Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis

Abstract

The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1 ؊ cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1 ؊ cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1 ؊ cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation. INTRODUCTIONPhagocytosis involves the uptake of large particles (typically 1 m in diameter) by phagocytic cells. In the human organism, phagocytic cells are responsible for the elimination of invasive microorganisms and thus participate in the immune defense. Phagocytosis is triggered by the recognition of particles by cell surface phagocyte receptors. This event results in the activation of signaling pathways, mainly depending on phosphatidyl inositol 3-kinase (Cox et al., 1999;Vieira et al., 2001) and syk tyrosine kinase (Durden and Liu, 1994;Greenberg et al., 1994;Cox et al., 1996) and leading to actin polymerization underneath the contact zone, pseudopodia extension, and particles sequestration in membranebound organelles called phagosomes. Phagosomes undergo a maturation process, which involves extensive recycling of proteins back to the cell surface and fusion with endocytic compartments. Whereas the fusion of endosomes with maturing phagosomes has been investigated in detail (for review, see Vieira et al., 2002), the mechanisms responsible for the transport of components into and out of phagosomes are still poorly understood.Transport of proteins in the endocytic and biosynthetic pathways occurs via vesicles coated with specific proteins (Mellman, 1996;Rothman and Wieland, 1996). One of the best-characterized coat proteins is clathrin, which participates in multiple transport steps (Hirst and Robinson, 1998;Smith and Pearse, 1999). There is some evidence that clathrin associates with the phagocytic cup in mammalian cells, although its precise role remains unclear (Aggeler and Werb, 1982;Montesano et al., 1983;Clerc and Sansonetti, 1989). Because adaptor protein complexes (APs) are the main components of clathrin-coated vesicles, a specific AP complex is likely to be associated to clathrin on phagosomes. One early study on APs has reported that AP-2 adaptor complexes...

AP-1 and ARF1 Control Endosomal Dynamics at Sites of FcR–mediated Phagocytosis

“…In addition, AP-1 was found on punctate endosomal structures in the cytoplasm underneath the forming phagosome, but did not decorate the plasma membrane or the membrane of the phagosome. This is consistent with the localization of AP-1 in mammalian phagocytes as reported previously in Lefkir et al (2004) and is different from the situation in Dictyostelium, where AP-1 was found on phagosomal membranes and could be identified on purified phagosomal fractions. The pattern of accumulation we observed below phagocytosed particles in murine macrophages is very different from the pattern of VAMP3 recruitment on forming phagosomes (Bajno et al, 2000;Niedergang et al, 2003), which indicates that the AP-1-positive membranes do not fuse with the plasma membrane, whereas VAMP3-positive membranes do so (see Figure 7).…”

“…These observations were made when we stained the cells with antibodies against clathrin or AP-2 or when we expressed clathrinDsRed or alpha-adaptin-GFP, which rules out a problem of detection by the antibodies used. This observation differs from some studies reporting the presence of clathrin in phagocytic cups (Aggeler and Werb, 1982;Takemura et al, 1986;Perry et al, 1999;Castellano et al, 2000;Lefkir et al, 2004). Many differences in the experimental systems might explain these discrepancies.…”

“…So far, only AP-1 has been connected to the phagocytic process. The implication of AP-1 has been reported in Dictyostelium discoideum, where AP-1 was shown to be necessary for efficient phagocytosis (Lefkir et al, 2004). The efficiency of uptake in the mu1 mutants was dependent on the size of the particles to internalize, indicating that the defect of phagocytosis in the mu1 mutant cells was at the stage of pseudopod extension.…”

WASH drives early recycling from macropinosomes and phagosomes to maintain surface phagocytic receptors

“…6F). phagosomal and macropinosome membranes, the specificity of protein internalization is unclear (49)(50)(51). To assess this lack of specificity, we labeled surface proteins by biotinylation and looked for their presence in purified early phagosomes.…”

Apoptotic cell and phagocyte interplay: recognition and consequences in different cell systems

“…Differently from what is observed in Drosophila, the clearance of dying cells in C. elegans , as well as in Dictyostelium discoideum (Arnoult et al 2001), is performed by non specialized neighboring cells. Only a few reports have been made on phagocytosis of dying cells in the D. discoideum (Arnoult et al 2001), the more recent being the involvement of the adaptor complex-1 (AP-1), normally participating in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes (Lefkir et al 2003); the authors generated Dictyostelium mutant cells, disrupted for AP-1 medium chain, and found impaired phagocytosis, mainly for large particles. There are no evidences, until now, of circulating molecules important for the process of engulfment in C. elegans.…”