O2-filled swimbladder employs monocarboxylate transporters for the generation of O2 by lactate-induced root effect hemoglobin.

The swimbladder volume is regulated by O(2) transfer between the luminal space and the blood In the swimbladder, lactic acid generation by anaerobic glycolysis in the gas gland epithelial cells and its recycling through the rete mirabile bundles of countercurrent capillaries are essential for local blood acidification and oxygen liberation from hemoglobin by the "Root effect." While O(2) generation is critical for fish flotation, the molecular mechanism of the secretion and recycling of lactic acid in this critical process is not clear. To clarify molecules that are involved in the blood acidification and visualize the route of lactic acid movement, we analyzed the expression of 17 members of the H(+)/monocarboxylate transporter (MCT) family in the fugu genome and found that only MCT1b and MCT4b are highly expressed in the fugu swimbladder. Electrophysiological analyses demonstrated that MCT1b is a high-affinity lactate transporter whereas MCT4b is a low-affinity/high-conductance lactate transporter. Immunohistochemistry demonstrated that (i) MCT4b expresses in gas gland cells together with the glycolytic enzyme GAPDH at high level and mediate lactic acid secretion by gas gland cells, and (ii) MCT1b expresses in arterial, but not venous, capillary endothelial cells in rete mirabile and mediates recycling of lactic acid in the rete mirabile by solute-specific transcellular transport. These results clarified the mechanism of the blood acidification in the swimbladder by spatially organized two lactic acid transporters MCT4b and MCT1b.

Histological demonstration of glucose transporters, fructose-1,6-bisphosphatase, and glycogen in gas gland cells of the swimbladder: is a metabolic futile cycle operating?

Luminal surface of the swimbladder is covered by gas gland epithelial cells and is responsible for inflating the swimbladder by generating O(2) from Root-effect hemoglobin that releases O(2) under acidic conditions. Acidification of blood is achieved by lactic acid secreted from gas gland cells, which are poor in mitochondria but rich in the glycolytic activity. The acidic conditions are locally maintained by a countercurrent capillary system called rete mirabile. To understand the regulation of anaerobic metabolism of glucose in the gas gland cells, we analyzed the glucose transporter expressed there and the fate of ATP generated by glycolysis. The latter is important because the ATP should be immediately consumed otherwise it strongly inhibits the glycolysis rendering the cells unable to produce lactic acid anymore. Expression analyses of glucose transporter (glut) genes in the swimbladder of fugu (Takifugu rubripes) by RT-PCR and in situ hybridization demonstrated that glut1a and glut6 are expressed in gas gland cells. Immunohistochemical analyses of metabolic enzymes demonstrated that a gluconeogenesis enzyme fructose-1,6-bisphosphatase (Fbp1) and a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Gapdh) are highly expressed in gas gland cells. The simultaneous catalyses of glycolysis and gluconeogenesis reactions suggest the presence of a futile cycle in gas gland cells to maintain the levels of ATP low and to generate heat that helps reduce the solubility of O(2).

Mechanism of development of ionocytes rich in vacuolar-type H(+)-ATPase in the skin of zebrafish larvae.

Mitochondrion-rich cells (MRCs), or ionocytes, play a central role in aquatic species, maintaining body fluid ionic homeostasis by actively taking up or excreting ions. Since their first description in 1932 in eel gills, extensive morphological and physiological analyses have yielded important insights into ionocyte structure and function, but understanding the developmental pathway specifying these cells remains an ongoing challenge. We previously succeeded in identifying a key transcription factor, Foxi3a, in zebrafish larvae by database mining. In the present study, we analyzed a zebrafish mutant, quadro (quo), deficient in foxi1 gene expression and found that foxi1 is essential for development of an MRC subpopulation rich in vacuolar-type H(+)-ATPase (vH-MRC). foxi1 acts upstream of Delta-Notch signaling that determines sporadic distribution of vH-MRC and regulates foxi3a expression. Through gain- and loss-of-function assays and cell transplantation experiments, we further clarified that (1) the expression level of foxi3a is maintained by a positive feedback loop between foxi3a and its downstream gene gcm2 and (2) Foxi3a functions cell-autonomously in the specification of vH-MRC. These observations provide a better understanding of the differentiation and distribution of the vH-MRC subtype.

Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains.

Deubiquitinating enzymes (DUBs) remove ubiquitin from conjugated substrates to regulate various cellular processes. The Zn(2+)-dependent DUBs AMSH and AMSH-LP regulate receptor trafficking by specifically cleaving Lys 63-linked polyubiquitin chains from internalized receptors. Here we report the crystal structures of the human AMSH-LP DUB domain alone and in complex with a Lys 63-linked di-ubiquitin at 1.2 A and 1.6 A resolutions, respectively. The AMSH-LP DUB domain consists of a Zn(2+)-coordinating catalytic core and two characteristic insertions, Ins-1 and Ins-2. The distal ubiquitin interacts with Ins-1 and the core, whereas the proximal ubiquitin interacts with Ins-2 and the core. The core and Ins-1 form a catalytic groove that accommodates the Lys 63 side chain of the proximal ubiquitin and the isopeptide-linked carboxy-terminal tail of the distal ubiquitin. This is the first reported structure of a DUB in complex with an isopeptide-linked ubiquitin chain, which reveals the mechanism for Lys 63-linkage-specific deubiquitination by AMSH family members.

MBD3 and HDAC1, two components of the NuRD complex, are localized at Aurora-A-positive centrosomes in M phase.

MBD3, a component of the histone deacetylase NuRD complex, contains the methyl-CpG-binding domain (MBD), yet does not possess appreciable mCpG-specific binding activity. The functional significance of MBD3 in the NuRD complex remains enigmatic, partly because of the limited availability of biochemical approaches, such as immunoprecipitation, to analyze MBD3. In this study, we stably expressed the FLAG-tagged version of MBD3 in HeLa cells. We found that MBD3-FLAG was incorporated into the NuRD complex, and the MBD3-FLAG-containing NuRD complex was efficiently immunoprecipitated by anti-FLAG antibodies. By exploiting this system, we found that MBD3 is phosphorylated in vivo in the late G(2) and early M phases. Moreover, we found that Aurora-A, a serine/threonine kinase active specifically in the late G(2) and early M phases, phosphorylates MBD3 in vitro, physically associates with MBD3 in vivo, and co-localizes with MBD3 at the centrosomes in the early M phase. Interestingly, HDAC1 is distributed at the centrosomes in a manner similar to MBD3. These results suggest the highly dynamic nature of the temporal and spatial distributions, as well as the biochemical modification, of the NuRD complex in M phase, probably through an interaction with kinases, including Aurora-A. These observations will contribute significantly to the elucidation of the yet-uncharacterized cell cycle-controlled functions of the NuRD complex.

Localization of inward rectifier potassium channel Kir7.1 in the basolateral membrane of distal nephron and collecting duct.

Inward rectifier potassium channels (Kir) play an important role in the K(+) secretion from the kidney. Recently, a new subfamily of Kir, Kir7.1, has been cloned and shown to be present in the kidney as well as in the brain, choroid plexus, thyroid, and intestine. Its cellular and subcellular localization was examined along the renal tubule. Western blot from the kidney cortex showed a single band for Kir7.1 at 52 kD, which was also observed in microdissected segments from the thick ascending limb of Henle, distal convoluted tubule (DCT), connecting tubule, and cortical and medullary collecting ducts. Kir7.1 immunoreactivity was detected predominantly in the DCT, connecting tubule, and cortical collecting duct, with lesser expression in the thick ascending limb of Henle and in the medullary collecting duct. Kir7.1 was detected by electron microscopic immunocytochemistry on the basolateral membrane of the DCT and the principal cells of cortical collecting duct, but neither type A nor type B intercalated cells were stained. The message levels and immunoreactivity were decreased under low-K diet and reversed by low-K diet supplemented with 4% KCl. By the double-labeling immunogold method, both Kir7.1 and Na(+), K(+)-ATPase were independently located on the basolateral membrane. In conclusion, the novel Kir7.1 potassium channel is located predominantly in the basolateral membrane of the distal nephron and collecting duct where it could function together with Na(+), K(+)-ATPase and contribute to cell ion homeostasis and tubular K(+) secretion.