Lack of acrosome formation in Hrb-deficient mice. (57/1021)

The sperm acrosome is essential for sperm-egg fusion and is often defective in men with nonobstructive infertility. Here we report that male mice with a null mutation in Hrb are infertile and display round-headed spermatozoa that lack an acrosome. In wild-type spermatids, Hrb is associated with the cytosolic surface of proacrosomic transport vesicles that fuse to create a single large acrosomic vesicle at step 3 of spermiogenesis. Although proacrosomic vesicles form in spermatids that lack Hrb, the vesicles are unable to fuse, blocking acrosome development at step 2. We conclude that Hrb is required for docking and/or fusion of proacrosomic vesicles during acrosome biogenesis.  (+info)

Endosomal proteolysis of internalized insulin at the C-terminal region of the B chain by cathepsin D. (58/1021)

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.  (+info)

Isolation and characterization of the two major intracellular Glut4 storage compartments. (59/1021)

In rat adipose cells, intracellular Glut4 resides in two distinct vesicular populations one of which contains cellugyrin whereas another lacks this protein (Kupriyanova, T. A., and Kandror, K. V. (2000) J. Biol. Chem. 275, 36263--36268). Cell surface biotinylated MPR and (125)I-labeled transferrin are accumulated in cellugyrin-positive vesicles and to a lesser extent in cellugyrin-negative vesicles. An average cellugyrin-positive vesicle carries not more than one molecule of either Glut4, insulin-responsive aminopeptidase (IRAP), or transferrin receptor (TfR), whereas cellugyrin-negative vesicles contain five to six molecules of Glut4, more than 10 molecules of IRAP, and still one molecule of TfR per vesicle. Cellugyrin-negative vesicles are translocated to the cell surface after insulin stimulation, whereas cellugyrin-positive vesicles maintain intracellular localization both in the absence and in the presence of insulin and, therefore, may be involved in interendosomal protein transport. Both cellugyrin-positive and cellugyrin-negative vesicles are present in extracts of non-homogenized cells and therefore may represent the major form of Glut4 storage in vivo.  (+info)

Regulation of vesicle trafficking, transcription, and meiosis: lessons learned from yeast regarding the disparate biologies of phosphatidylcholine. (60/1021)

Phosphatidylcholine (PtdCho) is the major phospholipid present in eukaryotic cell membranes generally comprising 50% of the phospholipid mass of most cells and their requisite organelles. PtdCho has a major structural role in maintaining cell and organelle integrity, and thus its synthesis must be tightly monitored to ensure appropriate PtdCho levels are present to allow for its coordination with cell growth regulatory mechanisms. One would also expect that there needs to be coordinated regulation of PtdCho synthesis with its transport from its site of synthesis to cellular organelles to ensure organellar structures and functions are maintained. Each of these processes need to be intimately coordinated with cellular growth decision making processes. To this end, it has recently been revealed that ongoing PtdCho synthesis is required for global transcriptional regulation of phospholipid synthesis. PtdCho is also a major component of intracellular transport vesicles and the synthesis of PtdCho is intimately involved in the regulation of vesicle transport from the Golgi apparatus to the cell surface and the vacuole (yeast equivalent of the mammalian lysosome). This review details some of the more recent advances in our knowledge concerning the role of PtdCho in the regulation of global lipid homeostasis through (i) its restriction of the trafficking of intracellular vesicles that distribute lipids and proteins from their sites of synthesis to their ultimate cellular destinations, (ii) its regulation of specific transcriptional processes that coordinate lipid biosynthetic pathways, and (iii) the role of PtdCho catabolism in the regulation of meiosis. Combined, these regulatory roles for PtdCho ensure vesicular, organellar, and cellular membrane biogenesis occur in a coordinated manner.  (+info)

Membrane traffic: exocyst III--makes a family. (61/1021)

Although many factors have been implicated in the docking steps that precede vesicle fusion with a target membrane, few similarities have been found between them. New evidence suggests that at least some of these factors form related multimeric complexes that may help to explain the mechanism of vesicle docking.  (+info)

Megalin and cubilin are endocytic receptors involved in renal clearance of hemoglobin. (62/1021)

The kidney is the main site of hemoglobin clearance and degradation in conditions of severe hemolysis. Herein it is reported that megalin and cubilin, two epithelial endocytic receptors, mediate the uptake of hemoglobin in renal proximal tubules. Both receptors were purified by use of hemoglobin-Sepharose affinity chromatography of solubilized renal brush-border membranes. Apparent dissociation constants of 1.7 microM for megalin and 4.1 microM for cubilin were determined by surface plasmon resonance analysis. The binding was calcium dependent in both cases. Uptake of fluorescence-labeled hemoglobin by BN-16 cells was inhibited by anti-megalin and anti-cubilin antibodies as well as by receptor-associated protein, a chaperone for LDL-receptor family proteins. Partial inhibition by myoglobin was observed, whereas bovine serum albumin, intrinsic factor-cobalamin complexes, and beta2-microglobulin did not affect the uptake. By use of immunohistochemistry, it was demonstrated that uptake of hemoglobin in proximal tubules of rat, mouse, and dog kidneys occurs under physiologic conditions. Studies on normal and megalin knockout mouse kidney sections showed that megalin is responsible for physiologic clearance of hemoglobin. Labeling intensities in kidneys from normal and cubilin-malexpressing dogs were similar, which suggests that, in the normal state, the role of cubilin in uptake of hemoglobin is rather limited. However, cubilin is likely to assist hemoglobin endocytosis in settings of hemoglobinuria. In conclusion, the study provides a molecular explanation for long-standing observations of hemoglobin uptake in renal proximal tubules that involve the endocytic receptors megalin and cubilin. The findings may prove to be essential for further research on the pathophysiology of hemoglobinuric acute renal failure and proteinuria-associated tubulointerstitial nephritis.  (+info)

Export from pericentriolar endocytic recycling compartment to cell surface depends on stable, detyrosinated (glu) microtubules and kinesin. (63/1021)

A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface.  (+info)

Repairing a torn cell surface: make way, lysosomes to the rescue. (64/1021)

Biological membranes are often described as 'self-sealing' structures. If indeed membranes do have an inherent capacity for repair, does this explain how a cell can rapidly reseal a very large (1-1000 microm(2)) disruption in its plasma membrane? It is becoming increasingly clear that, in nucleated animal cells, the cytoplasm plays an active and essential role in resealing. A rapid and apparently chaotic membrane fusion response is initiated locally in the cytoplasm by the Ca(2+) that floods in through a disruption: cytoplasmic vesicles are thereby joined with one another (homotypically) and with the surrounding plasma membrane (exocytotically). As a consequence, internal membrane is added to cell surface membrane at the disruption site. In the case of large disruptions, this addition is hypothesized to function as a 'patch'. In sea urchin eggs, the internal compartment used is the yolk granule. Several recent studies have significantly advanced our understanding of how cells survive disruption-inducing injuries. In fibroblasts, the lysosome has been identified as a key organelle in resealing. Protein markers of the lysosome membrane appear on the surface of fibroblasts at sites of disruption. Antibodies against lysosome-specific proteins, introduced into the living fibroblast, inhibit its resealing response. In gastric epithelial cells, local depolymerization of filamentous actin has been identified as a crucial step in resealing: it may function to remove a barrier to lysosome-plasma membrane contact leading to exocytotic fusion. Plasma membrane disruption in epithelial cells induces depolymerization of cortical filamentous actin and, if this depolymerization response is inhibited, resealing is blocked. In the Xenopus egg, the cortical cytoskeleton has been identified as an active participant in post-resealing repair of disruption-related damage to underlying cell cortex. A striking, highly localized actin polymerization response is observable around the margin of cortical defects. A myosin powered contraction occurring within this newly formed zone of F-actin then drives closure of the defect in a purse-string fashion.  (+info)