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J Gruenberg

Publications and source records attributed to J Gruenberg.

At least 73 records · Page 4Linked to original sources
PubMed1990

Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytic vesicles from MDCK cells.

The pathways of endocytosis from the apical and the basolateral domains of epithelial MDCK cells are known to converge at the level of late endosomes in vivo. We have now reconstituted the meeting process in a cell-free assay that measures the fusion of apically and basolaterally derived endocytic vesicles with late endosomes. Our results show that this in vitro process requires the presence of polymerized microtubules, as does the convergence of the two pathways in vivo, and also depends on the presence of microtubule binding proteins, in particular the mechanochemical motors kinesin and cytoplasmic dynein.

Adenosine Triphosphatases↗
PubMed1989

Subcellular fractionation of tissue culture cells.

Subcellular fractionation has two major steps, (1) the homogenization of the cells and (2) the subsequent separation of the organelles. The homogenization step is discussed with reference to the problems encountered using tissue culture cells. Promising techniques for the isolation of specific compartments are illustrated using the isolation of the endosomal compartment as the example.

Animals↗
PubMed1989

Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro.

We have investigated two aspects of membrane traffic at early stages of endocytosis: membrane fusion and microtubule-dependent transport. As a marker, we have used the trans-membrane glycoprotein G of vesicular stomatitis virus implanted into the plasma membrane and then internalized for different times at 37 degrees C. The corresponding endosomal fractions were immunoisolated using the cytoplasmic domain of the G protein as antigen. These fractions were then used in an in vitro assay to quantify the efficiency of fusion between endosomal vesicles. To identify the vesicular partners of the fusion, these in vitro studies were combined with in vivo biochemical and morphological experiments. Internalized molecules were delivered to early endosomal elements, which corresponded to a network of tubular and tubulovesicular structures. Rapid recycling back to the plasma membrane and routing to late stages of the pathway occurred from these early endosomal elements. These elements exhibited a high and specific fusion activity with each other in vitro, suggesting that individual elements of the early endosomal compartment interact with each other in vivo. After their appearance in the early endosome, the molecules destined to be degraded were observed at the next stage of the pathway in distinct spherical vesicles (0.5 micron diam) and then in late endosomes and lysosomes. When the microtubules were depolymerized with nocodazole, endocytosis proceeded as in control cells. However, internalized molecules remained in the spherical vesicles and did not appear in late endosomes or lysosomes. These spherical vesicles had relatively little fusion activity with each other or with early endosomal elements in vitro. Our observations suggest that the spherical vesicles mediate transport between the early endosome and late endosomes and that this process requires intact microtubules.

Animals↗
PubMed1989

Endocytosis in filter-grown Madin-Darby canine kidney cells.

In this paper, we have characterized the apical and basolateral endocytic pathways of epithelial MDCK cells grown on filters. The three-dimensional organization of the endocytic compartments was analyzed by confocal microscopy after internalization of a fluorescent fluid-phase marker from either side of the cell layer. After 5 min of internalization, distinct sets of apical and basolateral early endosomes were observed lining the plasma membrane domain from which internalization had occurred. At later time points, the apical and the basolateral endocytic pathways were shown to converge in the perinuclear region. Mixing of two different fluorescent markers could be detected after their simultaneous internalization from opposite sides of the cell layer. The extent of the meeting was quantitated by measuring the amount of complex formed intracellularly between avidin internalized from the apical side and biotinylated horseradish peroxidase (HRP) from the basolateral side. After 15 min, 14% of the avidin marker was complexed with the biotinylated HRP and this value increased to 50% during a subsequent chase of 60 min in avidin-free medium. We also determined the kinetics of fluid internalization, recycling, transcytosis, and intracellular retention using HRP as a marker. Fluid was internalized with the same rates from either surface domain (1.2 x 10(-4) microns 3/min per microns 2 of surface area). However, significant differences were observed for each pathway in the amounts and kinetics of marker recycled and transcytosed. The content of apical early endosomes was primarily recycled and transcytosed (45% along Bach route after 1 h internalization), whereas delivery to late endocytic compartments was favored from the basolateral early endosome (77% after 1 h). Our results demonstrate that early apical and basolateral endosomes are functionally and topologically distinct, but that the endocytic pathways converge at later stages in the perinuclear region of the cell.

Animals↗
PubMed1989

The structure of organelles of the endocytic pathway in hydrated cryosections of cultured cells.

The structure of cellular organelles, in particular those involved in endocytosis, was studied by electron microscopy with hydrated cryosections. In this technique no chemical treatment is used, and the native structure of organelles can be observed in sections viewed at temperatures below -140 degrees C, using a cold stage accessory on the electron microscope. The compartments of the endocytic pathway were prelabeled with gold markers in the living cell, facilitating the identification of different structures in the cryosections. The structure of most identifiable cellular organelles, including those involved in endocytosis, appeared very similar in the hydrated cryosections to that seen after conventional plastic and cryosections of chemically fixed cells. In particular, the internal membranes of the structure we refer to as the prelysosomal compartment (Griffiths et al., Cell 52, 329-341 (1988] could be clearly visualized in these sections indicating that the organization of these membranes is not a consequence of the chemical fixation process.

Animals↗
PubMed1987

Two threshold values of low pH block endocytosis at different stages.

The influence of low extracellular pH on endocytosis was studied in baby hamster kidney cells. When the extracellular medium was adjusted to pH 5.7, the intracellular pH decreased within 2 min to pH 6.2 and the endocytosis of horseradish peroxidase (HRP) in the fluid phase dropped to an undetectable level. With an external pH of 6.3, the internal pH dropped to pH 6.8 and HRP was internalized at a normal rate for 5 min but accumulation during longer incubation times did not occur. Morphologically, HRP was visualized in the lumen of a subpopulation of tubular and vesicular endosomes. These observations were confirmed by subcellular fractionation studies using free flow electrophoresis. Low extracellular pH also had an effect on the endocytosis of the membrane-spanning glycoprotein G of vesicular stomatitis virus which was implanted into the plasma membrane. The internalization of G-protein was quantitated by a surface fluoroimmunoassay. The endocytosis of G-protein was not affected when the external pH was dropped to 6.3, but was reduced at an external pH of 5.7. The intracellular ATP was not depleted and the reduction of endocytosis was reversible upon return to physiological pH. Clathrin coated pits were detected by electron microscopy at the plasma membrane of the low-pH-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1987

An internalized transmembrane protein resides in a fusion-competent endosome for less than 5 minutes.

We have used our assay for the cell-free reconstitution of vesicle fusion occurring in endocytosis to investigate the fusion competence of defined endosomal fractions containing the G protein of vesicular stomatitis virus, G protein was first implanted into the plasma membrane, and endocytosis was then allowed to proceed for defined periods of time. Endosomal fractions were prepared by "immuno-isolation" on a solid support with a monoclonal antibody against the cytoplasmic domain of the G protein. Maximal internalization of the G protein occurred within 5 min at 37 degrees C. From this early endosome the G molecules follow a branched pathway: 50% recycles to the cell surface, while 50% is transported along the endocytic route to the lysosomal compartment. The proportion of G protein following each pathway can be modulated. When the amount of implanted G protein was increased, the fraction of G molecules recycling to the cell surface was reduced. When the G molecules were cross-linked with an antibody prior to the internalization step, recycling to the cell surface was abolished. The cell-free analysis of vesicle fusion was carried out with endosomal fractions immuno-isolated after 5, 10, 15, and 30 min of G-protein internalization at 37 degrees C. Fusion competence was at a maximum with the fraction isolated 5 min after internalization and then decreased with a half-life of approximately equal to 3 min with fractions isolated at later time points. The fusion-competent compartment is the early endosome where sorting of the transmembrane G protein to recycling or degradation occurs.

Animals↗
PubMed1987

Endosomal compartment of toad bladder epithelium.

Apical exocytosis and increased permeability are induced by antidiuretic hormone (ADH). After this, endocytosis is also induced by ADH and is associated with the decline in ADH-induced water permeability at the apical surface of the toad urinary bladder (9, 19, 20). During this process, horseradish peroxidase (HRP), a fluid phase marker, is taken up from the mucosal solution into endocytic tubules and multivesicular bodies. We now report that we can introduce from the apical (mucosal) side, a viral transmembrane protein (the G-protein of VSV) and that this protein can be retrieved as an integral membrane protein in endocytic membranes. This was demonstrated by immunoisolation of endosomal vesicles loaded with HRP using a monoclonal antibody against the cytoplasmic domain of the G-protein.

Animals↗
PubMed1985

Immuno-isolation of vesicles using antigenic sites either located on the cytoplasmic or the exoplasmic domain of an implanted viral protein. A quantitative analysis.

In this study, we present a new general approach for immuno-isolation: a foreign integral membrane protein, the G-protein of vesicular stomatitis virus (VSV), is implanted into the plasma membrane for subsequent immuno-isolation. A quantitative analysis was accomplished using the erythrocyte plasma membrane as a model system. The virus was artificially bound to the membrane via a lectin and subsequently fused at low pH. Vesicles of two opposite orientations were prepared from erythrocytes with fused G-protein. Right-side-out and inside-out vesicles expose the exoplasmic and the cytoplasmic domains of the G-protein on their surfaces respectively. In immuno-isolation experiments antibodies against each of the domains of the G-protein were used. Vesicles were presented to an immunoadsorbent (ImAd) consisting of a solid support with appropriate antibodies bound to its surface. Two commonly used immunoadsorbents prepared from either polyacrylamide beads or fixed Staphylococcus aureus cells were compared and found to have identical immuno-isolation efficiencies. It was possible to control and quantitate the amount of implanted antigen. Therefore, we were able to show that the critical antigen density required for immuno-isolation is 50 G molecules/micron2 plasma membrane surface area for both types of vesicle/antibody couples. This analysis showed that vesicles presenting either the cytoplasmic or the exoplasmic domain of the G-protein are immuno-isolated with the same efficiency.

Animals↗
PubMed1985

Urobehavioral intervention in the rehabilitation of lower urinary tract dysfunction: a case report.

A case study illustrates the usefulness of behavioral analysis and paradoxical strategy in the treatment of life-long urgency incontinence in a cerebral palsy patient. Volitional increase in diurnal enuresis appeared to result in rapid (within 5 days) acquisition of continence. From pretreatment to 13 month follow-up interview, the weekly wetting frequency decreased from an average of 25 to 0.5 episodes. Symptom rehearsal is believed to have interrupted the typical pathobehavioral sequence between the urge to void and micturition.

Adult↗
PubMed1983

Isolation and characterization of the plasma membrane of human erythrocytes infected with the malarial parasite Plasmodium falciparum.

Human erythrocytes infected with the malarial parasite Plasmodium falciparum were labeled metabolically with a mixture of 15 radioactive amino acids. When synchronously growing parasites were at the schizont stage of development infected cells were concentrated and purified by using a Percoll-Hypaque gradient. The plasma membrane of the infected erythrocyte, isolated by binding cells to a solid support (Affi-Gel 731, Bio-Rad), was less than 1% contaminated with parasite membranes. Erythrocyte membrane proteins were analyzed by polyacrylamide gel electrophoresis and autoradiography. Despite the high sensitivity of the procedure, there was no evidence for the insertion of parasite proteins into the infected host cell membrane. One possible exception is a Mr 230,000 parasite protein present maximally as 9,000 copies per infected erythrocyte membrane. Moreover, no differences in the membrane proteins were observed between a highly knobby clone and a knobless clone of the same strain of P. falciparum. These findings appear to rule out the presence of parasite protein(s) playing a structural role in the formation of knobs on the erythrocyte surface and question whether the antigenic determinants on the P. falciparum-infected erythrocyte are of parasite origin or whether such antigens represent newly exposed or chemically modified erythrocyte determinants.

Erythrocyte Membrane↗
PubMed1983

Scanning electron microscope-analysis of the protrusions (knobs) present on the surface of Plasmodium falciparum-infected erythrocytes.

The nature of the surface deformations of erythrocytes infected with the human malaria parasite Plasmodium falciparum was analyzed using scanning electron microscopy at two stages of the 48-h parasite maturation cycle. Infected cells bearing trophozoite-stage parasites (24-36 h) had small protrusions (knobs), with diameters varying from 160 to 110 nm, and a density ranging from 10 to 35 knobs X micron-2. When parasites were fully mature (schizont stage, 40-44 h), knob size decreased (100-70 nm), whereas density increased (45-70 knobs X micron-2). Size and density of the knobs varied inversely, suggesting that knob production (a) occurred throughout intraerythrocytic parasite development from trophozoite to schizont and (b) was related to dynamic changes of the erythrocyte membrane. Variation in the distribution of the knobs over the red cell surface was observed during parasite maturation. At the early trophozoite stage of parasite development, knobs appeared to be formed in particular domains of the cell surface. As the density of knobs increased and they covered the entire cell surface, their lateral distribution was dispersive (more-than-random); this was particularly evident at the schizont stage. Regional surface patterns of knobs (rows, circles) were seen throughout parasite development. The nature of the dynamic changes that occurred at the red cell surface during knob formation, as well as the nonrandom distribution of knobs, suggested that the red cell cytoskeleton may have played a key role in knob formation and patterning.

Erythrocyte Membrane↗
PubMed1980

Renal function in critically ill postoperative patients: sequential assessment of creatinine osmolar and free water clearance.

Sequential assessment of renal function in 50 critically ill postoperative patients was done by simultaneous determinants of osmolar, free water, creatinine and sodium clearances, and fractional excretion of sodium. The traumatic and nontraumatic critically ill surgical patients had a characteristic pattern of creatinine clearance (Ccr) which was age related. This was manifested in the trauma patient by Ccr which were initially greater than expected (140-190 ml/min.1.73 M2). In the uncomplicated postoperative patients, a positive correlation was found between increased cardiac index and the indexed glomerular filtration rate. Decreases in free water clearance and Ccr were useful in predicting the onset of renal failure before the development of the oliguric state. The authors conclude that frequent assessment of those renal functions which are most adversely affected by accidental or surgical trauma is most appropriately monitored by serial measurement of creatinine and free water clearance.

Acute Kidney Injury↗
PubMed1980

Role of glycerol permeation in the bloodstream form of Trypanosoma brucei.

Under aerobic conditions, we have determined glycerol uptake in the long slender (LS) bloodstream form of Trypanosoma (Trypanozoon) brucei brucei by studying glycerophosphate accumulation in the parasites. The coupled enzyme theory applies to the permeation-phosphorylation sequence. Glycerol passage through the plasma membrane is asymmetric, the efflux process being favored over the influx process. No free diffusion of glycerol can be detected even under conditions under which free glycerol accumulates within the cells; most probably, glycerol permeation is mediated by a specific transport system. In the absence of respiratory activities, glycerol is known to be an end-product of T. brucei glycolysis; its production from glycerophosphate should allow ATP synthesis. The observed efflux of free glycerol following intracellular accumulation of glycerophosphate confirms the hypothesis that glycerol production occurs through reversal of glycerol kinase activity. We conclude that in vivo the role of the carrier-mediated asymmetric permeation process is to prevent inhibition of the reversal of the glycerol kinase-mediated reaction by removing free glycerol.

Anaerobiosis↗