Glomerular visceral epithelial cells (podocytes) contain interdigitated processes that form specialized intercellular junctions, termed slit diaphragms, which provide a selective filtration barrier in the renal glomerulus. Analyses of disease-causing mutations in familial nephrotic syndromes and targeted mutagenesis in mice have revealed critical roles of several proteins in the assembly of slit diaphragms. The nephrin–podocin complex is the main constituent of slit diaphragms. However, the molecular mechanisms regulating these proteins to maintain the slit diaphragms are still largely unknown. Here, we demonstrate that the PAR3–atypical protein kinase C (aPKC)–PAR6β cell polarity proteins co-localize to the slit diaphragms with nephrin. Furthermore, selective depletion of aPKCλ in mouse podocytes results in the disassembly of slit diaphragms, a disturbance in apico-basal cell polarity, and focal segmental glomerulosclerosis (FSGS). The aPKC–PAR3 complex associates with the nephrin–podocin complex in podocytes through direct interaction between PAR3 and nephrin, and the kinase activity of aPKC is required for the appropriate distribution of nephrin and podocin in podocytes. These observations not only establish a critical function of the polarity proteins in the maintenance of slit diaphragms, but also imply their potential involvement in renal failure in FSGS.
Diplonemid mitochondria are considered to have very eccentric structural genes. Coding regions of individual diplonemid mitochondrial genes are fragmented into small pieces and found on different circular DNAs. Short RNAs transcribed from each DNA molecule mature through a unique RNA maturation process involving assembly and three types of RNA editing (i.e., U insertion and A-to-I and C-to-U substitutions), although the molecular mechanism(s) of RNA maturation and the evolutionary history of these eccentric structural genes still remain to be understood. Since the gene fragmentation pattern is generally conserved among the diplonemid species studied to date, it was considered that their structural complexity has plateaued and further gene fragmentation could not occur. Here, we show the mitochondrial gene structure of Hemistasia phaeocysticola, which was recently identified as a member of a novel lineage in diplonemids, by comparison of the mitochondrial DNA sequences with cDNA sequences synthesized from mature mRNA. The genes of H. phaeocysticola are fragmented much more finely than those of other diplonemids studied to date. Furthermore, in addition to all known types of RNA editing, it is suggested that a novel processing step (i.e., secondary RNA insertion) is involved in the RNA maturation in the mitochondria of H. phaeocysticola. Our findings demonstrate the tremendous plasticity of mitochondrial gene structures.
Survival of deep-sea Calyptogena clams depends on organic carbon produced by symbiotic, sulfur-oxidizing, autotrophic bacteria present in the epithelial cells of the gill. To understand the mechanism underlying this symbiosis, the development of a long-term cultivation system is essential. We cultivated specimens of Calyptogena okutanii in an artificial chemosynthetic aquarium with a hydrogen sulfide (H2S) supply system provided by the sulfate reduction of dog food buried in the sediment. We studied morphological and histochemical changes in the clams' gills by immunohistochemical and energy-dispersive X-ray analyses. The freshly collected clams contained a high amount of elemental sulfur in the gill epithelial cells, as well as densely packed symbiotic bacteria. Neither elemental sulfur nor symbiotic bacteria was detected in any other organs except the ovaries, where symbiotic bacteria, but not sulfur, was detected. The longest survival of an individual clam in this aquarium was 151 days. In the 3 clams dissected on Days 57 and 91 of the experiment, no elemental sulfur was detected in the gills. The symbiotic bacteria content had significantly decreased by Day 57, and was absent by Day 91. For comparison, we also studied the deep-sea mussel Bathymodiolus septemdierum, which harbors a phylogenetically close, sulfur-oxidizing, symbiotic bacterium with similar sulfur oxidation pathways. Sulfur particles were not detected, even in the gills of the freshly collected mussels. We discuss the importance of the proportion of available H2S and oxygen to the bivalves for elemental sulfur accumulation. Storage of nontoxic elemental sulfur, an energy source, seems to be an adaptive strategy of C. okutanii.
The deep-sea clam Calyptogena okutanii possesses a large gill containing vertically transmitted symbiotic sulfur-oxidizing bacteria. It produces large amounts of highly viscoelastic mucus from the gill, which is thought to be a physical and chemical barrier. The mucus collected from the gill was shown to be composed of glycoproteins having the following sugar composition: Man (17.4%), GlcNAc (16.6%), GalNAc (15%), Glc (1.1%), Gal (29.9%), Xyl (3.0%), Fuc (14.4%), and unknown (2.6%), indicating that it contained mucin-like glycoproteins. In a monoclonal antibody library against the gill tissue, we found a monoclonal antibody (mAb), CokG-B3C10, reacting to the mucus. Western blot analysis using the mAb showed that it reacted to several glycoproteins in the mucus from the gill tissue, but not with those of other tissues such as the mantle, foot, and ovary, where mucus production has been reported in bivalves. Further, immunohistochemical analysis showed the CokG-B3C10 mAb reacting to glycoproteins was detected in the inner area of the gill, which was occupied by many bacteriocytes in the row of gill filaments. Strong mAb signals were found on the outer surface of the bacteriocytes facing the interfilamental space, and in the interfilamental spaces between filaments. Weaker signals were also observed in the bacteriocyte cells. These results indicate that the CokG-B3C10 mAbbinding mucus glycoproteins were produced from cells including bacteriocytes and nonbacteriocyte cells in the inner area of the gill filaments.
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