• Caveolin limits membrane microdomain mobility and integrin-mediated uptake of fibronectin-binding pathogens.

      Hoffmann, Christine; Berking, Anne; Agerer, Franziska; Buntru, Alexander; Neske, Florian; Chhatwal, G Singh; Ohlsen, Knut; Hauck, Christof R; Lehrstuhl Zellbiologie X908, Universität Konstanz, Universitätsstr. 10, 78457 Konstanz, Germany. (2010-12-15)
      Staphylococcus aureus, which is a leading cause of hospital-acquired infections, binds via fibronectin to integrin α5β1, a process that can promote host colonization in vivo. Integrin engagement induces actin cytoskeleton rearrangements that result in the uptake of S. aureus by non-professional phagocytic cells. Interestingly, we found that fibronectin-binding S. aureus trigger the redistribution of membrane microdomain components. In particular, ganglioside GM1 and GPI-linked proteins were recruited upon integrin β1 engagement, and disruption of membrane microdomains blocked bacterial internalization. Several membrane-microdomain-associated proteins, such as flotillin-1 and flotillin-2, as well as caveolin, were recruited to sites of bacterial attachment. Whereas dominant-negative versions of flotillin-2 did not affect bacterial attachment or internalization, cells deficient for caveolin-1 (Cav1(-/-)) showed increased uptake of S. aureus and other Fn-binding pathogens. Recruitment of membrane microdomains to cell-associated bacteria was unaltered in Cav1(-/-) cells. However, fluorescence recovery after photobleaching (FRAP) revealed an enhanced mobility of membrane-microdomain-associated proteins in the absence of caveolin-1. Enhanced membrane microdomain mobility and increased uptake of S. aureus was repressed by expression of wild-type caveolin-1, but not caveolin-1 G83S, which harbors a point mutation in the caveolin scaffolding domain. Similarly, chemical or physical stimulation of membrane fluidity led to increased uptake of S. aureus. These results highlight a crucial role for caveolin-1 in negative regulation of membrane microdomain mobility, thereby affecting endocytosis of bacteria-engaged integrins. This process might not only limit host cell invasion by integrin-binding bacterial pathogens, but might also be physiologically relevant for integrin-mediated cell adhesion.
    • Golgi-to-phagosome transport of acid sphingomyelinase and prosaposin is mediated by sortilin.

      Wähe, Anna; Kasmapour, Bahram; Schmaderer, Christoph; Liebl, David; Sandhoff, Konrad; Nykjaer, Anders; Griffiths, Gareth; Gutierrez, Maximiliano G; European Molecular Biology Laboratory, Postfach 102209, 69117 Heidelberg, Germany. (2010-07-15)
      Sortilin, also known as neurotensin receptor 3 (NTR3), is a transmembrane protein with a dual function. It acts as a receptor for neuromediators and growth factors at the plasma membrane, but it has also been implicated in binding and transport of some lysosomal proteins. However, the role of sortilin during phagosome maturation has not been investigated before. Here, we show that in macrophages, sortilin is mainly localized in the Golgi and transported to latex-bead phagosomes (LBPs). Using live-cell imaging and electron microscopy, we found that sortilin is delivered to LBPs in a manner that depends on its cytoplasmic tail. We also show that sortilin participates in the direct delivery of acid sphingomyelinase (ASM) and prosaposin (PS) to the phagosome, bypassing fusion with lysosomal compartments. Further analysis confirmed that ASM and PS are targeted to the phagosome by sortilin in a Brefeldin-A-sensitive pathway. Analysis of primary macrophages isolated from Sort1(-/-) mice indicated that the delivery of ASM and PS, but not pro-cathepsin D, to LBPs was severely impaired. We propose a pathway mediated by sortilin by which selected lysosomal proteins are transported to the phagosome along a Golgi-dependent route during the maturation of phagosomes.
    • Identification of an immune-regulated phagosomal Rab cascade in macrophages.

      Pei, Gang; Repnik, Urska; Griffiths, Gareth; Gutierrez, Maximiliano Gabriel; Helmholtz Centre for infection research, Inhoffenstr. 7 , D-38124 Braunschweig, Germany. (2014-05-01)
      Interferon-γ (IFN-γ) has been shown to regulate phagosome trafficking and function in macrophages, but the molecular mechanisms involved are poorly understood. Here, we identify Rab20 as part of the machinery by which IFN-γ controls phagosome maturation. We found that IFN-γ stimulates the association of Rab20 with early phagosomes in macrophages. By using imaging of single phagosomes in live cells, we found that Rab20 induces an early delay in phagosome maturation and extends the time for which Rab5a and phosphatidylinositol 3-phosphate (PI3P) remain associated with phagosomes. Moreover, Rab20 depletion in macrophages abrogates the delay in phagosome maturation induced by IFN-γ. Finally, we demonstrate that Rab20 interacts with the Rab5a guanine nucleotide exchange factor Rabex-5 (also known as RABGEF1) and that Rab20 knockdown impairs the IFN-γ-dependent recruitment of Rabex-5 and Rab5a into phagosomes. Taken together, here, we uncover Rab20 as a key player in the Rab cascade by which IFN-γ induces a delay in phagosome maturation in macrophages.