Reconstitution of recycling from the phagosomal compartment

LEIVA N.; PAVAROTTI M; COLOMBO MI; DAMIANI MT

EXPERIMENTAL CELL RESEARCH

Elsevier

Lugar: United States; Año: 2006 vol. 312 p. 1843 - 1855

0014-4827

By phagocytosis, macrophages engulf large particles, microorganisms and senescent cells invesicles called phagosomes. Many internalized proteins rapidly shuttle back to the plasmamembrane following phagosome biogenesis. Here, we report a new approach to the study ofrecycling from the phagosomal compartment: streptolysin O- (SLO) permeabilizedmacrophages. In this semi-intact cell system, energy and cytosol are required toefficiently reconstitute recycling transport. Addition of GDPβS strongly inhibits thistransport step, suggesting that a GTP-binding protein modulates the dynamics of cargoexit from the phagosomal compartment. GTPases of the Rab family control vesiculartrafficking, and Rab11 is involved in transferrin receptor recycling. To unravel the role ofRab11 in the phagocytic pathway, we added recombinant proteins to SLO-permeabilizedmacrophages. Rab11:S25N, a negative mutant, strongly diminishes the release of recycledproteins from phagosomes. In contrast, wild type Rab11 and its positive mutant (Rab11:Q70L) favor this vesicular transport event. Using biochemical and morphological assays, weconfirm that overexpression of Rab11:S25N substantially decreases recycling fromphagosomes in intact cells. These findings show the requirement of a functional Rab11for the retrieval to the plasma membrane of phagosomal content. SLO-permeabilizedmacrophages likely constitute a useful tool to identify new molecules involved in regulatingtransport along the phagocytic pathway.