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J Cell Biol. Dec 1, 1989; 109(6): 2703–2720.
PMCID: PMC2115901

A quantitative analysis of the endocytic pathway in baby hamster kidney cells

Abstract

A morphological analysis of the compartments of the endocytic pathway in baby hamster kidney (BHK) cells has been made using the fluid-phase marker horseradish peroxidase (HRP). The endocytic structures labeled after increasing times of endocytosis have been identified and their volume and surface densities measured. In the first 2 min of HRP uptake the volume density of the labeled structures increased rapidly and thereafter remained constant for the next 13-18 min. This plateau represents the volume density of endosome organelles and accounts for 0.65% of the cytoplasmic volume (or 6.8 microns 3 per cell). The labeled structures consist of tubular-cisternal elements which are frequently observed in continuity with 300-400 nm vesicles. After 15-20 min of internalization the volume density of HRP-labeled structures again increased rapidly and reached a second plateau between 30 and 60 min of labeling. This second increase corresponded to detectable levels of HRP reaching later, acid phosphatase (AcPase)-reactive compartments. These structures, comprising the prelysosomes and lysosomes, were mostly vesicular and collectively accounted for 3.5% of the cytoplasmic volume (or 37 microns 3 per cell). The absolute peripheral surface areas of the two classes of organelles (endosomes and prelysosomes/lysosomes) were estimated to be 430 and 370 microns 2 per cell, respectively. The volume of fluid internalized in the first 2 min of uptake was five- to sevenfold less than the volume of the compartment labeled in this time. To account for these results we propose that, after uptake from the cell surface, HRP is delivered to, and diluted in, endosomes that are preexisting organelles initially devoid of the marker. With increasing times of endocytosis the concentration of HRP in the early endosomes increases, as more of the marker enters this compartment. An elevation in HRP concentration in endosomes during the early time points was shown directly using anti- HRP antibodies and colloidal gold on cryosections. The stereological values given in the present study, in combination with earlier studies, provide a minimum estimate for both the total surface area of membranes and the rate of membrane synthesis in a BHK cell.

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Selected References

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  • Adams CJ, Maurey KM, Storrie B. Exocytosis of pinocytic contents by Chinese hamster ovary cells. J Cell Biol. 1982 Jun;93(3):632–637. [PMC free article] [PubMed]
  • Anderson RG, Brown MS, Goldstein JL. Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts. Cell. 1977 Mar;10(3):351–364. [PubMed]
  • Bergeron JJ, Cruz J, Khan MN, Posner BI. Uptake of insulin and other ligands into receptor-rich endocytic components of target cells: the endosomal apparatus. Annu Rev Physiol. 1985;47:383–403. [PubMed]
  • Besterman JM, Airhart JA, Woodworth RC, Low RB. Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation. J Cell Biol. 1981 Dec;91(3 Pt 1):716–727. [PMC free article] [PubMed]
  • Cope GH, Williams MA. Quantitative analyses of the constituent membranes of parotid acinar cells and of the changes evident after induced exocytosis. Z Zellforsch Mikrosk Anat. 1973 Dec 6;145(3):311–330. [PubMed]
  • de Chastellier C, Lang T, Ryter A, Thilo L. Exchange kinetics and composition of endocytic membranes in terms of plasma membrane constituents: a morphometric study in macrophages. Eur J Cell Biol. 1987 Aug;44(1):112–123. [PubMed]
  • Geuze HJ, Slot JW, Strous GJ, Lodish HF, Schwartz AL. Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell. 1983 Jan;32(1):277–287. [PubMed]
  • Goldstein JL, Brown MS, Anderson RG, Russell DW, Schneider WJ. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1985;1:1–39. [PubMed]
  • Green J, Griffiths G, Louvard D, Quinn P, Warren G. Passage of viral membrane proteins through the Golgi complex. J Mol Biol. 1981 Nov 15;152(4):663–698. [PubMed]
  • Griffiths G, Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. [PubMed]
  • Griffiths G, Quinn P, Warren G. Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus. J Cell Biol. 1983 Mar;96(3):835–850. [PMC free article] [PubMed]
  • Griffiths G, Warren G, Quinn P, Mathieu-Costello O, Hoppeler H. Density of newly synthesized plasma membrane proteins in intracellular membranes. I. Stereological studies. J Cell Biol. 1984 Jun;98(6):2133–2141. [PMC free article] [PubMed]
  • Griffiths G, McDowall A, Back R, Dubochet J. On the preparation of cryosections for immunocytochemistry. J Ultrastruct Res. 1984 Oct;89(1):65–78. [PubMed]
  • Griffiths G, Hoflack B, Simons K, Mellman I, Kornfeld S. The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell. 1988 Feb 12;52(3):329–341. [PubMed]
  • Griffiths G, Fuller SD, Back R, Hollinshead M, Pfeiffer S, Simons K. The dynamic nature of the Golgi complex. J Cell Biol. 1989 Feb;108(2):277–297. [PMC free article] [PubMed]
  • Gruenberg JE, Howell KE. Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system. EMBO J. 1986 Dec 1;5(12):3091–3101. [PMC free article] [PubMed]
  • Gruenberg J, Howell KE. An internalized transmembrane protein resides in a fusion-competent endosome for less than 5 minutes. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5758–5762. [PMC free article] [PubMed]
  • Gruenberg J, Griffiths G, Howell KE. Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J Cell Biol. 1989 Apr;108(4):1301–1316. [PMC free article] [PubMed]
  • Hopkins CR. Intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma A431 cells. Cell. 1983 Nov;35(1):321–330. [PubMed]
  • Hopkins CR, Trowbridge IS. Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells. J Cell Biol. 1983 Aug;97(2):508–521. [PMC free article] [PubMed]
  • Johanson V, Ofverholm T, Ericson LE. Turnover of apical plasma membrane in thyroid follicle cells of normal and thyroxine-treated rats. Eur J Cell Biol. 1984 Nov;35(2):165–170. [PubMed]
  • Kielian MC, Marsh M, Helenius A. Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus. EMBO J. 1986 Dec 1;5(12):3103–3109. [PMC free article] [PubMed]
  • Marsh M, Helenius A. Adsorptive endocytosis of Semliki Forest virus. J Mol Biol. 1980 Sep 25;142(3):439–454. [PubMed]
  • Marsh M, Bolzau E, Helenius A. Penetration of Semliki Forest virus from acidic prelysosomal vacuoles. Cell. 1983 Mar;32(3):931–940. [PubMed]
  • Marsh M, Griffiths G, Dean GE, Mellman I, Helenius A. Three-dimensional structure of endosomes in BHK-21 cells. Proc Natl Acad Sci U S A. 1986 May;83(9):2899–2903. [PMC free article] [PubMed]
  • Marsh M, Schmid S, Kern H, Harms E, Male P, Mellman I, Helenius A. Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis. J Cell Biol. 1987 Apr;104(4):875–886. [PMC free article] [PubMed]
  • McDowall A, Gruenberg J, Römisch K, Griffiths G. The structure of organelles of the endocytic pathway in hydrated cryosections of cultured cells. Eur J Cell Biol. 1989 Aug;49(2):281–294. [PubMed]
  • Mellman I, Fuchs R, Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. [PubMed]
  • Miller K, Beardmore J, Kanety H, Schlessinger J, Hopkins CR. Localization of the epidermal growth factor (EGF) receptor within the endosome of EGF-stimulated epidermoid carcinoma (A431) cells. J Cell Biol. 1986 Feb;102(2):500–509. [PMC free article] [PubMed]
  • Mueller SC, Hubbard AL. Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes. J Cell Biol. 1986 Mar;102(3):932–942. [PMC free article] [PubMed]
  • Murphy RF. Analysis and isolation of endocytic vesicles by flow cytometry and sorting: demonstration of three kinetically distinct compartments involved in fluid-phase endocytosis. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8523–8526. [PMC free article] [PubMed]
  • Novikoff AB, Novikoff PM. Cytochemical contributions to differentiating GERL from the Golgi apparatus. Histochem J. 1977 Sep;9(5):525–551. [PubMed]
  • Weibel ER, Paumgartner D. Integrated stereological and biochemical studies on hepatocytic membranes. II. Correction of section thickness effect on volume and surface density estimates. J Cell Biol. 1978 May;77(2):584–597. [PMC free article] [PubMed]
  • Pearse BM, Bretscher MS. Membrane recycling by coated vesicles. Annu Rev Biochem. 1981;50:85–101. [PubMed]
  • Schmid SL, Fuchs R, Male P, Mellman I. Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes. Cell. 1988 Jan 15;52(1):73–83. [PubMed]
  • Shio H, Haley NJ, Fowler S. Characterization of lipid-laden aortic cells from cholesterol-fed rabbits. III. Intracellular localization of cholesterol and cholesteryl ester. Lab Invest. 1979 Aug;41(2):160–167. [PubMed]
  • Simons K, Fuller SD. Cell surface polarity in epithelia. Annu Rev Cell Biol. 1985;1:243–288. [PubMed]
  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. [PubMed]
  • Steinman RM, Brodie SE, Cohn ZA. Membrane flow during pinocytosis. A stereologic analysis. J Cell Biol. 1976 Mar;68(3):665–687. [PMC free article] [PubMed]
  • Sullivan PC, Ferris AL, Storrie B. Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles. J Cell Physiol. 1987 Apr;131(1):58–63. [PubMed]
  • Swanson JA, Yirinec BD, Silverstein SC. Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. J Cell Biol. 1985 Mar;100(3):851–859. [PMC free article] [PubMed]
  • Swanson J, Bushnell A, Silverstein SC. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1921–1925. [PMC free article] [PubMed]
  • Thilo L. Quantification of endocytosis-derived membrane traffic. Biochim Biophys Acta. 1985 Sep 9;822(2):243–266. [PubMed]
  • Wall DA, Hubbard AL. Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments. J Cell Biol. 1985 Dec;101(6):2104–2112. [PMC free article] [PubMed]
  • Wall DA, Wilson G, Hubbard AL. The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell. 1980 Aug;21(1):79–93. [PubMed]

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