The Evolutionary History of Prosaposin: Two Successive Tandem-Duplication Events Gave Rise to the Four Saposin Domains in Vertebrates

Prosaposin is a multifunctional protein encoded by a single-copy gene. It contains four saposin domains (A, B, C, and D) occurring as tandem repeats connected by linker sequences. Because the saposin domains are similar to one another, it is deduced that they were created by sequential duplications of an ancestral domain. There are two types of evolutionary scenarios that may explain the creation of the four-domain gene: (1) two rounds of tandem internal gene duplication and (2) three rounds of duplications. An evolutionary and phylogenetic analysis of saposin DNA and amino acid sequences from human, mouse, rat, chicken, and zebrafish indicates that the first evolutionary scenario is the most likely. Accordingly, an ancestral saposin-unit duplication produced a two-domain gene, which, subsequently, underwent a second complete tandem duplication to give rise to the present four-domain structure of the prosaposin gene. Received: 8 February 2001 / Accepted: 29 June 2001     

Intracellular distribution and late endosomal effects of the ocular albinism type 1 gene product: consequences of disease-causing mutations and implications for melanosome biogenesis

To investigate the function of ocular albinism type 1 ( OA1 ), the gene responsible for X-linked ocular albinism, we employed a construct containing murine Oa1 fused to green fluorescent protein (GFP) in a heterologous COS cell expression system. The cellular distribution of wild-type (WT) Oa1 protein and Oa1 proteins reflecting mutations causing X-linked ocular albinism were examined. Comparison with different organelle markers revealed that Oa1-GFP localized to the late endolysosomal compartments. Some Oa1 mutant proteins failed to exit the endoplasmic reticulum (ER) (Class I mutants), while other mutants partially (Class II mutants) or fully (Class III mutants) exited the ER and trafficked to endolysosomal compartments. We observed that expression of WT Oa1-GFP in COS cells caused an apparent enlargement of late endosomes and a redistribution of the mannose-6-phosphate receptor (M6PR). None of the mutants displayed the full range of effects on the redistribution of M6PR exhibited by WT Oa1. The effects of Oa1 on late endosome structure and content are thus likely to reflect an important biological property of Oa1. We propose that OA1 is involved in reorganizing the endolysosomal compartment as a necessary step in ocular melanosome biogenesis.     

Ageing of mouse liver lysosomes

Summary Lysosomes in mouse liver parenchymal cells have been marked by intravenous injection of Thorotrast. They were subsequently followed in a time sequence from five hours up to sixteen weeks after injection. At two days after injection the majority of the lysosomes was heavily loaded with marker particles, while endocytosis was no longer observed. From six days after injection Thorotrast was partly accumulated in very large lysosomes (conglomerates) with mean diameters up to 2.5 m. As the time after injection advanced the Thorotrast content of the cells was reduced while most of the remaining marker substance became concentrated in the conglomerates. Many Thorotrast conglomerates were shown to contain acid phosphatase and some of them were able to fuse with functionally younger lysosomes which were marked with colloidal gold. Morphometric analysis showed an increase in the volume density of the dense body population between 0 and 2 days after injection, followed by a decrease between 2 and 11 days. The observed decrease is probably caused by exocytosis of the contents of Thorotrast containing lysosomes in bile capillaries.The author is highly indebted to Prof. Dr. J. James, Histological Laboratory, University of Amsterdam and Prof. Dr. W. Th. Daems, Laboratory for Electron Microscopy, University of Leiden for their stimulating advice and helpful criticism throughout this investigation. Advice on statistical analysis was given by Ir. J. J. Houtkooper, whose kind cooperation is gratefully acknowledged. Many thanks are due to the Application Laboratory of N. V. Philips at Eindhoven, The Netherlands, for facilities to make use of their equipment for X-ray microanalysis.     

A possible role for placental lysosomes in the formation of villous syncytiotrophoblast

Summary An ultrastructural and ultrahistochemical study of first trimester human placentae confirms previous reports that the cytotrophoblastic cells show a spectrum of differentiation, that dissolution of the limiting membrane of the cytotrophoblastic cells occurs and that fragments of free membrane can be found in the syncytiotrophoblast. There is an aggregation of primary lysosomes in the region of approximation of the cytotrophoblast to the syncytiotrophoblast, free lysosomal enzymes are found in the space between the two trophoblastic components, secondary lysosomes have been noted in the vicinity of fragmenting cytotrophoblastic cell membrane and the incorporation of a segment of free membrane into a vesicular structure has been noted. It is suggested that placental lysosomes mediate the dissolution of the cytotrophoblastic cell membranes that is a necessary prerequisite for their full differentiation into syncytiotrophoblast and it is further suggested that one of the principal roles of placental lysosomes is in the structural refashioning of the organ that occurs during the first trimester.     

Differential fixation of poly(L-arginine) and poly(L-lysine) by tannic acid and its application to the fixation of collagen in electron microscopy

A differential fixation of poly(L-arginine) and poly(L-lysine) has been demonstrated by means of cellulose acetate electrophoresis and colorimetric titration. Electrophoresis showed that at pH 3.0 and concentrations between 0.025% and 2% the reagent interacts with poly(L-arginine) but not with poly(L-lysine). at pH 7.5, however, poly(L-lysine) also reacts, although at a higher concentration of tannic acid than was required to fix poly(L-arginine) at this pH. Colorimetric titration revealed that for poly(L-arginine) the reaction with tannic acid commences at pH 3.0 and is complete at pH 4.1 whereas for poly(L-lysine) the reaction commences at pH 3.5 and is complete at pH 4.9. It is suggested that the reaction is predominantly electrostatic. The results are discussed in relation to the use of tannic acid as a protein fixative in electron microscopy.     

Ultrastructure of Chlamydia pneumoniae in cell culture

The electron microscopic appearance of Chlamydia pneumoniae elementary bodies with pear-shaped, loose outer membrane has been suggested as one criterion of its classification as a new chlamydial species. The study of the original strain TW 183 in LCL 929 and HL cells and a low-passage isolate of Kajaani-6 isolate in HL cells revealed spherical compact elementary bodies common to other chlamydia.     

AP-1 and retromer play opposite roles in the trafficking of sortilin between the Golgi apparatus and the lysosomes

Sortilin has been implicated in the sorting of one soluble hydrolase and two sphingolipid activator proteins to the lysosomes. While the GGA adaptor proteins have been demonstrated to play a role in the targeting of sortilin to the endosomes, the recycling of sortilin has not yet been elucidated. Here we examine the role of two adaptor protein complexes, AP-1 and retromer. Our results demonstrate that AP-1 is required for the transport of sortilin to the endosomes and retromer for the recycling of sortilin to the Golgi apparatus. While inhibition of AP-1 causes accumulation of sortilin in the Golgi apparatus, RNAi depletion of retromer results in retention of sortilin in the lysosomes. We also demonstrate that the interaction of sortilin with retromer occurs through a YXXΦ site in its cytosolic tail. In conclusion, our observations indicate that retromer and AP-1 play opposite roles in the trafficking of sortilin.     

Structure of yolk granules in oocytes and eggs of Blattella germanica and their interaction with vitellophages and endosymbiotic bacteria during granule degradation

Oocytes and embryos of the cockroach Blattella germanica were examined by optical and electron microscopy to study yolk granule degradation during embryo development. During vitellogenesis, progressively larger yolk granules are formed in the ooplasm and by chorionogenesis, the mature granules are packed so tightly that their shape is highly distorted. Throughout ovarian development, endosymbiotic bacteria lie at the follicle cell/oocyte interface. Just prior to chorionogenesis the endosymbionts transit the oocyte plasma membrane and cluster at the periphery. Bacteria become more numerous over the ventral region of the egg by day 1 postovulation and begin to invade the interior of the yolk mass from the ventral periphery. At that time, lysis of the nearby yolk granules occurs while those in the central ooplasm remain intact and free of bacteria up to day 4. Vitellophages become evident by day 2 postovulation. These cells are also distributed over the egg's periphery but are most numerous in the ventral region. Vitellophages, in association with the endosymbionts, protrude toward the yolk granules and extend filo- and lamellipodia over the granule surface. Portions of the yolk granules are then engulfed and sequestered as large vacuoles in the vitellophage's cytoplasm. The vacuoles then become vesiculated. As embryo development proceeds, the vesiculated portions partition into smaller multivesicular bodies. This study describes the dynamics of yolk granule-vitellophage interaction in embryos of B. germanica and suggests that yolk utilization entails the cooperative efforts of both vitellophages and endosymbiont bacteria.     

Cell interaction at the maternal-embryonic interface during implantation in the mouse

Summary The interaction between the trophoblast and the maternal epithelium at early implantation was studied by means of light and electron microscopy. The uterine horns were fixed in situ and a double-embedding method was used to locate implantation sites. Observations were made on mice killed at 2 hour intervals 90–116 h. post coitum which covered the following stages: pre-attachment (i) with zona pellucida intact (ii) with zona pellucida in dissolution (iii) after loss of the zona; attachment; adherence; and invasion.The intact zona pellucida was electron opaque and of uniform density. In the stage of apparent dissolution it became electron dense and was trapped between trophoblast and epithelium.At preattachment the trophoblast cells were round. Subsequently they became long and attenuated, often with lysosomes in the cytoplasm proximal to the epithelial layer. Epithelial cells, which could be seen in various stages of degeneration were apparently phagocytosed by the trophoblast. Occasional pyknotic epithelial cells were seen, as well as some apparently normal ones which contained cytosegresomes. The possible reasons for their presence are discussed.The microvilli of the epithelial cells changed from regular and pointed at preattachment to short and irregular at adherence and invasion.Research supported by the Lalor Foundation, Wilmington, Del, U.S.A. to I.B.W.We are grateful to Dr. H. M. Beaumont, and Dr. L. L. Franchi of the Anatomy Department, Birmingham, for helpful discussion and to Mr. J. Wallington for photographic assistance.     

Adenosine triphosphatase activity in the carotid body of the cat

Summary Three kinds of nucleoside phosphatases were demonstrated histochemically in the cat carotid body with nucleoside triphosphate, nucleoside disphosphate and nucleoside monophosphate as substrates. Each of these enzyme activities exhibited the substrate specificity respectively. The nucleoside triphosphatase activity showed specific localization in association with the parenchymal cells of the carotid body.The electronmicroscopy revealed that the reaction product was located on and between the two apposing plasma membranes of type I and type II cells, of a type II cell and its wrapping axons and of the intricate basal infolding of a type II cell itself.Some possible functions of the adenosine triphosphatase in the carotid body are discussed.     

Implementation of alternating excitation schemes in a biochip-reader for quasi-simultaneous multi-color single-molecule detection

We report here the development of a device for single-molecule biochip readout using fast alternating excitation. The technology extends standard imaging cytometry by offering additional color channels in excitation. To enable the study of mobile objects, e.g. actively transported vesicles in living cells or freely diffusing lipids in a lipid bilayer, the frequency of the illumination pulses was chosen high enough to virtually freeze the motion of the biomolecules, as they are shifted through the illuminated area. The synchronization of sample illumination, scanning and line-camera readout yield two quasi-simultaneously recorded images covering the same sample region. Diffraction-limited resolution and high localization precision for point-light sources down to approximately 10 nm was shown by scanning immobilized 100 nm fluorescence latex beads. Ultra-sensitivity was demonstrated by imaging single fluorescent streptavidin molecules diffusing in a fluid lipid bilayer. Two-color streptavidin labeled with Cy3 and Cy5 could be easily identified in the two respective excitation channels; high accordance in the dye positions confirms the applicability for colocalization studies of moving objects. Finally, scans of antibody-receptor interactions in large populations of live cells illustrate the feasibility of this method for biochip application.     

STAM-AMSH interaction facilitates the deubiquitination activity in the C-terminal AMSH

Signal transducing adaptor molecule (STAM) complexed with hepatocyte growth factor regulated tyrosine kinase substrate (Hrs) works on sorting of cargo proteins in multivesicular body (MVB) pathway. Associated molecule with SH3 domain of STAM (AMSH), a zinc-containing ubiquitin isopeptidase, is thought to play a role in regulation of ubiquitin-mediated degradation by binding to STAM. We have found that AMSH requires the conformation of Px(V/I)(D/N)RxxKP sequence to bind SH3 domain of STAM with approximately 7 microM affinity, and that the isolated C-terminal domain of AMSH contains the isopeptidase activity. Deubiquitination by AMSH was assisted when ubiquitins were bound to STAM which can bind to AMSH simultaneously. With the specificity toward K63-linked ubiquitins, this facilitated ubiquitin processing activity of AMSH may imply a distinct regulatory mechanism for sorting and degradation through STAM binding.     

Ultrastructural changes in the rat carotid body in severe haemorrhagia

Summary Rat carotid bodies were studied electron microscopically after short-term severe hypovolaemia, which is known to induce a marked chemoreceptor activation in the carotid body. Altogether 84 nerve-endings in the hypovolaemic rats' carotid bodies and 91 nerve-endings in the control carotid bodies were investigated. An increased accumulation of the glomus cell granular vesicles near the synaptic specializations of the nerve-endings was observed after hypovolaemia. Moreover, a statistically significant increase in the contacts between the nerve-ending synaptic specializations and the glomus cell granular vesicles was observed after hypovolaemia. A suggestion was made that the glomus cells might act as modulating, probably inhibitory, interneurones, whose catecholamines are responsible for the inhibition.The authors are greatly indebted to lecturer Pekka Korkala Ph.L. from the Department of Psychology for his skilful statistical analysis of the results.     

Regions of putative acetylcholine receptors at synaptic contacts between neurons maintained in culture and subsequently fixed in solutions containing tannic acid

Summary Spinal cord neurons from 9-day chick embryos were maintained in culture for up to 35 days and then fixed in 4% cacodylate-buffered glutaraldehyde containing 2% tannic acid. After about 15 days in culture a small percentage of the synaptic specializations present were characterized by striking electron-dense striations averaging 15 nm in width, oriented perpendicular to the postsynaptic membrane. These structures increased in frequency with time in culture (to a maximum of about 10% of all synapses in the oldest cultures); they were asymmetrical, protruding approximately 8 nm into the synaptic cleft, and more deeply (approximately 15–18 nm), into the postsynaptic cytoplasm. On the basis of earlier work by Sealock (1980) they are interpreted as concentrations of acetylcholine receptors.Similar membrane differentiations were also seen associated with active-zone areas of a few presynaptic membranes, and the possibility that these represent presynaptic acetylcholine receptors is discussed. Additional observations reported are (1) the presence of striations resembling those seen at the postsynaptic membrane in the membranes of some postsynaptic vesicles, and (2) filamentous links between the striations and cytoskeletal elements of the postsynaptic cell.     

Cellular localization and interaction of ABCA1 and caveolin-1 in aortic endothelial cells after HDL incubation

The goal of this study was to investigate the cellular localization and the interaction between caveolin-1 and ABCA1 in cholesterol-loaded aortic endothelial cells after HDL incubation. Immunofluorescence confocal microscopy showed that ABCA1 was found primarily on the cell surface, whereas caveolin-1 was revealed on the cell surface and in the cytoplasm. The HDL appeared to colocalize with ABCA1 and caveolin-1 on the cell surface. No free HDL was revealed in the cytoplasm. The HDL was colocalized neither with early endosome marker (CD71) nor with late endosome marker (LAMP2). The chemical cross-linking and immunoprecipitation analysis revealed that ABCA1 binds directly to both HDL and caveolin-1, whereas HDL does not bind directly to caveolin-1. The studies provide evidence for a direct interaction between ABCA1 and HDL, ABCA1 and caveolin-1, but not HDL and caveolin-1, indicating that ABCA1 may act as a structural platform between HDL and caveolin-1 on the cell surface during cellular cholesterol efflux.     

Three-dimensional organization of a transcellular tubulocisternal endoplasmic reticulum in epithelial cells of Reissner's membrane in the guinea-pig

Summary The ultrastructure of the epithelial cells of Reissner's membrane (membrana vestibularis) in the guinea-pig is described following vascular perfusion with glutaraldehyde of live, anaesthetised and artificially respirated animals. Postfixation in a solution containing O_sO₄ and potassium ferricyanide revealed a well-developed tubulocisternal endoplasmic reticulum, not previously described, the continuity of which has been mapped by serial sectioning and reconstruction. Large disc-shaped subsurface cisternae lining the cell membrane, but separated from it by a space approximately 10 nm wide, are in continuity with the smooth endoplasmic reticulum, forming an elaborated transcellular canalicular pathway. This structure is compared to that found in solute-transporting epithelia, e.g., renal proximal tubule, gall bladder, small intestine and choroid plexus. The fixation method used in the present study is compared to other techniques used for preservation of Reissner's membrane. Each epithelial cell of Reissner's membrane is endowed with one kinocilium, one to four multivesicular bodies, and a number of intercalated bodies. The functional significance of the canalicular pathway is discussed.