Preferential electrical coupling regulates neocortical lineage-dependent microcircuit assembly.

Radial glial cells are the primary neural progenitor cells in the developing neocortex. Consecutive asymmetric divisions of individual radial glial progenitor cells produce a number of sister excitatory neurons that migrate along the elongated radial glial fibre, resulting in the formation of ontogenetic columns. Moreover, sister excitatory neurons in ontogenetic columns preferentially develop specific chemical synapses with each other rather than with nearby non-siblings. Although these findings provide crucial insight into the emergence of functional columns in the neocortex, little is known about the basis of this lineage-dependent assembly of excitatory neuron microcircuits at single-cell resolution. Here we show that transient electrical coupling between radially aligned sister excitatory neurons regulates the subsequent formation of specific chemical synapses in the neocortex. Multiple-electrode whole-cell recordings showed that sister excitatory neurons preferentially form strong electrical coupling with each other rather than with adjacent non-sister excitatory neurons during early postnatal stages. This preferential coupling allows selective electrical communication between sister excitatory neurons, promoting their action potential generation and synchronous firing. Interestingly, although this electrical communication largely disappears before the appearance of chemical synapses, blockade of the electrical communication impairs the subsequent formation of specific chemical synapses between sister excitatory neurons in ontogenetic columns. These results suggest a strong link between lineage-dependent transient electrical coupling and the assembly of precise excitatory neuron microcircuits in the neocortex.

Protein kinase Cδ-mediated phosphorylation of Connexin43 gap junction channels causes movement within gap junctions followed by vesicle internalization and protein degradation.

Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.

Atomic force microscopy shows connexin26 hemichannel clustering in purified membrane fragments.

Connexin proteins form hexameric assemblies known as hemichannels. When docked to form gap junction (GJ) channels, hemichannels play a critical role in cell-cell communication and cellular homeostasis, but often are functional entities on their own in unapposed cell membranes. Defects in the Connexin26 (Cx26) gene are the major cause of hereditary deafness arising from dysfunctional hemichannels in the cochlea. Structural studies of Cx26 hemichannels properly trafficked and inserted in plasma membranes, including their clustering that forms a plaque-like feature in whole gap junctions, are limited. We used atomic force microscopy (AFM) to study the surface topography of Cx26 hemichannels using two different membrane preparations. Rat Cx26 containing appended carboxy terminal V5 and hexahistidine tags were expressed in baculovirus/Sf9 cell systems. The expressed Cx26 proteins form hemichannels in situ in Sf9 cells that were then purified either as (1) Sf9 membrane fragments containing Cx26 hemichannels or (2) solubilized hemichannels. The latter were subsequently reconstituted in liposomes. AFM images of purified membrane fragments showed clusters of protein macromolecular structures in the membrane that at higher magnification corresponded to Cx26 hemichannels. Hemichannels reconstituted into DOPC bilayers displayed two populations of channel heights likely resulting from differences in orientations of inserted hemichannels. Hemichannels in the protein rich portions of purified membranes also showed a reduced channel height above the bilayer compared to membranes with reconstituted hemichannels perhaps due to reduced AFM probe access to the lipid bilayer. These preparations of purified membranes enriched for connexin hemichannels that have been properly trafficked and inserted in membranes provide a platform for high-resolution AFM imaging of the structure, interconnexon interactions, and cooperativity of properly trafficked and inserted noncrystalline connexin hemichannels.

Trafficking and recycling of the connexin43 gap junction protein during mitosis.

During the cell cycle, gap junction communication, morphology and distribution of connexin43 (Cx43)-containing structures change dramatically. As cells round up in mitosis, Cx43 labeling is mostly intracellular and intercellular coupling is reduced. We investigated Cx43 distributions during mitosis both in endogenous and exogenous expressing cells using optical pulse-chase labeling, correlated light and electron microscopy, immunocytochemistry and biochemical analysis. Time-lapse imaging of green fluorescent protein (GFP)/tetracysteine tagged Cx43 (Cx43-GFP-4C) expressing cells revealed an early disappearance of gap junctions, progressive accumulation of Cx43 in cytoplasmic structures, and an unexpected subset pool of protein concentrated in the plasma membrane surrounding the midbody region in telophase followed by rapid reappearance of punctate plaques upon mitotic exit. These distributions were also observed in immuno-labeled endogenous Cx43-expressing cells. Photo-oxidation of ReAsH-labeled Cx43-GFP-4C cells in telophase confirmed that Cx43 is distributed in the plasma membrane surrounding the midbody as apparent connexons and in cytoplasmic vesicles. We performed optical pulse-chase labeling and single label time-lapse imaging of synchronized cells stably expressing Cx43 with internal tetracysteine domains through mitosis. In late telophase, older Cx43 is segregated mainly to the plasma membrane while newer Cx43 is intracellular. This older population nucleates new gap junctions permitting rapid resumption of communication upon mitotic exit.

Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation.

Fibrosis following myocardial infarction is associated with increases in arrhythmias and sudden cardiac death. Initial steps in the development of fibrosis are not clear; however, it is likely that cardiac fibroblasts play an important role. In immune cells, ATP release from pannexin 1 (Panx1) channels acts as a paracrine signal initiating activation of innate immunity. ATP has been shown in noncardiac systems to initiate fibroblast activation. Therefore, we propose that ATP release through Panx1 channels and subsequent fibroblast activation in the heart drives the development of fibrosis in the heart following myocardial infarction. We identified for the first time that Panx1 is localized within sarcolemmal membranes of canine cardiac myocytes where it directly interacts with the postsynaptic density 95/Drosophila disk large/zonula occludens-1-containing scaffolding protein synapse-associated protein 97 via its carboxyl terminal domain (amino acids 300-357). Induced ischemia rapidly increased glycosylation of Panx1, resulting in increased trafficking to the plasma membrane as well as increased interaction with synapse-associated protein 97. Cellular stress enhanced ATP release from myocyte Panx1 channels, which, in turn, causes fibroblast transformation to the activated myofibroblast phenotype via activation of the MAPK and p53 pathways, both of which are involved in the development of cardiac fibrosis. ATP release through Panx1 channels in cardiac myocytes during ischemia may be an early paracrine event leading to profibrotic responses to ischemic cardiac injury.

Pannexin1 and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other.

Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other.

Analysis of four connexin26 mutant gap junctions and hemichannels reveals variations in hexamer stability.

Connexin26 is a ubiquitous gap junction protein that serves critical homeostatic functions. Four single-site mutations found in the transmembrane helices (M1-M4) cause different types of dysfunctional channels: 1), Cx26T135A in M3 produces a closed channel; 2), Cx26M34A in M1 severely decreases channel activity; 3), Cx26P87L in M2 has been implicated in defective channel gating; and 4), Cx26V84L in M2, a nonsyndromic deafness mutant, retains normal dye coupling and electrophysiological properties but is deficient in IP(3) transfer. These mutations do not affect Cx26 trafficking in mammalian cells, and make normal-appearing channels in baculovirus-infected Sf9 membranes when imaged by negative stain electron microscopy. Upon dodecylmaltoside solubilization of the membrane fraction, Cx26M34A and Cx26V84L are stable as hexamers or dodecamers, but Cx26T135A and Cx26P87L oligomers are not. This instability is also found in Cx26T135A and Cx26P87L hemichannels isolated from mammalian cells. In this work, coexpression of both wild-type Cx26 and Cx26P87L in Sf9 cells rescued P87L hexamer stability. Similarly, in paired Xenopus oocytes, coexpression with wild-type restored function. In contrast, the stability of Cx26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating.

The cytoplasmic accumulations of the cataract-associated mutant, Connexin50P88S, are long-lived and form in the endoplasmic reticulum.

Mutant connexins have been linked to hereditary congenital cataracts. One such mutant causes a proline-to-serine substitution at position 88 in human connexin 50 (CX50P88S). In transfected cells, CX50P88S does not form gap junctions, but localizes in cytoplasmic multilamellar structures. We studied the dynamics of formation and the stability of these structures in HeLa cells stably transfected with CX50P88S containing a tetracysteine motif appended to its C-terminus (HeLa-CX50P88S(Cys)(4) cells). The tetracysteine motif binds the membrane-permeable biarsenical compounds, FlAsH and ReAsH, which become fluorescent upon binding allowing detection of CX50P88S(Cys)(4) by fluorescence microscopy or by transmission electron microscopy after the ReAsH-driven fluorescent photoconversion of diaminobenzidine. CX50P88S structures were long-lived. Pulse labeling of HeLa-CX50P88S(Cys)(4) cells with FlAsH followed by a chase and ReAsH labeling showed a differential distribution of the labels, with older CX50P88S surrounded by newly synthesized protein. Formation of CX50P88S accumulations was not affected by treatments that block ER-to-Golgi transport. Transmission electron microscopy and tomographic reconstruction revealed that CX50P88S accumulations corresponded to closely apposed circular or semicircular membrane stacks that were sometimes continuous with the rough endoplasmic reticulum. These results suggest that CX50P88S accumulations originate from the rough endoplasmic reticulum and that mutant protein is sequentially added resulting in long-lived cytoplasmic particles. The persistence of these particles in the lens may cause light scattering and the pulverulent cataracts observed in affected individuals.

Computational modeling of three-dimensional electrodiffusion in biological systems: application to the node of Ranvier.

A computational model is presented for the simulation of three-dimensional electrodiffusion of ions. Finite volume techniques were used to solve the Poisson-Nernst-Planck equation, and a dual Delaunay-Voronoi mesh was constructed to evaluate fluxes of ions, as well as resulting electric potentials. The algorithm has been validated and applied to a generalized node of Ranvier, where numerical results for computed action potentials agree well with cable model predictions for large clusters of voltage-gated ion channels. At smaller channel clusters, however, the three-dimensional electrodiffusion predictions diverge from the cable model predictions and show a broadening of the action potential, indicating a significant effect due to each channel's own local electric field. The node of Ranvier complex is an elaborate organization of membrane-bound aqueous compartments, and the model presented here represents what we believe is a significant first step in simulating electrophysiological events with combined realistic structural and physiological data.

Electron tomographic analysis of cytoskeletal cross-bridges in the paranodal region of the node of Ranvier in peripheral nerves.

The node of Ranvier is a site for ionic conductances along myelinated nerves and governs the saltatory transmission of action potentials. Defects in the cross-bridging and spacing of the cytoskeleton are a prominent pathological feature in diseases of the peripheral nerve. Electron tomography was used to examine cytoskeletal-cytoskeletal, membrane-cytoskeletal, and heterologous cell connections in the paranodal region of the node of Ranvier in peripheral nerves. Focal attachment of cytoskeletal filaments to each other and to the axolemma and paranodal membranes of the Schwann cell via narrow cross-bridges was visualized in both neuronal and glial cytoplasm. A subset of intermediate filaments associates with the cytoplasmic surfaces of supramolecular complexes of transmembrane structures that are presumed to include known and unknown junctional proteins. Mitochondria were linked to both microtubules and neurofilaments in the axoplasm and to neighboring smooth endoplasmic reticulum by narrow cross-bridges. Tubular cisternae in the glial cytoplasm were also linked to the paranodal glial cytoplasmic loop juxtanodal membrane by short cross-bridges. In the extracellular matrix between axon and Schwann cell, junctional bridges formed long cylinders linking the two membranes. Interactions between cytoskeleton, membranes, and extracellular matrix associations in the paranodal region are likely critical not only for scaffolding, but also for intracellular and extracellular communication.

Visualizing flock house virus infection in Drosophila cells with correlated fluorescence and electron microscopy.

Virus assembly occurs in a complex environment and is dependent upon viral and cellular components being properly correlated in time and space. The simplicity of the flock house virus (FHV) capsid and the extensive structural, biochemical and genetic characterization of the virus make it an excellent system for studying in vivo virus assembly. The tetracysteine motif (CCPGCC), that induces fluorescence in bound biarsenical compounds (FlAsH and ReAsH), was genetically inserted in the coat protein, to visualize this gene product during virus infection. The small size of this modification when compared to those made by traditional fluorescent proteins minimizes disruption of the coat proteins numerous functions. ReAsH not only fluoresces when bound to the tetracysteine motif but also allows correlated electron microscopy (EM) of the same cell following photoconversion and osmium staining. These studies demonstrated that the coat protein was concentrated in discrete patches in the cell. High pressure freezing (HPF) followed by freeze substitution (FS) of infected cells showed that these patches were formed by virus particles in crystalline arrays. EM tomography (EMT) of the HPF/FS prepared samples showed that these arrays were proximal to highly modified mitochondria previously established to be the site of RNA replication. Two features of the mitochondrial modification are approximately 60 nm spherules that line the outer membrane and the large chamber created by the convolution induced in the entire organelle.

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications.

The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.

The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodies.

The C-terminus of the most abundant and best-studied gap-junction protein, connexin43, contains multiple phosphorylation sites and protein-binding domains that are involved in regulation of connexin trafficking and channel gating. It is well-documented that SDS/PAGE of NRK (normal rat kidney) cell lysates reveals at least three connexin43-specific bands (P0, P1 and P2). P1 and P2 are phosphorylated on multiple, unidentified serine residues and are found primarily in gap-junction plaques. In the present study we prepared monoclonal antibodies against a peptide representing the last 23 residues at the C-terminus of connexin43. Immunofluorescence studies showed that one antibody (designated CT1) bound primarily to connexin43 present in the Golgi apparatus, whereas the other antibody (designated IF1) labelled predominately connexin43 present in gap junctions. CT1 immunoprecipitates predominantly the P0 form whereas IF1 recognized all three bands. Peptide mapping, mutational analysis and protein-protein interaction experiments revealed that unphosphorylated Ser364 and/or Ser365 are critical for CT1 binding. The IF1 paratope binds to residues Pro375-Asp379 and requires Pro375 and Pro377. These proline residues are also necessary for ZO-1 interaction. These studies indicate that the conformation of Ser364/Ser365 is important for intracellular localization, whereas the tertiary structure of Pro375-Asp379 is essential in targeting and regulation of gap junctional connexin43.

Structural organization of gap junction channels.

Gap junctions were initially described morphologically, and identified as semi-crystalline arrays of channels linking two cells. This suggested that they may represent an amenable target for electron and X-ray crystallographic studies in much the same way that bacteriorhodopsin has. Over 30 years later, however, an atomic resolution structural solution of these unique intercellular pores is still lacking due to many challenges faced in obtaining high expression levels and purification of these structures. A variety of microscopic techniques, as well as NMR structure determination of fragments of the protein, have now provided clearer and correlated views of how these structures are assembled and function as intercellular conduits. As a complement to these structural approaches, a variety of mutagenic studies linking structure and function have now allowed molecular details to be superimposed on these lower resolution structures, so that a clearer image of pore architecture and its modes of regulation are beginning to emerge.

Connexin43 Forms Supramolecular Complexes through Non-Overlapping Binding Sites for Drebrin, Tubulin, and ZO-1.

Gap junctions are membrane specialization domains identified in most tissue types where cells abut each other. The connexin channels found in these membrane domains are conduits for direct cell-to-cell transfer of ions and molecules. Connexin43 (Cx43) is the most ubiquitous connexin, with critical roles in heart, skin, and brain. Several studies described the interaction between Cx43 and the cytoskeleton involving the actin binding proteins Zonula occludens (ZO-1) and drebrin, as well as with tubulin. However, a direct interaction has not been identified between drebrin and Cx43. In this study, co-IP and NMR experiments were used to demonstrate that the Cx43-CT directly interacts with the highly conserved N-terminus region of drebrin. Three Cx43-CT areas were found to be involved in drebrin binding, with residues 264-275 being critical for the interaction. Mimicking Src phosphorylation within this region (Y265) significantly disrupted the interaction between the Cx43-CT and drebrin. Immunofluorescence showed colocalization of Cx43, drebrin, and F-actin in astrocytes and Vero cells membrane, indicating that Cx43 forms a submembrane protein complex with cytoskeletal and scaffolding proteins. The co-IP data suggest that Cx43 indirectly interacts with F-actin through drebrin. Along with the known interaction of the Cx43-CT with ZO-1 and tubulin, the data presented here for drebrin indicate non-overlapping and separated binding sites for all three proteins for which simultaneous binding could be important in regulating cytoskeleton rearrangements, especially for neuronal migration during brain development.

Isolation and characterization of gap junctions from tissue culture cells.

The purification of membrane proteins in a form and amount suitable for structural or biochemical studies still remains a great challenge. Gap junctions have long been studied using electron microscopy and X-ray diffraction. However, only a limited number of proteins in the connexin family have been amenable to protein or membrane purification techniques. Molecular biology techniques for expressing large gap junctions in tissue culture cells combined with improvements in electron crystallography have shown great promise for determining the channel structure to better than 10 A resolution. Here, we have isolated two-dimensional (2D) gap junction crystals from HeLa Cx26 transfectants. This isoform has never been isolated in large fractions from tissues. We characterize these preparations by SDS-PAGE, Western blotting, negative stain electron microscopy and atomic force microscopy. In our preparations, the Cx26 is easily detected in the Western blots and we have increased expression levels so that connexin bands are visible on SDS-PAGE gels. Preliminary assessment of the samples by electron cryo-microscopy shows that these 2D crystals diffract to at least 22 A. Atomic force microscopy of these Cx26 gap junctions show exquisite surface modulation at the extracellular surface in force dissected gap junctions. We also applied our protocol to cell lines such as NRK cells that express endogenous Cx43 and NRK and HeLa cell lines transfected with exogenous connexins. While the gap junction membrane channels are recognizable in negatively stained electron micrographs, these lattices are disordered and the gap junction plaques are smaller. SDS-PAGE and Western blotting revealed expression of connexins, but at a lower level than with our HeLa Cx26 transfectants. Therefore, the purity and morphology of the gap junction plaques depends the size and abundance of the gap junctions in the cell line itself.

Development of a model for microphysiological simulations: small nodes of ranvier from peripheral nerves of mice reconstructed by electron tomography.

The node of Ranvier is a complex structure found along myelinated nerves of vertebrate animals. Specific membrane, cytoskeletal, junctional, extracellular matrix proteins and organelles interact to maintain and regulate associated ion movements between spaces in the nodal complex, potentially influencing response variation during repetitive activations or metabolic stress. Understanding and building high resolution three dimensional (3D) structures of the node of Ranvier, including localization of specific macromolecules, is crucial to a better understanding of the relationship between its structure and function and the macromolecular basis for impaired conduction in disease. Using serial section electron tomographic methods, we have constructed accurate 3D models of the nodal complex from mouse spinal roots with resolution better than 7.5 nm. These reconstructed volumes contain 75-80% of the thickness of the nodal region. We also directly imaged the glial axonal junctions that serve to anchor the terminal loops of the myelin lamellae to the axolemma. We created a model of an intact node of Ranvier by truncating the volume at its midpoint in Z, duplicating the remaining volume and then merging the new half volume with mirror symmetry about the Z-axis. We added to this model the distribution and number of Na+ channels on this reconstruction using tools associated with the MCell simulation program environment. The model created provides accurate structural descriptions of the membrane compartments, external spaces, and formed structures enabling more realistic simulations of the role of the node in modulation of impulse propagation than have been conducted on myelinated nerve previously.

Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics.

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.

Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane.

Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.

A comparative antibody analysis of pannexin1 expression in four rat brain regions reveals varying subcellular localizations.

Pannexin1 (Panx1) channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus, and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on-line viewing, and download.