PLASMA AND PHAGOSOME MEMBRANES OF <i>ACANTHAMOEBA CASTELLANII </i>

Ulsamer, Andrew G.; Wright, Philip L.; Wetzel, Mary G.; Korn, Edward D.

doi:10.1083/jcb.51.1.193

Cited by 116 publications

(65 citation statements)

References 64 publications

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“…Cytochemical reactions, such as the cerium capture used for glucose-6-phosphatase determinations, are not as specific as gold immunoelectron microscopy, and may reflect a minor nonspecific contribution of other enzymes, particularly in tissues with low glucose-6-phosphatase activity. Hydrolysis of glucose 6-phosphate can also be catalyzed by other enzymes, including alkaline and acid phosphatases, which are enriched in the plasma membrane and lysosomes, respectively (43)(44)(45). In addition, the resolution of the images obtained by enzymatic staining is lower than that achieved by the use of immunogold techniques, and some reaction products might diffuse into nearby organelles.…”

Section: Discussionmentioning

confidence: 94%

Selective Fusion of Azurophilic Granules with Leishmania-containing Phagosomes in Human Neutrophils

Mollinedo

Janssen

Iglesia-Vicente

et al. 2010

Journal of Biological Chemistry

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Leishmania parasites use polymorphonuclear neutrophils as intermediate hosts before their ultimate delivery to macrophages following engulfment of parasite-infected neutrophils. This leads to a silent and unrecognized entry of Leishmania into the macrophage host cell. Neutrophil function depends on its cytoplasmic granules, but their mobilization and role in how Leishmania parasites evade intracellular killing in neutrophils remain undetermined. Here, we have found by ultrastructural approaches that neutrophils ingested Leishmania major promastigotes, and azurophilic granules fused in a preferential way with parasite-containing phagosomes, without promoting parasite killing. Azurophilic granules, identified by the granule marker myeloperoxidase, also fused with Leishmania donovaniengulfed vacuoles in human neutrophils. In addition, the azurophilic membrane marker CD63 was also detected in the vacuole surrounding the parasite, and in the fusion of azurophilic granules with the parasite-engulfed phagosome. Tertiary and specific granules, involved in vacuole acidification and superoxide anion generation, hardly fused with Leishmania-containing phagosomes. L. major interaction with neutrophils did not elicit production of reactive oxygen species or mobilization of tertiary and specific granules. By using immunogold electron microscopy approaches in the engulfment of L. major and L. donovani by human neutrophils, we did not find a significant contribution of endoplasmic reticulum to the formation of Leishmania-containing vacuoles. Live Leishmania parasites were required to be optimally internalized by neutrophils. Our data suggest that Leishmania promastigotes modulate their uptake by neutrophils, and regulate granule fusion processes in a rather selective way to favor parasite survival in human neutrophils.

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Section: Discussionmentioning

confidence: 94%

Selective Fusion of Azurophilic Granules with Leishmania-containing Phagosomes in Human Neutrophils

Mollinedo

Janssen

Iglesia-Vicente

et al. 2010

Journal of Biological Chemistry

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“…(a) The coincidence of cellulase activity with ion-activated ATPase in one sharp peak was seen consistently. (b) The density of 1 .16 to 1 .17 g/cm3, at which the latent cellulase activity was found, corresponds to the density of plasma membrane fractions in other systems (1,2,6,7,18,19). (c) The membrane fraction containing the particulate cellulase activity also showed the highest proportion of plasma membranes as judged by the PACP stain, which is selective for the plasma membrane of bean abscission zones (Fig.…”

Section: Discussionmentioning

confidence: 99%

Association of Latent Cellulase Activity with Plasma Membranes from Kidney Bean Abscission Zones

Koehler

Leonard²,

VanDerWoude³

et al. 1976

Plant Physiol.

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Membranes isolated from abscission zones of Phaseolus vulgaris L., cv. Red Kidney, contained cellulase activity. This particulate activity was enhanced 10-to 20-fold by treatment with Triton X-100. Sucrose density gradient analyses of cell fractions showed that the membranes with which celiulase was associated had a peak equilibrium density of 1.16 to 1.17 g/ cm3 which coincided with that of ion-activated ATPase, a marker for plasma membranes. The membrane fraction having the highest cellulase activity also contained a high proportion of plasma membranes as shown by electron microscopy of sucrose density gradient fractions after staining by periodic acid-chromic acid-phosphotungstic acid. It was concluded that the particulate celiulase was associated with the plasma membrane.Abscission zones from kidney bean seedlings contained cellulase which existed in two forms (9,14). An intracellular form with a p13 of 4.5 was present at all times, and an extracellular form with a pl of 9.5 increased during abscission or after treatment with ethylene (10). The 4.5-pI form was buffer-soluble whereas the 9.5-pI form was extracted from cell walls by high salt. The 9.5-pl form hydrolyzed cellulose (9) and appeared to cause breakdown of cell walls during abscission.The relationship between the forms of cellulase and their function during abscission is not known. One possibility is that the 4.5-pI form is transported out of the cell to give rise to the 9.5-pI form which functions in cell wall breakdown. As a first step to testing this hypothesis, we have determined that cellulase is associated with cell membranes and identified the membrane system with which it is associated. The results show that from 5 to 10% of the total cellulase activity in the tissue was associated with the plasma membrane whereas no significant activity was found in other membranes. MATERIALS AND METHODSPlant Material and Cell Fractionation. Seeds of Phaseolus vulgaris L., cv. Red Kidney, were germinated in a mixture of vermiculite and pea gravel (3:1) and grown for 10 to 12 days at 24 C and a light intensity of 2,000 ft-c. One-cm sections of petiole including the laminar abscission zone were excised into a chilled beaker and homogenized at a ratio of 3 ml buffer/I g fresh weight of tissue in a polytron (Brinkmann Instruments) at 2 C for 45 sec at a setting of 5. The homogenization buffer contained 20 mm tris-MES, pH 7.2, 0.3 M sucrose, 3 mM EDTA, and 0.1 % BSA. The homogenate was squeezed through a 50-,um mesh nylon cloth, and cell fractions were collected by centrifugation at 2,000g for 10 min and 13,000g for 15 min at 2 C. These pellets were washed by suspending in homogenization buffer and centrifuging at the original forces. An additional cell fraction was obtained by centrifuging the 13,000g supernatant at 80,000g for 45 min onto a cushion of 55% (w/w) sucrose.This fraction was washed by suspending in homogenization buffer without sucrose and pelleting again onto a sucrose cushion.Sucrose Density Gradients. Linear sucrose density gradients we...

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“…What makes the membranes of the lysosomal system different from other subcellular membranes in this regard is not known at the present time. However, this would appear to be an attractive system for future investigation, since Acanthamoeba's phagolysosomal membrane is chemically and enzymatically very similar to the plasma membrane (27), which appears to have relatively few components (13). In addition, a considerable amount of chemical and enzymatic data on these membranes has already been obtained by E. D. Korn and his group (4,11,13,27).…”

Section: Oates and Touster In Vitro Fusion Of Phagolysosomes 335mentioning

confidence: 99%

“…swELLING: When washing was completed (final recovery of cells, 85 90%), each group was recombined in 40 ml of GM into a separate tube and pelleted, accelerating to 1,500 rpm (530 g) at 1 min. The cell pellets (approximately 2.5 ml each) were each resuspended to 40 ml with ice-cold, 0.02 M Tris-HCl buffer, pH 7.4, and the cells were allowed to swell at 0~ for 5-15 min (27). HOMOGENIZATION" The cells were then pelleted as above and the pellets were resuspended to 6 ml with 0.02 M Tris and transferred to two 8-ml Dounce homogenizers on ice.…”

Section: In Vitro Fusion Experimentsmentioning

confidence: 99%

In vitro fusion of Acanthamoeba phagolysosomes. I. Demonstration and quantitation of vacuole fusion in Acanthamoeba homogenates.

Oates¹,

Touster²

1976

The Journal of Cell Biology

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Fusion of phagolysosomes (PLs) has been demonstrated to occur in vitro. Two separate cell homogenates of the ameba Acanthamoeba sp. (Neff) were prepared, each rich in PLs labeled with distinctive particulate markers. Portions of each were incubated together in vitro and fusion occurred as evidenced by the appearance of PLs containing both types of markers. Fusion was confirmed by electron microscopy, including serial sectioning. The membranes of fused vacuoles excluded the dye eosin Y. Surviving cells in the homogenates were not responsible for the observed fusion. Fusion was obtained using either synthetic markers (polystyrene and polyvinyltoluene latex) or biological markers (autoclaved yeast cells and glutaraldehyde-fixed goat red blood cells), or a combination of both. The specificity of PL fusion in vivo appeared to be maintained in vitro.As determined by light and electron microscopy, the fusion reaction was dependent on time and temperature, and on the initial presence of membrane around both marker particles. A minimum of 10% of the vacuoles fused by l0 min of incubation at 30~ and no rupture of the vacuoles was detected during this time. After l0 min of incubation, vacuole rupture began and fusion ceased. At a constant initial vacuole concentration, the extent of PL fusion in vitro was quantitatively reproducible. This appears to be a promising system for further investigation of membrane fusion in the lysosomal system.

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PLASMA AND PHAGOSOME MEMBRANES OF ACANTHAMOEBA CASTELLANII

Cited by 116 publications

References 64 publications

Selective Fusion of Azurophilic Granules with Leishmania-containing Phagosomes in Human Neutrophils

Selective Fusion of Azurophilic Granules with Leishmania-containing Phagosomes in Human Neutrophils

Association of Latent Cellulase Activity with Plasma Membranes from Kidney Bean Abscission Zones

In vitro fusion of Acanthamoeba phagolysosomes. I. Demonstration and quantitation of vacuole fusion in Acanthamoeba homogenates.

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