Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Jan 1;377(Pt 1):159–169. doi: 10.1042/BJ20031253

Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis.

Joanna Rejman 1, Volker Oberle 1, Inge S Zuhorn 1, Dick Hoekstra 1
PMCID: PMC1223843  PMID: 14505488

Abstract

Non-phagocytic eukaryotic cells can internalize particles <1 microm in size, encompassing pathogens, liposomes for drug delivery or lipoplexes applied in gene delivery. In the present study, we have investigated the effect of particle size on the pathway of entry and subsequent intracellular fate in non-phagocytic B16 cells, using a range of fluorescent latex beads of defined sizes (50-1000 nm). Our data reveal that particles as large as 500 nm were internalized by cells via an energy-dependent process. With an increase in size (50-500 nm), cholesterol depletion increased the efficiency of inhibition of uptake. The processing of the smaller particles was significantly perturbed upon microtubule disruption, while displaying a negligible effect on that of the 500 nm beads. Inhibitor and co-localization studies revealed that the mechanism by which the beads were internalized, and their subsequent intracellular routing, was strongly dependent on particle size. Internalization of microspheres with a diameter <200 nm involved clathrin-coated pits. With increasing size, a shift to a mechanism that relied on caveolae-mediated internalization became apparent, which became the predominant pathway of entry for particles of 500 nm in size. At these conditions, delivery to the lysosomes was no longer apparent. The data indicate that the size itself of (ligand-devoid) particles can determine the pathway of entry. The clathrin-mediated pathway of endocytosis shows an upper size limit for internalization of approx. 200 nm, and kinetic parameters may determine the almost exclusive internalization of such particles along this pathway rather than via caveolae.

Full Text

The Full Text of this article is available as a PDF (296.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson H. A., Chen Y., Norkin L. C. MHC class I molecules are enriched in caveolae but do not enter with simian virus 40. J Gen Virol. 1998 Jun;79(Pt 6):1469–1477. doi: 10.1099/0022-1317-79-6-1469. [DOI] [PubMed] [Google Scholar]
  2. Aoki T., Nomura R., Fujimoto T. Tyrosine phosphorylation of caveolin-1 in the endothelium. Exp Cell Res. 1999 Dec 15;253(2):629–636. doi: 10.1006/excr.1999.4652. [DOI] [PubMed] [Google Scholar]
  3. Benmerah A., Bayrou M., Cerf-Bensussan N., Dautry-Varsat A. Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J Cell Sci. 1999 May;112(Pt 9):1303–1311. doi: 10.1242/jcs.112.9.1303. [DOI] [PubMed] [Google Scholar]
  4. Benmerah A., Lamaze C., Bègue B., Schmid S. L., Dautry-Varsat A., Cerf-Bensussan N. AP-2/Eps15 interaction is required for receptor-mediated endocytosis. J Cell Biol. 1998 Mar 9;140(5):1055–1062. doi: 10.1083/jcb.140.5.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dangoria N. S., Breau W. C., Anderson H. A., Cishek D. M., Norkin L. C. Extracellular simian virus 40 induces an ERK/MAP kinase-independent signalling pathway that activates primary response genes and promotes virus entry. J Gen Virol. 1996 Sep;77(Pt 9):2173–2182. doi: 10.1099/0022-1317-77-9-2173. [DOI] [PubMed] [Google Scholar]
  6. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Finlay B. B., Ruschkowski S., Dedhar S. Cytoskeletal rearrangements accompanying salmonella entry into epithelial cells. J Cell Sci. 1991 Jun;99(Pt 2):283–296. doi: 10.1242/jcs.99.2.283. [DOI] [PubMed] [Google Scholar]
  8. Godbey W. T., Wu K. K., Mikos A. G. Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):5177–5181. doi: 10.1073/pnas.96.9.5177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grimmer Stine, van Deurs Bo, Sandvig Kirsten. Membrane ruffling and macropinocytosis in A431 cells require cholesterol. J Cell Sci. 2002 Jul 15;115(Pt 14):2953–2962. doi: 10.1242/jcs.115.14.2953. [DOI] [PubMed] [Google Scholar]
  10. Hafez I. M., Maurer N., Cullis P. R. On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids. Gene Ther. 2001 Aug;8(15):1188–1196. doi: 10.1038/sj.gt.3301506. [DOI] [PubMed] [Google Scholar]
  11. Hed J., Hallden G., Johansson S. G., Larsson P. The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J Immunol Methods. 1987 Jul 16;101(1):119–125. doi: 10.1016/0022-1759(87)90224-9. [DOI] [PubMed] [Google Scholar]
  12. Hopwood D., Spiers E. M., Ross P. E., Anderson J. T., McCullough J. B., Murray F. E. Endocytosis of fluorescent microspheres by human oesophageal epithelial cells: comparison between normal and inflamed tissue. Gut. 1995 Nov;37(5):598–602. doi: 10.1136/gut.37.5.598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Innes N. P., Ogden G. R. A technique for the study of endocytosis in human oral epithelial cells. Arch Oral Biol. 1999 Jun;44(6):519–523. doi: 10.1016/s0003-9969(99)00027-8. [DOI] [PubMed] [Google Scholar]
  14. Iwabuchi K., Handa K., Hakomori S. Separation of "glycosphingolipid signaling domain" from caveolin-containing membrane fraction in mouse melanoma B16 cells and its role in cell adhesion coupled with signaling. J Biol Chem. 1998 Dec 11;273(50):33766–33773. doi: 10.1074/jbc.273.50.33766. [DOI] [PubMed] [Google Scholar]
  15. Joiner K. A., Fuhrman S. A., Miettinen H. M., Kasper L. H., Mellman I. Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts. Science. 1990 Aug 10;249(4969):641–646. doi: 10.1126/science.2200126. [DOI] [PubMed] [Google Scholar]
  16. Kilsdonk E. P., Yancey P. G., Stoudt G. W., Bangerter F. W., Johnson W. J., Phillips M. C., Rothblat G. H. Cellular cholesterol efflux mediated by cyclodextrins. J Biol Chem. 1995 Jul 21;270(29):17250–17256. doi: 10.1074/jbc.270.29.17250. [DOI] [PubMed] [Google Scholar]
  17. Larkin J. M., Brown M. S., Goldstein J. L., Anderson R. G. Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts. Cell. 1983 May;33(1):273–285. doi: 10.1016/0092-8674(83)90356-2. [DOI] [PubMed] [Google Scholar]
  18. Lencer W. I., Hirst T. R., Holmes R. K. Membrane traffic and the cellular uptake of cholera toxin. Biochim Biophys Acta. 1999 Jul 8;1450(3):177–190. doi: 10.1016/s0167-4889(99)00070-1. [DOI] [PubMed] [Google Scholar]
  19. Liu Nancy Q., Lossinsky Albert S., Popik Waldemar, Li Xia, Gujuluva Chandrasekhar, Kriederman Benjamin, Roberts Jaclyn, Pushkarsky Tatania, Bukrinsky Michael, Witte Marlys. Human immunodeficiency virus type 1 enters brain microvascular endothelia by macropinocytosis dependent on lipid rafts and the mitogen-activated protein kinase signaling pathway. J Virol. 2002 Jul;76(13):6689–6700. doi: 10.1128/JVI.76.13.6689-6700.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liu P., Anderson R. G. Spatial organization of EGF receptor transmodulation by PDGF. Biochem Biophys Res Commun. 1999 Aug 11;261(3):695–700. doi: 10.1006/bbrc.1999.1082. [DOI] [PubMed] [Google Scholar]
  21. Matlin K. S., Reggio H., Helenius A., Simons K. Pathway of vesicular stomatitis virus entry leading to infection. J Mol Biol. 1982 Apr 15;156(3):609–631. doi: 10.1016/0022-2836(82)90269-8. [DOI] [PubMed] [Google Scholar]
  22. Ohtani Y., Irie T., Uekama K., Fukunaga K., Pitha J. Differential effects of alpha-, beta- and gamma-cyclodextrins on human erythrocytes. Eur J Biochem. 1989 Dec 8;186(1-2):17–22. doi: 10.1111/j.1432-1033.1989.tb15171.x. [DOI] [PubMed] [Google Scholar]
  23. Orlandi P. A., Fishman P. H. Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains. J Cell Biol. 1998 May 18;141(4):905–915. doi: 10.1083/jcb.141.4.905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Parton R. G., Joggerst B., Simons K. Regulated internalization of caveolae. J Cell Biol. 1994 Dec;127(5):1199–1215. doi: 10.1083/jcb.127.5.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pedroso de Lima M. C., Simões S., Pires P., Faneca H., Düzgüneş N. Cationic lipid-DNA complexes in gene delivery: from biophysics to biological applications. Adv Drug Deliv Rev. 2001 Apr 25;47(2-3):277–294. doi: 10.1016/s0169-409x(01)00110-7. [DOI] [PubMed] [Google Scholar]
  26. Pitha J., Irie T., Sklar P. B., Nye J. S. Drug solubilizers to aid pharmacologists: amorphous cyclodextrin derivatives. Life Sci. 1988;43(6):493–502. doi: 10.1016/0024-3205(88)90150-6. [DOI] [PubMed] [Google Scholar]
  27. Puri V., Watanabe R., Singh R. D., Dominguez M., Brown J. C., Wheatley C. L., Marks D. L., Pagano R. E. Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways. J Cell Biol. 2001 Jul 30;154(3):535–547. doi: 10.1083/jcb.200102084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ridley A. J. Membrane ruffling and signal transduction. Bioessays. 1994 May;16(5):321–327. doi: 10.1002/bies.950160506. [DOI] [PubMed] [Google Scholar]
  29. Rodal S. K., Skretting G., Garred O., Vilhardt F., van Deurs B., Sandvig K. Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol Biol Cell. 1999 Apr;10(4):961–974. doi: 10.1091/mbc.10.4.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ross P. C., Hui S. W. Lipoplex size is a major determinant of in vitro lipofection efficiency. Gene Ther. 1999 Apr;6(4):651–659. doi: 10.1038/sj.gt.3300863. [DOI] [PubMed] [Google Scholar]
  31. Rothberg K. G., Heuser J. E., Donzell W. C., Ying Y. S., Glenney J. R., Anderson R. G. Caveolin, a protein component of caveolae membrane coats. Cell. 1992 Feb 21;68(4):673–682. doi: 10.1016/0092-8674(92)90143-z. [DOI] [PubMed] [Google Scholar]
  32. Schnitzer J. E., Oh P., Pinney E., Allard J. Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J Cell Biol. 1994 Dec;127(5):1217–1232. doi: 10.1083/jcb.127.5.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shin J. S., Abraham S. N. Caveolae as portals of entry for microbes. Microbes Infect. 2001 Jul;3(9):755–761. doi: 10.1016/s1286-4579(01)01423-x. [DOI] [PubMed] [Google Scholar]
  34. Shin J. S., Gao Z., Abraham S. N. Involvement of cellular caveolae in bacterial entry into mast cells. Science. 2000 Aug 4;289(5480):785–788. doi: 10.1126/science.289.5480.785. [DOI] [PubMed] [Google Scholar]
  35. Stang E., Kartenbeck J., Parton R. G. Major histocompatibility complex class I molecules mediate association of SV40 with caveolae. Mol Biol Cell. 1997 Jan;8(1):47–57. doi: 10.1091/mbc.8.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Subtil A., Gaidarov I., Kobylarz K., Lampson M. A., Keen J. H., McGraw T. E. Acute cholesterol depletion inhibits clathrin-coated pit budding. Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6775–6780. doi: 10.1073/pnas.96.12.6775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Swanson J. A., Watts C. Macropinocytosis. Trends Cell Biol. 1995 Nov;5(11):424–428. doi: 10.1016/s0962-8924(00)89101-1. [DOI] [PubMed] [Google Scholar]
  38. Wang L. H., Rothberg K. G., Anderson R. G. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J Cell Biol. 1993 Dec;123(5):1107–1117. doi: 10.1083/jcb.123.5.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Werling D., Hope J. C., Chaplin P., Collins R. A., Taylor G., Howard C. J. Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells. J Leukoc Biol. 1999 Jul;66(1):50–58. doi: 10.1002/jlb.66.1.50. [DOI] [PubMed] [Google Scholar]
  40. Wrobel I., Collins D. Fusion of cationic liposomes with mammalian cells occurs after endocytosis. Biochim Biophys Acta. 1995 May 4;1235(2):296–304. doi: 10.1016/0005-2736(95)80017-a. [DOI] [PubMed] [Google Scholar]
  41. Xu Y., Hui S. W., Frederik P., Szoka F. C., Jr Physicochemical characterization and purification of cationic lipoplexes. Biophys J. 1999 Jul;77(1):341–353. doi: 10.1016/S0006-3495(99)76894-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Xu Y., Szoka F. C., Jr Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry. 1996 May 7;35(18):5616–5623. doi: 10.1021/bi9602019. [DOI] [PubMed] [Google Scholar]
  43. Zabner J., Fasbender A. J., Moninger T., Poellinger K. A., Welsh M. J. Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem. 1995 Aug 11;270(32):18997–19007. doi: 10.1074/jbc.270.32.18997. [DOI] [PubMed] [Google Scholar]
  44. Zhang Y. P., Reimer D. L., Zhang G., Lee P. H., Bally M. B. Self-assembling DNA-lipid particles for gene transfer. Pharm Res. 1997 Feb;14(2):190–196. doi: 10.1023/a:1012000711033. [DOI] [PubMed] [Google Scholar]
  45. Zuhorn I. S., Hoekstra D. On the mechanism of cationic amphiphile-mediated transfection. To fuse or not to fuse: is that the question? J Membr Biol. 2002 Oct 1;189(3):167–179. doi: 10.1007/s00232-002-1015-7. [DOI] [PubMed] [Google Scholar]
  46. Zuhorn Inge S., Kalicharan Ruby, Hoekstra Dick. Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis. J Biol Chem. 2002 Mar 1;277(20):18021–18028. doi: 10.1074/jbc.M111257200. [DOI] [PubMed] [Google Scholar]
  47. Zuhorn Inge S., Visser Willy H., Bakowsky Udo, Engberts Jan B. F. N., Hoekstra Dick. Interference of serum with lipoplex-cell interaction: modulation of intracellular processing. Biochim Biophys Acta. 2002 Feb 18;1560(1-2):25–36. doi: 10.1016/s0005-2736(01)00448-5. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES