Characterization of endocytic compartments after holo-high density lipoprotein particle uptake in HepG2 cells

Clemens Röhrl, Tamara A. Pagler, Witta Strobl, Adolf Ellinger, Josef Neumüller, Margit Pavelka, Herbert Stangl, Claudia Meisslitzer-Ruppitsch

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)

Abstract

Holo-high density lipoprotein (HDL) particle uptake, besides selective lipid uptake, constitutes an alternative pathway to regulate cellular cholesterol homeostasis. In the current study, the cellular path of holo-HDL particles was investigated in human liver carcinoma cells (HepG2) using combined light and electron microscopical methods. The apolipoprotein moiety of HDL was visualized with different markers: horseradish peroxidase, colloidal gold and the fluorochrome Alexa 568, used in fluorescence microscopy and after photooxidation correlatively at the ultrastructural level. Time course experiments showed a rapid uptake of holo-HDL particles, an accumulation in endosomal compartments, with a plateau after 1-2 h of continuous uptake, and a clearance 1-2 h upon replacement by unlabeled HDL. Correlative microscopy, using HDL-Alexa 568-driven diaminobenzidine (DAB) photooxidation, identified the fluorescent organelles as DAB-positive multivesicular bodies (MVBs) in the electron microscope; their luminal contents but not the internal vesicles were stained. Labeled MVBs increased in numbers and changed shapes along with the duration of uptake, from polymorphic organelles with multiple surface domains and differently shaped protrusions dominating at early times of uptake to compact bodies with mainly tubular appendices and densely packed vesicles after later times. Differently shaped and labeled surface domains and appendices, as revealed by three dimensional reconstructions, as well as images of homotypic fusions indicate the dynamics of the HDL-positive MVBs. Double staining visualized by confocal microscopy, along with the electron microscopic data, shows that holo-HDL particles after temporal storage in MVBs are only to a minor degree transported to lysosomes, which suggests that different mechanisms are involved in cellular HDL clearance, including resecretion.

Original languageEnglish
Pages (from-to)261-272
Number of pages12
JournalHistochemistry and Cell Biology
Volume133
Issue number3
DOIs
Publication statusPublished - Mar 2010
Externally publishedYes

Keywords

  • Correlative microscopy
  • Diaminobenzidine (DAB)-photooxidation
  • Electron tomography
  • High density lipoprotein (HDL)
  • Holo particle uptake
  • Multivesicular bodies
  • Horseradish Peroxidase/chemistry
  • Humans
  • Endosomes/chemistry
  • Gold/chemistry
  • Hep G2 Cells
  • Particle Size
  • Endocytosis
  • Lipoproteins, HDL/chemistry

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