Abstracts (complete list) - Wissenschaft Online

Nirmal Robinson, Tom Li Stephen, Sonja Meemboor, Wiltrud M. Kalka-Moll, Georg Plum
Phagosomal and endosomal trafficking studies by adenoviral
Rab-GFP fusion proteins in primary cells
Antigen presentation by major histocompatibility complex (MHC) class II on antigen
presenting cells (APCs) is facilitated after a sequence of events that starts with a
phagocytic process, followed by phagosomal processing of the antigen, phagolysosomal
degradation of the phagocytosed material and loading of antigenic peptides onto MHC
molecules. A detailed picture of this process termed phagosome maturation is beginning
to emerge, involving regulators of membrane trafficking in mammalian cells and
phagosomal interactions with endosomal organelles and the trans-Golgi network. So far,
cell biology studies of antigen presentation can be performed in primary cells after
fixation and permeabilization for staining with specific antibodies or probes for other
endosomal pathway . Investigation in live cells is solely possible in cell lines after
transfection with expression vectors for GFP-tagged endosomal marker proteins. In an
effort to open up ways to elucidate the phagosomal processing events after antigen
uptake in live primary antigen presenting cells of murine and human origin we have
developed a set of adenoviral Rab-GFP fusion protein expression vectors that allow
observation and tracking individual phagosomes. Transfection with the early endosomal
marker Rab5-GFP, late endosomal marker Rab7-GFP, and transferrin receptor recycling
marker Rab11-GFP was achieved in 92% to 93% of the mouse dendritic cells, in 89% to
94% of human macrophages, and in 70% to 82% of mouse macrophages. Rab-GFP
fusion proteins were readily expressed in these primary cells using these highly efficient
adenoviral constructs allowing phagosome and endosome trafficking by fluorescently
labeled fluid phase marker. Our data show that the intracellular endocytic trafficking
events preceeding antigen presentation in primary APCs can be effectively studied in
live cells by using our adenoviral expression systems.