Pathway and kinetics of vitellogenin-gold internalization in the Xenopus oocyte

After in vitro incubation of Xenopus oocytes with vitellogenin (VTG)-gold conjugate, the gold particles are distributed on the whole plasma membrane. Their concentration in coated pits still occurs at 0 degrees C. At +20 degrees C the label quickly (30 sec) appears in multi-vesicular endosomes (MVE) which segregate together with primary endocytic vesicles into distinct clusters below the plasma membrane. From this step up to crystallization of the yolk platelets, the gold particles stay in the same compartment. During 5.5 h the label progressively increases along the MVE membrane, first (1.5 h) by fusion of primary endocytic vesicles with consecutively enlarging endosomes, then (4 h) by decreasing of the MVE membrane. As concerns the yolk platelet formation, concentration of primordial yolk platelets (PYP) occurs at 5.5 h from the incubation onset, the labeling of preexisting yolk platelets starts at 7 h, while crystallization of PYP begins only after 12-13 h. Our results indicate that VTG receptors are not preclustered in coated pits and their lateral translation is not inhibited at 0 degrees C. The yolk protein processing takes place within one compartment only. The VTG condensation begins with a long concentration phase of receptor-VTG complexes still integrated in the endosome membrane. It occurs in MVE by: i) a repeated fusion of primary endocytic vesicles; ii) removing part of the endosome membrane by internal vesiculation. Fusion between endosomes occurs only after VTG has dissociated from its receptors and VTG dissociates only when when the density of the VTG-receptor complexes in the endosome membrane is sufficient. Crystallization begins after a 7-8 h delay. The endosome migration into the oocyte is also controlled by the binding of VTG to its receptors. Our results also demonstrate that binding of VTG colloidal gold modifies neither the vitellogenic pathway nor the duration of the vitellogenin internalization. However when vitellogenin is bound to colloidal gold, dissociation of ligand-receptor complexes is delayed because the amount of ligand in the incubation medium is necessarily low.     

Ferromagnetic isolation of endosomes: a novel method for subcellular fractionation of Xenopus oocytes

A novel method has been developed using ferric particles to label endosomes, and to achieve magnetic sorting of the various endocytic compartments involved in lipoprotein uptake into cells. Ferric particles conjugated to a receptor-recognized ligand are bound to coated membrane pits and become internalized into the cytoplasm inside coated vesicles. After apparent fusion of the vesicles to tubular endosomes, the conjugates accumulate and finally discharge into multivesicular endosomes. Pulse-chase experiments elucidate the pathway of internalized conjugates and allow both early compartments (pinosomes and tubular endosomes) and late compartments (multivesicular endosomes and storage organelles) to be selectively labelled. After ferroloading of the various transport compartments, the cells are homogenized and subcellularly fractionated. Sorting of labelled endosomes is performed by a specially designed "free-flow" magnetic chamber. Prophase I-arrested oocytes of the toad Xenopus laevis are used as a model system for studying the transport pathway and the conversion of the yolk precursor vitellogenin. It is possible to follow the route of internalization of vitellogenin-iron conjugates via coated pits, coated vesicles, uncoated vesicles, tubular endosomes, multivesicular endosomes, and light primordial yolk platelets. These endosomes shuttle the ferric particles together with the vitellogenin from oolemma to performed heavy yolk organelles which are still growing. In addition, these various compartments can be isolated according to their function and subjected to electron microscopy and to gel electrophoresis for detailed characterization of their limiting membranes as well as their contents.     

Ferromagnetic isolation of endosomes involved in vitellogenin transfer into Xenopus oocytes

The transport pathway of the yolk precursor vitellogenin (VTG) has been followed using the techniques of ferrolabeling and ferromagnetic sorting, coupled with electron microscopic visualization. Vitellogenin conjugated to colloidal ferric particles of ca. 11 nm is selectively transported from the oolemma to the yolk platelets of vitellogenic Xenopus oocytes after gonadotropin stimulation of the female. Several cortical membrane compartments, labeled or unlabeled with ferric particles, are involved in the internalization and the transfer of vitellogenin to the yolk platelets. 1) Coated pits apparently fuse with coated vesicles, and coated vesicles fuse with each other in the outermost cortical cytoplasm. 2) Vesicles, depleted of their clathrin coat, fuse with cortical tubular endosomes and discharge their contents into yolk endosomes. 3) These endosomes are the direct precursors of the yolk organelles. 4) Endocytic vesicles fuse only with primordial yolk platelets of type I and not with type II or fully grown yolk platelets. After pulse-chase loading with ferric particles conjugated to vitellogenin and subsequent subcellular fractionation of the oocytes, ferromagnetic sorting of the various vesicle populations has been performed by using a "free-flow magnetic chamber". This novel method enables specification and characterization of purified endosomal compartments that accumulate protein yolk in Xenopus oocytes.     

Ultrastructural observations of previtellogenic ovarian follicles of dove

Dove ovarian follicle is a complex structure composed of oocyte surrounded by a somatic compartment consisting of theca externa, theca interna and granulosa. The structure of ovarian follicle (1 and 2 mm) of dove was studied by electron microscopy. The granulosa was pseudostratified in the 1-mm-diameter follicles and stratified with two or three irregular rows of cells in the 2-mm-diameter follicles. In the larger follicle indentations between oocyte and granulosa cells become more numerous and the microvilli of granulosa cell elongated to form a zona radiata with similarly elongated oocyte microvilli. Lining bodies were present at the tips of granulosa microvilli and in the cortical region of the oocyte. In the oocyte cortex were observed coated pits, coated vesicles, dense tubules, multivesicular bodies and primordial yolk spheres. Primordial yolk spheres may contain lining bodies and were observed fused with dense tubules and multivesicular bodies or associated with smooth cisternae.     

Endosomes transfer yolk proteins to lysosomes in the vitellogenic oocyte of the trout

The internalization of the yolk proteins has been investigated by electron microscopy and cytochemistry in the oocyte of the trout which stores up large quantities of yolk. The oocyte evolution has been followed for 18 months in a homogeneous group of animals. Anionic ferritin has been injected during vitellogenesis. The results indicate that as in other oocytes the yolk proteins are absorbed by coated vesicles during vitellogenesis. But unlike most other oocytes the yolk proteins are then transferred via typical endosomes to a conspicuous lysosomal compartment built up very early at the onset of the cytoplasmic differentiation of the oocyte e.i. 10 months earlier. During vitellogenesis yolk progressively accumulates in this lysosomal compartment. Injected anionic ferritin follows the same pathway of internalization. These findings indicate that in this oocyte, the whole yolk cycle presumably represents an adaptation of a general cellular activity, the receptor-mediated endocytosis, largely amplified, sequenced and spread over several months.     

Multivesicular bodies play a key role in vitellogenin endocytosis by Xenopus oocytes

A combination of electron microscopic tracers and subcellular fractionation has been used to examine the endocytic pathway of the yolk protein precursor, vitellogenin (VG), in Xenopus oocytes. VG was adsorbed to colloidal gold, and the organelles traversed by newly internalized ligand were examined at various time intervals after endocytosis. VG-Au enters oocytes via coated pits and vesicles and then appears rapidly in tubular endosomes and multivesicular bodies (MVBs). MVBs play a central role in VG processing for storage; the large majority of newly internalized VG enters this compartment, remaining there for up to several hours. Condensation of VG into crystalline bodies begins in MVBs, and continues with growth of the crystals until typical platelets are formed. When oocytes are exposed to high [VG], MVBs containing large amounts of internalized VG are morphologically indistinguishable from the primordial yolk platelets described earlier (Dumont, 1978). The use of VG-Au particles of two sizes demonstrates that gold particles in early MVBs were generally associated with the limiting membrane of these organelles, while older MVB compartments have gold particles well separated from the limiting membranes, suggesting that dissociation of VG from its receptor occurs in this compartment. Newly internalized ligand preferentially forms a new MVB, rather than fusing and mixing with previously formed MVBs. Progressive yolk protein condensation gradually transforms MVBs into yolk platelets over a period of several hours. Analysis of 125I-VG-Au behavior after sucrose gradient fractionation of oocytes allowed correlation of biochemical compartments with those observed in the electron microscope. MVBs containing yolk in progressive stages of condensation were found at densities from 1.16 up to 1.21 g/cc. The final, rate-limiting step in VG transport is a shift of ligand from light (1.21 g/cc) to heavy (1.23 g/cc) platelet compartments (Wall and Meleka, 1985). The morphological correlate of this process is movement of VG-Au from small (less than 3-4 microns diameter) to large (greater than 4 microns diameter) platelets.     

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.     

Chicken oocyte growth: receptor-mediated yolk deposition

During the rapid final stage of growth, chicken oocytes take up massive amounts of plasma components and convert them to yolk. The oocyte expresses a receptor that binds both major yolk lipoprotein precursors, vitellogenin (VTG) and very low density lipoprotein (VLDL). In the present study, in vivo transport tracing methodology, isolation of coated vesicles, ligand- and immuno-blotting, and ultrastructural immunocytochemistry were used for the analysis of receptor-mediated yolk formation. The VTG/VLDL receptor was identified in coated profiles in the oocyte periphery, in isolated coated vesicles, and within vesicular compartments both outside and inside membrane-bounded yolk storage organelles (yolk spheres). VLDL particles colocalized with the receptor, as demonstrated by ultrastructural visualization of VLDL-gold following intravenous administration, as well as by immunocytochemical analysis with antibodies to VLDL. Lipoprotein particles were shown to reach the oocyte surface by passage across the basement membrane, which possibly plays an active and selective role in yolk precursor accessibility to the oocyte surface, and through gaps between the follicular granulosa cells. Following delivery of ligands from the plasma membrane into yolk spheres, proteolytic processing of VTG and VLDL by cathepsin D appears to correlate with segregation of receptors and ligands which enter disparate sub-compartments within the yolk spheres. In small, quiescent oocytes, the VTG/VLDL receptor was localized to the central portion of the cell. At onset of the rapid growth phase, it appears that this pre-existing pool of receptors redistributes to the peripheral region, thereby initiating yolk formation. Such a redistribution mechanism would obliterate the need for de novo synthesis of receptors when the oocyte's energy expenditure is to be utilized for plasma membrane synthesis, establishment and maintenance of intracellular topography and yolk formation, and preparation for ovulation.     

Specific proteolysis regulates fusion between endocytic compartments in Xenopus oocytes

We examined the role of proteolytic ligand modification in endosomal targeting using vitellogenin (VTG) uptake by Xenopus oocytes as a model system. Non-cleavable VTG is internalized, but does not appear in yolk platelets. We identified two inhibitors of VTG processing into the yolk proteins: the ionophore monensin and pepstatin A, a specific inhibitor of cathepsin D. Pepstatin neither affected ligand binding and internalization, nor inhibited the degradation of nonspecifically incorporated proteins, whereas monensin inhibited all of these processes. Inhibiting VTG processing prevented its deposition into yolk platelets by strongly interfering with endosome-yolk platelet fusion. Monensin treatment resulted in morphologically abnormal endosomes, while pepstatin only inhibited VTG cleavage and the subsequent fusion of endosomes with yolk platelets. Since VTG cleavage is initiated prior to its deposition in platelets, we postulate that ligand proteolysis could be necessary for normal endosomal targeting.     

Hemopexin joins transferrin as representative members of a distinct class of receptor-mediated endocytic transport systems

Receptor-mediated transport of heme by hemopexin in vivo and in vitro results in catabolism of heme but not the protein, suggesting that intact apohemopexin recycles from cells. However, until now, the intracellular transport of hemopexin by receptor-mediated endocytosis remained to be established. Biochemical studies on cultured human HepG2 and mouse Hepa hepatoma cells demonstrate that hemopexin is transported to an intracellular location and, after endocytosis, is subsequently returned intact to the medium. During incubation at 37 degrees C, hemopexin accumulated intracellularly for ca. 15 min before reaching a plateau while surface binding was saturated by 5 min. No internalization of ligand took place during incubation at 4 degrees C. These and other data suggest that hemopexin receptors recycle, and furthermore, incubation with monensin significantly inhibits the amount of cell associated of heme-[125I]hemopexin during short-term incubation at 37 degrees C, consistent with a block in receptor recycling. Ammonium chloride and methylamine were less inhibitory. Electron microscopic autoradiography of heme-[125I]hemopexin showed the presence of hemopexin in vesicles of the classical pathway of endocytosis in human HepG2 hepatoma cells, confirming the internalization of hemopexin. Colloidal gold-conjugated hemopexin and electron microscopy showed that hemopexin bound to receptors at 4 degrees C is distributed initially over the entire cell surface, including microvilli and coated pits. After incubation at 37 degrees C, hemopexin-gold is located intracellularly in coated vesicles and then in small endosomes and multivesicular bodies. Colocalization of hemopexin and transferrin intracellularly was shown in two ways. Radioiodinated hemopexin was observed in the same subcellular compartment as horseradish peroxidase conjugates of transferrin using the diaminobenzidine-induced density shift assay. In addition, colloidal gold derivatives of heme-hemopexin and diferric transferrin were found together in coated pits, coated vesicles, endosomes and multivesicular bodies. Therefore, hemopexin and transferrin act by a similar receptor-mediated mechanism in which the transport protein recycles after endocytosis from the cell to undergo further rounds of intracellular transport.     

Biochemical and immunocytochemical analysis of vitellogenesis in the olive fruit fly Dacus (bactrocera) oleae (Diptera: Tephritidae)

Synthesis and selective accumulation of the major yolk proteins in the developing oocytes of the species Dacus oleae (Diptera: Tephritidae) was studied biochemically and by immunoelectron microscopy. In the hemolymph of adult females, two yolk proteins precursors (or vitellogenins) have been detected. They each exhibit a similar molecular weight and isoelectric point to their respective mature yolk proteins (or vitellins), while electrophoretic analysis of their synthetic profile shows that their levels in the hemolymph increase rapidly during development. Immunogold electron microscopy of ovarian sections, revealed that the hemolymph vitellogenins reach the oocyte through enlarged inter-follicular spaces and demonstrated vitellogenin synthesis by the follicle cells of the vitellogenic follicles. The newly synthesized vitellogenins follow a distinct secretory pathway into these cells as compared to other components being synthesized at the same time (e.g. the vitelline envelope proteins), since they were found in secretory vesicles that appeared to be differentiated from those destined to participate in the vitelline envelope. The vitellogenin-containing vesicles exocytose their contents directionally into the follicle cell/vitelline envelope boundary, and subsequently the vitellogenins diffuse among the gaps of the forming vitelline envelope and reach the oocyte plasma membrane. Their internalization by the oocyte includes the formation of an endocytic complex consisting of coated pits, coated vesicles, endosomes, transitional yolk bodies, and finally mature yolk bodies, in which the storage of the vitellins and other yolk proteins occur. These results are discussed in relation to data obtained from other Dipteran species.     

A network system for vitellogenin synthesis in the mussel Mytilus galloprovincialis (L.)

The aim of this study is to assess, by RT‐PCR, in situ hybridization, electron microscopy, and immunohistochemistry, the site/s of vitellogenin (VTG) synthesis in the mussel Mytilus galloprovincialis. Our investigations demonstrate that, among the analyzed tissues, the synthesis of VTG occurs only in the female gonad, that is, within the oocyte and follicle and connective cells. Such a synthesis is just evident in early vitellogenic oocytes, whose cytoplasm is characterized by numerous RER cisternae and an extended Golgi complex surrounded by nascent yolk platelets. The synthesis of VTG goes on in vitellogenic oocytes assuming a pear form, and progressively reduces once the oocyte shows the pear or polygonal form, typical of those oocytes that have concluded the growth. The expression of VTG occurs also within follicle (auxiliary) and connective cells. In particular, it is noteworthy that follicle cells are characterized by numerous RER cisternae and an active Golgi complex surrounded by numerous vesicles and vacuoles containing electron dense material. The same material is also present along their plasma membrane, within the intercellular space between oocyte and follicle cells, and finally within invaginations of the oocyte surface, thus suggesting a VTG transfer to the oocyte via endocytosis. Differently, no VTG synthesis was observed within digestive gland. All together the findings here reported strongly suggest that in M. galloprovincialis, inside the gonad, the VTG synthesis occurs in the oocyte (autosynthesis) and in the follicle and adipogranular cells (heterosynthesis). J. Cell. Physiol. 228: 547–555, 2013. © 2012 Wiley Periodicals, Inc.     

Intracellular localization and endocytosis of brush border enzymes in the enterocyte-like cell line Caco-2.

Immunoelectron microscopy was used to localize the brush border hydrolases sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPPIV) in the human colon carcinoma cell line Caco-2. Both enzymes were detected at the microvillar membrane, in small vesicles and multivesicular bodies (MVBs), and in lysosomal bodies. In addition, DPPIV was found in the Golgi apparatus, a variety of apical vesicles and tubules, and at the basolateral membrane. To investigate whether the hydrolases present in the lysosomal bodies were endocytosed from the apical membrane, endocytic compartments were marked with the endocytic tracer cationized ferritin (CF). After internalization from the apical membrane through coated pits, CF was first recovered in apical vesicles and tubules, and larger electronlucent vesicles (early endosomes), and later accumulated in MVBs (late endosomes) and lysosomal bodies. DPPIV was localized in a subpopulation of both early and late endocytic vesicles, which contained CF after 3 and 15 min of uptake, respectively. Also, internalization of the specific antibody against DPPIV and gold labeling on cryosections showed endocytosed DPPIV in both early and late endosomes. However, unlike CF, no accumulation of DPPIV was seen in MVBs or lysosomal bodies after longer chase times. The results indicate that in Caco-2 cells the majority of brush border hydrolases present in lysosomal bodies are not endocytosed from the brush border membrane. Furthermore, the labeling patterns obtained, suggest that late endosomes may be involved in the recycling of endocytosed DPPIV to the microvilli.     

An unusual lysosome compartment involved in vitellogenin endocytosis by Xenopus oocytes

We have investigated the lysosomal compartment of Xenopus oocytes to determine the possible role of this organelle in the endocytic pathway of the yolk protein precursor, vitellogenin. Oocytes have lysosome-like organelles of unusual enzymatic composition at all stages of their development, and the amount of hydrolase activity increases steadily throughout oogenesis. These unusual lysosomes appear to be located primarily in a peripheral zone of oocyte cytoplasm. At least two distinct populations of lysosomal organelles can be identified after sucrose density gradient fractionation of vitellogenic oocytes. Most enzyme activity resides in a compartment of large size and high density that appears to be a subpopulation of yolk platelets that are less dense than most platelets within the cell. The appearance of this high density peak of lysosomal enzyme activity coincides with the time of onset of vitellogenin endocytosis during oocyte development. The data suggest that endocytic vesicles that contain vitellogenin fuse with modified lysosomes shortly after their internalization by the oocyte. Pulse-chase experiments with radiolabeled vitellogenin suggest that the ligand passes through the low density platelet compartment en route to the heavy platelets. The accumulation of yolk proteins apparently results from a failure of these molecules to undergo complete digestion after their entry into an unusual lysosomal compartment. The yolk platelets that these proteins finally enter for prolonged storage appear to be a postlysosomal organelle.     

Ultrastructural visualization of the internalization of low density lipoprotein by human placental cells

Summary Low density lipoproteins (LDL) were conjugated to colloidal gold to visualize the route for internalization of LDL in the cultured cells of human term placenta. Cells were obtained from placental villi (caesarian section) by a standard trypsin-DNase dispersion method followed in some cases by a Percoll gradient centrifugation step. Employing electron microscopy it was observed that after 3 days of culture, cells obtained by trypsin-DNAse dispersion were a mixture of macrophages, mononucleated cells and large multinucleated cells. When the cells were incubated for 3 days after the Percoll purification, essentially multinucleated cells identical to the syncytiotrophoblast were present. The number of LDL receptor was increased by preincubation in medium with lipoprotein depleted serum. In binding experiments cells incubated at 4° C for 2 h with medium containing gold LDL conjugates showed gold LDL attached to the plasma membrane without characteristic localization. After incubation with gold LDL at 37° C for various times, the three cellular types showed ligand internalization. Gold LDL endocytosis involved first coated pits but also uncoated plasmalemmal invaginations. Then gold LDL was further observed in coated and non coated vesicles, smooth walled endosomes, multivesicular bodies and tubular vesicles. Lastly free gold particles were observed in lysosome like dense bodies. These results prove the internalization of gold LDL conjugates by human cultured placental cells, particularly by syncytiotrophoblast like multinucleated cells. This accumulation of LDL (the major cholesterol carrying protein in humans) is recognised to be responsable for the exogenous cholesterol supply indispensable to the progesterone biosynthesis and cellular growth of the placenta.     

Ultrastructural visualization of the internalization of low density lipoprotein by human placental cells

Low density lipoproteins (LDL) were conjugated to colloidal gold to visualize the route for internalization of LDL in the cultured cells of human term placenta. Cells were obtained from placental villi (caesarian section) by a standard trypsin-DNase dispersion method followed in some cases by a Percoll gradient centrifugation step. Employing electron microscopy it was observed that after 3 days of culture, cells obtained by trypsin-DNase dispersion were a mixture of macrophages, mononucleated cells and large multinucleated cells. When the cells were incubated for 3 days after the Percoll purification, essentially multinucleated cells identical to the syncytiotrophoblast were present. The number of LDL receptor was increased by preincubation in medium with lipoprotein depleted serum. In binding experiments cells incubated at 4 degrees C for 2 h with medium containing gold LDL conjugates showed gold LDL attached to the plasma membrane without characteristic localization. After incubation with gold LDL at 37 degrees C for various times, the three cellular types showed ligand internalization. Gold LDL endocytosis involved first coated pits but also uncoated plasmalemmal invaginations. Then gold LDL was further observed in coated and non coated vesicles, smooth walled endosomes, multivesicular bodies and tubular vesicles. Lastly free gold particles were observed in lysosome like dense bodies. These results prove the internalization of gold LDL conjugates by human cultured placental cells, particularly by syncytiotrophoblast like multinucleated cells. This accumulation of LDL (the major cholesterol carrying protein in humans) is recognised to be responsible for the exogenous cholesterol supply indispensable to the progesterone biosynthesis and cellular growth of the placenta.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages

Endocytosis of immunoglobulin G (IgG)-coated colloidal gold particles in cultured mouse peritoneal macrophages was studied by scanning and transmission electron microscopy. At 4 degrees C, the tracers adhered to the plasma membrane and accumulated in coated pits located in the bottom of furrows or deep invaginations on the cell surface. In the presence of an excess of unlabeled mouse IgG, cellular binding of the tracer was reduced by 80 to 90%. After warming to 37 degrees C, surface-bound tracer particles were rapidly ingested and transported to small and large vesicles lacking membrane coat. From here, they were then passed over to multivesicular bodies and lysosomes characterized by their content of myelin-like figures and other inclusions. Double-labeling experiments with IgG-coated colloidal gold particles of two different sizes (20 and 5 nm diameter) indicated that the plasma membrane was depleted of binding sites after uptake of a polyvalent ligand. The restoration of the binding capacity was a slow process requiring ongoing protein synthesis. On the basis of these observations, a model for endocytosis of immune complexes in macrophages is presented. It includes the following four steps: IgG-containing macromolecular aggregates bind to specific receptors in the plasma membrane. These appear to be preclustered in coated pits or able to move laterally within the membrane even at 4 degrees C. The receptor-ligand complexes are internalized and transferred sequentially to larger uncoated vesicles or endosomes, multivesicular bodies, and lysosomes with inclusions of varying appearance. Receptors and ligands are degraded within the lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular transport of vitellogenin in Xenopus oocytes: An autoradiographic study

The role of primordial yolk platelets (PYPs) in the transport of the yolk precursor vitellogenin to the yolk platelets in Xenopus laevis oocytes has been demonstrated by electron microscopic autoradiography. Within 20 min after exposure of the oocyte to ³H-labeled-vitellogenin, silver grains are associated with small PYPs which are formed by the fusion of endosomes. At 40 min after incorporation of ³H-labeled vitellogenin, autoradiographic silver grains are associated with larger PYPs and with the superficial layer of yolk platelets. Thus, the results demonstrate that PYPs are an intermediate in the transport of vitellogenin from endosomes to yolk platelets. These observations are consonant with the general hypothesis that vitellogenin first associates (binds?) with the plasma membrane, then is incorporated by endocytosis into endosomes which fuse to form PYPs, and finally the contents of the PYPs are eventually deposited into yolk platelets.     

Internalized proteins directed into accumulative compartments of mosquito oocytes by the specific ligand, vitellogenin

We have investigated the internalization pathways for a specific protein, vitellogenin, and a non-specific protein, horseradish peroxidase, in the mosquito oocyte in vivo. The internalized proteins were localized by electron microscopical immunocytochemistry or autoradiography; the relationship of their destination compartments with lysosomes was monitored by visualization of acid phosphatase. Proteins internalized by the oocyte follow either a specific accumulative route or a lysosomal degradative route. Via coated vesicles, both proteins enter the same compartment, the endosome, where they dissociate from membrane-binding sites. The route to their final destination depends on the presence of the specific ligand. In its absence, the degradative route is followed, and the endosome with non-specific protein fuses with lysosomes. In the presence of the specific ligand, the accumulative route is followed, and both specific and non-specific proteins are delivered into an accumulative compartment, the transitional yolk body. During the transformation of the transitional yolk body into the final storage compartment, a mature yolk body, vitellogenin undergoes crystallization, whereas the non-specific protein is concentrated in small vesicular extensions of the compartmental membrane. These vesicles are separated from the yolk bodies and apparently deliver the non-specific protein into the lysosomal system. We concluded that any protein bound to the membrane would be internalized by the oocyte, but only binding of the specific ligand to its receptor serves as a transmembrane signal stimulating the formation of accumulative compartments.     

Plasminogen activator: Morphological evidence of binding, internalization and delivery to lysosomes in 3T3 mouse fibroblasts

Summary Using a direct conjugate of urokinase and ferritin, the binding has been followed at the plasma membrane and the internalization of urokinase into BALB/C-3T3 fibroblasts, cultured in plasminogen-free conditions. At 0° C, the conjugate was observed bound on both coated and uncoated cell surface regions as singlets, and small and large clusters. No binding was observed in the presence of excess native urokinase. The binding was impaired by preincubation of the conjugate with a competitive inhibitor of the catalytic site, suggesting an interaction between the receptor and the catalytic site of the enzyme.Within 1 min at 37° C, urokinase clustered on coated regions of the plasma membrane. At 5 min after warming, ferritin was found on deeply indented coated pits and in both coated and uncoated vesicles close to the cell surface. By 10 min at 37° C, ferritin particles were present in uncoated endosomes and in multivesicular bodies in the Golgi area. Within 10 min, the receptors on the surface strongly decreased. New receptors were observed on the membrane after 20 min at 37° C. At this time, ferritin was observed both in endosomes or multivesicular bodies and in vesicles close to the plasma membrane.