The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons

G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation.     

Punctin, a novel ADAMTS-like molecule, ADAMTSL-1, in extracellular matrix.

Punctin (ADAMTSL-1) is a secreted molecule resembling members of the ADAMTS family of proteases. Punctin lacks the pro-metalloprotease and the disintegrin-like domain typical of this family but contains other ADAMTS domains in precise order including four thrombospondin type I repeats. Punctin is the product of a distinct gene on human chromosome 9p21-22 and mouse chromosome 4 that is expressed in adult skeletal muscle. His-tagged punctin expressed in stably transfected High-Five(TM) insect cells was purified to apparent homogeneity by Ni-chromatography of conditioned medium. The NH(2) terminus is not blocked and has the sequence EEDRD and so forth as determined by Edman degradation, demonstrating signal peptidase processing. Recombinant epitope-tagged punctin has a calculated mass of 59,991 Da but exhibits major molecular species of 61970 +/- 6 Da and 62131 +/- 5 Da as measured by liquid chromatography electrospray mass spectrometry. Punctin is a glycoprotein based on carbohydrate staining and liquid chromatography electrospray mass spectrometry glycopeptide analysis. Glycosylation occurs at a single N-linked site as demonstrated by altered electrophoretic migration of punctin expressed in the presence of tunicamycin A. Punctin contains disulfide bonds based on antibody accessibility and electrophoretic migration under reducing versus nonreducing conditions. Rotary shadowing demonstrates that punctin is hatchet-shaped having a globular region attached to a short stem. In transfected COS-1 cells, punctin is deposited in the cell substratum in a punctate fashion and is excluded from focal contacts. Punctin is the first member of a novel family of ADAMTS-like proteins that may have important functions in the extracellular matrix.     

Immunohistochemical evidence for the existence of rat cytosolic sialidase in rat skeletal muscles

 Histochemical evidence is required to demonstrate the presence of biochemically defined cytosolic sialidase. To meet this requirement, we examined the immunohistochemical localization of the enzyme in rat skeletal muscles. Sections of chemically fixed tissues were incubated with a polyclonal antibody raised against a synthetic peptide which corresponded to a part of the enzyme protein. After incubation with the primary antibody, cryosections for fluorescence microscopy and resin sections for electron microscopy were incubated with a fluorochrome- and colloidal gold-labeled secondary antibody, respectively. Immunofluorescence was diffusely distributed in the muscle fibers and was also found in the perimysium and blood vessels. Many immunogold particles were scattered over the sarcoplasm, myofibrils, nucleoplasm, and matrix of mitochondria. The immunogold particles were also found in the equivalent compartments of axons, Schwann cells, and cells of endomysium and blood vessels. The specificity of the primary antibody was elucidated by immunoblotting and an immunoprecipitation test. These findings clearly indicate that this type of sialidase is essentially located in the cytosolic compartment. Consequently, the name, cytosolic sialidase, will be appropriate for this enzyme. Additionally it is indicated that this enzyme is also present in cells other than skeletal muscle fibers. Accepted: 29 January 1997     

A histological comparison of the development of pollen and female gametophytes in fertile and sterile Cryptomeria japonica

To determine a possible mechanism causing male and female sterility in Cryptomeria japonica male and female cones were collected from a C. japonica, tree, ShinDai2, that lacks pollen release and fertile seeds and specimens were processed to examine the development of pollen and female gametophytes using light microscopy and field emission scanning electron microscopy. Pre-meiotic development proceeded normally, but the formation of aberrant meiotic products was observed in cones of both sexes. In sterile microsporangia, heterogeneous microspore populations ranging from monads to polyads gave rise to mature pollen grains of non-uniform size. These pollen grains were covered with an amorphous layer and adhered to each other. In addition, they remained in the microsporangia and were not released even after the onset of pollen dissemination from fertile trees. In the ovules of sterile female cones, megaspores with abnormal shapes, numbers, and sizes formed, and the development of female gametophytes was arrested at the free nuclear or archegonium formation stages. These gametophytes collapsed, and no fertile embryo was generated. Results indicate that meiotic defects are important in the sterility mechanism.     

Role of a putative third subunit YhcB on the assembly and function of cytochrome bd-type ubiquinol oxidase from Escherichia coli

Recent proteome studies on the Escherichia coli membrane proteins suggested that YhcB is a putative third subunit of cytochrome bd-type ubiquinol oxidase (CydAB) (F. Stenberg, P. Chovanec, S.L. Maslen, C.V. Robinson, L.L. Ilag, G. von Heijne, D.O. Daley, Protein complexes of the Escherichia coli cell envelope. J. Biol. Chem. 280 (2005) 34409-34419). We isolated and characterized cytochrome bd from the ΔyhcB strain, and found that the formation of the CydAB heterodimer, the spectroscopic properties of bound hemes, and kinetic parameters for the ubiquinol-1 oxidation were identical to those of cytochrome bd from the wild-type strain. Anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis showed that YhcB was not associated with the cytochrome bd complex. We concluded that YhcB is dispensable for the assembly and function of cytochrome bd. YhcB, which is distributed only in γ-proteobacteria, may be a part of another membrane protein complex or may form a homo multimeric complex.     

Molecular cloning and biochemical characterization of two novel Neu3 sialidases, neu3a and neu3b, from medaka (Oryzias latipes)

Mammalian Neu3 sialidases are involved in various biological processes, such as cell death and differentiation, through desialylation of gangliosides. The enzymatic profile of Neu3 seems to be highly conserved from birds to mammals. In fish, the functional properties of Neu3 sialidase are not clearly understood, with the partial exception of the zebrafish form. To cast further light on the molecular evolution of Neu3 sialidase, we identified the encoding genes in the medaka Oryzias latipes and investigated the properties of the enzyme. PCR amplification using medaka brain cDNA allowed identification of two novel medaka Neu3 genes, neu3a and neu3b. The YRIP, VGPG motif and Asp-Box, characteristic of consensus motifs of sialidases, were well conserved in the both medaka Neu3 sialidases. When each gene was transfected into HEK293 to allow cell lysates for the use of enzymatic characterization, two Neu3 sialidases showed strict substrate specificity toward gangliosides, similar to mammalian Neu3. The optimal pH values were at pH 4.2 and pH 4.0, respectively, and neu3b in particular showed a broad optimum. Immunofluorescence assays indicated neu3a localization at plasma membranes, while neu3b was found in cytosol. The tissue distribution of two genes was then investigated by estimation of mRNA expression and sialidase activity, both being dominantly expressed in the brain. In neu3a gene-transfected neuroblastoma cells, the enzyme was found to positively regulate retinoic acid-induced differentiation with the elongation of axon length. On the other hand, neu3b did not affect neurite formation. These results and phylogenetic analysis suggested that the medaka neu3a is an evolutionally conserved sialidase with regard to enzymatic properties, whereas neu3b is likely to have originally evolved in medaka.