Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 is regulated by actin filaments and 4.1N in neuronal dendrites

Inositol 1,4,5-trisphosphate receptor type1 (IP3R1) plays an important role in neuronal functions; however, the lateral diffusion of IP3R1 on the endoplasmic reticulum membrane and its regulation in the living neurons remain unknown. We expressed green fluorescent protein-tagged IP3R1 in cultured rat hippocampal neurons and observed the lateral diffusion by the fluorescence recovery after photobleaching technique. IP3R1 showed lateral diffusion with an effective diffusion constant of approximately 0.3 microm2/s. Depletion of actin filaments increased the diffusion constant of IP3R1, suggesting that the diffusion of IP3R1 is regulated negatively through actin filaments. We also found that protein 4.1N, which binds to IP3R1 and contains an actin-spectrin-binding region, was responsible for this actin regulation of the IP3R1 diffusion constant. Overexpression of dominant-negative 4.1N and blockade of 4.1N binding to IP3R1 increased the IP3R1 diffusion constant. The diffusion of IP3R type 3 (IP3R3), one of the isoforms of IP3Rs lacking the binding ability to 4.1N, was not dependent on actin filaments but became dependent on actin filaments after the addition of a 4.1N-binding sequence. These data suggest that 4.1N serves as a linker protein between IP3R1 and actin filaments. This actin filament-dependent regulation of IP3R1 diffusion may be important for the spatiotemporal regulation of intracellular Ca2+ signaling.     

InsP₃receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca2? entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP?Rs (InsP? receptors). The co-localization between Orai1 and IP?Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca2? store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca2? store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP?Rs and a basolateral pool that interacts with STIM1 following the Ca2? store depletion. Experiments on IP?R knockout animals demonstrated that the apical Orai1 localization does not require IP?Rs and that IP?Rs are not necessary for the activation of SOCE. However, the InsP?-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP?Rs could have an inhibitory effect on this Ca2? entry mechanism.     

Doc2gamma, a third isoform of double C2 protein, lacking calcium-dependent phospholipid binding activity

The Doc2 (double C2) family consists of two isoforms (Doc2alpha and Doc2beta) characterized by an N-terminal Munc13-1 interacting domain (Mid) and two C2 domains that interact with Ca(2+) and phospholipid at the C-terminus. This Ca(2+)-binding property is thought to be important to the regulation of neurotransmitter release. In this paper, we report a third isoform of mouse Doc2, named Doc2gamma. Doc2gamma also contains a putative Mid domain and two C2 domains, and it is 45.6 and 43.2% identical to mouse Doc2alpha and Doc2beta, respectively, at the amino acid level. In contrast to the other Doc2 isoforms, the C2 domains of Doc2gamma impair Ca(2+)-dependent phospholipid binding activity. The highest expression of Doc2gamma mRNA was found in the heart, but occurs ubiquitously, the same as Doc2beta. These findings indicate that Doc2gamma may also function as an effector for Munc13-1 and that it may be involved in the regulation of vesicular trafficking.     

Striatum-specific expression of Cre recombinase using the Gpr88 promoter in mice

We generated a transgenic (Tg) mouse line expressing Cre recombinase under the control of the Gpr88 promoter within a bacterial artificial chromosome clone. We crossed the established Tg mice with reporter mice (CAG-CAT-Z Tg), which express Escherichia coli lacZ in response to Cre-mediated excision of the loxP-flanked chloramphenicol acetyltransferase gene, and examined the Cre activity in the Tg mouse brains by assessing β-galactosidase activity. Cre activity was specifically detected in the caudate-putamen, nucleus accumbens, and olfactory tubercle of the Gpr88-Cre Tg mouse brain. Medium spiny neurons within the caudate-putamen exhibited Cre activity. Thus, Gpr88-Cre Tg mice could be a useful tool for analyzing the function of the basal ganglia by using Cre/loxP systems.     

Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae

Activation of various receptors by extracellular ligands induces an influx of Ca2+ through the plasma membrane, but its molecular mechanism remains elusive and seems variable in different cell types. In the present study, we utilized mAbs generated against the cerebellar type I inositol 1,4,5-trisphosphate (InsP3) receptor and performed immunocytochemical and immunochemical experiments to examine its localization in several non-neuronal cells. By immunogold electron microscopy of ultrathin frozen sections as well as permeabilized tissue specimens, we found that a mAb to the type I InsP3 receptor (mAb 4C11) labels the plasma membrane of the endothelium, smooth muscle cell and keratinocyte in vivo. Interestingly, the labeling with the antibody was confined to caveolae, smooth vesicular inpocketings of the plasma membrane. The reactive protein, with an M(r) of 240,000 by SDS-PAGE, could be biotinylated with a membrane-impermeable reagent, sulfo-NHS-biotin, in intact cultured endothelial cells, and recovered by streptavidin-agarose beads, which result further confirmed its presence on the cell surface. The present findings indicate that a protein structurally homologous to the type I InsP3 receptor is localized in the caveolar structure of the plasma membrane and might be involved in the Ca2+ influx.     

Molecular cloning and expression profile of Xenopus calcineurin A subunit(1)

We have cloned a cDNA encoding a catalytic subunit of calcineurin (CnA) expressed in Xenopus oocytes. The deduced amino acid sequence indicates 96.3% and 96.8% identities with the mouse and human CnAalpha isoforms, respectively. Xenopus CnA (XCnA) RNA and protein are expressed as maternal and throughout development. Recombinant XCnA protein interacted with calmodulin in the presence of Ca(2+). Deletion of calmodulin binding domain and auto-inhibitory domain revealed calcium independent phosphatase activity, thereby showing that XCnA is likely to be modulated by both calmodulin and calcium.     

Characterization of Microtubule-Associated Protein 2 from Mouse Brain and Its Localization in the Cerebellar Cortex

Microtubule-associated protein (MAP) 2 was purified from the microtubule fraction of mouse brain by heat treatment and BioGel A-5m gel filtration. The purified preparation showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis using both a gradient gel (3.75-12.5%) and a low-percentage gel (5%), a finding indicating that MAP2B was absent under the conditions used. Amino acid analysis revealed that mouse MAP2 was an acidic protein with an isoelectric point (pI 4.5) and amino acid composition similar to those of porcine brain MAP2. Immunoblot analysis indicated that the antigens that reacted with MAP2 antiserum were present in large quantities in mouse brain. However, we also found a weak reaction in various tissues other than brain, and the major antigens involved were recognized to be common molecular species with the same molecular mass, 162 and 170 kilodaltons. Using antiserum against mouse brain MAP2, the developmental localization patterns of MAP2 in the mouse cerebellar cortex were studied by immunohistochemistry. MAP2 was mainly localized in the neuronal cells throughout development, with the expression in Purkinje cell dendrites being especially remarkable in the growth of arborization from postnatal day 3 to day 20. At the mature stage, the reaction was strong in the dendritic tree but very weak in the proximal dendrites and cell bodies.     

JNK phosphorylates Ser332 of doublecortin and regulates its function in neurite extension and neuronal migration

Doublecortin (DCX) is expressed in young neurons and functions as a microtubule‐associated protein. DCX is essential for neuronal migration because humans with mutations in the DCX gene exhibit cortical lamination defects known as lissencephaly in males and subcortical laminar heterotopia (or double cortex syndrome) in females. Phosphorylation of DCX alters its affinity for tubulin and may modulate neurite extension and neuronal migra tion. Previous in vitro phosphorylation experiments revealed that cyclin‐dependent kinase 5 (Cdk5) phosphorylates multiple sites of DCX, including Ser332, (S332). However, phosphorylation at only Ser297 has been shown in vivo. In the present study, we examined phosphorylation of S332 of DCX in the Cdk5?/? mouse brain and results found, unexpectedly, indicate an increased DCX phosphorylation at S332. We found that JNK, not Cdk5, phosphorylates DCX at S332 in vivo. To examine the physiological significance of S332 phosphorylation of DCX in neuronal cells, we transfected cells with either GFP, GFP‐DCX‐WT, or GFP‐DCX‐S332A and analyzed neurite extension and migration. Introduction of GFP‐DCX‐WT enhanced neurite extension and migration. These effects of DCX introduction were suppressed when we used GFP‐DCX‐S332A. Treatment of neurons with JNK inhibitor increased the amount of DCX that bound to tubulin. Interestingly, amount of DCX that bound to tubulin decreased in Cdk5?/? brain homogenates, which indicates that phosphorylation of DCX by JNK is critical for the regulation of DCX binding to tubulin. These results suggest the physiological importance of phosphorylation of DCX for its function. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 929–942, 2010     

2-Deoxyglucose Incorporation in the Cerebellum of Weaver and Nervous Mutant Mice

Abstract: The 2-deoxyglucose autoradiographic method has been used to study activity in cerebellum of the weaver and nervous mutant mice. Patterns of 2-deoxyglucose incorporation into the cerebral hemispheres from weaver and nervous strains did not differ significantly from those of the controls. In the normal cerebellum, 2-deoxyglucose incorporation was maximal in the granular layer, where mossy fibers form synapses with the dendrites of granule cells. In the cerebellum of nervous mice, which lacks Purkinje cells, the incorporation of the 2-deoxyglucose was maximal in the granular layer, but the incorporation into the molecular layer appeared less than in the control. The incorporation into the cerebellum from weaver, which lacks granule cells, was much higher than that of the control, the maximal incorporation being found in the Purkinje cell layer and in cell masses located in the white matter. These data suggest that the heterologous synapses that mossy fibers or climbing fibers form with the cells in the Purkinje cell layer and the cells in the white matter in the weaver cerebellum are functional.