Characterization of pinocytic vesicles from CHO cells: Resolution of pinosomes from lysosomes by analytical centrifugation

Pool, R.R.; Maurey, K M; Storrie, Brian

doi:10.1016/0309-1651(83)90076-0

Cited by 33 publications

(39 citation statements)

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“…In a second experiment, no appearance of lysosomally incorporated radioiodine at the plasma membrane was detected after a 2-h chase (data not shown). For fibroblasts, Golgi apparatus is well resolved from lysosomes in 10% Percoll gradients (11,16). No accumulation of radioiodine The overnight uptake period used for the accumulation of HRP or LPO in lysosomes resulted in the unambiguous localization of internalized peroxidase in lysosomes.…”

Section: Resultsmentioning

confidence: 99%

“…Alkaline phosphodiesterase and P-hexosaminidase were assayed as described previously (16). Incorporation of radioactivity into cell proteins was determined by acid precipitation onto glass fiber disks (8).…”

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confidence: 99%

“…For plasma membrane radioiodination, washed cells were incubated at 4°C with 100 ,ug of LPO per ml and radioiodination substrates as previously described (20 (16).…”

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confidence: 99%

“…In experiments in which marker enzyme activity was measured, postnuclear supernatants (16) were prepared and diluted to 0.05 M sucrose with deionized water to lyse organelles. The samples were then incubated on ice for 20 min and centrifuged at 100,000 x gav for 1 h at 2 to 4°C to give a membrane pellet and a soluble supernatant fraction.…”

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confidence: 99%

“…Cells were homogenized by N2 cavitation, and total postnuclear supernatants were prepared and fractionated in 10% Percoll gradients (16,21).…”

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confidence: 99%

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Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

Storrie¹,

Singh²,

Viers³

1984

Mol. Cell. Biol.

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We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t112 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000x g under conditions in which >80% of the lysosomal marker enzyme 1-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37°C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.Recent observations have suggested that many endocytic components of the vacuolar apparatus of mammalian cells are bidirectional or exocytic compartments with respect to the intracellular medium or cell surface. Studies have shown that fluid-phase pinocytosis is a reversible process, with exocytosis of endocytic contents occurring from a pinosomal compartment over a time span of a few minutes (1, 2). Later endocytic compartments than pinosomes may also be exocytic. Over brief time spans (.30 min), Chinese hamster ovary (CHO) cells retain horseradish peroxidase (HRP) internalized into a lysosomal compartment (1). However, over a longer time span, lysosomes may be exocytic. Besterman et al. (2), using sucrose, a small molecule, as a marker, found that lysosomes are a slowly (t112 = 3 to 10 h) exocytic compartment in both macrophages and fibroblasts. Pollack et al. (15) found that HeLa cells very slowly (t112 = 70 h) exocytose ouabain, another small molecule. used lactoperoxidase (LPO) covalently coupled to latex beads to radioiodinate phagolysosome membranes in macrophages. These investigators reported a rapid bidirectional flow of radioiodinated membrane proteins from phagolysosomes to plasma membranes and back which occurs over a period of a few minutes and may occur even at 4°C.In the present work, we have addressed directly the question of whether lysosomes are a slowly exocytic compartment for macromolecules. As ,ug of LPO per ml and radioiodination substrates as previously described (20

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

“…For plasma membrane radioiodination, washed cells were incubated at 4°C with 100 ,ug of LPO per ml and radioiodination substrates as previously described (20 (16).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

“…Cells were homogenized by N2 cavitation, and total postnuclear supernatants were prepared and fractionated in 10% Percoll gradients (16,21).…”

mentioning

confidence: 99%

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Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

Storrie¹,

Singh²,

Viers³

1984

Mol. Cell. Biol.

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Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles

Sullivan

Ferris

Storrie

1987

Journal Cellular Physiology

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We investigated the effects of reduced temperature, the pH elevators NH4Cl, monensin, and HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) buffer, as well as the metabolic poisons NaF/KCN on transport of the fluid phase pinocytic marker, horseradish peroxidase (HRP), to lysosomes in Chinese hamster ovary (CHO) cells. In cell fractionation experiments, these agents appeared to block HRP transit at specific point(s) from "early" to "late" (i.e., low to high density) prelysosomal vesicles and lysosomes. Reduced temperature (17 degrees C) most strongly inhibited HRP transport from low density, early endosomes to lysosomes. In long-term HRP uptakes at 17 degrees C, marked peroxidase accumulation occurred both in early endosomes and in lysosomes. Loss (reversible pinocytosis) of HRP from "very early" endosomes occurred at 17 degrees C. All three pH elevators including the common media supplement HEPES buffer inhibited transit of internalized HRP into lysosomes. For all three pH elevators, inhibition was most pronounced at the "early" endosome stage. The respiratory inhibitors NaF/KCN also inhibited transport most strongly at the early endosome stage. Together these results suggest that "early" steps in the endocytic transport of HRP are the most sensitive and that the conditions tested may exert direct effects on the processing of endocytic vesicles.

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Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments

Park

Sullivan

Storrie

1988

Journal Cellular Physiology

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Incubation of animal cells with hypertonic sucrose and polyethylene glycol (PEG) 1,000 renders endosomes sensitive in situ to hypotonic shock (Okada and Rechsteiner, 1982). We found that: 1) in vitro endosomes were osmotically insensitive; and 2) hypertonic sucrose inhibited transport from very early endosomes to lysosomes. Endocytic vesicles were labeled by incubating Chinese hamster ovary (CHO) cells for 1-10 min at 37 degrees C with horseradish peroxidase (HRP) and/or fluorescein isothiocyanate-conjugated dextran (FITC-dextran). Cell fractions prepared in 0.25 M sucrose were hypotonically shocked by dilution with 5 mM Na phosphate buffer, pH 6.7, to a final sucrose concentration of 0.05 M. After hypotonic shock, endocytized HRP and FITC-dextran pelleted with membrane while lysosomal hydrolases did not. The HRP activity in the pellet was latent, suggesting that endosomes were resistant to osmotic shock. Uptake in the presence of hypertonic sucrose had little effect on the subsequent osmotic sensitivity of the endosomes. Uptake in the presence of hypertonic sucrose and PEG 1,000 rendered endosomes fragile to cell homogenization. Unexpectedly, the inclusion of hypertonic sucrose in the uptake and chase media inhibited the appearance of HRP in lysosomes. HRP internalized during a 10-min uptake appeared as if it were present in two physically distinct compartments, one accessible to transport inhibition by exogenous sucrose ("very early" endosomes) and the other not ("early" endosomes). After a brief uptake (1-3 min), postincubation of CHO cells in 0.25 M sucrose-containing media completely blocked transport of internalized HRP to lysosomes. This blockage could be partially relieved by cointernalization of invertase with HRP. These results suggest that transport between multiple early endosome populations is sensitive to intraorganellar osmotic conditions.

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Characterization of pinocytic vesicles from CHO cells: Resolution of pinosomes from lysosomes by analytical centrifugation

Cited by 33 publications

References 12 publications

Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles

Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments

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