Loss of γ-tubulin, GCP-WD/NEDD1 and CDK5RAP2 from the Centrosome of Neurons in Developing Mouse Cerebral and Cerebellar Cortex.

It has been recently reported that the centrosome of neurons does not have microtubule nucleating activity. Microtubule nucleation requires γ-tubulin as well as its recruiting proteins, GCP-WD/NEDD1 and CDK5RAP2 that anchor γ-tubulin to the centrosome. Change in the localization of these proteins during in vivo development of brain, however, has not been well examined. In this study we investigate the localization of γ-tubulin, GCP-WD and CDK5RAP2 in developing cerebral and cerebellar cortex with immunofluorescence. We found that γ-tubulin and its recruiting proteins were localized at centrosomes of immature neurons, while they were lost at centrosomes in mature neurons. This indicated that the loss of microtubule nucleating activity at the centrosome of neurons is due to the loss of γ-tubulin-recruiting proteins from the centrosome. RT-PCR analysis revealed that these proteins are still expressed after birth, suggesting that they have a role in microtubule generation in cell body and dendrites of mature neurons. Microtubule regrowth experiments on cultured mature neurons showed that microtubules are nucleated not at the centrosome but within dendrites. These data indicated the translocation of microtubule-organizing activity from the centrosome to dendrites during maturation of neurons, which would explain the mixed polarity of microtubules in dendrites.

Redox-dependent structural ambivalence of the cytoplasmic domain in the inner ear-specific cadherin 23 isoform.

Cadherin 23 (Cdh23), an essential factor in inner ear mechano-electric transduction, exists in two alternatively spliced forms, Cdh23(+68) and Cdh23(-68), depending on the presence and absence of exon 68. Cdh23(+68) is inner ear-specific. The exon 68-corresponding region confers an alpha-helical configuration upon the cytoplasmic domain (Cy) and includes a cysteine residue, Cys(3240). We demonstrate here that Cy(+68) as well as the transmembrane (TM) plus Cy(+68) region is present in two different forms in transfected cells, reduced and non-reduced, the latter existing in more compact configuration than the former. The observed characteristic of Cy(+68) was completely abolished by Cys(3240)Ala substitution. Treatment of TMCy(+68)-transfected cells with diethyl maleate, a glutathione depleting reagent, resulted in conversion of the non-reduced to the reduced form of TMCy(+68), suggesting glutathione to be a Cys(3240)-binding partner. Multiple alignment of mammalian Cdh23Cy sequences indicated the occurrence of conformation-inducible Cys in Cdh23Cy of mammals, but not lower vertebrates. The implications of Cys-dependent structural ambivalence of Cdh23 in inner ear mechanosensation are discussed.

Fates of Cdh23/CDH23 with mutations affecting the cytoplasmic region.

BUS/Idr mice carrying a mutant waltzer allele (vbus) are characterized by splayed hair bundles in inner ear sensory cells, providing a mouse homolog of USH1D/DFNB12. RT-PCR-based screening for the presence of mutations in mouse Cdh23, the gene responsible for the waltzer phenotype, has identified a G>A mutation in the donor splice site of intron 67 (Cdh23:c.9633+1G>A: GenBank AF308939.1), indicating that two altered Cdh23 molecules having intron-derived COOH-terminal structures could be generated in BUS mouse tissues. Immunochemical analyses with anti-Cdh23 antibodies showed, however, no clear Cdh23-related proteins in vbus/vbus tissues, while the antibodies immunoreacted with approximately 350 kDa proteins in control mice. Immunofluorescent experiments revealed considerable weakening of Cdh23 signals in sensory hair cell stereocilia and Reissner's membrane in the vbus/vbus inner ear, and transmission electron microscopy demonstrated abundant autophagosome/autolysosome vesicles, suggesting aberrant Cdh23:c.9633+1G>A-derived protein-induced acceleration of lysosomal bulk degradation of proteins. In transfection experiments, signal sequence-preceded FLAG-tagged transmembrane plus cytoplasmic regions (TMCy) of tissue-specific Cdh23(+/-68) isoforms were localized to filamentous actin-rich protrusions and the plasma membrane of cultured cells, whereas FLAG-TMCy:c.9633+1G>A proteins were highly insoluble and retained in the cytoplasm. In contrast, FLAG-tagged TMCy:p.Arg3175His and human TMCy:c.9625_9626insC forms were both localized to the plasma membrane in cultured cells, allowing prediction that USH1D-associated CDH23:p.Arg3175His and CDH23:c.9625_9626insC proteins could be transported to the plasma membrane in vivo. The present results thus suggest different fates of CDH23/Cdh23 with mutations affecting the cytoplasmic region.

Two-step consumption of yolk granules during the development of quail embryos.

The mechanism of yolk consumption was studied morphologically and biochemically in Japanese quail Coturnix japonica. The amount of yolk granules in the yolk (or 'yolk cell') decreased in two steps during embryonic development. In the first step, during days 0-4 of incubation, the yolk-granule weight decreased at a rate of 13 mg/day. This decrease was due to segregation by endodermal cells that were newly formed in the developing yolk sac. In the second step after day 6, the decrease was drastic at a rate of 29.8 mg/day during days 6-12 and very slow thereafter. The decrease at the second step was due to the enzymatic digestion of yolk granules by cathepsin D that coexisted in yolk spheres. This digesting reaction was triggered by the solubilization of the granules with high concentrations of salts that were supplied after disruption of the limiting membrane of yolk spheres. The 'yolk cell' seemed to die around day 5 of incubation. Thus the digestion products might be taken up together with yolk lipids by endocytosis into the endodermal cells and transported to blood vessels.

Vitellogenin transport and yolk formation in the quail ovary.

Morphological and biochemical investigations were made on the yolk formation in ovaries of the quail Coturnix japonica. Morphologically, two ways of nutrient uptake were observed in follicles. In small oocytes of white follicles, vitellogenin (VTG) was taken up through fluid-phase endocytosis which was assisted by follicular lining bodies. The lining bodies were produced in follicle cells. They adhered to the lateral cell membrane, moved along the membrane in the direction of the enclosed oocyte and were posted to the tips of the microvilli. These tips, now with lining bodies, were pinched off from the main cell body, engulfed by indented cell membranes of the oocyte, and transported to yolk spheres. In large oocytes of yellow follicles, VTG and very-low-density lipoproteins (VLDL) were taken up through receptor-mediated endocytosis. The VTG and VLDL particles diffused through the huge interspaces between follicle cells, and once in oocytes were transported to yolk spheres via coated vesicles. Immunohistochemistry showed that the VTG resides on or near the surface of the follicle cell membrane at the zona radiata whereas the cathepsin D resides at or near the oocytic cell membranes. Tubular and round vesicles in the cortical cytoplasm of oocytes were also stained with both antisera, suggesting that these vesicles are the sites where the VTG is enzymatically processed by cathepsin D. Upon analysis by SDS-PAGE, a profile similar to that of yolk-granule proteins was produced by incubating VTG with a quail cathepsin D of 40 kD.

Possible involvement of myosin-X in intercellular adhesion: importance of serial pleckstrin homology regions for intracellular localization.

Subcellular fractionation experiments with mouse hepatocytes, combined with sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)-immunoblot analysis using antibodies against two different tail regions of mouse myosin-X demonstrated a 240 kDa molecular mass to be associated with the plasma membrane-rich P2 fraction. The basolateral plasma membrane fraction, but not the brush border fraction, isolated from renal cortices also contained the 240 kDa form of myosin-X. In an attempt to assess relative contributions of possible functional domains in the tail of myosin-X to localization and function, cDNA corresponding to all three pleckstrin homology (PH) domains and different regions (PH1, 2 and 3, and the two subdomains of PH1: PHS1 and PHS2), as well as the myosin tail homology 4 domain (MyTH4) and the band4.1/ezrin/radixin/moesin-like domain (FERM) were separately inserted into the pEGFP vector and expressed in cultured COS-1 cells. As a result, two distinct regions responsible for localization were identified with regard to PH: one covers all three forms that tends to localize to regions of dynamic actin, such as membrane ruffles, lamellipodia and thick cortical actin bundles at the sites of cell-cell adhesion in a Rac- and Cdc42-dependent manner. The other covers PHS1 and PH2 that localizes to filopodia, filopodial puncta and the sites of intercellular adhesion in a Cdc42-dependent manner. Expression of green fluorescent protein (GFP)-MyTH4 fusion protein resulted in formation of phalloidin-positive granules, while GFP-FERM affected the actin cytoskeletal system in a distinctly different way. Taken altogether, the results lend support to the view that myosin-X is involved in cell-cell adhesion-associated signaling-linked membrane and/or cytoskeleton reorganization.

Localization of two conserved cis -acting enhancer regions for the filensin gene promoter that direct lens-specific expression.

Filensin is a unique eye lens intermediate filament protein, and a major component of 'beaded filament' cytoskeletal network. Expression is restricted to lens fiber cells and is believed to be functionally important for lens development by maintaining lens fiber cell shape conformation and lens transparency. The mouse filensin gene promoter core was identified by the promoter activity analysis and found to cover the region from -56 to +4. A reporter vector driven by the filensin promoter core was constructed and DNA fragments derived from the 5'-flanking regions of the gene were cloned into the vector to identify putative cis -acting promoter enhancing activities. The activity of these constructs was monitored by transfection into the chicken embryonic lens cell culture system. Two fragments were identified with cis -acting enhancer activity that re-conferred the lens fiber cell specific expression of the promoter. The first was a 1.7-kbp region located 7.5 kbp upstream from the transcriptional start point while a second 2.1-kbp region was found adjacent to the promoter core. Both fragments specifically directed the lens cell specific expression of only the filensin promoter and not other heterologous promoters. Comparison of 10 kbp of human and mouse 5'-upstream sequences of the filensin promoter revealed two highly conserved sequences that corresponded in their spacing to the putative enhancer regions. These studies have identified the first conserved regulatory sequences important in the lens specific regulation of the filensin gene.

Salt concentration-dependency of vitellogenin processing by cathepsin D in Xenopus laevis.

The mechanism by which cathepsin D produces only limited proteolysis of vitellogenins (VTG) was studied in Xenopus oocytes. We first examined mature oocytes for the existence of cathepsin D; immunoblot and biochemical analyses revealed the existence of a 43kDa enzyme protein and its proteolytic activity in oocytes during and after the vitellogenesis. By determining the proteolytic activity of the fractions after subcellular fractionation of oocytes, we confirmed that cathepsin D is preserved in the yolk plasma of mature yolk platelets. The reaction of VTG with cathepsin D was examined in vitro at pH 5.6 as a function of NaCl concentrations. Lipovitellins generated from the VTG were preserved for several days at 37°C in the presence of the enzyme if the NaCl concentration was 0.15 mol/L or lower. The amount of lipovitellins decreased with increased molarity of the salt and at 0.5 mol/L NaCl they were rapidly degraded. The precipitates, growing in the reaction tube with 0.15 mol/L NaCl, included all constituents of yolk proteins and were ultrastructurally shown to have crystal structures perforated by empty cavities. No precipitates appeared at 0.5 mol/L NaCl. The results indicate that the limitation on proteolysis of the VTG by cathepsin D is due to the insolubility of yolk proteins at physiological salt concentrations, which explains why yolk can be stored stably in the presence of acid hydrolases over a long period.

Cathepsin D Activity in the Vitellogenesis of Xenopus laevis: (Xenopus/oocyte/vitellogenin cleavage/cathepsin D/immunohistochemisty).

An ovarian extract of Xenopus laevis exhibited in SDS-PAGE analyses an activity cleaving vitellogenin to lipovitellins under mildly acidic conditions. This activity was pepstatin-sensitive and inhibited by monospecific anti-rat liver cathepsin D antibody and thus identified as cathepsin D. Immunoblot analysis showed that two proteins of 43 kDa and 36 kDa immunoreacted with the antibody. Immunocytochemical staining revealed that the enzyme was located in the cortical cytoplasm of stage I and II oocytes and in small yolk platelets and nascent forms of large yolk platelets in the cortical cytoplasm of stage III oocytes. In stage IV and V oocytes, small yolk platelets retained the immuno-staining but large yolk platelets decreased it. No immuno-positive signals were observed in oocytes at stage VI. When examined by immunoelectron microscopy, gold particles indicated that cathepsin D was located on dense lamellar bodies in the cortical cytoplasm of stage I and II oocytes. The particles were located on primordial yolk platelets and on the superficial layer of small yolk platelets in stage III oocytes, while they were sparse or not present at all on large yolk platelets in stage IV and V oocytes. These results indicate that cathepsin D plays a key role in vitellogenesis by cleaving endocytosed vitellogenin to yolk proteins in developing oocytes.

Changes with Development in the Expression of Cathepsin E in the Fetal Rat Stomach: (cathepsin E/gastric gland/cell differentiation/functional roles).

The changes with development in the expression of cathepsin E in the fetal rat stomach were examined immunochemically and immunohistochemically. The activity of acid proteinase in fetal gastric extracts increased dramatically during late gestational stages, rising from 0.017 units per mg of protein on day 15 of gestation to 0.591 units per mg of protein on day 21 of gestation. Electrophoretic analysis, combined with immunological tests, showed that the increase was due exclusively to increases in the activity of the monomeric and dimeric forms of cathepsin E, while SDS-PAGE-immunoblot analysis revealed that both forms are present as a 43-kDa proenzyme. Immunohistochemically, cathepsin E was localized in the cytoplasm of all proliferating epithelial cells of pars glandularis on day 16 of gestation or later. As revealed by conventional histological methods, surface mucous cells and parietal cells appeared for the first time in specimens on day 19 of gestation, and all of these cells were immunopositive for cathepsin E. The present study further indicated that cathepsin E is the predominant aspartic proteinase in the stomach of young rats, until pepsinogen C appears. Based on these results, possible roles of gastric cathepsin E are discussed.