs), bring together the components of a complex to orchestrate precise spatial and temporal regulation of cellular processes. One such protein is AKAP5 (previously AKAP79), which contains SLiMs that anchor PKA and Calcineurin, and recruit substrate (the TRPV1 receptor). Calcineurin is anchored to AKAP5 by a well-characterised PxIxIT SLiM. Here we show, using a combination of biochemical and biophysical approaches, that the Calcineurin PxIxIT-binding groove also recognises several hitherto unknown lower-affinity SLiMs in addition to the PxIxIT motif. We demonstrate that the assembly is in reality a complex system with conserved SLiMs spanning a wide affinity range. The capture is analogous to that seen for many DNA-binding proteins that have a weak non-specific affinity for DNA outside the canonical binding site, but different in that it involves (i) two proteins, and (ii) hydrophobic rather than electrostatic interactions. It is also compatible with the requirement for both stable anchoring of the enzyme and responsive downstream signalling. We conclude that the AKAP5 C-terminus is enriched in lower-affinity/mini-SLiMs that, together with the canonical SLiM, maintain a structurally disordered but tightly regulated signalosome.

Autoantibodies produced at the site of tissue damage provide evidence of humoral autoimmunity in inclusion body myositis.

Inclusion body myositis (IBM) belongs to a group of muscle diseases known as the inflammatory myopathies. The presence of antibody-secreting plasma cells in IBM muscle implicates the humoral immune response in this disease. However, whether the humoral immune response actively contributes to IBM pathology has not been established. We sought to investigate whether the humoral immune response in IBM both in the periphery and at the site of tissue damage was directed towards self-antigens. Peripheral autoantibodies present in IBM serum but not control serum recognized self-antigens in both muscle tissue and human-derived cell lines. To study the humoral immune response at the site of tissue damage in IBM patients, we isolated single plasma cells directly from IBM-derived muscle tissue sections and from these cells, reconstructed a series of recombinant immunoglobulins (rIgG). These rIgG, each representing a single muscle-associated plasma cell, were examined for reactivity to self-antigens. Both, flow cytometry and immunoblotting revealed that these rIgG recognized antigens expressed by cell lines and in muscle tissue homogenates. Using a mass spectrometry-based approach, Desmin, a major intermediate filament protein, expressed abundantly in muscle tissue, was identified as the target of one IBM muscle-derived rIgG. Collectively, these data support the view that IBM includes a humoral immune response in both the periphery and at the site of tissue damage that is directed towards self-antigens.

IgSF8: a developmentally and functionally regulated cell adhesion molecule in olfactory sensory neuron axons and synapses.

Here, we investigated an Immunoglobulin (Ig) superfamily protein IgSF8 which is abundantly expressed in olfactory sensory neuron (OSN) axons and their developing synapses. We demonstrate that expression of IgSF8 within synaptic neuropil is transitory, limited to the period of glomerular formation. Glomerular expression decreases after synaptic maturation and compartmental glomerular organization is achieved, although expression is maintained at high levels within the olfactory nerve layer (ONL). Immunoprecipitations indicate that IgSF8 interacts with tetraspanin CD9 in the olfactory bulb (OB). CD9 is a component of tetraspanin-enriched microdomains (TEMs), specialized microdomains of the plasma membrane known to regulate cell morphology, motility, invasion, fusion and signaling, in both the nervous and immune systems, as well as in tumors. In vitro, both IgSF8 and CD9 localize to puncta within axons and growth cones of OSNs, consistent with TEM localization. When the olfactory epithelium (OE) was lesioned, forcing OSN regeneration en masse, IgSF8 was once again able to be detected in OSN axon terminals as synapses were reestablished. Finally, we halted synaptic maturation within glomeruli by unilaterally blocking functional activity and found that IgSF8 did not undergo exclusion from this subcellular compartment and instead continued to be detected in adult glomeruli. These data support the hypothesis that IgSF8 facilitates OSN synapse formation.

Tenascin-C is an inhibitory boundary molecule in the developing olfactory bulb.

We recently described the boundary-like expression pattern of the extracellular matrix molecule tenascin-C (Tnc) in the developing mouse olfactory bulb (OB) (Shay et al., 2008). In the present study, we test the hypothesis that Tnc inhibits olfactory sensory neuron (OSN) axon growth in the developing OB before glomerulogenesis. The period of time before glomerular formation begins, when axons remain restricted to the developing olfactory nerve layer (ONL), is crucial for axon sorting. Here, we show with in vitro analyses that OSN neurite outgrowth is inhibited by Tnc in a dose-dependent manner and that, in stripe assays, axons preferentially avoid Tnc. Using Tnc-null mice, we also show that that glomerular development is delayed in the absence of Tnc. In wild-type mice, OSN axons coalesce into immature or protoglomeruli, which further differentiate and segregate into glomeruli. Glomeruli are first identifiable as discrete structures at birth. In null mice, glomeruli appear immature at birth, remain fused to the ONL, and have a significantly larger diameter compared with wild-type controls. By postnatal day 4, null glomeruli are indistinguishable from controls. Thus, OSN axons appear delayed in their coalescence into glomerular structures. These data correlate with behavioral reports of Tnc-null mice, which are delayed by 24 h in their acquisition of an olfactory behavior (de Chevigny et al., 2006). Collectively, these data demonstrate that Tnc is an inhibitory boundary molecule in the developing OB during a key period of development.

Pannexin expression in the cerebellum.

Pannexin1 and pannexin2 are members of the pannexin gene family which are widely expressed in the central nervous system. Here we present an overview of pannexin expression and distribution in the mouse cerebellum. Pannexin1 and pannexin2 are expressed in the Purkinje cells and in some cells of the granule cell layer. Pannexin2 is also expressed in the stellate cells of the molecular layer. A differential expression of pannexin1 and pannexin2 mRNA is observed during cerebellar development. These findings constitute the first indication of the involvement of pannexin molecules in the developing cerebellum. Although the functional relevance of these molecules remains currently unknown, the abundance of pannexins in the Purkinje cells suggests that they may contribute to the generation of cerebellar rhythms.

Site-specific and developmental expression of pannexin1 in the mouse nervous system.

Until recently, members of the connexin gene family were believed to comprise the sole molecular component forming gap junction channels in vertebrates. The recent discovery of the pannexin gene family has challenged this view, as these genes may encode for a putative second class of gap junction proteins in vertebrates. The expression of pannexin genes overlaps with those cellular networks known to exhibit a high degree of gap junctional coupling. We investigated the spatio-temporal mRNA distribution of one member of this gene family, pannexin1 (Panx1), in the brain and retina of mice using quantitative real-time polymerase chain reaction and a combination of in situ hybridization and immunohistochemistry for cellular resolution. Our results demonstrate a widespread expression of Panx1 in the brain, retina and other non-neuronal tissues. In the cortex, cerebellum and eye, Panx1 is expressed at early embryonic time points and peaks around embryonic day 18 followed by a decline towards adulthood. Most notably, Panx1 is detectable in neurons of many brain nuclei, which are known to be coupled by gap junctions as well as in previously unrecognized areas. Abundant expression was found in the adult hippocampal and neocortical pyramidal cells and interneurons, neurons of the reticular thalamus, the inferior olive, magnocellular hypothalamic neurons, midbrain and brain stem motoneurons, Purkinje cells and the retina.

Expression of neural connexins and pannexin1 in the hippocampus and inferior olive: a quantitative approach.

Electrical synapses (or neuronal gap junctions) are thought to be essential for the generation of synchronous oscillatory activities in various areas of the brain. In this study, we quantified the steady state mRNA expression levels of two neuronal gap junction proteins, connexin36 (Cx36) and connexin45 (Cx45), as well as of pannexin1, a member of a novel class of communicative junction forming proteins, and of connexin47 (Cx47) which is expressed in oligodendrocytes. The expression levels of these genes were compared in two regions known for oscillatory activity and which are equipped with electrically coupled neurons. Assessment of the levels of mRNA expression in the hippocampus and the nuclear complex of the inferior olive (IO) was achieved by means of laser microdissection (LMM) in combination with real time RT-PCR. Our results demonstrate the differential expression of Cx36, Cx45, pannexin1 and Cx47 in the hippocampus, with pannexin1 showing the highest level of expression followed by Cx36, Cx47, and Cx45. In the IO, pannexin1 showed a comparable expression level as in the hippocampus, but connexin expression levels were increased. Upon direct comparison, the combination of LMM and real time RT-PCR data generated specific, robust and reproducible results consistent with recent data reported about connexin expression in the nervous system. We conclude that the analytical strategy shown here provides a technological solution to overcome the less sensitive and notoriously less specific analysis of connexin expression by in situ hybridization.